Sample records for fii g20210a fv

  1. The frequencies of FV Leiden and FII G20210A mutations in patients with different clinical manifestations of venous thromboembolism: Experience from large Serbian cohort

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    Tomić Branko


    Full Text Available Venous thromboembolism is a multifactorial disorder with two manifestations: deep-vein thrombosis and pulmonary embolism. Pulmonary embolism is usually considered as the complication of deep-vein thrombosis, but there are reported cases of isolated pulmonary embolism. FV Leiden and FII G20210A mutations are most common genetic risk factors for the venous thromboembolism. Several studies reported "FV Leiden paradox": lower prevalence of FV Leiden mutation among patients with isolated pulmonary embolism than among those with deep-vein thrombosis. The aim of this study was to determine FV Leiden and FII G20210A mutations frequency in thrombophilic patients in Serbian population. We tested prevalence of these mutations carriers in 1427 individuals divided in three groups of patients (with deep-vein thrombosis, deep-vein thrombosis/ pulmonary embolism and isolated pulmonary embolism and control group. All subjects were tested for these mutations using PCR-RFLP analysis. Detected frequency of FV Leiden heterozygous carriers in patients with isolated pulmonary embolism was 6.9% (for FII G20210A 11.6%, while in other two groups of patients with deep-vein thrombosis and deep vein thrombosis/pulmonary embolism, frequency was 18.6% (for FII G20210A mutation were 11.6% and 8.3%, respectively. Our results showed that FV Leiden mutation is less frequent in patients with isolated pulmonary embolism than in patients with deep-vein thrombosis or deep-vein thrombosis accompanied with pulmonary embolism, confirming "FV Leiden paradox". On the other hand, detected frequency of FII G20210A mutation carriers was similar in all three groups of patients. [Projekat Ministarstva nauke Republike Srbije, br. 173008

  2. [Prevalence of thrombophilic mutations of FV Leiden, prothrombin G20210A and PAl-1 4G/5G and their combinations in a group of 1450 healthy middle-aged individuals in the Prague and Central Bohemian regions (results of FRET real-time PCR assay)]. (United States)

    Kvasnicka, Jan; Hájková, Jaroslava; Bobcíková, Petra; Kvasnicka, Tomás; Dusková, Daniela; Poletínová, Sárka; Kieferová, Veronika


    Factor V Leiden (G1691A) and prothrombin gene (FII G20210A) mutations are independent risk factors of venous thrombosis and this risk is further increased by the combined genotype 4G/4G PAI-1. The primary objective was to identify the frequency of mutations of minor alleles and genotypes of FVL, FII G20210A and PAI-1 4G/5G in healthy Caucasians in the Prague and Central Bohemia regions. The secondary objective was to identify the occurrence of their mutual combinations. Genotyping was performed in 1,450 healthy individuals (blood donors, 981 men and 469 women) using robotic DNA isolation and subsequent PCR and melting curve analysis (Light Cycler 480 System, Roche). The minor allele frequencies in FV Leiden and FII G20210A mutations were 4.5% and 1.3% respectively. The frequency of the 4G PAI-1 allele was 55.9%. The genotype frequencies were as follows: GG 91.10%, GA 8.83% and AA 0.07% for FV Leiden; GG 97.38%, GA 2.55% and AA 0.07% for FII G20210A and 4G/4G 30.69%, 4G/5G 50.34% and 5G/5G 18.97% for PAI-1. No differences in these frequencies were found between the genders. The occurrence of the combined heterozygous FII and heterozygous FV Leiden mutations was 0.14%. The PAI-1 4G/4G genotype was combined with the heterozygous FV leiden mutation in 2.83% of cases and with the heterozygous FII mutation in 0.62% of cases. The found frequencies of genotypes and alleles confirm a relatively high prevalence of hereditary thrombophilia in the Czech Republic.

  3. Prevalence of factor V Leiden and prothrombin G20210A mutation in a large French population selected for nonthrombotic history: geographical and age distribution. (United States)

    Mazoyer, Elisabeth; Ripoll, Laurent; Gueguen, René; Tiret, Laurence; Collet, Jean-Philippe; dit Sollier, Claire Bal; Roussi, Jacqueline; Drouet, Ludovic


    Among inherited risk factors for venous thrombosis, the most common are the FV-G1691A and FII-G20210A polymorphisms. The FV-G1691A polymorphism is preferentially observed in Europe, with differences between European countries. The FII-G20210A polymorphism is observed all over the world. The study was designed to compare the prevalence of the FV-G1691A and FII-G20210A polymorphisms in a large French population of unrelated individuals with no thrombotic disease history and to determine the age and geographical distributions. Over a period of 18 months, 6154 individuals were included throughout France and FV-G1691A and FII-G20210A polymorphisms were determined. The FV-G1691A prevalence was 3.84% (95% confidence interval 3.35-4.33) and the FII-G20210A prevalence was 3.07% (95% CI 2.63-3.51). A north-east/south-west gradient was observed in the FV-G1691A geographical distribution. No difference was observed in the geographical distribution of FII-G20210A polymorphism nor in the age distribution of the two polymorphisms. The prevalence of the two polymorphisms was similar whatever the blood group (O or non-O). Plasma D-dimers were significantly higher in healthy individuals with FV-G1691A but not in individuals with FII-G20210A. Thirty percent of variation in plasma prothrombin level was explained by environmental factors (serum cholesterol, age, oral contraception, hormonal replacement therapy, body mass index, sex) and genetic factors (FII-G20210A). As expected, individuals with FII-G20210A displayed higher plasma prothrombin level compared with individuals with wild type. However, this was not associated with a modification of the fibrin clot elastic modulus. This study shows a differential distribution of the two polymorphisms among the French territory. These polymorphisms confer a very mild hypercoagulable state as shown by the limited increased in basal D-dimers in mutated FV-G1691A populations and only a trend that does not reach statistical significance for FII-G

  4. Meta-analysis of factor V Leiden and prothrombin G20210A polymorphism in migraine. (United States)

    Lippi, Giuseppe; Mattiuzzi, Camilla; Cervellin, Gianfranco


    Migraine is a frequent and disabling condition, which exhibits a substantial genetic background and is frequently associated with abnormalities of primary and secondary hemostasis. We performed a systematic literature search and a meta-analysis of available data about the potential associations between migraine and factor V (FV) Leiden or prothrombin (FII) G20210A gene polymorphism. The final number of studies included was 15 (all cross-sectional) about migraine and FV Leiden, and 12 (all cross-sectional) about migraine and FII G20210A polymorphism, with broad inter-study heterogeneity (I², 82 and 85%). The cumulative prevalence of the FV 1691A allele was found to be similar between cases (n = 1450; 4.9%) and controls (n = 3468; 4.7%; P = 0.74). The cumulative prevalence of the FII 20210A allele was also found to be similar between cases (n = 1226; 4.2%) and controls (n = 3144; 4.5%; P = 0.59). Nevertheless, sub-analysis of studies in adults and children revealed that both polymorphisms were not associated with migraine in adults, but FV Leiden and the FII 20210A allele were approximately two-fold more prevalent in children with migraine than in those without. In conclusion, despite migraine exhibits a clear neurovascular origin and is frequently associated with thrombotic disorders, isolate thrombophilic mutations seem to play a negligible pathogenetic role in this condition in adults, whereas the increased prevalence of FV Leiden and the FII 20210A allele in children with migraine deserves further scrutiny.

  5. Prevalence of factor V G1691A (Leiden) and prothrombin G20210A polymorphisms among apparently healthy Jordanians. (United States)

    Nusier, Mohamad K; Radaideh, Abdelrahman M; Ababneh, Nida'a A; Qaqish, Bara'ah M; Alzoubi, Renad; Khader, Yousef; Mersa, Janet Y; Irshaid, Nidal M; El-Khateeb, Mohammed


    Factor V Leiden and prothrombin G20210A are related genetic risk factors for venous thromboembolism (VTE). Analysis for both mutations is increasingly being performed on patients exhibiting hypercoagulability. The objective of this study was to determine the prevalence of factor V Leiden (FVL), prothrombin-G20210A (PT-G20210A) polymorphisms and their coexistence among apparently healthy Jordanians. One thousand apparently healthy individuals from representative regions of Jordan with no previous history of VTE participated in this study. The mean age of participants was 28.5+/-6.4 years (age range 18-45 years). Two hundred and eighteen subjects were APC resistant with an APC-R mean of 85.52+/-15.35 seconds; the non-resistant subjects had an APC-R mean of 159.90+/-26.96 seconds. A multiplex polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for the simultaneous detection of FVL and prothrombin G20210A was used to analyze the 218 DNA samples that were APC-R resistant. Both mutations generate HindIII RFLPs and the prothrombin amplicon contains an invariant HindIII recognition sites. The multiplex PCR-RFLP of Factor V for those 218-samples was: 41 wild-type, 169 heterozygous mutant, and eight homozygous mutant individuals. For prothrombin G20210A, the multiplex PCR-RFLP identified 215 wild-type and three heterozygous mutant individuals. Factor V positive individuals (n=50) had a mean F-V activity of 78.04%+/-25.81. F-V activity among wild type (n=41), F-V Leiden heterozygous (n=169) and F-V Leiden homozygous (n=8) were 92.93%+/-16.17, 87.02%+/-15.21 and 96.14%+/-12.32, respectively. Factor II positive subjects (n=47) had a mean factor II activity of 127.96%+/-21.37. F-II activity among carriers (heterozygous, n=3) and non-carriers (normal, n=215) of PT-G20210A mutation were 107.67%+/-9.29 and 105.00%+/-17.79, respectively. The prevalence of FVL was 21.8% and there is a little likelihood of the co-inheritance of the FVL and PT-G20210A among

  6. A Multiplex Allele Specific Polymerase Chain Reaction (MAS-PCR) for the Detection of Factor V Leiden and Prothrombin G20210A. (United States)

    Bagheri, Morteza; Rad, Isa Abdi


    In order to determine the frequencies of factor V Leiden and prothrombin G20210A point mutations in the Iranian population with Azeri Turkish origin. 120 unrelated individuals from general population randomly selected and were examined for factor V Leiden and prothrombin G20210A mutations using a multiplex allele specific polymerase chain reaction (MAS-PCR) assayOutcomes: The frequency of prothrombin G20210A mutation was 2.08%, which means 5 chromosomes out of 240 chromosomes had prothrombin G20210A mutation. The distribution of prothrombin 20210 GG, GA, AA genotypes and prothrombin 20210A allele were 37(92.5%), 3(7.5%), 0(0%) and 3(3.75%) in males and 78(97.5%), 2(2.5%), 0(0%) and 2(1.25%) in females, respectively. Factor V Leiden was not found in our tested group (zero chromosomes out of 240 chromosomes). Analysis of the observed frequencies in the studied groups indicates that there is no statistically significant difference between females and males, regarding prothrombin G20210A mutation (p value>0.05). This is the first study in its own kind in this population and implies that the frequency of Factor V Leiden G1691A (R506Q, FV-Leiden) allele is extremely low but the prothrombin G20210A mutation is more frequent in the tested group.

  7. Low prevalence of Factor V Leiden and the prothrombin G20210A mutation in a healthy population from the central-south region of Chile Baixa prevalência do Fator V Leiden e da mutação da protrombina G20210A em uma população sã da região centro-sul do Chile

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    Iván Palomo


    Full Text Available Thrombosis is a result of the interaction between predisposing genetic polymorphisms and acquired risk factors. The two prothrombotic polymorphisms which are most frequently associated with thrombosis are factor V (FV Leiden and the prothrombin (PT G20210A mutation. The objective of this work was to investigate the prevalence of both factors in the central-south region of Chile. Determination of the frequency was carried out by means of a genetic analysis of 1200 healthy, non-native individuals. The mutation of FV Leiden was found in 1.25% of the population and the PT G20210A mutation in 1.33%. None of the individuals were homozygosis or had both polymorphisms. The prevalences of FV Leiden and the PT G20210A mutation are less common in the healthy population.A trombose pode ser o resultado da interação de polimorfismo genético e fatores de riscos adquiridos. Os polimosfirmos protrombóticos mais frequentes são fator V (FV Leiden e a mutação da protrombina (PT G20210A. O objetivo deste trabalho foi investigar a prevalência de ambos os polimorfismos na região centro-sul do Chile. Foram realizadas análises genéticas (PCR RFLP de 1.200 pessoas saudáveis, não nativas da região. Foram encontrados 1,25% de mutação do Fator V Leiden e 1,33% da mutação da protrombina G20210A. Não foi detectada homozigose em ambos os polimorfismos. A prevalência de FV Leiden e da mutação G20210A é baixa na população estudada.

  8. Prevalence of factor Ⅴ Leiden and prothrombin G20210A in patients with gastric cancer

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    Sandra Battistelli; Massimo Stefanoni; Alberto Genovese; Aurelio Vittoria; Roberto Cappelli; Franco Roviello


    AIM: To analyze the prevalence of the two commonest thrombophilic mutations, factor Ⅴ Leiden and prothrombin G20210A, in patients with gastric cancer.METHODS: One hundred and twenty-one patients with primary gastric carcinoma and 130 healthy subjects,comparable for age and sex, were investigated. Factor Ⅴ Leiden was detected by using polymerase chain reaction and restriction enzyme digestion, and prothrombin G20210A gene mutation by allele-specific PCR.RESULTS: Among the 121 cancer patients, factor Ⅴ Leiden was found in 4 cases (GA genotype: 3.3%) and prothrombin G20210A in 10 cases (GA genotype: 8.3%).Of the 130 control subjects, factor Ⅴ Leiden was detected in 6 cases (GA genotype: 4.6%) and prothrombin G20210A in 8 cases (GA genotype: 6.1%). No double heterozygous carriers of both mutations were found in either group. The prevalence of both factor Ⅴ Leiden and prothrombin G20210A variant was not statistically different between the cancer patients and the healthy subjects.CONCLUSION: Our study suggests that, in gastric cancer, the risk factors of thrombophilic cancer state are on acquired rather than on a genetic basis and that prothrombin G20210A does not seem to be a cofactor in gastric cancer pathogenesis.

  9. Epidemiology of prothrombin G20210A polymorphism in the Southern Iran

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    Mohadeseh Arabnejad


    Conclusion: Results of the present study might be important in understanding the distribution of PTH G20210A polymorphism in the Southern Iran. Minor allele frequency in this population is higher than in the Iranian and European population but similar to the prevalence in the Western Iran, Iranian Jews, American, Irish, Tunisian and Bahraini population.

  10. Venous thrombosis with both heterozygous factor V Leiden (R507Q) and factor II (G20210A) mutations. (United States)

    Bhaijee, Feriyl; Jordan, Brenda; Pepper, Dominique J; Leacock, Rodney; Rock, William A


    Both hereditary and acquired factors increase the risk of venous thromboembolism, thus the clinical management of affected patients involves evaluation of genetic factors that predispose to hypercoagulability. Factor V Leiden (R507Q) and factor II (prothrombin) mutation (G20210A) are the two most common inherited hypercoagulability disorders among populations of European origin. Both factor V Leiden and factor II mutation (G20210A) represent gain-of-function mutations: factor V Leiden causes resistance to activated protein C, and factor II mutation (G20210A) results in higher levels of plasma prothrombin. Herein, we present an uncommon case of combined factor V Leiden mutation (R507Q) and factor II mutation (G20210A), and discuss the prevalence and features of each entity, as well as their role in the clinical management of affected patients.

  11. Analytic validity of genetic tests to identify factor V Leiden and prothrombin G20210A. (United States)

    Emadi, Ashkan; Crim, Matthew T; Brotman, Daniel J; Necochea, Alejandro J; Samal, Lipika; Wilson, Lisa M; Bass, Eric B; Segal, Jodi B


    The objective of this study is to systematically review methods for detecting Factor V Leiden or prothrombin G20210A. English-language literature from MEDLINE, EMBASE, The Cochrane Library, the Cumulative Index to Nursing and Allied Health Literature, PsycInfo(c), 2000-December 2008. Studies assessed methods for detection of these mutations in at least 10 human blood samples and reported concordance, discordance, or reproducibility. Two investigators abstracted data on the sample selection criteria, test operators, DNA extraction, experimental test, reference standard, commercial instruments, concordance rates, explanation of any discordance, and whether discordance resolved after repetition. We assessed strength of the evidence using the GRADE criteria. We reviewed 7,777 titles and included 66 articles. The majority of the reviewed studies used PCR-RFLP or AS-PCR as the reference standard. The studies demonstrated that commercially available and precommercial tests have high analytic validity with all having greater than 99% concordance with the reference standard. With a few exceptions, discordance resolved with repetition of the test, suggesting operator or administrative errors were responsible for the discordant results. In the quality assurance studies, greater than 98% of laboratories demonstrated high, even perfect, accuracy when asked to diagnose a sample with a known mutation. The majority of errors came from a limited number of laboratories. Although not all methods may be accurate, there is high-grade evidence that genetic tests for the detection of FVL and prothrombin G20210A have excellent analytic validity. There is high-grade evidence that most, but not all, clinical laboratories test for FVL and prothrombin G20210A accurately.

  12. An unusual cause of cerebral venous sinus thrombosis: prothrombin G20210A gene mutation. (United States)

    Porres-Aguilar, Mateo; Square, Jaime H; Storey, Raul; Rodriguez-Dunn, Simon; Mohamed-Aly, Mohamed S


    Cerebral venous sinus thrombosis represents less than 1% of all strokes, being an uncommon entity with a wide spectrum of clinical scenarios. We present a 45-year-old Hispanic female with a history of long-term oral contraceptive use who was diagnosed with cerebral venous sinus thrombosis due to a heterozygous carrier mutation in the prothrombin G20210A gene. The patient was successfully managed with intravenous heparin with favorable clinical results without adverse effects. The prevalence of inherited primary thrombophilia increases with additional risk factors such as the use of oral contraceptives that can trigger or prothrombotic events in any vascular bed. An increased prevalence in the prothrombin G20210 gene mutation has been demonstrated in the Mexican-Mestizo population. Controversy exists regarding therapy of cerebral venous sinus thrombosis; according to experts, heparin remains the cornerstone of therapy with acceptable outcomes. More clinical trials are required to evaluate long-term outcomes in this subgroup of patients.

  13. Do Factor V Leiden and Prothrombin G20210A Mutations Predict Recurrent Venous Thromboembolism in Older Patients? (United States)

    Méan, Marie; Limacher, Andreas; Stalder, Odile; Angelillo-Scherrer, Anne; Alberio, Lorenzo; Fontana, Pierre; Beer, Hans-Jürg; Rodondi, Nicolas; Lämmle, Bernhard; Aujesky, Drahomir


    The value of genetic thrombophilia testing in elderly patients with an unprovoked venous thromboembolism is unclear. We assessed whether the Factor V Leiden and the prothrombin G20210A mutation are associated with recurrent venous thromboembolism in elderly patients in a prospective multicenter cohort study. We genotyped the Factor V Leiden and the prothrombin G20210A mutation in 354 consecutive in- and outpatients aged ≥65 years with a first unprovoked venous thromboembolism from 9 Swiss hospitals. Patients and managing physicians were blinded to testing results. The outcome was recurrent symptomatic venous thromboembolism during follow-up. We examined the association between the Factor V Leiden and the prothrombin G20210A mutation and venous thromboembolism recurrence using competing risk regression, adjusting for age, sex, and periods of anticoagulation as a time-varying covariate. Overall, 9.0% of patients had a Factor V Leiden and 3.7% had a prothrombin G20210A mutation. At 36 months of follow-up, patients with a Factor V Leiden and a prothrombin G20210A mutation had a cumulative incidence of recurrent venous thromboembolism of 12.9% (95% confidence interval [CI], 5.1%-30.8%) and 18.5% (95% CI, 4.9%-56.5%), respectively, compared with 16.7% (95% CI, 12.5%-22.1%) of patients without mutation (P = .91 by the log-rank test). After adjustment, neither the Factor V Leiden (sub-hazard ratio 0.98; 95% CI, 0.35-2.77) nor the prothrombin G20210A mutation (sub-hazard ratio 1.15; 95% CI, 0.25-5.19) was associated with recurrent venous thromboembolism. Our results suggest that testing for genetic thrombophilia may not be beneficial in elderly patients with a first unprovoked venous thromboembolism. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. [Recurrent vascular access trombosis associated with the prothrombin mutation G20210A in a adult patient in haemodialysis]. (United States)

    Quintana, L F; Coll, E; Monteagudo, I; Collado, S; López-Pedret, J; Cases, A


    Vascular access-related complications are a frequent cause of morbidity in haemodialysis patients and generate high costs. We present the case of an adult patient with end-stage renal disease and recurrent vascular access thrombosis associated with the prothrombin mutation G20210A and renal graft intolerance. The clinical expression of this heterozygous gene mutation may have been favoured by inflammatory state, frequent in dialysis patients. In this patient, the inflammatory response associated with the renal graft intolerance would have favored the development of recurrent vascular access thrombosis in a adult heterozygous for prothrombin mutation G20210A. In the case of early dysfunction of haemodialysis vascular access and after ruling out technical problems, it is convenient to carry out a screening for thrombophilia.

  15. Mutação G20210A no gene da protrombina, fator V de Leiden e anticorpos anticardiolipina não influenciam a sobrevida do enxerto renal após o transplante Prothrombin G20210A gene mutation, factor V Leiden and anticardiolipin antibodies do not influence renal graft survival after transplantation

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    Luis Klaus A. da Rocha


    Full Text Available INTRODUÇÃO: Complicações tromboembólicas são importantes fatores de risco para perda do enxerto e pior evolução após o transplante renal. Pacientes com defeito trombofílico apresentam maior risco de complicações tromboembólicas. Foram analisados, entre receptores de transplante renal, a prevalência de defeito trombofílico e o risco atribuído a esta condição para a perda do enxerto e para o desenvolvimento de tromboses intravasculares. MÉTODOS: Estudo do tipo coorte incluindo 388 receptores adultos analisados quanto à presença de trombofilia de acordo com a pesquisa de anticorpos anticardiolipina (aCL por ELISA e das mutações G1691A no gene do fator V (FV e G20210A no gene da protrombina (PT por PCR multiplex. RESULTADOS: Defeito trombofílico foi identificado em 25,8% dos pacientes. As taxas de sobrevida de 2 anos do enxerto foram semelhantes entre os pacientes com e sem defeito trombofílico (94% versus 94%, p = 0,53, bem como a sobrevida dos enxertos livres de tromboses intravasculares (97% versus 97%, p = 0,83. Pacientes com defeito trombofílico apresentaram prevalência de tromboses intravasculares semelhante à do grupo-controle (3% versus 3,5%, p = 0,82. O transplante renal anterior foi associado a maior risco de perda de enxerto (OR 20,8, p INTRODUCTION: Thromboembolic complications are important risk factors for graft failure and worse renal transplantation outcome. Patients with thrombophilic disorders have a higher risk of thromboembolic complications. The prevalence of thrombophilic disorders and the associated risk for graft failure and for intravascular thrombosis were analyzed in renal transplant recipients. METHODS: This is a cohort study of 388 adult recipients investigated regarding the presence of thrombophilia, through the search for anticardiolipin antibodies (aCL via ELISA and FV G1691A and PT G20210A gene mutations by multiplex PCR. RESULTS: Thrombophilic disorders were identified in 25.8% of the

  16. Risk of recurrent venous thrombosis in homozygous carriers and double heterozygous carriers of factor V Leiden and prothrombin G20210A. (United States)

    Lijfering, Willem M; Middeldorp, Saskia; Veeger, Nic J G M; Hamulyák, Karly; Prins, Martin H; Büller, Harry R; van der Meer, Jan


    Homozygous or double heterozygous factor V Leiden and/or prothrombin G20210A is a rare inherited thrombophilic trait. Whether individuals with this genetic background have an increased risk of recurrent venous thrombosis is uncertain. A case-control design within a large cohort of families with thrombophilia was chosen to calculate the risk of recurrent venous thrombosis in individuals with homozygosity or double heterozygosity of factor V Leiden and/or prothrombin G20210A. Cases were individuals with recurrent venous thrombosis, and controls were those with only 1 venous thrombosis. The cohort consisted of 788 individuals with venous thrombosis; 357 had factor V Leiden, 137 had prothrombin G20210A, 27 had factor V Leiden and/or prothrombin G20210A homozygosity, and 49 had double heterozygosity for both mutations. We identified 325 cases with recurrent venous thrombosis and 463 controls with only 1 venous thrombosis. Compared with noncarriers, crude odds ratio for recurrence was 1.2 (95% confidence interval, 0.9 to 1.6) for heterozygous carriers of factor V Leiden, 0.7 (95% confidence interval, 0.4 to 1.2) for prothrombin G20210A, 1.2 (95% confidence interval, 0.5 to 2.6) for homozygous carriers of factor V Leiden and/or prothrombin G20210A, and 1.0 (95% confidence interval, 0.6 to 1.9) for double heterozygotes of both mutations. Adjustments for age, sex, family status, first event type, and concomitance of natural anticoagulant deficiencies did not alter the risk estimates. In this study, individuals with homozygous factor V Leiden and/or homozygous prothrombin G20210A or double heterozygous carriers of factor V Leiden and prothrombin G20210A did not have a high risk of recurrent venous thrombosis.

  17. Clinical characteristics of patients with factor V Leiden or prothrombin G20210A and a first episode of venous thromboembolism. Findings from the RIETE Registry. (United States)

    Gadelha, Telma; Roldán, Vanessa; Lecumberri, Ramón; Trujillo-Santos, Javier; del Campo, Raquel; Poggio, Renzo; Monreal, Manuel


    The clinical characteristics of patients with factor V Leiden or prothrombin G20210A presenting with a first episode of venous thromboembolism (VTE) have not been thoroughly studied. RIETE is an ongoing registry of consecutive patients with acute VTE. We compared the clinical characteristics of patients with factor V Leiden, prothrombin G20210A, or no thrombophilia, at presentation with a first episode of VTE. As of May 2009, 22428 patients had been enrolled with a first episode of VTE. Of these, 345 had factor V Leiden, 261 had prothrombin G20210A, and 2399 tested negative. Sixty-two percent of the VTE episodes in women with factor V Leiden or prothrombin G20210A (40% in men) were associated with an acquired risk factor. Among women, pregnancy or contraceptive use accounted for 63% and 67% of such risk factors. Patients with factor V Leiden presented with pulmonary embolism (PE) less likely than those with prothrombin G20210A (31% vs. 51%; pFactor V Leiden presented with hypoxaemia (Sat O(2) levelsfactor V Leiden or prothrombin G20210A were associated with an acquired risk factor (mostly pregnancy or contraceptive use). Only 4.5% of patients with factor V Leiden presenting with acute PE had hypoxaemia. Copyright © 2010 Elsevier Ltd. All rights reserved.

  18. The Frequency of Factor V Leiden, Prothrombin G20210A and Methylenetetrahydrofolate Reductase C677T Mutations in Migraine Patients

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    Ruhsen Öcal


    Full Text Available OBJECTIVE: Migraine is an independent risk factor for ischemic stroke, but its pathophysiology is still unclear. Genetic factors that predispose patients to thrombosis have been studied in patients with migraine to highlight the pathogenesis, but the results remain controversial. In this study, the frequencies of factor V Leiden (FVL, prothrombin (Pt G20210A and methylenetetrahydrofolate reductase (MTHFR C677T mutations were investigated. METHODS: One hundred and sixty patients aged of 15 to 55 years with no history of systemic disease and who had been diagnosed as migraine according to the International Headache Society (IHS diagnostic criteria at Baskent University Hospital Neurology Outpatient Clinics were investigated for FVL, Pt G20210A and MTHFR C677T mutations from their genomic DNA, and the results were compared with those of healthy controls. RESULTS: One hundred and fifty five (96.9% of 160 migraine patients were homozygote normal, 5 (3.1% were heterozygote and none of them were homozygote mutant for FVL. The control group had 9.8% heterozygote individuals but the difference between the percentages was not statistically significant (p> 0.05. There were no homozygote mutant individuals in the Turkish population study in normal subjects like our study. Thirty nine (24.4% of 160 migraine patients were heterozygote and 8 (5% were homozygote mutant for MTHFR C677T. The control group had 37 (34.9% heterozygote and 6 (5.6% homozygote mutant individuals. The difference between the percentages was not statistically significant (p= 0.15. Three (1.9% of 160 migraine patients were heterozygote and 5 (2.9% of the control group were heterozygote mutant for Pt G20210A mutation. The control group had 37 (34.9% heterozygote and 6 (5.6% homozygote mutant individuals. The difference between the percentages was not statistically significant (p= 0.420. CONCLUSION: Our study indicates that FVL, Pt G20210A and MTHFR C677T gene mutations, which are considered

  19. Molecular characterization of factor V leiden G1691A and prothrombin G20210A mutations in Saudi newborns with stroke. (United States)

    Gawish, Gihan E-H


    This study examined a possible association between the mutations related to Factor V Leiden and Factor II (prothrombin) and stroke in Saudi neonates. A multiplex PCR was established to detect Factor V Leiden G1691A and prothrombin G20210A mutations in 72 neonatal stroke subjects and 70 healthy adult controls with no family history of thromboembolic diseases. The frequency of the homozygous normal genotype (GG) of both genes was found to be significantly lower in the stroke subjects than in the controls (P Factor II heterozygous mutant form (GA) and the homozygous normal Factor V (GG) (P Factor V and the homozygous normal Factor II genotypes (GG) (P = 0.0) than controls. The study concluded that prothrombin and Factor V Leiden may be important risk factors for neonatal stroke in Saudi children.

  20. Mesenteric vein thrombosis in a patient heterozygous for factor V Leiden and G20210A prothrombin genotypes. (United States)

    Karmacharya, Paras; Aryal, Madan Raj; Donato, Anthony


    Mesenteric venous thrombosis (MVT) is a rare but life threatening form of bowel ischemia. It is implicated in 6%-9% of all cases of acute mesenteric ischemia. The proportion of patients with primary (or idiopathic) MVT varies from 0% to 49%, with a decrease in frequency secondary to more recent availability of newer investigations for hypercoagulability. The presence of factor V Leiden (FVL) and prothrombin G20210A mutations (PGM) have been well documented in these cases. However, there have been scarce case reports describing MVT in heterozygotes of both these mutations occurring simultaneously and its implications on long term management. Our case describes acute MVT in a previously asymptomatic young patient with no prior history of venous thromboembolism. The patient was found to be heterozygous for FVL and PGM and treated with lifelong anticoagulation with warfarin (goal international normalized ratio: 2-3) and avoidance of hormonal contraceptives.

  1. Risk of Recurrent Venous Thrombosis in Homozygous Carriers and Double Heterozygous Carriers of Factor V Leiden and Prothrombin G20210A

    NARCIS (Netherlands)

    Lijfering, Willem M.; Middeldorp, Saskia; Veeger, Nic J. G. M.; Hamulyak, Karly; Prins, Martin H.; Bueller, Harry R.; van der Meer, Jan


    Background-Homozygous or double heterozygous factor V Leiden and/or prothrombin G20210A is a rare inherited thrombophilic trait. Whether individuals with this genetic background have an increased risk of recurrent venous thrombosis is uncertain. Methods and Results-A case-control design within a lar

  2. Prevalence of coagulation factor II G20210A and factor V G1691A Leiden polymorphisms in Chechans, a genetically isolated population in Jordan. (United States)

    Dajani, Rana; Fatahallah, Raja; Dajani, Abdelrahman; Al-Shboul, Mohammad; Khader, Yousef


    Coagulation factor II G20210A and coagulation factor V (Leiden) G1691A single nucleotide polymorphisms (SNPs) are major inherited risk factors of venous thromboembolism. In view of the heterogeneity in their world distribution and lack of sufficient information about their distribution among Chechans, we addressed the prevalence of these SNPs in the Chechan population in Jordan, a genetically isolated population. Factor II G20210A and factor V Leiden SNPs were analysed by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) method and Amplification refractory mutation detection system (ARMS) respectively in 120 random unrelated subjects from the Chechan population in Jordan. Among the subjects studied for factor II G20210A mutation there were three individuals carrying this mutation as heterozygous (one female and two male), giving a prevalence of 2.5 % and an allele frequency of 1.25 %. No homozygous factor II allele was found. Factor V Leiden G1691A mutation was detected as heterozygous in 22 of 120 of individuals (17 female and five male) indicating a prevalence of 18.3 % and allele frequency of 9.2 %. No homozygous allele was found. Our results indicated that prevalence of factor II G20210A mutation in the Chechan population is similar to prevalence in Jordan and Caucasian populations (1-6 %) while the prevalence of factor V Leiden was higher in the Chechan population compared to Jordan and Caucasian populations (2-15 %).

  3. Risk of Budd-Chiari syndrome associated with factor V Leiden and G20210A prothrombin mutation: a meta-analysis.

    Directory of Open Access Journals (Sweden)

    Peijin Zhang

    Full Text Available BACKGROUND: Various studies have demonstrated that factor V Leiden (FVL and G20210A prothrombin mutation contribute to the risk of Budd-Chiari syndrome (BCS, while other studies provided conflicting findings. In order to derive more precise estimations of the relationships, a meta-analysis was performed. METHODS: Eligible articles were identified through search of databases including Pubmed, Chinese Biomedical Database (CBM, Chinese, and Chinese National Knowledge Infrastructure (CNKI, Chinese. Odd ratios (ORs with 95% confidence intervals (CIs were calculated using random- or fixed- model. RESULTS: Finally, twelve studies were included for FVL and nine studies were included for G20210A prothrombin mutation. With respect to FVL, significantly increased BCS risk was found in the overall population (OR = 6.29, 95%CI = 4.23-9.36. Subgroup analyses suggested that FVL was associated with an increased risk of BCS in the population with high background mutation prevalence (>1% in the normal population. No significant association was found between BCS and G20210A prothrombin mutation (OR = 1.78, 95%CI = 0.77-4.11. CONCLUSION: The presence of FVL should be evaluated in patients with BCS. Conversely, G20210A prothrombin mutation is not significantly associated with risk of BCS. Large-scale well designed studies are necessary to be conducted to further confirm or refute the observed association.

  4. Factor V Leiden, prothrombin G20210A, and methylene tetrahydrofolate reductase mutations and stillbirth: the Stillbirth Collaborative Research Network. (United States)

    Silver, Robert M; Saade, George R; Thorsten, Vanessa; Parker, Corette B; Reddy, Uma M; Drews-Botsch, Carey; Conway, Deborah; Coustan, Donald; Dudley, Donald J; Bukowski, Radek; Rowland Hogue, Carol J; Pinar, Halit; Varner, Michael W; Goldenberg, Robert; Willinger, Marian


    An evaluation for heritable thrombophilias is recommended in the evaluation of stillbirth. However, the association between thrombophilias and stillbirth remains uncertain. We sought to assess the association between maternal and fetal/placental heritable thrombophilias and stillbirth in a population-based, case-control study in a geographically, racially, and ethnically diverse population. We conducted secondary analysis of data from the Stillbirth Collaborative Research Network, a population-based case-control study of stillbirth. Testing for factor V Leiden, prothrombin G20210A, methylene tetrahydrofolate reductase C677T and A1298C, and plasminogen activating inhibitor (PAI)-1 4G/5G mutations was done on maternal and fetal (or placental) DNA from singleton pregnancies. Data analyses were weighted for oversampling and other aspects of the design. Odds ratios (OR) were generated from univariate models regressing stillbirth/live birth status on each thrombophilia marker. Results were available for ≥1 marker in 488 stillbirths and 1342 live birth mothers and 405 stillbirths and 990 live birth fetuses. There was an increased odds of stillbirth for maternal homozygous factor V Leiden mutation (2/488; 0.4% vs 1/1380; 0.0046%; OR, 87.44; 95% confidence interval, 7.88-970.92). However, there were no significant differences in the odds of stillbirth for any other maternal thrombophilia, even after stratified analyses. Fetal 4G/4G PAI-1 (OR, 0.63; 95% confidence interval, 0.43-0.91) was associated with decreased odds of stillbirth. Other fetal thrombophilias were similar among groups. Most maternal and fetal thrombophilias were not associated with stillbirth. Maternal factor V Leiden was weakly associated with stillbirth, and the fetal PAI-1 4G/4G polymorphism was associated with live birth. Our data do not support routine testing for heritable thrombophilias as part of an evaluation for possible causes of stillbirth. Copyright © 2016. Published by Elsevier Inc.

  5. Combined heterozygosity of factor V leiden and the G20210A prothrombin gene mutation in a patient with cerebral cortical vein thrombosis. (United States)

    Liu, X Y; Gabig, T G; Bang, N U


    Cerebral venous thrombosis (CVT) is a rare type of stroke with a variety of causes. Several reports have suggested that either factor V Leiden or G20210A prothrombin gene mutation is associated with an increased risk of CVT. The genetic thrombophilias are typically associated with other predisposing factors. We report a unique case of CVT in a patient with both the factor V Leiden and the G20210A prothrombin gene mutations without other identifiable precipitating factors in a 28-year-old white male in good health. MRI and cerebral arterial angiography showed cerebral cortical venous thrombosis. This case suggests that combined heterozygous individuals may be particularly prone to spontaneous thrombosis, like CVT.

  6. Lack of association between factor V Leiden and prothrombin G20210A polymorphisms in Tunisian subjects with a history of myocardial infarction. (United States)

    Berredjeb Ben Slama, Dhouha; Fekih-Mrissa, Najiba; Haggui, Abdeddayem; Nsiri, Brahim; Baraket, Nadia; Haouala, Habib; Gritli, Nasreddine


    Myocardial infarction is a multifactorial disease. It is provoked by occlusions in the coronary arteries resulting from exposure to multiple risk factors. To study the risk of myocardial infarction associated with the gene polymorphisms of factor V Leiden and factor II (G20210A). Cases consisted of 100 myocardial infarction patients who were hospitalized in the Principal Military Hospital of Tunis and 200 control subjects with no history of myocardial infarction. The prevalence of the factor V Leiden was higher in myocardial infarction patients (9%) than in control subjects (6%) with an OR=1.55 (95% CI=0.58-4.12), whereas the prevalence of prothrombin G20210A mutation was 3% and 2.5% in the patient and control groups, respectively [OR=1.21 (95% CI=0.22-5.94)]. Our results indicate that neither factor V Leiden nor the prothrombin G20210A contributed to the risk factors for myocardial infarction. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Prothrombin G20210A mutation is associated with young-onset stroke: the genetics of early-onset stroke study and meta-analysis. (United States)

    Jiang, Baijia; Ryan, Kathleen A; Hamedani, Ali; Cheng, Yuching; Sparks, Mary J; Koontz, Deborah; Bean, Christopher J; Gallagher, Margaret; Hooper, W Craig; McArdle, Patrick F; O'Connell, Jeffrey R; Stine, O Colin; Wozniak, Marcella A; Stern, Barney J; Mitchell, Braxton D; Kittner, Steven J; Cole, John W


    Although the prothrombin G20210A mutation has been implicated as a risk factor for venous thrombosis, its role in arterial ischemic stroke is unclear, particularly among young adults. To address this issue, we examined the association between prothrombin G20210A and ischemic stroke in a white case-control population and additionally performed a meta-analysis. From the population-based Genetics of Early Onset Stroke (GEOS) study, we identified 397 individuals of European ancestry aged 15 to 49 years with first-ever ischemic stroke and 426 matched controls. Logistic regression was used to calculate odds ratios (ORs) in the entire population and for subgroups stratified by sex, age, oral contraceptive use, migraine, and smoking status. A meta-analysis of 17 case-control studies (n=2305 cases <55 years) was also performed with and without GEOS data. Within GEOS, the association of the prothrombin G20210A mutation with ischemic stroke did not achieve statistical significance (OR=2.5; 95% confidence interval [CI]=0.9-6.5; P=0.07). However, among adults aged 15 to 42 years (younger than median age), cases were significantly more likely than controls to have the mutation (OR=5.9; 95% CI=1.2-28.1; P=0.03), whereas adults aged 42 to 49 years were not (OR=1.4; 95% CI=0.4-5.1; P=0.94). In our meta-analysis, the mutation was associated with significantly increased stroke risk in adults ≤55 years (OR=1.4; 95% CI=1.1-1.9; P=0.02), with significance increasing with addition of the GEOS results (OR=1.5; 95% CI=1.1-2.0; P=0.005). The prothrombin G20210A mutation is associated with ischemic stroke in young adults and may have an even stronger association among those with earlier onset strokes. Our finding of a stronger association in the younger young adult population requires replication.

  8. The frequency of factor V Leiden and prothrombin G20210A mutations in Slovak and Roma (Gypsy) ethnic group of Eastern Slovakia. (United States)

    Bôžiková, Alexandra; Gabriková, Dana; Sovičová, Adriana; Behulová, Regina; Mačeková, Soňa; Boroňová, Iveta; Petrejčíková, Eva; Soták, Miroslav; Bernasovská, Jarmila; Bernasovský, Ivan


    Factor V Leiden and prothrombin G20210A are the two most prevalent causes of inherited thrombophilia. The prevalence of these mutations varies widely in healthy Caucasian population. The aim of our study was to determine the frequency of factor V Leiden and prothrombin G20210A mutations in Slovak and Roma ethnic group from Eastern Slovakia. We analyzed 540 asymptomatic individuals (269 individuals of Slovak ethnicity and 271 individuals of Roma ethnicity) by real-time PCR method. The detected allele frequencies were 2.97 versus 6.64 % for factor V Leiden (p = 0.0049), and 0.74 versus 0.92 % for prothrombin mutation (p = 0.7463) in Slovak and Roma population, respectively. The Roma ethnic group had significantly higher prevalence of factor V Leiden mutation when compared to Slovak ethnic group. The allele frequency of factor V Leiden in ethnic Romanies from Eastern Slovakia was one of the highest in Europe. Our results confirm an uneven geographical and ethnic distribution of factor V Leiden.

  9. 维吾尔族静脉血栓栓塞症与凝血酶原G20210A和凝血因子V Leiden 突变的相关性研究%Association of polymorphism gene G20210A mutation or factor V Leiden mutation andactivated protein C resistance in Uyghur patients with Venous thromboembolism

    Institute of Scientific and Technical Information of China (English)

    胡雪梅; 艾力曼·马合木提; 夏玉宁; 古丽尼格尔·吾布力; 古丽米热·吐尼牙孜


    目的:探讨凝血酶原基因 G20210A(FⅡG20210A)和凝血因子 V基因 Leiden 突变(FVL)单核苷酸多态性(SNP)及活化蛋白C抵抗(APCR)与维吾尔族(维族)静脉血栓栓塞症(VTE)的关系。方法选择2008年1月-2013年6月在新疆医科大学第一附属医院住院的维族 VTE患者178例(病例组)及本院同期同民族住院体检者217例(健康对照组)。采用聚合酶链式反应-限制性片段长度多态性(PCR-PFLP)技术检测 FⅡG20210A 和FVL多态性,应用凝固法于SYSMEX CS-2000型全自动血液凝固仪检测APCR,并分析FⅡG20210A和FVL多态性及 APCR与 VTE的关联性。结果(1)所有受检对象均未检出 FⅡG20210A基因突变,其变异频率为0。(2)健康对照组中只有 GG基因型,而未见 GA和 AA基因型,A等位基因频率为0。病例组中 FVL多态性 GG型与GA/AA型分布频率差异有统计学意义(P =0.018),且A等位基因分布频率差异亦有统计学意义(P =0.018)。(3)病例组 APCR阳性率较健康对照组高(11.24%与4.60%),其差异有统计学意义(P <0.05)。但 VTE患者FVL多态性各基因型间 APCR阳性率比较差异无统计学意义(P >0.05)。(4)非条件 logistic回归分析后,肥胖(OR=1.599,95%CI 1.383~1.849,P =0.000)、高脂血症(OR =1.087,95%CI 1.035~1.141,P =0.001)和APCR阳性(OR =1.922,95%CI 1.880~5.561,P =0.033)是 VTE患者的独立危险因素。结论凝血酶原G20210A基因突变在新疆维族 VTE患者及正常人群中罕见甚至缺如。APCR及 FVL多态性是维吾尔族 VTE患者的危险因素,但 FVL多态性不是引起 APCR的主要因素。%Objective To investigate the association between the G20210A mutation of Prothrombin gene (FII G20210A),factor V Leiden mutation (FVL)and activated protein C resistance (APCR)in Uyghur patients with

  10. The Prothrombin G20210A Mutation is Associated with Young-Onset Stroke: The Genetics of Early Onset Stroke Study and Meta-Analysis (United States)

    Jiang, Baijia; Ryan, Kathleen A.; Hamedani, Ali; Cheng, Yuching; Sparks, Mary J.; Koontz, Deborah; Bean, Christopher J.; Gallagher, Margaret; Hooper, W. Craig; McArdle, Patrick F.; O'Connell, Jeffrey R.; Stine, O. Colin; Wozniak, Marcella A.; Stern, Barney J.; Mitchell, Braxton D.; Kittner, Steven J.; Cole, John W.


    Background and Purpose Although the prothrombin G20210A mutation has been implicated as a risk factor for venous thrombosis, its role in arterial ischemic stroke is unclear, particularly among young-adults. To address this issue, we examined the association between prothrombin G20210A and ischemic stroke in a Caucasian case-control population and additionally performed a meta-analysis Methods From the population-based Genetics of Early Onset Stroke (GEOS) study we identified 397 individuals of European ancestry aged 15-49 years with first-ever ischemic stroke and 426 matched-controls. Logistic regression was used to calculate odds ratios in the entire population and for subgroups stratified by gender, age, oral contraceptive use, migraine and smoking status. A meta-analysis of 17 case-control studies (n=2305 cases stroke did not achieve statistical significance (OR=2.5,95%CI=0.9-6.5,p=0.07). However, among adults aged 15-42 (younger than median age), cases were significantly more likely than controls to have the mutation (OR=5.9,95%CI=1.2-28.1,p=0.03), whereas adults ages 42-49 were not (OR=1.4,95%CI=0.4-5.1,p=0.94). In our meta-analysis, the mutation was associated with significantly increased stroke risk in adults stroke in young-adults and may have an even stronger association among those with earlier onset strokes. Our finding of a stronger association in the younger-young adult population requires replication. PMID:24619398

  11. The relationship between factor V Leiden, prothrombin G20210A, and MTHFR mutations and the first major thrombotic episode in polycythemia vera and essential thrombocythemia. (United States)

    Trifa, Adrian P; Cucuianu, Andrei; Popp, Radu A; Coadă, Camelia A; Costache, Roxana M; Militaru, Mariela S; Vesa, Ştefan C; Pop, Ioan V


    Arterial and venous thrombosis are the most frequent complications in patients with polycythemia vera and essential thrombocythemia. We sought to demonstrate a possible contribution of the factor V Leiden, prothrombin G20210A, and methylenetetrahydrofolate reductase (MTHFR) 677 C > T and 1298 A > C mutations to the thrombotic risk in patients with polycythemia vera and essential thrombocythemia along with other biological features of these patients. We included 86 patients with polycythemia vera, of which 34 (39.5 %) had major thrombosis and 95 patients with essential thrombocythemia, of which 22 (23.1 %) had major thrombosis. In the whole cohort of patients, only the factor V Leiden mutation was significantly associated with both arterial and venous thrombosis in univariate and multivariate analysis (odds ratio (OR) = 4.3; 95 % confidence interval (CI) = 1.5-12.5; p = 0.008 and OR = 4.3; 95 % CI = 1.2-15.9; p = 0.02, respectively). Other factors significantly associated with thrombosis in both univariate and multivariate analysis were male sex (OR = 2.8, 95 % CI = 1.4-5.4, p = 0.002 and OR = 3.5, 95 % CI = 1.6-7.6, p = 0.002, respectively) and the JAK2 V617F mutation (OR = 5.5, 95 % CI = 2.1-15, p = 0.0001 and OR = 6.9, 95 % CI = 2.2-21.2, p = 0.001, respectively). In conclusion, among the four mutations analyzed (factor V Leiden, prothrombin G20210A, and MTHFR 677 C > T and 1298 A > C), only factor V Leiden is a major contributor to thrombosis in polycythemia vera and essential thrombocythemia.

  12. Primary thrombophilia in Mexico. II. Factor V G1691A (Leiden), prothrombin G20210A, and methylenetetrahydrofolate reductase C677T polymorphism in thrombophilic Mexican mestizos. (United States)

    Ruiz-Argüelles, G J; Garcés-Eisele, J; Reyes-Núñez, V; Ramírez-Cisneros, F J


    We have shown that in Mexican mestizo patients with clinical features of primary thrombophilia, 39% have activated protein C resistance phenotype, 5% protein C deficiency, and 2% protein S deficiency. In the present study, in a group of 37 thrombophilic Mexicans and 50 normal controls, we assessed the factor V G1691A (Leiden), the prothrombin G20210A, and the methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphisms. Four patients were found to be heterozygous for factor V Leiden, 5 heterozygous for the prothrombin 20210, 16 heterozygous, and 6 homozygous for the MTHFR 677. There were four individuals with co-segregation of alleles: two heterozygotes for the factor V Leiden/prothrombin 20210, one heterozygote for prothrombin 20210/MTHFR 677, and one heterozygote for prothrombin 20210/homozygote for MTHFR 677. For factor V Leiden, prothrombin 20210, and MTHFR 677 mutations, the allele frequencies were respectively 1% (+/-0.2%, alpha = 0.05), 7.66 (P mestizo thrombophilic patients, the low prevalence of the factor V Leiden mutation (10.8%) and the high prevalence of the prothrombin 20210 mutation (13.5%) contrast with those identified in Caucasian thrombophilic patients (21% and 6%, respectively; P mestizo patients.

  13. Case control study of the factor V Leiden and factor II G20210A mutation frequency in women with recurrent pregnancy loss. (United States)

    Teremmahi Ardestani, Majid; Nodushan, Hossein Hadi; Aflatoonian, Abbas; Ghasemi, Nasrin; Sheikhha, Mohammad Hasan


    Recurrent pregnancy loss (RPL) caused by various genetic and non-genetic factors. After chromosome abnormality, thrombophilia is one of the most important genetic factors that could cause RPL. Factor V Leiden and factor II G20210A mutation were the most common mutations cause thrombophilia in the world. The purpose of this study was to determine the frequency of factor V Leiden and prothrombine gene mutations in women with RPL compared with women who had uneventful pregnancies. This case control study evaluates the frequency of factor V-Leiden and factor II G20210 genotypes in 80 women with two or more pregnancy losses, compared with 80 women without adverse pregnancy outcome. The mutations were assessed by PCR-RFLP. Frequency of the factor V Leiden among cases was 2.5%, which was higher than controls (1.25%), but the difference was not significant. No factor II G20210 mutation was found among cases and controls. These data did not confirm that factor V Leiden and factor II G20210 mutation might play a role in recurrent pregnancy loss in Iranian women.

  14. Factor V G1691A (Leiden and prothrombin G20210A gene mutation status, and thrombosis in patients with chronic myeloproliferative disorders

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    Nur Soyer


    Full Text Available Objective: The aim of this study was to examine Factor V G1691A (Leiden (FVL and prothrombin G20210A (PT gene mutation status, and their relationship with thrombosis in patients with chronic myeloproliferative disorders (CMPDs.Materials and Methods: The study included 160 patients with a CMPD that were regularly followed-up between 1993 and 2009. FVL and PT mutation status was established based on blood samples analyzed via PCR using specific primers.Results: The frequency of FVL and PT mutation was 12.5% and 4.4%, respectively. In total, 27 episodes of thrombosis occurred in 24 (15% of the patients, and there wasn’t an association between the observed thrombotic events, and FVL or PT mutations. Hepatic vein thrombosis was noted in 3 patients that had FVL mutation, of which 1 also had PT mutation.Conclusion: We did not observe a relationship between thrombosis, and FVL or PT mutations in CMPD patients; however, 3 of the patients that had hepatic vein thrombosis also had FVL mutation. Larger studies are needed to more clearly determine if all CMPD patients with hepatic vein thrombosis need be investigated for FVL and PT mutation.

  15. Thrombophilic risk of individuals with rare compound factor V Leiden and prothrombin G20210A polymorphisms: an international case series of 100 individuals. (United States)

    Lim, Ming Y; Deal, Allison M; Kim, Steven; Musty, Michael D; Conard, Jacqueline; Simioni, Paolo; Dutrillaux, Fabienne; Eid, Suhair S; Middeldorp, Saskia; Halbmayer, Walter M; Boneu, Bernard; Moia, Marco; Moll, Stephan


    The risk of thrombosis in individuals with rare compound thrombophilias, homozygous factor V Leiden (FVL) plus heterozygous prothrombin G20210A (PTM), homozygous PTM plus heterozygous FVL, and homozygous FVL plus homozygous PTM, is unknown. We identified, worldwide, individuals with these compound thrombophilias, predominantly through mailing members of the International Society on Thrombosis and Haemostasis. Physicians were sent a clinical questionnaire. Confirmatory copies of the genetic results were obtained. One hundred individuals were enrolled; 58% were female. Seventy-one individuals had a venous thrombosis (includes superficial and deep vein thrombosis, and pulmonary embolism), 4 had an arterial thrombosis and 6 had both. Nineteen individuals had never had a thrombotic event. Thrombosis-free survival curves demonstrated that 50% of individuals had experienced a thrombotic event by 35 yrs of age, while 50% had a first venous thromboembolic event (VTE; includes all venous thrombosis except superficial thrombosis) by 41 yrs of age; 38.2% of first VTEs were unprovoked. 37% of patients had at least one VTE recurrence. Seventy percent of first pregnancies carried to term and not treated with anticoagulation were thrombosis-free. In conclusion, patients with these rare compound thrombophilias are not exceedingly thrombogenic, even though they have a substantial risk for VTE. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  16. Clinical impact of factor V Leiden, prothrombin G20210A, and MTHFR C677T mutations among sickle cell disease patients of Central India. (United States)

    Nishank, Sudhansu Sekhar; Singh, Mendi Prema Shyam Sunder; Yadav, Rajiv


    It is known that patients with sickle cell disease (SCD) present activation of the blood coagulation and fibrinolytic systems, especially during vaso-occlusive crises and also during the steady state of the disease. We determined whether the presence of the factor prothrombin gene G20210A variant, factor V gene G1691A mutation (factor V Leiden), and methylenetetrahydrofolate reductase (MTHFR) C677T polymorphisms may be risk factors for vascular complications in individuals with SCD. The study involved 150 patients with sickle cell anemia and 150 healthy controls of Central India. Genotyping of three thrombophilic mutations was carried out by PCR-RFLP methods using MnlI, Hind III, and Hinf I, respectively, for factor V Leiden, prothrombin, and MTHFR mutations. Patients with SCD had significantly higher prevalence of mutant variants of MTHFR gene (28.0% heterozygotes and 14.6% homozygotes) and FVL gene (14.6% heterozygotes) as compared to normal/control individuals, but complete absence of mutant variants of prothrombin gene. The patients with SCD having mutant variants of MTHFR and FVL genes showed higher incidence of pain in chest, abdomen, and bone joints along with early age of onset of clinical manifestations as well as frequent dependence on blood transfusion than those patients with SCD having wild variants of these thrombotic genes. As compared to control subjects, SCD individuals having mutant variants of FVL and MTHFR genes had significant association with higher levels of prothrombin fragment (F1+2), D-dimer, thrombin-antithrombin (TAT), and lower level of protein C. MTHFR C677T and FVL G1691A polymorphisms may be risk factors for increased vascular complications in patient with SCD. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. 凝血酶原基因G20210A突变检测对肺血栓栓塞症的预测价值%Predictive value of prothrombin G20210A mutation detection in pulmonary thromboembolism

    Institute of Scientific and Technical Information of China (English)

    张佳; 赵凤芹; 谭平; 季红


    目的:探讨凝血酶原基因 G20210A(FⅡG20210A)突变在中国东北地区肺血栓栓塞症(PTE)患者中的发生频率,阐明检测该突变基因对中国东北地区人群 PTE的预测价值。方法:选择经核素肺灌注显像和(或)螺旋CT肺动脉造影(CTPA)结合临床症状确诊的PTE患者60例(病例组)和同期来自相同地区、性别和年龄相匹配的正常健康人80名(对照组)。应用蛋白酶消化及乙醇抽提获得2组研究对象的 DNA。采用聚合酶链式反应(PCR)、HindⅢ限制性内切酶片段多态性分析(RFLP )和琼脂糖凝胶电泳检测病例组和对照组研究对象FⅡG20210A基因突变情况。结果:经HindⅢ酶切后,病例组仅出现407和99 bp 2个条带,FⅡG20210A突变频率为0%,与对照组比较差异无统计学意义(P>0.05);病例组和对照组均未发现 FⅡG20210A基因杂合子或纯合子突变。结论:FⅡ G20210A基因突变在我国东北地区 PTE患者中发生率低,FⅡG20210A基因突变检测可能对中国东北地区人群 PTE无预测价值。%Objective To study the incidence frequency of prothrombin G20210A (FⅡ G20210A)mutation in the patients with pulmonary thromboembolism(PTE)in northeast China,and to clarify the predictive value of FⅡG20210A mutation detection in PTE of the population in northeast China.Methods 60 PTE patients(PTE group) and 80 sex-matched healthy controls(control group)from the same geographic area were selected.All the patients were diagnosed by lung ventilation/perfusion scan and/or multi-slice CT pulmonary angiography(CTPA)as well as medical history.The genome DNA was extracted from the whole blood using alcohol.Polymerase chain reaction (PCR),restriction fragment length polymorphisms(RFLP)analysis with HindⅢ restriction enzyme and sepharose gel electrophoresis were used to identify the F Ⅱ G20210A mutation in PTE group and control group. Results After digested by Hind

  18. Análise da mutação G20210A no gene da protrombina (fator II em pacientes com suspeita de trombofilia no sul do Brasil Analysis of prothrombin G20210A mutation (factor II in patients with suspected trombophilia in Southern Brazil

    Directory of Open Access Journals (Sweden)

    Marcos Edgar Herkenhoff


    Full Text Available INTRODUÇÃO: A protrombina (fator II é uma proteína sanguínea sintetizada no fígado com a presença de vitamina K. É a precursora da trombina, que induz a formação de fibrina. Foi descrita uma mutação no gene da protrombina G20210A, associada diretamente a altos níveis de protrombina no sangue e, consequentemente, à trombofilia. Essa variante alélica consiste em mutação pontual, também chamada de polimorfismo de nucleotídeo simples (SNP, ocasionando a troca de uma guanina por uma adenina no nucleotídeo 20210, localizado em um sítio de clivagem do precursor do ácido ribonucleico mensageiro (mRNA. Essa troca caracteriza o alelo A e a ausência da mutação do alelo G. OBJETIVO: Quantificar o número de indivíduos homozigotos para alelo G, homozigotos para alelo A e heterozigotos, cujas amostras foram enviadas para o laboratório Genolab Análises Genéticas, abrangendo os estados do Paraná e Santa Catarina, no período de 1º de janeiro de 2009 a 10 de outubro de 2010. MÉTODOS: Análise de mutação pontual por reação em cadeia da polimerase em tempo real (RT-PCR. RESULTADOS: Obtivemos o número de 243 indivíduos e desse total 51,03% eram oriundos do estado do Paraná, enquanto 48,97%, oriundos do estado de Santa Catarina. Do total analisado, 88,89% possuíam o genótipo para homozigoto G, e nenhum indivíduo foi encontrado com mutação para homozigoto A. Apenas 11,11% possuíam genótipo heterozigoto. O estado de Santa Catarina apresentou frequência superior para genótipo heterozigoto em relação ao Paraná. CONCLUSÃO: Este estudo mostrou que é recomendável a identificação do genótipo para esse gene em pacientes com suspeita de trombofilia nos dois estados.INTRODUCTION: Prothrombin (factor II is a blood protein synthesized in the liver in the presence of vitamin K. It is a thrombin precursor, which induces fibrin formation. Prothrombin G20210A mutation and high prothrombin levels have been closely associated

  19. Avaliação da incidência das mutações G1691A no gene do fator V (fator V Leiden e G20210A no gene da protrombina em pacientes com diabetes mellitus tipo 2 Incidence of G1691A factor V gene (factor V Leiden and G20210A prothrombin gene mutations in type 2 diabetes patients

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    Anna L. Soares


    Full Text Available Em virtude da alta prevalência de Diabetes mellitus tipo 2 (DM2 na população mundial e da alta taxa de mortalidade decorrente de eventos trombóticos, é de extrema importância o conhecimento das alterações no sistema hemostático em pacientes portadores deste distúrbio. A mutação no gene do fator V (G1691A - fator V Leiden em heterozigose ou homozigose confere aos portadores o fenótipo de resistência à proteína C ativada, situação que aumenta em sete vezes o risco de desenvolver uma trombose. A mutação G20210A no gene da protrombina resulta no quadro de hiperprotrombinemia, aumentando o risco de trombose em três vezes. A pesquisa dessas mutações de interesse em trombofilia é de grande relevância considerando que a presença das mesmas pode exacerbar o estado de hipercoagulabilidade acelerando as complicações no diabetes. O presente estudo teve como objetivo avaliar a incidência dessas mutações em indivíduos hígidos (Controle, n=16, pacientes com DM2 (n=7, com hipertensão (HAS, n=12 e com DM2+HAS (n=18, através da técnica de PCR-RFLP. As freqüências encontradas nos grupos estudados foram baixas e similares àquelas observadas na população brasileira em geral. Não foi possível estabelecer correlação entre a presença da mutação e características específicas de cada grupo. Dessa forma, ainda não está claro se há ou não uma maior prevalência dessas mutações em indivíduos diabéticos e se a presença das mesmas contribui para o aumento do risco de desenvolver trombose nesses indivíduos, sendo necessário estudos mais amplos para a elucidação da questão.Because of the high prevalence of type 2 diabetes mellitus (DM2 worldwide and the high mortality rate due to thrombotic events, it is extremely important to know about changes in the hemostatic system of such patients. Factor V mutation (G1691A - factor V Leiden in either heterozygosis or homozygosis confers the activated protein C resistant

  20. Endothelial Nitric Oxide Synthase T-786C Mutation, Prothrombin Gene Mutation (G-20210-A and Protein S Deficiency Could Lead to Myocardial Infarction in a Very Young Male Adult

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    Milka Klincheva


    Full Text Available INTRODUCTION: Myocardial infarction is a rare medical event in young people. The main reasons include congenital coronary abnormalities, coronary artery spasm, and coronary thrombosis due to hypercoagulable states (hereditary and acquired. AIM: We present a case of a young male adult with myocardial infarction caused by a combination of gene mutations and anticoagulation protein deficiency. CASE PRESENTATION: A 19 years old young man was admitted to our hospital complaining of chest pain during the last two weeks. The patient did not have any known cardiovascular risk factors, except a positive family anamnesis. Subacute inferior nonST segment myocardial infarction was diagnosed according to the patient’s history, electrocardiographic and laboratory findings. Coronary angiography revealed suboclusive thrombus in the proximal, medial and distal part of the right coronary artery (TIMI 2. Percutaneous coronary intervention was performed. Anticoagulant and antiagregant therapy (heparin, acetilsalicilic acid and clopidogrel according to protocol was started. The hospital stay was uneventful. Homozygous endothelial nitric oxid synthase (eNOS T-786-C mutation, heterozygote prothrombin gene mutation (G-20210-A, and protein S deficiency were verified from the thrombophilia testing. Other trombophilic tests were normal. Three months after discharge from hospital another coronary angiography was performed. It revealed normal coronary arteries. Four years after the attack, the patient is free of symptoms and another cardiovascular event. CONCLUSION: Combination of genetic mutations and anticoagulation protein deficiency could be a reasonable cause for myocardial infarction in a very young male adult without any other cardiovascular risk factors.

  1. A Meta-analysis of prothrombin G20210A polymorphism and its risk for sudden sensorineural hearing loss%凝血酶原基因G20210A多态性与突发性聋发病风险的Meta分析

    Institute of Scientific and Technical Information of China (English)

    刘博; 冯文静; 彭晓霞; 杨剑


    目的 采用Meta分析的方法探讨凝血酶原基因G20210A多态性与突发性聋发病风险的相关性.方法 计算机检索PUBMED、EMBASE、Cochrane图书馆(英文)和中国生物医学文献数据库(CBM),查找凝血酶原基因G20210A多态性与突发性聋发病相关的临床研究,对纳入的文献进行严格方法学质量评价,应用Review Manager Version 4.2软件进行统计分析.结果 共有9篇文献纳入研究,累计病例组735例,对照组1230例.方法学质量评价采用三方面8项计分(满分为8星),结果显示纳入的文献总体质量较高,其中2篇为8星,3篇为7星,1篇为6星,1篇为5星,2篇为4星.Meta分析统计结果提示凝血酶原基因G20210A多态性在基因型水平[OR=1.79,95% CI=(1.06,3.01),P=0.03]和等位基因水平[0R=1.77,95% CI=(1.06,2.97),P=0.03]均可能增加突发性聋的发病风险.结论 凝血酶原基因G20210A多态性可能增加突发性聋的发病风险,但仍需大样本的高质量研究进一步证实.%Objective To investigate the correlation between prothrombin G20210A polymorphism and the risk for idiopathic sudden sensorineural hearing loss (ISSNHL) using Meta-analysis methodology.Methods Databases,including PUBMED,EMBASE,Cochrane Library and CBM,were searched to collect the case control studies on the correlation between prothrombin G20210A polymorphism and idiopathic sudden sensorineural hearing loss.Only high quality studies were included.All analysis were conducted with Review Manager Version 4.2 software.Results A total of 9 studies were included,involving 735 cases and 1230 controls.The quality assessment involved 3 parts,8 scores (totally 8 stars).The results showed the included studies were high-quality.Two studies were 8 stars of quality,three studies were 7 stars,one study was 6 stars,one study was 5 stars,and two studies were 4 stars.Meta-analysis showed that the prothrombin G20210A mutation frequencies of the genotypes and alleles showed significant

  2. Prothrombotic gene variants as risk factors of acute myocardial infarction in young women

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    Tomaiuolo Rossella


    Full Text Available Abstract Background Acute myocardial infarction (AMI in young women represent an extreme phenotype associated with a higher mortality compared with similarly aged men. Prothrombotic gene variants could play a role as risk factors for AMI at young age. Methods We studied Factor V Leiden, FII G20210A, MTHFR C677T and beta-fibrinogen -455G>A variants by real-time PCR in 955 young AMI (362 females and in 698 AMI (245 females patients. The data were compared to those obtained in 909 unrelated subjects (458 females from the general population of the same geographical area (southern Italy. Results In young AMI females, the allelic frequency of either FV Leiden and of FII G20210A was significantly higher versus the general population (O.R.: 3.67 for FV Leiden and O.R.: 3.84 for FII G20210A; p Discussion and conclusion Our data confirm that young AMI in females is a peculiar phenotype with specific risk factors as the increased plasma procoagulant activity of FV and FII. On the contrary, the homozygous state for the 677T MTHFR variant may cause increased levels of homocysteine and/or an altered folate status and thus an increased risk for AMI, particularly in males. The knowledge of such risk factors (that may be easily identified by molecular analysis may help to improve prevention strategies for acute coronary diseases in specific risk-group subjects.

  3. Analysis of thrombophilic genetic mutations in patients with Sheehan's syndrome: is thrombophilia responsible for the pathogenesis of Sheehan's syndrome? (United States)

    Gokalp, Deniz; Tuzcu, Alpaslan; Bahceci, Mithat; Ayyildiz, Orhan; Yurt, Murat; Celik, Yusuf; Alpagat, Gulistan


    The gene mutations of Factor V R506Q (FV-Leiden), prothrombin (FII G20210A), methylene tetrahydrofolate reductase (MTHFR) C677T and A1298C and PAI-1 4G/5G are well-established risk factors for thrombosis. We aimed to investigate the prevalence of these gene mutations and their possible impact on the development of pathogenesis in patients with Sheehan's syndrome (SS). 40 female patients with SS compared to a control group of 45 healthy women. The presence of FV-Leiden, FII G20210A, MTHFR C677T, MTHFR A1298C and PAI-1 4G/5G gene mutations were assessed by polymerase chain reaction analysis with a light cycler analyzer. An odds ratio of greater than one is considered to increase the risk of SS disease as found in Factor V Leiden, FII G20210A, MTHFR C677T, MTHFR A1298C and PAI-1 4G/5G polymorphism, as follows respectively: 1.13, 1.85, 6.00, 8.14 and 1.45. MTHFR C677T and MTHFR A1298C polymorphism were found significantly higher in SS patients than the control group (P0.05). The level of plasma total homocysteine (tHcy) was significantly higher in patients with SS than in the control group (P<0.001). We suggest that the genetic mutations of FV-Leiden, FII G20210A, MTHFR C677T, MTHFR A1298C and PAI-1 4G/5G increase the risk of SS. Also, high plasma tHcy levels may be a risk factor for the development of SS.

  4. A frequency domain radar interferometric imaging (FII) technique based on high-resolution methods (United States)

    Luce, H.; Yamamoto, M.; Fukao, S.; Helal, D.; Crochet, M.


    In the present work, we propose a frequency-domain interferometric imaging (FII) technique for a better knowledge of the vertical distribution of the atmospheric scatterers detected by MST radars. This is an extension of the dual frequency-domain interferometry (FDI) technique to multiple frequencies. Its objective is to reduce the ambiguity (resulting from the use of only two adjacent frequencies), inherent with the FDI technique. Different methods, commonly used in antenna array processing, are first described within the context of application to the FII technique. These methods are the Fourier-based imaging, the Capon's and the singular value decomposition method used with the MUSIC algorithm. Some preliminary simulations and tests performed on data collected with the middle and upper atmosphere (MU) radar (Shigaraki, Japan) are also presented. This work is a first step in the developments of the FII technique which seems to be very promising.

  5. Extended radar observations with the frequency radar domain interferometric imaging (FII) technique (United States)

    Luce, H.; Yamamoto, M.; Fukao, S.; Crochet, M.


    In this paper, we present high-resolution observations obtained with the Middle and Upper Atmosphere (MU) radar (Shigaraki, Japan, /34.85°N, /136.10°E) using the frequency radar domain interferometric imaging (FII) technique. This technique has recently been introduced for improving the range resolution capabilities of the mesosphere-stratosphere-troposphere (MST) radars which are limited by their minimum pulse length. The Fourier-based imaging, the Capon method have been performed with 5 equally spaced frequencies between 46.25 and 46.75MHz and with an initial range resolution of 300m. These results have been compared firstly to results obtained using the frequency domain interferometry (FDI) technique with Δf=0.5MHz and, secondly, to results from a classical Doppler beam swinging (DBS) mode applied with a range resolution of 150m. Thin echoing structures could be tracked owing to the improved radar range resolution and some complex structures possibly related to Kelvin Helmholtz instabilities have been detected. Indeed, these structures appeared within the core of a wind shear and were associated with intense vertical wind fluctuations. Moreover, a well-defined thin echo layer was found in an altitude range located below the height of the wind shear. The radar observations have not been fully interpreted yet because the radar configuration was not adapted for this kind of study and because of the lack of complementary information provided by other techniques when the interesting echoing phenomena occurred. However, the results confirm the high potentialities of the FII technique for the study of atmospheric dynamics at small scales.

  6. Genetic modulation of the FV(Leiden)/normal FV ratio and risk of venous thrombosis in factor V Leiden heterozygotes. (United States)

    Segers, O; Simioni, P; Tormene, D; Bulato, C; Gavasso, S; Rosing, J; Castoldi, Elisabetta


    The factor (F)V Leiden mutation causes activated protein C (APC) resistance by decreasing the susceptibility of FVa to APC-mediated inactivation and by impairing the APC-cofactor activity of FV in FVIIIa inactivation. However, APC resistance and the risk of venous thromboembolism (VTE) vary widely among FV Leiden heterozygotes. Common F5 genetic variation probably contributes to this variability. APC resistance was determined in 250 FV Leiden heterozygotes and 133 normal relatives using the prothrombinase-based assay, which specifically measures the susceptibility of plasma FVa to APC. The effects of 12 F5 single-nucleotide polymorphisms (SNPs) on the normalized APC sensitivity ratio (nAPCsr) and on FV levels were determined by multiple regression analysis. In FV Leiden heterozygotes,VTE risk increased with increasing nAPCsr, reaching an odds ratio (OR) of 9.9 (95% confidence interval [CI] 1.2–80.5) in the highest nAPCsr quartile. The minor alleles of several F5 SNPs, including 327 A/G (Q51Q), 409 G/C (D79H), 2663 A/G(K830R, T2 haplotype), 6533 T/C (M2120T) and 6755 A/G (D2194G, R2 haplotype), increased the nAPCsr in FV Leiden heterozygotes, but not in their normal relatives. Most of these effects could be attributed to a shift in the FV(Leiden)/normal FV ratio. Four FV Leiden heterozygotes with extremely high nAPCsr turned out to be pseudo-homozygotes, i.e. they carried a deleterious mutation on the non-Leiden allele. In FV Leiden heterozygotes, the prothrombinase-based nAPCsr is a marker of VTE risk and is modulated by common F5 SNPs that affect the FV(Leiden)/normal FV ratio in plasma.

  7. Construction of multiform scFv antibodies using linker peptide

    Institute of Scientific and Technical Information of China (English)

    Shihua Wang; Cengjie Zheng; Ying Liu; Huirong Zheng; Zonghua Wang


    Multiform single chain variable fragments (acFvs) including different length linker scFvs and bispecific seFv were constructed. The linker lengths of 0, 3, 5, 8, 12, and 15 amino acids between VH and VL of antideoxynivalenol (anti-DON) scFv were used to analyze the affinities of scFvs. The affinity constants of these scFvs increased when the linker was lower than 12 amino acids. The affinity constant would not change when the linker was longer than 12 amino acids. Fusion gene of anti-DON seFv and antizearalenone (anti-ZEN) scFv was also constructed through eormection by a short peptide tinker DNA to express a bispecific scFv. The affinity constants assay showed that the two scFvs of fusion bispecific scFv remained their own affinity compared to their parental scFvs. Competitive direct enzyme linked immunosorbent assay was used to detect DON and ZEN in contaminated wheat (Triticum aestivum L.) samples, and the results indicated that this bispecifie acFv was applicable in DON and ZEN detection. This work confirmed that bispecific scFv could be successfully obtained, and might also have an application in diagnosing fungal infection, and breeding transgertic plants.

  8. Effective single chain antibody (scFv) concentrations in vivo via adenoviral vector mediated expression of secretory scFv

    NARCIS (Netherlands)

    Arafat, WO; Gomez-Navarro, J; Buchsbaum, DJ; Xiang, J; Casado, E; Barker, SD; Mahasreshti, PJ; Haisma, HJ; Barnes, MN; Siegal, GP; Alvarez, RD; Hemminki, A; Nettelbeck, DM; Curiel, DT


    Single chain antibodies (scFv) represent powerful interventional agents for the achievement of targeted therapeutics. The practical utility of these agents have been limited, however, by difficulties related to production of recombinant scFv and the achievement of effective and sustained levels of s

  9. Chemical Chaperones Increasing Expression Level of Soluble Single-chain Fv Antibody(scFv2F3)

    Institute of Scientific and Technical Information of China (English)


    Glutathione peroxidase (GPX) is a selenoenzyme that protects the biomembrane and other cellular components against oxidative damage. The selenium-containing single chain Fv fragment of monoclonal antibody 2F3 (SescFv2F3 ) is a kind of GPX mimic and it has a wide clinical applications because of its high activity and low antigenicity. Se-scFv2F3 is generated by the chemical modification of the single chain Fy fragment of monoclonal antibody 2F3 ( scFv2F3 ), which can be expressed in E. coli. In this article, the effect of chemical chaperones, such as glycerol, glucose, and β-cyclodextrin added to the culture medium, on the expression of soluble scFv2F3 was investigated. The expression level was evaluated by the determination of soluble scFv2F3 contents in the whole cell lysates.The results suggest that both glycerol and β-cyclodextrin greatly increase the expression level of soluble scFv2F3, and β-cyclodextrin is found to be more effective compared with glycerol. Glucose has a slight effect on the expression level of soluble scFv2F3. This is the first example, wherein β-cyclodextrin has been used as a chemical chaperone during the cell culture to improve the expression level of recombinant proteins. In addition, chemical chaperones are found to decrease the toxic effect of IPTG on cells.

  10. Microcin B17, a novel tool for preparation of maxicells: identification of polypeptides encoded by the IncFII minireplicon pMccB17. (United States)

    Mayo, O; Hernández-Chico, C; Moreno, F


    The DNA replication inhibitor peptide microcin B17 is shown to be a useful tool for preparing Escherichia coli maxicells. To illustrate its usefulness, we have identified polypeptides synthesized from pMccB17 and R100 IncFII miniplasmids. After comparing the respective polypeptides and the miniplasmid restriction maps, we concluded that these plasmids share extensive homology in the basic replicon but are different for an adjacent region (parD) that is involved in plasmid stability and maintenance.

  11. Different outcome of six homozygotes for prothrombin A20210A gene variant

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    Angiolillo Antonella


    Full Text Available Abstract Prothrombin G20210A gene variant (FII G20210A is a risk factor for venous thrombotic disease while conflicting results have been reported for the risk of arterial thrombotic events. However, vascular episodes were absent in up to 40% of the 67 homozygotes for the G20210A described so far, which indicates that the clinical expression depends on additional risk/trigger factors. We describe six homozygotes for the G20210A variant, among which the first pair of siblings (cases n. 3 and 4 reported so far that displayed a strongly heterogeneous clinical outcome. Case 1, a female of 27 years, developed a full thrombosis of common femoral, superficial and popliteal veins. She assumed oral contraceptives in the last two years. Case n. 2, 34 years old, suffered of recurrent pregnancy loss in absence of any causative alteration. Cases n. 3 and n. 5 experienced arterial thrombotic disease, i.e., juvenile myocardial infarction (40 years old and stroke (48 years old, respectively, in absence of other risk factors. Finally, cases n. 4 and 6 identified as homozygotes for the FII G20210A variant being consanguineous of symptomatic subjects bearing the variant, did not experience any episode of venous nor arterial disease. Both of them have chronic liver disease with an impairement of the prothrombin time INR. Thus, homozygotes for the G20210A are at risk for arterial (in addition to venous thromobotic events; chronic liver disease might modulate this risk.

  12. Cloning, bacterial expression and crystallization of Fv antibody fragments (United States)

    E´, Jean-Luc; Boulot, Ginette; Chitarra, V´ronique; Riottot, Marie-Madeleine; Souchon, H´le`ne; Houdusse, Anne; Bentley, Graham A.; Narayana Bhat, T.; Spinelli, Silvia; Poljak, Roberto J.


    The variable Fv fragments of antibodies, cloned in recombinant plasmids, can be expressed in bacteria as functional proteins having immunochemical properties which are very similar or identical with those of the corresponding parts of the parent eukaryotic antibodies. They offer new possibilities for the study of antibody-antigen interactions since the crystals of Fv fragments and of their complexes with antigen reported here diffract X-rays to a higher resolution that those obtained with the cognate Fab fragments. The Fv approach should facilitate the structural study of the combining site of antibodies and the further characterization of antigen-antibody interactions by site-directed mutagenesis experiments.

  13. Soluble Expression, Purification and Characterization of Single-chain Fv Catalytic Antibody(sFv-2F3)

    Institute of Scientific and Technical Information of China (English)

    SUN Ye; LI Wei-jia; MA Ji-sheng; MU Ying; DU Xiu-bo; YAN Gang-lin; LUO Gui-min


    To find optimal conditions for expressing the soluble form of sFv-2F3 and to study the purification and property of its derivative Se-sFv-2F3, the preferred expression conditions were investigated by means of orthogonal design. These culture conditions included incubation temperature, inducer concentration, induction time and cell concentration. The evaluation of expression was accomplished by the analysis of whole cell lysates and the yield of soluble sFv-2F3 was calculated according to the analysis of Profinder(FTI-500,Pharmacia). The purification procedure was carried out via a two-step purification procedure consisting of ion-exchange chromatography, followed by immobilized metal affinity chromatography(IMAC). The antioxidant efficacy of Se-sFv-2F3 was demonstrated by the determination of the content of the main product of lipid peroxidation, MDA, the viability of cells and the activity of LDH. We obtained the preferred culture conditions to grow the engineered bacteria and the procedure for preparing soluble sFv-2F3 and confirmed the antioxidant efficacy of Se-sFv-2F3.

  14. A single-chain Fv reactive with the Goodpasture antigen. (United States)

    Ross, C N; Turner, N; Savage, P; Cashman, S J; Spooner, R A; Pusey, C D


    Goodpasture's disease is defined by the presence of autoantibodies to the glomerular basement membrane and characterized clinically by rapidly progressive glomerulonephritis and pulmonary hemorrhage. P1, a murine monoclonal antibody to the Goodpasture antigen (the noncollagenous domain of the alpha 3 chain of type IV collagen, alpha 3(IV)NC1), has been a valuable reagent in investigating the pathogenesis of this disorder. The purpose of this study was to generate and characterize a recombinant form of P1 as a single-chain Fv (scFv). First strand cDNA was made from RNA extracted from the P1 hybridoma cell line, and DNA encoding the antibody light and heavy chain variable domains was amplified by polymerase chain reaction, using universal oligonucleotides. The purified products were ligated sequentially into an expression plasmid separated by a sequence encoding a 15 amino acid flexible oligopeptide linker. The resulting scFv was expressed in E. coli. Functional scFv, designated HBR-3, was obtained by denaturing and refolding the expressed product. HBR-3 was shown by ELISA, immunoblotting, and immunohistologic techniques, to have the same specificity for alpha 3(IV)NC1 as P1 and autoantibodies from patients with Goodpasture's disease. HBR-3 and P1 were shown to have similar affinity for their mutual ligand. On sections of normal human kidney, the scFv bound only to glomerular basement membrane and distal tubular basement membrane. It did not bind to the glomerular basement membrane of patients with Alport's syndrome, in whom the Goodpasture antigen is often not expressed in an antigenic form. We have, therefore, generated a scFv which reproduces the specific binding properties of the parent monoclonal antibody, P1. The potential of HBR-3 as a diagnostic reagent in Alport's syndrome has been demonstrated. The development of this recombinant molecule should permit new approaches to the investigation of Goodpasture's disease.

  15. Homology modelling and bivalent single-chain Fv construction of anti-HepG2 single-chain immunoglobulin Fv fragments from a phage display library

    Indian Academy of Sciences (India)

    Ming Ni; Bing Yu; Y U Huang; Zhenjie Tang; Ping Lei; Xin Shen; Wei Xin; Huifen Zhu; Guanxin Shen


    We prepared single-chain immunoglobulin Fv fragments (scFv) SLH10 specific for the HepG2 cell line after biopanning from a large human-naïve phage display library (Griffin. 1 Library). The three-dimensional (3D) structure of SLH10 was modelled by the Insight II molecule simulation software. The structure was refined using the molecular dynamics method. The structures with the least steric clashes and lowest energy were determined finally. The optimized structures of heavy (VH) and light (VL) variable chains of SLH10 scFv were obtained. Then SLH10 bivalent single-chain Fv (BsFv) was constructed that would be suitable for high-affinity targeting. SLH10 BsFv was generated by linking scFvs together and identified by sequencing. Its expression products were confirmed by western blot analysis. The relative molecular masses of scFv and BsFv were approximately 30 kDa and 60 kDa, respectively. Flow cytometry revealed that SLH10 BsFv bound the selected cell lines with greater signal intensity than the parental scFv. The improved antigen binding of SLH10 BsFv may be useful for immunodiagnostics or targeted gene therapy for liver cancer.

  16. Light quality regulates flowering in FvFT1/FvTFL1 dependent manner in the woodland strawberry Fragaria vesca

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    Marja eRantanen


    Full Text Available Control of flowering in the perennial model, the woodland strawberry (Fragaria vesca L., involves distinct molecular mechanisms that result in contrasting photoperiodic flowering responses and growth cycles in different accessions. The F. vesca homolog of TERMINAL FLOWER1 (FvTFL1 functions as a key floral repressor that causes short-day (SD requirement of flowering and seasonal flowering habit in the SD strawberry. In contrast, perpetual flowering F. vesca accessions lacking functional FvTFL1 show FLOWERING LOCUS T (FvFT1-dependent early flowering specifically under long-days (LD. We show here that the end-of-day far-red (FR and blue (B light activate the expression of FvFT1 and the F. vesca homolog of SUPPRESSOR OF THE OVEREXPRESSION OF CONSTANS (FvSOC1 in both SD and LD strawberries, whereas low expression levels are detected in red (R and SD treatments. By using transgenic lines, we demonstrate that FvFT1 advances flowering under FR and B treatments compared to R and SD treatments in the LD strawberry, and that FvSOC1 is specifically needed for the B light response. In the SD strawberry, flowering responses to these light quality treatments are reversed due to up-regulation of the floral repressor FvTFL1 in parallel with FvFT1 and FvSOC1. Our data highlights the central role of FvFT1 in the light quality dependent flower induction in the LD strawberry and demonstrates that FvTFL1 reverses not only photoperiodic requirements but also light quality effects on flower induction in the SD strawberry.

  17. Light quality regulates flowering in FvFT1/FvTFL1 dependent manner in the woodland strawberry Fragaria vesca. (United States)

    Rantanen, Marja; Kurokura, Takeshi; Mouhu, Katriina; Pinho, Paulo; Tetri, Eino; Halonen, Liisa; Palonen, Pauliina; Elomaa, Paula; Hytönen, Timo


    Control of flowering in the perennial model, the woodland strawberry (Fragaria vesca L.), involves distinct molecular mechanisms that result in contrasting photoperiodic flowering responses and growth cycles in different accessions. The F. vesca homolog of TERMINAL FLOWER1 (FvTFL1) functions as a key floral repressor that causes short-day (SD) requirement of flowering and seasonal flowering habit in the SD strawberry. In contrast, perpetual flowering F. vesca accessions lacking functional FvTFL1 show FLOWERING LOCUS T (FvFT1)-dependent early flowering specifically under long-days (LD). We show here that the end-of-day far-red (FR) and blue (B) light activate the expression of FvFT1 and the F. vesca homolog of SUPPRESSOR OF THE OVEREXPRESSION OF CONSTANS (FvSOC1) in both SD and LD strawberries, whereas low expression levels are detected in red (R) and SD treatments. By using transgenic lines, we demonstrate that FvFT1 advances flowering under FR and B treatments compared to R and SD treatments in the LD strawberry, and that FvSOC1 is specifically needed for the B light response. In the SD strawberry, flowering responses to these light quality treatments are reversed due to up-regulation of the floral repressor FvTFL1 in parallel with FvFT1 and FvSOC1. Our data highlights the central role of FvFT1 in the light quality dependent flower induction in the LD strawberry and demonstrates that FvTFL1 reverses not only photoperiodic requirements but also light quality effects on flower induction in the SD strawberry.

  18. Antibody engineering using phage display with a coiled-coil heterodimeric Fv antibody fragment.

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    Xinwei Wang

    Full Text Available A Fab-like antibody binding unit, ccFv, in which a pair of heterodimeric coiled-coil domains was fused to V(H and V(L for Fv stabilization, was constructed for an anti-VEGF antibody. The anti-VEGF ccFv showed the same binding affinity as scFv but significantly improved stability and phage display level. Furthermore, phage display libraries in the ccFv format were constructed for humanization and affinity maturation of the anti-VEGF antibody. A panel of V(H frameworks and V(H-CDR3 variants, with a significant improvement in affinity and expressibility in both E. coli and yeast systems, was isolated from the ccFv phage libraries. These results demonstrate the potential application of the ccFv antibody format in antibody engineering.

  19. Construction of single chain Fv antibody against transferrin receptor and its protein fusion with alkaline phosphatase

    Institute of Scientific and Technical Information of China (English)

    Dao-Feng Yang; Hui-Fen Zhu; Zhi-Hua Wang; Guan-Xin Shen; De-Ying Tian


    AIM: To construct fusion protein of a single-chain antibody(scFv) against transferrin receptor (TfR) with alkalinephosphatase (AP).METHODS: The VH-linker-VL, namely scFv gene, wasprepared by amplifying the VH and VL genes from plasmid pGEM-T-VH and pGEM-T-VL with splicing overlap extension polymerase chain reaction (SOE PCR). After the ScFv gene was modified by SfiⅠ and NotⅠ, it was subcloned into the secretory expression vector pUC19/119, and then was transformed into E. coli TG1. The positive colonies were screened by colony PCR and their expressions were induced by IPTG. ScFv gene was gained by digesting ScFv expression vector pUC19/119 with Sfi I and NotⅠ restriction enzymes, then subcloned into expression vector pDAP2, followed by transformation in E. coli TG1. The positive colonies were selected by bacterial colony PCR. The expression of fusion protein (scFv-AP) was induced by IPTG. Its activity was detected by enzyme immunoassay. The molecular weights of scFv and scFv-AP were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).RESULTS: The product of SOE PCR formed a band of 700 bp in agarose gel electrophoresis. SDS-PAGE demonstrated the molecular weight of scFv was 27 ku. Immunofluorescent assay (IFA) demonstrated its reactivity with TfR. The molecular weight of scFv-AP was 75 ku. Enzyme immunoassay showed that scFv-AP could specifically bind to human TfR and play AP activity.CONCLUSION: We have successfully prepared the antihuman TfR scFv and constructed the fusion protein of scFv and AP. It is promising for immunological experiments.

  20. Toward low-cost affinity reagents: lyophilized yeast-scFv probes specific for pathogen antigens.

    Directory of Open Access Journals (Sweden)

    Sean A Gray

    Full Text Available The generation of affinity reagents, usually monoclonal antibodies, remains a critical bottleneck in biomedical research and diagnostic test development. Recombinant antibody-like proteins such as scFv have yet to replace traditional monoclonal antibodies in antigen detection applications, in large part because of poor performance of scFv in solution. To address this limitation, we have developed assays that use whole yeast cells expressing scFv on their surfaces (yeast-scFv in place of soluble purified scFv or traditional monoclonal antibodies. In this study, a nonimmune library of human scFv displayed on the surfaces of yeast cells was screened for clones that bind to recombinant cyst proteins of Entamoeba histolytica, an enteric pathogen of humans. Selected yeast-scFv clones were stabilized by lyophilization and used in detection assay formats in which the yeast-scFv served as solid support-bound monoclonal antibodies. Specific binding of antigen to the yeast-scFv was detected by staining with rabbit polyclonal antibodies. In flow cytometry-based assays, lyophilized yeast-scFv reagents retained full binding activity and specificity for their cognate antigens after 4 weeks of storage at room temperature in the absence of desiccants or stabilizers. Because flow cytometry is not available to all potential assay users, an immunofluorescence assay was also developed that detects antigen with similar sensitivity and specificity. Antigen-specific whole-cell yeast-scFv reagents can be selected from nonimmune libraries in 2-3 weeks, produced in vast quantities, and packaged in lyophilized form for extended shelf life. Lyophilized yeast-scFv show promise as low cost, renewable alternatives to monoclonal antibodies for diagnosis and research.

  1. HI SNRs, FV Wings, and the Structure of the ISM (United States)

    Koo, B.-C.


    We discuss the statistics of old supernova remnants (SNRs) with expanding schi shells, or ``schi SNRs", in the Galaxy. Systematic schi 21-cm emission line studies have detected fast-moving atomic gas associated with 25 SNRs, which is about 10% of the known radio/X-ray SNRs. This number is much less than the expected number of visible schi SNRs if the interstellar medium is filled with warm neutral medium. A plausible explanation is that most of the schi SNRs are too faint to be visible in radio or X-ray. Large-scale (ℓ, v) diagrams of the schi 21-cm emission in the Galactic plane show broad wings which extend to velocities forbidden by the Galactic rotation. Many of these ``forbidden-velocity (FV)" wings are not associated with known SNRs or known external galaxies and could be candidates for heretofore unidentified old schi SNRs. We discuss the properties of the FV wings and the implications for the structure of the interstellar medium.

  2. Baculovirus display of single chain antibody (scFv using a novel signal peptide

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    Gonzalez Gaëlle


    Full Text Available Abstract Background Cells permissive to virus can become refractory to viral replication upon intracellular expression of single chain fragment variable (scFv antibodies directed towards viral structural or regulatory proteins, or virus-coded enzymes. For example, an intrabody derived from MH-SVM33, a monoclonal antibody against a conserved C-terminal epitope of the HIV-1 matrix protein (MAp17, was found to exert an inhibitory effect on HIV-1 replication. Results Two versions of MH-SVM33-derived scFv were constructed in recombinant baculoviruses (BVs and expressed in BV-infected Sf9 cells, N-myristoylation-competent scFvG2/p17 and N-myristoylation-incompetent scFvE2/p17 protein, both carrying a C-terminal HA tag. ScFvG2/p17 expression resulted in an insoluble, membrane-associated protein, whereas scFvE2/p17 was recovered in both soluble and membrane-incorporated forms. When coexpressed with the HIV-1 Pr55Gag precursor, scFvG2/p17 and scFvE2/p17 did not show any detectable negative effect on virus-like particle (VLP assembly and egress, and both failed to be encapsidated in VLP. However, soluble scFvE2/p17 isolated from Sf9 cell lysates was capable of binding to its specific antigen, in the form of a synthetic p17 peptide or as Gag polyprotein-embedded epitope. Significant amounts of scFvE2/p17 were released in the extracellular medium of BV-infected cells in high-molecular weight, pelletable form. This particulate form corresponded to BV particles displaying scFvE2/p17 molecules, inserted into the BV envelope via the scFv N-terminal region. The BV-displayed scFvE2/p17 molecules were found to be immunologically functional, as they reacted with the C-terminal epitope of MAp17. Fusion of the N-terminal 18 amino acid residues from the scFvE2/p17 sequence (N18E2 to another scFv recognizing CD147 (scFv-M6-1B9 conferred the property of BV-display to the resulting chimeric scFv-N18E2/M6. Conclusion Expression of scFvE2/p17 in insect cells using a BV

  3. Expression and bioactivity identification of soluble MG7 scFv

    Institute of Scientific and Technical Information of China (English)

    Zhao-Cai Yu; Jie Ding; Bo-Rong Pan; Dai-Ming Fan; Xue-Yong Zhang


    AIM: To examine the molecular mass and identify thebioactivity of MG7 scFv for its application as a targetingmediator in gene therapy of gastric cancer.METHODS: Two strongly positive recombinant phage clonesscreened from MG7 recombinant phage antibody library wereseparately transfected into E. coli TG1. Plasmid wasisolated from the transfected E. coli TG1 and digested byEcoR Ⅰ and Hind Ⅲ to examine the length of exogenousscFv gene. Then, the positive recombinant phage cloneswere individually transfected into E. coli HB2151. Thetransfectant was cultured and induced by IPTG. Perplasmicextracts was prepared from the induced transfectant byosmotic shock. ELISA was used to examine the antigen-binding affinity of the soluble MG7 scFv. Immunodottingassay was adopted to evaluate the yield of soluble MG7 scFvproduced by transfected E. coli HB2151. Western blot wasused to examine the molecular mass of MG7 scFv. Finally,the nucleotide sequence of MG7 scFv was examined by DNAsequencing.RESULTS: Two positive recombinant phage clones werefound to contain the exogenous scFv gene. ELISA showedthat MG7 scFv had strong antigen-binding affinity.Immuodotting assay showed that transfected E. coli HB2151could successfully produce the soluble MG7 scFv with highyield via induction by IPTG. The molecular mass of MG7scFv was 30 kDa by western blot. DNA sequencingdemonstrated that the VH and VL genes of MG7 scFv were363bp and 321 bp, respectively.CONCLUSION: We have successfully developed the solubleMG7 scFv which possessed strong antigen-binding affinity.

  4. The relationship between FV Leiden and pulmonary embolism

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    Hooper W Craig


    Full Text Available Abstract Pulmonary embolism (PE is one of the leading causes of in-patient hospital deaths. As a consequence, the identification of hemostatic variables that could identify those at risk would be important in reducing mortality. It has previously been thought that deep vein thrombosis and PE are a single disease entity and would, therefore, have the same risk factors. This view is changing, however, with the realization that the prevalence of FV Leiden, a recognized genetic risk factor for deep vein thrombosis, may be a 'milder' genetic risk factor for PE. These observations suggest that PE is not only associated with a different set of risk factors, but may be reflective of a different clot structure.

  5. Ischemic stroke in Ukrainian population: possible involvement of the F2 G20210A, F5 G1691A and MTHFR C677T gene variants

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    Kuznetsova S. M.


    Full Text Available Aim. To evaluate a possible involvement of the F2, F5, MTHFR gene variants into ischemic stroke pathogenesis in population of Ukraine. Methods. Polymorphic variants were analyzed in unrelated 183 stroke patients, 100 individuals from the general population of Ukraine and 88 healthy individuals elder than 65 years using PCR followed by RFLP analysis. Results. Unfavourable polymorphic variants F2 20210A, F5 1691A and MTHFR 677T were observed more frequently in patients with ischemic stroke comparing to control groups. Conclusions. F5 1691A and MTHFR 677T polymorphic variants are associated with the occurrence of ischemic stroke in women. F2 20210A is associated with the occurrence of ischemic stroke in men. Cumulative risk factor for stroke development is revealed in a combination of unfavorable polymorphic variants 20210A, 1691A and 677T of F2, F5 and MTHFR genes.

  6. Clinical significance of factor V leiden and prothrombin G20210A-mutations in cerebral venous thrombosis - comparison with arterial ischemic stroke. (United States)

    Beye, Aida; Pindur, Gerhard


    Cerebrovascular diseases are considered in a different way concerning their etiology with regard to arterial and venous occlusion. The role of thrombophilia in this context remains undetermined. For this reason, a case-control study was conducted including a total of 202 patients (154 females, 48 males) aged from 18 to 76 years (mean: 39.8 years) suffering either from cerebral sinus venous thrombosis (n = 101) or from arterial ischemic stroke (n = 101). Study groups were evaluated on the basis of age- and gender-matched pairs. Gene mutations of factor V-1691 (factor V Leiden) and prothrombin-20210 being considered as the most common thrombophilia markers were analyzed in this study. Factor V Leiden-mutations were found in 16.8% of patients with cerebral sinus venous thrombosis (CVT) and in 17.8% of patients with arterial ischemic stroke (AIS), which was significantly more frequent than in controls at a rate of 4.95% (ORs: 3.89 and 4.16). Prothrombin-mutations were significantly more frequent in CVT at a rate of 14.9% versus 2.97% in controls (OR: 5.70). This does not apply for AIS showing a rate of 4.95% prothrombin-mutations. Rates of factor V Leiden-mutations are not different in CVT compared with AIS. In contrast, however, prothrombin-mutations were significantly more frequent in CVT than in AIS with a rate of 14.9% versus 4.95% (OR 3.35). Furthermore, 3 cases with combined heterozygosity of factor V Leiden- and prothrombin-mutation have been identified in CVT, but not in AIS or controls. All of the above mentioned mutations were exclusively heterozygous. We conclude from these data that thrombophilia in terms of factor V Leiden genotype is a risk factor for both CVT and AIS in equal measure. In contrast, prothrombin-20210-mutations were different playing a significant role in the pathogenesis of cerebral sinus vein- thrombosis, but not in arterial ischemic stroke. Also, the combined occurrence of heterozygous prothrombin- and factor V Leiden-mutation clearly favors the emergence of cerebral sinus venous thrombosis. Therefore, in terms of thrombophilia such as investigated in this study, pathogenesis of arterial and venous occlusions in cerebrovascular disease has to be regarded as different.

  7. Design and analysis of an intelligent public FV lighting system; Diseno y analisis de un alumbrado publico FV inteligente

    Energy Technology Data Exchange (ETDEWEB)

    Hanessian D, Ana V.; Gordon, Manuel [Universidad Autonoma Metropolitana, Unidad Azcapotzalco, Mexico, D.F. (Mexico)


    In the Mexico's National Energy Balance of 2008, it is considered that of the total of the electrical power consumption in our country, 18% is dedicated to lighting. Conscious of the necessity of saving energy in public lighting in this article is presented the design, construction and analysis of the power consumption of a public light fed with electricity of photovoltaic cells and the control of intensity on the light in inverse way of the natural light. A lamp constructed based in light emitting diodes (LEDs) is used. This has the quality of consuming very little energy that could be provided by the sun and be stored to use it at night. With this system, proven at scale, energy savings are obtained superior to 50% of the conventional one and, in relation to the commercial photovoltaic (FV) luminaries up to 30%. [Spanish] Del Balance Nacional de Energia, de Mexico, de 2008, se considera que del total del consumo de energia electrica en nuestro pais, el 18% esta dedicado a la iluminacion. Conscientes de la necesidad de ahorrar energia en alumbrado publico, en este articulo se presenta el diseno de construccion y analisis del consumo energetico de una luminaria publica alimentada con electricidad de celdas fotovoltaicas y el control de intensidad de la luz de manera inversa a la luz natural. Se utiliza un foco construido a base a los diodos emisores de luz (LEDs, por sus siglas en ingles). Estos tienen la cualidad de consumir muy poca energia que podra ser suministrada por el sol y almacenada para utilizarla en la noche. Con este sistema, probado a escala, se logran ahorros de energia superiores al 50% del convencional y, en relacion a las luminarias fotovoltaicas (FV) comerciales, hasa el 30%.

  8. Flow cytometry-based methods for assessing soluble scFv activities and detecting pathogen antigens in solution

    Energy Technology Data Exchange (ETDEWEB)

    Gray, Sean; Weigel, Kris M.; Miller, Keith D.; Ndung' u, Joseph; Buscher, Philippe; Tran, Thao N.; Baird, Cheryl L.; Cangelosi, Gerard A.


    Novel methods are reported for evaluating and utilizing single chain fragment variable (scFv) antibodies derived from yeast-display libraries. Yeast-display was used to select scFv specific to invariant surface glycoproteins (ISG) of Trypanosoma brucei. A limiting step in the isolation of scFv from nonimmune libraries is the conversion of highly active yeast-displayed scFv into soluble antibodies that can be used in standard immunoassays. Challenges include limited solubility or activity following secretion and purification of scFv. For this reason, few scFv derived from yeast-display platforms have moved into development and implementation as diagnostic reagents. To address this problem, assays were developed that employ both yeastdisplayed and secreted scFv as analytical reagents. The first is a competitive inhibition flow cytometry (CIFC) assay that detects secreted scFv by virtue of its ability to competitively inhibit the binding of biotinylated antigen to yeast-displayed scFv. The second is an epitope binning assay that uses secreted scFv toidentify additional yeast-displayed scFv that bind nonoverlapping or noncompeting epitopes on an antigen. The epitope binning assay was used not only to identify sandwich assay pairs with yeast-displayed scFv, but also to identify active soluble scFv present in low concentration in a crude expression extract. Finally, a CIFC assay was developed that bypasses entirely the need for soluble scFv expression, by using yeast displayed scFv to detect unlabeled antigen in samples. These methods will facilitate the continued development and practical implementation of scFv derived from yeast-display libraries.

  9. Evolution of the retroviral restriction gene Fv1: inhibition of non-MLV retroviruses.

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    Melvyn W Yap


    Full Text Available Fv1 is the prototypic restriction factor that protects against infection by the murine leukemia virus (MLV. It was first identified in cells that were derived from laboratory mice and was found to be homologous to the gag gene of an endogenous retrovirus (ERV. To understand the evolution of the host restriction gene from its retroviral origins, Fv1s from wild mice were isolated and characterized. Most of these possess intact open reading frames but not all restricted N-, B-, NR-or NB-tropic MLVs, suggesting that other viruses could have played a role in the selection of the gene. The Fv1s from Mus spretus and Mus caroli were found to restrict equine infectious anemia virus (EIAV and feline foamy virus (FFV respectively, indicating that Fv1 could have a broader target range than previously thought, including activity against lentiviruses and spumaviruses. Analyses of the Fv1 sequences revealed a number of residues in the C-terminal region that had evolved under positive selection. Four of these selected residues were found to be involved in the novel restriction by mapping studies. These results strengthen the similarities between the two capsid binding restriction factors, Fv1 and TRIM5α, which support the hypothesis that Fv1 defended mice against waves of retroviral infection possibly including non-MLVs as well as MLVs.

  10. Protein Fv produced during vital hepatitis is a novel activator of human basophils and mast cells. (United States)

    Patella, V; Bouvet, J P; Marone, G


    Protein Fv is found in the normal liver and is released in the stools of patients suffering from viral hepatitis. Protein Fv isolated from five patients stimulated the release of histamine and sulfidopeptide leukotriene C4 from purified and unpurified peripheral blood basophils. Protein Fv absorbed with protein A-Sepharose coated with polyclonal IgG did not induce histamine secretion, whereas removal of putative contaminating Ig did not modify the releasing activity. The characteristics of the release reaction were similar to those of rabbit IgG anti-Fc fragment of human IgE (anti-IgE). There was an excellent correlation (Spearman rank coefficient (rs) = 0.83; p ADZ) blocked both anti-IgE- and protein Fv-induced releases, whereas human polyclonal IgG and a monoclonal IgG purified from another myeloma patient (patient ZEG) selectively blocked protein Fv-induced secretion. Protein Fv also induced the release of preformed (histamine and tryptase) and de novo synthesized mediators (sulfidopeptide leukotriene C4 and/or PGD2) from mast cells purified from human lung parenchyma and skin tissues. There was a significant correlation between the maximal percent histamine release induced by protein Fv and anti-IgE from skin mast cells (rs = 0.63; p < 0.01). There was also an excellent correlation between histamine and tryptase release caused by protein Fv from both lung (rs = 0.80; p < 0.001) and skin mast cells (rs = 0.70; p < 0.01). Thus, we established that protein Fv acts as a novel activator of human basophils and mast cells presumably by interacting with the VH domain of the IgE.

  11. Synergistic capture of Clostridium botulinum Type A neurotoxin by scFv antibodies to novel epitopes

    Energy Technology Data Exchange (ETDEWEB)

    Gray, Sean A.; Barr, John R.; Kalb, Suzanne R.; Marks, James D.; Baird, Cheryl L.; Cangelosi, Gerard A.; Miller, Keith D.; Feldhaus, Michael J.


    A non-immune library of human single chain fragment variable (scFv) antibodies displayed on Saccharomyces cerevisiae was screened for binding to the Clostridium botulinum neurotoxin serotype A binding domain [BoNT/A (Hc)] with the goal of identifying scFv to novel epitopes. To do this, an antibody-mediated labeling strategy was used in which antigen-binding yeast clones were selected after labeling with previously characterized monoclonal antibodies (MAbs) specific to the Hc. Twenty unique scFv clones were isolated that bound Hc. Of these, three also bound to full-length BoNT/A toxin complex with affinities ranging from 5 nM to 170 nM. Epitope binning showed that the three unique clones recognized at least two epitopes that were distinct from one another and from the detection MAbs. After production in E. coli, the scFv were coupled to magnetic particles and tested for their ability to capture BoNT/A holotoxin using an Endopep-MS assay. In this assay, toxin captured by scFv coated magnetic particles was detected by incubation of the complex with a peptide containing a BoNT/A-specific cleavage sequence. Mass spectrometry was used to detect the ratio of intact peptide to cleavage products as evidence for toxin capture. When tested individually, each of the scFv showed a weak positive Endopep-MS result. However, when the particles were coated with all three scFv simultaneously, they exhibited significantly higher Endopep-MS activity, consistent with synergistic binding. These results demonstrate novel approaches toward the isolation and characterization of scFv antibodies specific to unlabeled antigen. They also provide evidence that distinct scFv antibodies can work synergistically to increase the efficiency of antigen capture onto a solid support.

  12. Fv-1 locus restriction of mouse retroviruses in glucocorticoid-treated cells

    Energy Technology Data Exchange (ETDEWEB)

    Tennant, R.W.; Jones, S.C.; Otten, J.A.; Yang, W.K.; Brown, A.


    Treatment of mouse embryo cells with hydrocortisone (10/sup -6/M) or dexamethasone (10/sup -4/ to 10/sup -6/M) increases virus synthesis whether the cells are permissive or restrictive at the Fv-1 locus. However, the number of cells infected was not increased in either permissive or restrictive cells by treatment with either glucocorticoid, and the two-hit titration pattern in restrictive cells remained unaltered. Therefore, the enhancement of virus replication by the glucocorticoids is independent of Fv-1 restriction and appears to occur after the Fv-1 locus-sensitive step in virus synthesis.


    Hausmann, Jennifer C; Wack, Allison N; Allender, Matthew C; Cranfield, Mike R; Murphy, Kevin J; Barrett, Kevin; Romero, Jennell L; Wellehan, James F X; Blum, Stella A; Zink, M Christine; Bronson, Ellen


    The Maryland Zoo in Baltimore experienced an outbreak of Frog virus-3 (FV3)-like ranavirus during the summer of 2011, during which 14 of 27 (52%) of its captive eastern box turtles (Terrapene carolina carolina) survived. To assess survival, immunity, and viral shedding, an experimental challenge study was performed in which the surviving, previously infected turtles were reinfected with the outbreak strain of FV3-like ranavirus. Seven turtles were inoculated with virus intramuscularly and four control turtles received saline intramuscularly. The turtles were monitored for 8 wk with blood and oral swabs collected for quantitative polymerase chain reaction (qPCR). During that time, one of seven (14%) inoculated turtles and none of the controls (0%) died; there was no significant difference in survival. Clinical signs of the inoculated turtles, except for the turtle that died, were mild compared to the original outbreak. Quantitative PCR for FV3-like ranavirus on blood and oral swabs was positive for all inoculated turtles and negative for all controls. The turtle that died had intracytoplasmic inclusion bodies in multiple organs. Three inoculated and two control turtles were euthanized at the end of the study. No inclusion bodies were present in any of the organs. Quantitative PCR detected FV3-like ranavirus in the spleen of a control turtle, which suggested persistence of the virus. The surviving five turtles were qPCR-negative for FV3-like ranavirus from blood and oral swabs after brumation. Quantitative PCR for Terrapene herpesvirus 1 found no association between ranavirus infection and herpesvirus loads. In conclusion, previously infected eastern box turtles can be reinfected with the same strain of FV3-like ranavirus and show mild to no clinical signs but can shed the virus from the oral cavity.

  14. AFSC/ABL: Origins of salmon seized from the F/V Arctic Wind (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Samples of chum (Oncorhynchus keta), sockeye (O. nerka), and chinook salmon (O. tshawytscha) seized from the F/V Arctic Wind were analyzed to determine their region...

  15. The inhibition of lung cancer cell growth by intracellular immunization with LC-1 ScFv

    Institute of Scientific and Technical Information of China (English)


    A monoclonal antibody, LC-l, recognizing lung cancer associated common antigens was obtained in authors' laboratory. Its single chain Fv fragment (ScFv) named LC-1 ScFv was constructed based on recombinant phage displayed techniques. For expression on cell membrane, LC-1 ScFv was cloned into pDisplay vector, which directed the cloned gene to express as cell membrane bound protein. The resulting plasmid was sequenced and then introduced by the lipofectin method into a lung adenocarcinoma cell line SPC-A-1. G418 resistant cells were obtained by G418 selection. After transfection, LC-1 ScFv expression was observed by Western blot analysis and the expression of cognate antigens was down-regulated as shown in ELISA assay. SPC-A-1-pDisplay-ScFv cells grew in vitro at lower speed than the control intact cells and the cells transfected with vacant vector. Flow cytometry analysis detected a substantial increase in G1 phase and decrease in S phase in population of SPC-A-1-pDisplay-ScFv cells compared to SPC-A-1 and SPC-A1-pDisplay cells. Semi-quantitative RT-PCR analysis showed that c-myc expression was down-regulated in SPC-A-1-pDisplay-ScFv cells. It seems that the antigens recognized by LC-1 may be in some way involved in a growth stimulating pathway and the antibody blocking of the function of the antigens shut down the pathway and thus down-regulate the expression of c-myc and growth of the cells.

  16. High Specific Selectivity and Membrane-Active Mechanism of Synthetic Cationic Hybrid Antimicrobial Peptides Based on the Peptide FV7. (United States)

    Tan, Tingting; Wu, Di; Li, Weizhong; Zheng, Xin; Li, Weifen; Shan, Anshan


    Hybrid peptides integrating different functional domains of peptides have many advantages, such as remarkable antimicrobial activity, lower hemolysis and ideal cell selectivity, compared with natural antimicrobial peptides. FV7 (FRIRVRV-NH₂), a consensus amphiphilic sequence was identified as being analogous to host defense peptides. In this study, we designed a series of hybrid peptides FV7-LL-37 (17-29) (FV-LL), FV7-magainin 2 (9-21) (FV-MA) and FV7-cecropin A (1-8) (FV-CE) by combining the FV7 sequence with the small functional sequences LL-37 (17-29) (LL), magainin 2 (9-21) (MA) and cecropin A (1-8) (CE) which all come from well-described natural peptides. The results demonstrated that the synthetic hybrid peptides, in particular FV-LL, had potent antibacterial activities over a wide range of Gram-negative and Gram-positive bacteria with lower hemolytic activity than other peptides. Furthermore, fluorescent spectroscopy indicated that the hybrid peptide FV-LL exhibited marked membrane destruction by inducing outer and inner bacterial membrane permeabilization, while scanning electron microscopy (SEM) and transmission electron microscopy (TEM) demonstrated that FV-LL damaged membrane integrity by disrupting the bacterial membrane. Inhibiting biofilm formation assays also showed that FV-LL had similar anti-biofilm activity compared with the functional peptide sequence FV7. Synthetic cationic hybrid peptides based on FV7 could provide new models for combining different functional domains and demonstrate effective avenues to screen for novel antimicrobial agents.

  17. High Specific Selectivity and Membrane-Active Mechanism of Synthetic Cationic Hybrid Antimicrobial Peptides Based on the Peptide FV7 (United States)

    Tan, Tingting; Wu, Di; Li, Weizhong; Zheng, Xin; Li, Weifen; Shan, Anshan


    Hybrid peptides integrating different functional domains of peptides have many advantages, such as remarkable antimicrobial activity, lower hemolysis and ideal cell selectivity, compared with natural antimicrobial peptides. FV7 (FRIRVRV-NH2), a consensus amphiphilic sequence was identified as being analogous to host defense peptides. In this study, we designed a series of hybrid peptides FV7-LL-37 (17–29) (FV-LL), FV7-magainin 2 (9–21) (FV-MA) and FV7-cecropin A (1–8) (FV-CE) by combining the FV7 sequence with the small functional sequences LL-37 (17–29) (LL), magainin 2 (9–21) (MA) and cecropin A (1–8) (CE) which all come from well-described natural peptides. The results demonstrated that the synthetic hybrid peptides, in particular FV-LL, had potent antibacterial activities over a wide range of Gram-negative and Gram-positive bacteria with lower hemolytic activity than other peptides. Furthermore, fluorescent spectroscopy indicated that the hybrid peptide FV-LL exhibited marked membrane destruction by inducing outer and inner bacterial membrane permeabilization, while scanning electron microscopy (SEM) and transmission electron microscopy (TEM) demonstrated that FV-LL damaged membrane integrity by disrupting the bacterial membrane. Inhibiting biofilm formation assays also showed that FV-LL had similar anti-biofilm activity compared with the functional peptide sequence FV7. Synthetic cationic hybrid peptides based on FV7 could provide new models for combining different functional domains and demonstrate effective avenues to screen for novel antimicrobial agents. PMID:28178190

  18. Xenopus-FV3 host-pathogen interactions and immune evasion. (United States)

    Jacques, Robert; Edholm, Eva-Stina; Jazz, Sanchez; Odalys, Torres-Luquis; Francisco, De Jesús Andino


    We first review fundamental insights into anti-ranavirus immunity learned with the Xenopus laevis/ranavirus FV3 model that are generally applicable to ectothermic vertebrates. We then further investigate FV3 genes involved in immune evasion. Focusing on FV3 knockout (KO) mutants defective for a putative viral caspase activation and recruitment domain-containing (CARD)-like protein (Δ64R-FV3), a β-hydroxysteroid dehydrogenase homolog (Δ52L-FV3), and an immediate-early18kDa protein (FV3-Δ18K), we assessed the involvement of these viral genes in replication, dissemination and interaction with peritoneal macrophages in tadpole and adult frogs. Our results substantiate the role of 64R and 52L as critical immune evasion genes, promoting persistence and dissemination in the host by counteracting type III IFN in tadpoles and type I IFN in adult frogs. Comparably, the substantial accumulation of genome copy numbers and exacerbation of type I and III IFN gene expression responses but deficient release of infectious virus suggests that 18K is a viral regulatory gene. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Structural dynamics of a single-chain Fv antibody against (4-hydroxy-3-nitrophenyl)acetyl. (United States)

    Sato, Yusui; Tanaka, Yusuke; Inaba, Satomi; Sekiguchi, Hiroshi; Maruno, Takahiro; Sasaki, Yuji C; Fukada, Harumi; Kobayashi, Yuji; Azuma, Takachika; Oda, Masayuki


    Protein structure dynamics are critical for understanding structure-function relationships. An antibody can recognize its antigen, and can evolve toward the immunogen to increase binding strength, in a process referred to as affinity maturation. In this study, a single-chain Fv (scFv) antibody against (4-hydroxy-3-nitrophenyl)acetyl, derived from affinity matured type, C6, was designed to comprise the variable regions of light and heavy chains connected by a (GGGGS)3 linker peptide. This scFv was expressed in Escherichia coli in the insoluble fraction, solubilized in the presence of urea, and refolded by stepwise dialysis. The correctly refolded scFv was purified, and its structural, physical, and functional properties were analyzed using analytical ultracentrifugation, circular dichroism spectrometry, differential scanning calorimetry, and surface plasmon resonance biosensor. Thermal stability of C6 scFv increased greatly upon antigen binding, due to favorable enthalpic contributions. Antigen binding kinetics were comparable to those of the intact C6 antibody. Structural dynamics were analyzed using the diffracted X-ray tracking method, showing that fluctuations were suppressed upon antigen binding. The antigen binding energy determined from the angular diffusion coefficients was in good agreement with that calculated from the kinetics analysis, indicating that the fluctuations detected at single-molecule level are well reflected by antigen binding events.

  20. Fast conversion of scFv to Fab antibodies using type IIs restriction enzymes. (United States)

    Sanmark, Hanna; Huovinen, Tuomas; Matikka, Tero; Pettersson, Tiina; Lahti, Maria; Lamminmäki, Urpo


    Single chain variable fragment (scFv) antibody libraries are widely used for developing novel bioaffinity reagents, although Fab or IgG molecules are the preferred antibody formats in many final applications. Therefore, rapid conversion methods for combining multiple DNA fragments are needed to attach constant domains to the scFv derived variable domains. In this study we describe a fast and easy cloning method for the conversion of single framework scFv fragments to Fab fragments using type IIS restriction enzymes. All cloning steps excluding plating of the Fab transformants can be done in 96 well plates and the procedure can be completed in one working day. The concept was tested by converting 69 scFv clones into Fab format on 96 well plates, which resulted in 93% success rate. The method is particularly useful as a high-throughput tool for the conversion of the chosen scFv clones into Fab molecules in order to analyze them as early as possible, as the conversion can significantly affect the binding properties of the chosen clones.

  1. Annual parallax and a dimming event of a Mira variable star, FV Bootis (United States)

    Kamezaki, Tatsuya; Nakagawa, Akiharu; Omodaka, Toshihiro; Inoue, Kan-ichiro; Chibueze, James O.; Nagayama, Takumi; Ueno, Yuji; Matsunaga, Noriyuki


    We present the first measurement of the trigonometric parallax of water masers associated with a Mira star, FV Bootis (FV Boo) using VLBI Exploration of Radio Astrometry (VERA). Based on our multi-epoch VERA observations, we derived the parallax to be 0.97 ± 0.06 mas, which corresponds to a distance of 1.03^{+0.07}_{-0.06} kpc. The water masers around FV Boo were spatially distributed over an area of 41 au × 41 au, and their internal motions indicate the presence of an outflow. Using the Kagoshima University 1 m optical/infrared telescope, we determined the period to be 305.6 d and the mean apparent magnitude to be +2.91 mag in the K'-band. On the period-luminosity plane, the obtained period and K'-band magnitude puts FV Boo slightly below the sequence of Miras, possibly due to circumstellar reddening. Combining our photometric data with COBE and 2MASS datasets spanning over 20 years, we found in the near infrared that FV Boo was significantly fainter in 2005 compared with preceding and later phases. Its color, however, did not show a large variation through this change. We infer that the dimming could be caused by an eclipse due to a cloud in a binary system.

  2. Construction of Human ScFv Phage Display Library against Ovarian Tumor

    Institute of Scientific and Technical Information of China (English)

    XIA Jinsong; BI Hao; YAO Qin; QU Shen; ZONG Yiqiang


    In order to construct a single chain fragment variable (ScFv) phage display library against ovarian tumor, by using RT-PCR, the human heavy chain variable region genes (VH) and light chain variable region genes (VL) were amplified from lymphocytes of ovarian tumor patients and subsequently assembled into ScFv genes by SOE. The resulting ScFv genes were electrotransformed into E.coli TG1 and amplified with the co-infection of helper phage M13KO7 to obtain phage display library. The capacity and titer of the resulting library were detected. The phage antibody library with a capacity of approximately 3 × 109 cfu/μg was obtained. After amplification with helper phage, the titer of antibody library reached 5 × 1012 cfu/mL. Human ScFv library against ovarian tumor was constructed successfully, which laid a foundation for the screening of ovarian tumor specific ScFv for the radioimmunoimaging diagnosis of ovarian tumor.

  3. Isolation of anti-T cell receptor scFv mutants by yeast surface display. (United States)

    Kieke, M C; Cho, B K; Boder, E T; Kranz, D M; Wittrup, K D


    Yeast surface display and sorting by flow cytometry have been used to isolate mutants of an scFv that is specific for the Vbeta8 region of the T cell receptor. Selection was based on equilibrium binding by two fluorescently labeled probes, a soluble Vbeta8 domain and an antibody to the c-myc epitope tag present at the carboxy-terminus of the scFv. The mutants that were selected in this screen included a scFv with threefold increased affinity for the Vbeta8 and scFv clones that were bound with reduced affinities by the anti-c-myc antibody. The latter finding indicates that the yeast display system may be used to map conformational epitopes, which cannot be revealed by standard peptide screens. Equilibrium antigen binding constants were estimated within the surface display format, allowing screening of isolated mutants without necessitating subcloning and soluble expression. Only a relatively small library of yeast cells (3 x 10[5]) displaying randomly mutagenized scFv was screened to identify these mutants, indicating that this system will provide a powerful tool for engineering the binding properties of eucaryotic secreted and cell surface proteins.

  4. Preparation of human single chain Fv antibody against hepatitis C virus E2 protein and its identification in immunohistochemistry

    Institute of Scientific and Technical Information of China (English)

    Yan-Wei Zhong; Jun Cheng; Gang Wang; Shuang-Shuang Shi; Li Li; Ling-Xia Zhang; Ju-Mei Chen


    AIM: To screen human single chain Fv antibody (scFv)against hepatitis C virus E2 antigen and identify its applicationin immunohistochemistry.METHODS: The phage antibody library was panned by HCVE2 antigen, which was coated in microtiter plate. After fiverounds of biopanning,56 phage clones were identified specificto HCV E2 antigen. The selected scFv clones were digestedby SfiI/NotI and DNA was sequenced. Then it was subclonedinto the vector pCANTABSE for expression as E-taggedsoluble scFv. The liver tissue sections from normal personand patients with chronic hepatitis B and chronic hepatitis Cwere immunostained with HCV E2 scFv antibody.RESULTS: The data of scFv-E2 DNA digestion and DNAsequencing showed that the scFv gene is composed of 750bp. ELISA and immunohistochemistry demonstrated that thehuman single chain Fy antibody against hepatitis C E2 antigenhas a specific binding character with hepatitis virus E2 antigenand paraffin-embedded tissue, but did not react with liver tissuesfrom healthy persons or patients with chronic hepatitis B.CONCLUSION: We have successfully screened andidentified HCV E2 scFv and the scFv could be used in theimmunostaining of liver tissue sections from patients withchronic hepatitis C.

  5. High efficiency recovery and epitope specific sorting of an scFv yeast display library

    Energy Technology Data Exchange (ETDEWEB)

    Siegel, Robert W.; Coleman, James R.; Miller, Keith D.; Feldhaus, Michael


    In order to more productively utilize the rich source of antigen specific reagents present in the previously described non-immune scFv yeast display library (Feldhaus et al., 2003) one must be able to efficiently isolate and characterize clones within the library. To this end, we have developed and validated a magnetic bead sorting technique utilizing the Miltenyi MacsTm system to recover greater than 90% of the antigen specific clones present in the library. In combination with flow cytometry, we rapidly reduced diversity and enriched for antigen specific clones in three rounds of selection. Furthermore, we demonstrate the use of pre-existing monoclonal antibodies (mAbs) for antigen labeling and subsequent flow cytometric sorting and characterization of epitope specific scFv. Combining these two improvements in library screening allowed isolation and characterization of 3 epitope specific scFv (including a previously uncharacterized epitope) to a 6 kd protein, epidermal growth factor EGF.

  6. Isolation and characterization of anti c-met single chain fragment variable (scFv) antibodies. (United States)

    Qamsari, Elmira Safaie; Sharifzadeh, Zahra; Bagheri, Salman; Riazi-Rad, Farhad; Younesi, Vahid; Abolhassani, Mohsen; Ghaderi, Sepideh Safaei; Baradaran, Behzad; Somi, Mohammad Hossein; Yousefi, Mehdi


    The receptor tyrosine kinase (RTK) Met is the cell surface receptor for hepatocyte growth factor (HGF) involved in invasive growth programs during embryogenesis and tumorgenesis. There is compelling evidence suggesting important roles for c-Met in colorectal cancer proliferation, migration, invasion, angiogenesis, and survival. Hence, a molecular inhibitor of an extracellular domain of c-Met receptor that blocks c-Met-cell surface interactions could be of great thera-peutic importance. In an attempt to develop molecular inhibitors of c-Met, single chain variable fragment (scFv) phage display libraries Tomlinson I + J against a specific synthetic oligopeptide from the extracellular domain of c-Met receptor were screened; selected scFv were then characterized using various immune techniques. Three c-Met specific scFv (ES1, ES2, and ES3) were selected following five rounds of panning procedures. The scFv showed specific binding to c-Met receptor, and significantly inhibited proliferation responses of a human colorectal carcinoma cell line (HCT-116). Moreover, anti- apoptotic effects of selected scFv antibodies on the HCT-116 cell line were also evaluated using Annexin V/PI assays. The results demonstrated rates of apoptotic cell death of 46.0, 25.5, and 37.8% among these cells were induced by use of ES1, ES2, and ES3, respectively. The results demonstrated ability to successfully isolate/char-acterize specific c-Met scFv that could ultimately have a great therapeutic potential in immuno-therapies against (colorectal) cancers.

  7. Survival advantage of heterozygous fV Leiden carriers in murine sepsis (United States)

    Kerschen, Edward; Hernandez, Irene; Zogg, Mark; Maas, Matthias; Weiler, Hartmut


    Summary Background The high allelic frequency of the prothrombotic Leiden polymorphism in human blood coagulation factor V (fV) has been speculated to reflect positive selection during evolution. Heterozygous Leiden carriers enrolled in the placebo arm of the PROWESS sepsis trial, and heterozygous Leiden mice challenged with endotoxin both showed reduced mortality, whereas homozygous Leiden mice were not protected from lethal endotoxemia. Follow-up analyses of clinical outcomes, and of mouse models of infection with various pathogens remained inconclusive. Objective To establish whether aPC-resistance of fV Leiden modifies the outcome of bacterial infection in murine sepsis models. Methods Homozygous and heterozygous fV Leiden mice were subjected to gram-positive (S.aureus) or gram-negative (Y.pestis; E.coli) septic peritonitis, or polymicrobial, focal septic peritonitis induced by cecal ligation and puncture (CLP); and the effect of fV Leiden on 7-day survival and bacterial dissemination was assessed. Outcomes were compared to the sepsis survival of mice with genetically impaired hemostasis (hemophilia A, thrombocytopenia, thrombin receptor PAR4 deficiency, protein C receptor ProcR/EPCR-deficiency). Results Heterozygous, but not homozygous Leiden mice were protected from lethal infection with highly virulent S.aureus and Y.pestis strains. FV Leiden did not affect the outcome of sepsis induced by CLP, staphylokinase-deficient S.aureus, Pla-deficient Y.pestis, or E.coli. Thrombocytopenia, deficiency of PAR1 or PAR4 did not affect S.aureus sepsis survival, whereas hemophilia A increased mortality. ProcR-deficiency selectively abolished the survival advantage of heterozygous Leiden mice. Conclusions In mice, heterozygous fV Leiden carriers are protected from sepsis mortality after infection with clinically relevant human bacterial pathogens. PMID:25690763

  8. Specific cell RNA mediators and the mechanism of Fv-1 gene restriction

    Energy Technology Data Exchange (ETDEWEB)

    Tennant, R. W.; Yang, W. K.; Rascati, R. J.; Hsu, I. C.; Brown, A.


    Cells which are normally permissive for both N- and B-tropic viruses (Fv-1/sup -/) can be made resistant to these viruses by treatment with phenol-extracted fractions of mouse cells with the appropriate Fv-1 genotype (Fv-1/sup bb/ or Fv-1/sup nn/). The ability of RNase, but not DNase or pronase, to inhibit resistance transfer indicated that resistance is mediated by an RNA molecule. The assay for resistance transfer is based upon XC-plaque reduction in DEAE-dextran treated cells. By this assay method stored pools of cellular RNA have shown statistically significant specific activity at concentrations of 5 to 10 However, the maximum plaque reduction is 60 to 80 percent and appears to be related to the ability of cells to incorporate RNA. Specific activity has been localized in the 18 to 22 S region of sucrose gradients. In addition, it can be detected in H/sub 2/O eluates from poly(U)Sepharose columns suggesting that the RNA is polyadenylated. The time course of resistance transfer indicates that the restriction mediated by RNA functions only if added within six hours after virus infection. By the technique of DNA transfection, high-molecular-weight DNA extracted from SC-1 cells infected with either N- or B-tropic virus have been found to infect cells of Fv-1/sup nn/ and Fv-1/sup bb/ genotypes equally well. Similar findings have been obtained with the Hirt extract supernatant DNA isolated from the infected SC-1 cells.

  9. A humanized anti-M2 scFv shows protective in vitro activity against influenza

    Energy Technology Data Exchange (ETDEWEB)

    Bradbury, Andrew M [Los Alamos National Laboratory; Velappan, Nileena [Los Alamos National Laboratory; Schmidt, Jurgen G [Los Alamos National Laboratory


    M2 is one of the most conserved influenza proteins, and has been widely prospected as a potential universal vaccine target, with protection predominantly mediated by antibodies. In this paper we describe the creation of a humanized single chain Fv from 14C2, a potent monoclonal antibody against M2. We show that the humanized scFv demonstrates similar activity to the parental mAb: it is able to recognize M2 in its native context on cell surfaces and is able to show protective in vitro activity against influenza, and so represents a potential lead antibody candidate for universal prophylactic or therapeutic intervention in influenza.

  10. Factor V Leiden mutation and high FVIII are associated with an increased risk of VTE in women with breast cancer during adjuvant tamoxifen - results from a prospective, single center, case control study. (United States)

    Kovac, Mirjana; Kovac, Zeljko; Tomasevic, Zorica; Vucicevic, Slavko; Djordjevic, Valentina; Pruner, Iva; Radojkovic, Dragica


    Estimates of the risk ratio of tamoxifen-associated venous thromboembolism (VTE) in breast cancer patients range from 2.4 to 7.1. The occurrence of thrombosis in patients with breast cancer complicates the clinical condition and causes a change of treatment. Our study was conducted in order to investigate the influence of patient-related risk factors for thrombosis development in breast cancer patients whose treatment included adjuvant tamoxifen. The prospective, single center, case control study included 150 breast cancer women, 50 whom developed venous thrombosis during adjuvant tamoxifen and 100 whom did not have thrombosis, as a control group. Patient-related risk factors such as: age, body mass index, previous VTE, varicose veins, concomitant diseases, the presence of prothrombotic mutations (FV Leiden, FII G20210A) and FVIII activity were evaluated in both groups. In respect of prothrombotic mutations, the FV Leiden mutation was present in a higher number of women from the VTE group (10/50 vs 7/100; P=0.020). Additionally, FVIII activity was significantly higher in the VTE group; median (IQR), of 1.79 (0.69) vs 1.45 (0.55); P1.5IU/ml, who were carriers of prothrombotic mutations, an OR of 3.76 (CI 95% 1.276-11.096; P=0.016) was obtained for VTE. The results of our study showed that the factor V Leiden mutation and high FVIII are associated with an increased risk of VTE in women with breast cancer during adjuvant tamoxifen. Copyright © 2014. Published by Elsevier B.V.

  11. Dynamics of Apis mellifera Filamentous Virus (AmFV) Infections in Honey Bees and Relationships with Other Parasites. (United States)

    Hartmann, Ulrike; Forsgren, Eva; Charrière, Jean-Daniel; Neumann, Peter; Gauthier, Laurent


    Apis mellifera filamentous virus (AmFV) is a large double stranded DNA virus of honey bees, but its relationship with other parasites and prevalence are poorly known. We analyzed individual honey bees from three colonies at different times post emergence in order to monitor the dynamics of the AmFV gut colonization under natural conditions. Prevalence and loads of microsporidia and trypanosomes were also recorded, as well as five common honey bee RNA viruses. The results show that a high proportion of bees get infected with AmFV during the first week post-emergence (75%) and that AmFV DNA levels remained constant. A similar pattern was observed for microsporidia while trypanosomes seem to require more time to colonize the gut. No significant associations between these three infections were found, but significant positive correlations were observed between AmFV and RNA viruses. In parallel, the prevalence of AmFV in France and Sweden was assessed from pooled honey bee workers. The data indicate that AmFV is almost ubiquitous, and does not seem to follow seasonal patterns, although higher viral loads were significantly detected in spring. A high prevalence of AmFV was also found in winter bees, without obvious impact on overwintering of the colonies.

  12. Cereal crops as viable production and storage systems for pharmaceutical scFv antibodies. (United States)

    Stöger, E; Vaquero, C; Torres, E; Sack, M; Nicholson, L; Drossard, J; Williams, S; Keen, D; Perrin, Y; Christou, P; Fischer, R


    This report describes the stable expression of a medically important antibody in the staple cereal crops rice and wheat. We successfully expressed a single-chain Fv antibody (ScFvT84.66) against carcinoembryonic antigen (CEA), a well characterized tumor-associated marker antigen. scFv constructs were engineered for recombinant antibody targeting to the plant cell apoplast and ER. Up to 30 microg/g of functional recombinant antibody was detected in the leaves and seeds of wheat and rice. We confirmed that transgenic dry seeds could be stored for at least five months at room temperature, without significant loss of the amount or activity of scFvT84.66. Our results represent the first transition from model plant expression systems, such as tobacco and Arabidopsis, to widely cultivated cereal crops, such as rice and wheat, for expression of an antibody molecule that has already shown efficacy in clinical applications. Thus, we have established that molecular pharming in cereals can be a viable production system for such high-value pharmaceutical macromolecules. Our findings provide a strong foundation for exploiting alternative uses of cereal crops both in industrialized and developing countries.

  13. Integrating scFv into xMAP Assays for the Detection of Marine Toxins

    Directory of Open Access Journals (Sweden)

    Lisa C. Shriver-Lake


    Full Text Available Marine toxins, such as saxitoxin and domoic acid are associated with algae blooms and can bioaccumulate in shell fish which present both health and economic concerns. The ability to detect the presence of toxin is paramount for the administration of the correct supportive care in case of intoxication; environmental monitoring to detect the presence of toxin is also important for prevention of intoxication. Immunoassays are one tool that has successfully been applied to the detection of marine toxins. Herein, we had the variable regions of two saxitoxin binding monoclonal antibodies sequenced and used the information to produce recombinant constructs that consist of linked heavy and light variable domains that make up the binding domains of the antibodies (scFv. Recombinantly produced binding elements such as scFv provide an alternative to traditional antibodies and serve to “preserve” monoclonal antibodies as they can be easily recreated from their sequence data. In this paper, we combined the anti-saxitoxin scFv developed here with a previously developed anti-domoic acid scFv and demonstrated their utility in a microsphere-based competitive immunoassay format. In addition to detection in buffer, we demonstrated equivalent sensitivity in oyster and scallop matrices. The potential for multiplexed detection using scFvs in this immunoassay format is demonstrated.

  14. Antioxidant Effect of Human Selenium-containing Single-chain Fv in Rat Cardiac Myocytes

    Institute of Scientific and Technical Information of China (English)

    HUO Rui; SHI Yi; XU Jun-jie; YAN Fei; L(U) Shao-wu; SU Jia-ming; DUAN Yu-jing; FAN Jia; NING Bo; CONG Deng-li; YAN Gang-lin; LUO Gui-min; WEI Jing-yan


    Reactive oxygen species(ROS) plays a key role in human heart diseases.Glutathione peroxidase(GPX) functions as an antioxidant as it catalyzes the reduction of hydroperoxide.In order to investigate the antioxidant effect of human selenium-containing single-chain Fv(Se-scFv-B3),a new mimic of GPX,a model system of hydrogen peroxide(H2O2)-induced rat cardiac myocyte damage was established.The cardiac myocyte damage was characterized in terms of cell viability,lipid peroxidation,cell membrane integrity,and intracellular H2O2 level.The Se-scFv-B3 significantly reduced H2O2-induced cell damage as shown by the increase of cell viability,the decline of malondialdehyde(MDA) production,lactate dehydrogenase(LDH) release,and intracellular H2O2 level.So Se-scFvB3 may have a great potential in the treatment of human heart diseases induced by ROS.

  15. Cloning, expression, purification, and characterization of LC-1 ScFv with GFP tag

    Institute of Scientific and Technical Information of China (English)

    LU Min; GONG Xing-guo; YU Hong; LI Jian-yong


    Total RNA was isolated from the hybridoma cell line (LC-1), which secretes anti-lung adenocarcinoma monoclonal antibody, and was transferred into cDNA. Based on the FR1 (framework region 1) and FR4 conserved regions of LC-I gene, the variable regions of heavy chain (Vh) and light chain (Vl) were amplified, and the Vh and modified Vl were connected to single chain Fv (ScFv) by SOE-PCR (splice overlap extension PCR). The modified ScFv was fused with green fluorescent protein (GFP)and introduced into E. coli JMi09. The fusion protein induced by IPTG (Isopropylthiogalactoside) was about 57000 on a 10%SDS-PAGE gel (10% Sds Polyacrylamide Gel Electrophoresis), and primarily manifested as inclusion bodies. The renatured protein purified by Ni-NTA Superflow resins showed ability to bind to antigen on SPC-A-1 lung adenocarcinoma. In addition, the induced host cells fluoresced bright green under 395 nm wavelength, which indicated that the expected protein with dual activity was expressed in the prokaryotic system. The ScFv with GFP tag used in this research can be applied as a new reagent to detect immunological dye, and provide a feasible way to detect adenocarcinoma in a clinical setting.

  16. Generation of a recombinant single-chain variable fragment (scFv) targeting 5-methyl-2'-deoxycytidine. (United States)

    Ohshima, Motohiro; Tadakuma, Tomomi; Hayashi, Hideki; Inoue, Kazuyuki; Itoh, Kunihiko


    We generated a single-chain variable fragment (scFv) against 5-methyl-2'-deoxycytidine (m(5)dCyd) using phage display technology. The heavy and light chain variable region genes were amplified by the polymerase chain reaction (PCR) from hybridoma cell line FMC9 and assembled as an scFv fragment with a flexible linker (Gly(4)-Ser)(3). The scFv DNA fragment was then cloned into pCANTAB-5E, and a phage displaying the scFv was produced. Antigen-positive phage clones were successfully selected by enzyme-linked immunosorbent assay (ELISA). The scFv was modified with FLAG and His tags for detection and purification. The scFv reacted strongly with m(5)dCyd and weakly with 5-methylcytidine (m(5)Cyd) but not with cytidine (Cyd) and 1-methyladenosine in a manner similar to the monoclonal antibody (MoAb). Although the specificities of scFv and MoAb were almost identical, the sensitivity of the scFv (IC(50) 0.054 microg/ml) was approximately 80 times higher than that of the parent MoAb (IC(50) 4.27 microg/ml), determined by inhibition ELISA. As a biochemical application of this scFv, we quantified the m(5)dCyd content of genomic DNA by enzymatic hydrolysis using inhibition ELISA. The cancer cell lines HeLa, HeLa S3 and MDA-MB-453 contained approximately 1% of the methylated DNA in total genomic DNA, as did peripheral blood cell genomic DNA from healthy volunteers, but HT29 and T-47D showed hypomethylation compared with the HeLa, HeLa S3 and MDA-MB-453 cell lines. The scFv generated here may be applicable to the assessment of cellular DNA methylation levels and is more sensitive than the MoAb.

  17. Construction, Expression and In Vitro Biological Behaviors of Ig scFv Fragment in Patients with Chronic B Cell Leukemia

    Institute of Scientific and Technical Information of China (English)

    ZHU Lijuan; LIAO Wenjun; ZHU Huifen; LEI Ping; WANG Zhihua; SHAO Jingfang; ZHANG Yue; SHEN Guanxin


    The expression vector of SmIg scFv fragment was constructed in patient with B cell chronic lymphocyte leukemia (B-CLL) and expressed in E. coli to obtain scFv fragment, and the effect of the protein on the proliferation of stimulated peripheral blood mononuclear cells (PBMC) was investigated in vitro. Two pairs of primers were designed, and variable region genes of light chain and heavy chain were amplified by PCR respectively from the pGEM-T vectors previously constructed in our laboratory which containing light chain gene or Fd fragment of heavy chain gene. The PCR product was digested, purified and inserted into pHEN2 vector to construct the soluble expression vector pHEN2-scFv. After the induction by IPTG, the scFv protein was identified by SDSPAGE electrophoresis and purified by Ni-NTA-Chromatography. MTT was used to determine the effect of purified protein on the proliferation of stimulated PBMC in vitro. Plasmid PCR and restriction enzyme digestion of pHEN2-scFv revealed the pHEN2-scFv vector was constructed successfully. Id-scFv protein was expressed in positive clone after induced by IPTG. SDS-PAGE analysis showed that the relative molecular weight of fusion protein was about 30 kD (1 kD=0.9921 ku),which was consistent with the theoretically predicted value. Proliferation of PBMC could be induced by purified Id-scFv. It was suggested that the expression vector of SmIg scFv fragment was constructed successfully, and scFv protein was expressed and secreted from E. coli, which could induce proliferation of PBMC. This may lay an experimental foundation for further research of IdHSP complex vaccine for B-CLL.

  18. Recombinant norovirus-specific scFv inhibit virus-like particle binding to cellular ligands

    Directory of Open Access Journals (Sweden)

    Hardy Michele E


    Full Text Available Abstract Background Noroviruses cause epidemic outbreaks of gastrointestinal illness in all age-groups. The rapid onset and ease of person-to-person transmission suggest that inhibitors of the initial steps of virus binding to susceptible cells have value in limiting spread and outbreak persistence. We previously generated a monoclonal antibody (mAb 54.6 that blocks binding of recombinant norovirus-like particles (VLP to Caco-2 intestinal cells and inhibits VLP-mediated hemagglutination. In this study, we engineered the antigen binding domains of mAb 54.6 into a single chain variable fragment (scFv and tested whether these scFv could function as cell binding inhibitors, similar to the parent mAb. Results The scFv54.6 construct was engineered to encode the light (VL and heavy (VH variable domains of mAb 54.6 separated by a flexible peptide linker, and this recombinant protein was expressed in Pichia pastoris. Purified scFv54.6 recognized native VLPs by immunoblot, inhibited VLP-mediated hemagglutination, and blocked VLP binding to H carbohydrate antigen expressed on the surface of a CHO cell line stably transfected to express α 1,2-fucosyltransferase. Conclusion scFv54.6 retained the functional properties of the parent mAb with respect to inhibiting norovirus particle interactions with cells. With further engineering into a form deliverable to the gut mucosa, norovirus neutralizing antibodies represent a prophylactic strategy that would be valuable in outbreak settings.

  19. Analysis on the co-expression of hydrophobin Fv-Hyd1 and its potential transcription factor Fv-Rtg3 in Flammulina velutipes%金针菇疏水蛋白Fv-Hyd1及其潜在的转录因子Fv-Rtg3的共表达分析

    Institute of Scientific and Technical Information of China (English)

    陈仁良; 李肖; 龙莹; 张磊; 严俊杰; 黄千慧; 熊宏民; 谢宝贵


    疏水蛋白(hydrophobin,Hyd)普遍存在于真菌的气生菌丝、侵染结构或子实体表面,且存在时空性高丰度表达。不同真菌疏水蛋白在其各个发育阶段发挥不同的功能,如气生菌丝的生长、子实体原基的形成及其形态分化等。虽然已有众多报道指出 H yd与生物或非生物环境刺激所产生的抗逆性有关,但是对它与细胞分裂分化等生物学功能及其信号转导调控的关系仍了解甚少。通过培养金针菇(Flammulina velutipes)菌丝以及金针菇出菇实验,结合基因组及转录组数据、蛋白结构预测、系统发育进化树聚类分析等生物信息学分析鉴定金针菇中一个编码疏水蛋白基因Fv-Hyd1,并利用其上游序列预测该疏水蛋白基因潜在的调控转录因子,辅以实时荧光定量 PCR(real time fluorescence quantitative polymerase chain reaction,qRT-PCR)数据和皮尔森相关系数鉴定它们的表达关系。结果表明,Fv-Hyd1与金针菇菌丝扭结和子实体原基的形成具有一定的相关性,与其潜在的转录因子 Fv-Rtg3之间存在较强的共表达规律,并以此结合蛋白磷酸化位点预测,推测基因Fv-Hyd1的表达调控机制。%Summary Flammulina velutipes,with high edible and medicinal value,was widely cultivated in China and Southeast Asia.Being worthy to be noted,the fruiting body formation and development of F.velutipes were influenced by many factors,such as the internal factors including transcription factor,protein kinase,sparse water protein and the cell pigment,and environmental factors including light,temperature and carbon dioxide.It is very important to understand the response of genes to the influencing factors in transcription level,translation level and environmental stimuli for studying the regulation of F.velutipes fruiting body growth and development. Among them,the hydrophobins were commonly found in fungal hyphae,infection structure or surface of the fruiting body,and there

  20. A Successful Mother and Neonate Outcome for a Woman with Essential Thrombocytosis and FV Leiden Heterozygosity

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    Marianna Politou


    Full Text Available Essential thrombocytosis (ET and FV Leiden heterozygosity represent an acquired and hereditable hypercoagulable state, respectively. An uncommon case of coexistence of ET and FV Leiden heterozygosity in a 36-year-old pregnant woman and her successful pregnancy outcome is described. She was considered to be at high risk of thrombosis during her pregnancy and she was treated with both prophylactic dose of LMWH and aspirin daily throughout her pregnancy and for a 6-week period postpartum. The efficacy of the anticoagulation treatment was monitored in various time points not only by measuring anti-Xa levels and D-Dimers but also with new coagulation methods such as rotation thromboelastometry and multiplate. Global assessment of coagulation using additional newer laboratory tests might prove useful in monitoring coagulation pregnancies at high risk for thrombosis.

  1. Generation and Characterization of an scFv Directed against Site II of Rabies Glycoprotein

    Directory of Open Access Journals (Sweden)

    Shukra M. Aavula


    Full Text Available Recombinant antibody phage display technology is a vital tool that facilitates identification of specific binding molecules to a target enabling the rapid generation and selection of high affinity, fully human, or mouse antibody product candidates essentially directed towards disease target appropriate for antibody therapy. In this study, a recombinant single-chain Fv antibody fragment (scFv A11 was isolated from immune spleen cells obtained from mice immunized with inactivated rabies virus (Pasteur strain using standard methodology and was characterized for its specificity towards the rabies virus glycoprotein. Epitope mapping using peptide libraries and truncated glycoprotein polypeptides suggested that A11 bound to the antigenic site II of rabies glycoprotein against which a majority of rabies virus neutralizing antibodies are directed. The use of the above technology could, therefore, allow development of scFvs with different specificities against the rabies glycoprotein as an alternative to the more cumbersome protocols used for the development of monoclonal antibodies.

  2. Development and Applications of the FV3 GEOS-5 Adjoint Modeling System (United States)

    Holdaway, Daniel; Kim, Jong G.; Lin, Shian-Jiann; Errico, Ron; Gelaro, Ron; Kent, James; Coy, Larry; Doyle, Jim; Goldstein, Alex


    GMAO has developed a highly sophisticated adjoint modeling system based on the most recent version of the finite volume cubed sphere (FV3) dynamical core. This provides a mechanism for investigating sensitivity to initial conditions and examining observation impacts. It also allows for the computation of singular vectors and for the implementation of hybrid 4DVAR. In this work we will present the scientific assessment of the new adjoint system and show results from a number of research application of the adjoint system.

  3. Characterization of a Single Chain Fv Antibody that Reacts with Free Morphine

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    Kazuhisa Sugimura


    Full Text Available An immune phage library derived from mice, hyperimmunized with morphine-conjugated BSA, was used to isolate a single-chain Fv (scFv clone, M86, with binding activity to morphine-conjugated thyroglobulin (morphine-C-Tg but not to codeine-, cocaine-, or ketamine-conjugated Tg. Surface plasmon resonance analysis using a morphine-C-Tg-coupled CM5 sensor chip showed that the Kd value was 1.26 × 10−8 M. To analyze its binding activity to free morphine and related compounds, we performed a competitive ELISA with M86 and morphine-C-Tg in the absence or presence of varying doses of free morphine and related compounds. IC50 values for opium, morphine, codeine, and heroin were 257 ng/mL, 36.4, 7.3, and 7.4 nM, respectively. Ketamine and cocaine exhibited no competitive binding activity to M86. Thus, we established a phage library-derived scFv, M86, which recognized not only free morphine and codeine as opium components but also heroin. This characteristic of M86 may be useful for developing therapeutic reagents for opiate addiction and as a free morphine-specific antibody probe.

  4. Production, purification, and characterization of scFv TNF ligand fusion proteins. (United States)

    Fick, Andrea; Wyzgol, Agnes; Wajant, Harald


    Single-chain variable fragments (scFvs) specific for tumor-associated cell surface antigens are the most broadly used reagents to direct therapeutic or diagnostic effector molecules, such as toxins, radioisotopes, and CD3-stimulating scFvs, to tumors. One novel class of effector molecules that can be targeted to tumors by scFvs are ligands of the tumor necrosis factor (TNF) family. Typically, these molecules have apoptosis inducing and/or immune stimulating properties and are therefore highly attractive for cancer treatment. N-terminal fusion of scFvs does not interfere with the receptor binding capabilities of TNF ligands and thus allows the straightforward generation of scFv TNF ligand fusion proteins. We report here a protocol for the purification of eukaryotically produced scFv TNF ligand fusion proteins based on affinity chromatography on anti-Flag agarose and further describe assays for the determination of the targeting index of this type of scFv-targeted proteins.

  5. Toward Performance Portability of the FV3 Weather Model on CPU, GPU and MIC Processors (United States)

    Govett, Mark; Rosinski, James; Middlecoff, Jacques; Schramm, Julie; Stringer, Lynd; Yu, Yonggang; Harrop, Chris


    The U.S. National Weather Service has selected the FV3 (Finite Volume cubed) dynamical core to become part of the its next global operational weather prediction model. While the NWS is preparing to run FV3 operationally in late 2017, NOAA's Earth System Research Laboratory is adapting the model to be capable of running on next-generation GPU and MIC processors. The FV3 model was designed in the 1990s, and while it has been extensively optimized for traditional CPU chips, some code refactoring has been required to expose sufficient parallelism needed to run on fine-grain GPU processors. The code transformations must demonstrate bit-wise reproducible results with the original CPU code, and between CPU, GPU and MIC processors. We will describe the parallelization and performance while attempting to maintain performance portability between CPU, GPU and MIC with the Fortran source code. Performance results will be shown using NOAA's new Pascal based fine-grain GPU system (800 GPUs), and for the Knights Landing processor on the National Science Foundation (NSF) Stampede-2 system.

  6. [Preparation of monoclonal antibody against 4-amylphenol and homology modeling of its Fv fragment]. (United States)

    Cheng, Lei; Wu, Haizhen; Fei, Jing; Zhang, Lujia; Ye, Jiang; Zhang, Huizhan


    Objective To prepare and characterize a monoclonal antibody (mAb) against 4-amylphenol (4-AP), clone its cDNA sequence and make homology modeling for its Fv fragment. Methods A high-affinity anti-4-AP mAb was generated from a hybridoma cell line F10 using electrofusion between splenocytes from APA-BSA-immunized mouse and Sp2/0 myeloma cells. Then we extracted the mRNA of F10 cells and cloned the cDNA of mAb. The homology modeling and molecular docking of its Fv fragment was conducted with biological software. Results Under the optimum conditions, the ic-ELISA equation was y=A2+(A1-A2)/(1+(x/x0)(p)) (A1=1.28; A2=-0.066; x0=12560.75; p=0.74) with a correlation coefficient (R(2)) of 0.997. The lowest detectable limit was 0.65 μg/mL. The heavy and light chains of mAb respectively belonged to IgG1 and Kappa. The homology modeling and molecular docking studies revealed that the binding of 4-Ap and mAb was attributed to the hydrogen bond and hydrophobic interactions. Conclusion The study successfully established a stable 4-AP mAb-secreting hybridoma cell line. The study on spatial structure of Fv fragment using homology modeling provided a reference for the development and design of single chain variable fragments.

  7. Cloning and expression of functional single-chain Fv antibodies directed against NIa and coat proteins of potato virus Y. (United States)

    Rouis, Souad; Lafaye, Pierre; Jaoua-Aydi, Leila; Sghaier, Zidani; Ayadi, Hammadi; Gargouri-Bouzid, Radhia


    Three single-chain variable fragment (scFv) antibodies recognizing the nuclear inclusion a (NIa) and capsid proteins of potato virus Y were obtained from two mouse derived hybridoma clones secreting, respectively, an anti-NIa (22-1) and an anti-coat protein (136-13) monoclonal antibodies. The first monoclonal antibody was able to inhibit in vitro the PVY polyprotein cleavage by blocking the NIa protease activity. The amplified scFv cDNAs were first inserted into the TOPO vector and then sequenced. Several recombinant E. coli clones carrying the accurate scFv sequences were selected and the corresponding cDNAs were subcloned in pHEN phagemid and transferred in E. coli strain. The expressed scFv fragments showed an antibody activity that recognized the viral target proteins in infected tissues. Their activity was comparable to the parental monoclonal antibodies.

  8. In vivo production of scFv-displaying biopolymer beads using a self-assembly-promoting fusion partner. (United States)

    Grage, Katrin; Rehm, Bernd H A


    Recombinant production and, in particular, immobilization of antibody fragments onto carrier materials are of high interest with regard to diagnostic and therapeutic applications. In this study, the recombinant production of scFv-displaying biopolymer beads intracellularly in Escherichia coli was investigated. An anti-beta-galactosidase scFv (single chain variable fragment of an antibody) was C-terminally tagged with the polymer-synthesizing enzyme PhaC from Cupriavidus necator by generating the respective hybrid gene. The functionality of the anti-beta-galactosidase scFv-PhaC fusion protein was assessed by producing the respective soluble fusion protein in an Escherichia coli AMEF mutant strain. AMEF (antibody-mediated enzyme formation) strains contain an inactive mutant beta-galactosidase, which can be activated by binding of an anti-beta-galactosidase antibody. In vivo activation of AMEF beta-galactosidase indicated that the scFv is functional with the C-terminal fusion partner PhaC. It was further demonstrated that polymer biosynthesis and bead formation were mediated by the scFv-PhaC fusion protein in the cytoplasm of recombinant E. coli when the polymer precursor was metabolically provided. This suggested that the C-terminal fusion partner PhaC acts as a functional insolubility partner, providing a natural cross-link to the bead and leading to in vivo immobilization of the scFv. Overproduction of the fusion protein at the polymer bead surface was confirmed by SDS-PAGE and MALDI-TOF/MS analysis of purified beads. Antigen binding functionality and specificity of the beads was assessed by analyzing the binding of beta-galactosidase to scFv-displaying beads and subsequently eluting the bound protein at pH 2.7. A strong enrichment of beta-galactosidase suggested the functional display of scFv at the bead surface as well as the applicability of these beads for antigen purification. Binding of beta-galactosidase to the scFv-displaying beads was quantitatively

  9. Negative effects of low dose atrazine exposure on the development of effective immunity to FV3 in Xenopus laevis. (United States)

    Sifkarovski, Jason; Grayfer, Leon; De Jesús Andino, Francisco; Lawrence, B Paige; Robert, Jacques


    The recent dramatic increase of the prevalence and range of amphibian host species and populations infected by ranaviruses such as Frog Virus 3 (FV3) raises concerns about the efficacies of amphibian antiviral immunity. In this context, the potential negative effects of water contaminants such as the herbicide atrazine, at environmentally relevant levels, on host antiviral immunity remains unclear. Here we describe the use of the amphibian Xenopus laevis as an ecotoxicology platform to elucidate the consequences of exposure to ecologically relevant doses of atrazine on amphibian antiviral immunity. X. laevis were exposed at tadpole and adult stages as well as during metamorphosis to atrazine (range from 0.1 to 10.0 ppb) prior to infection with FV3. Quantitative analysis of gene expression revealed significant changes in the pro-inflammatory cytokine, TNF-α and the antiviral type I IFN gene in response to FV3 infection. This was most marked in tadpoles that were exposed to atrazine at doses as low 0.1 ppb. Furthermore, atrazine exposure significantly compromised tadpole survival following FV3 infections. In contrast, acute atrazine exposure of mature adult frogs did not induce detectable effects on anti-FV3 immunity, but adults that were exposed to atrazine during metamorphosis exhibited pronounced defects in FV3-induced TNF-α gene expression responses and slight diminution in type I IFN gene induction. Thus, even at low doses, atrazine exposure culminates in impaired development of amphibian antiviral defenses.

  10. Identification of Fusarium virguliforme FvTox1-Interacting Synthetic Peptides for Enhancing Foliar Sudden Death Syndrome Resistance in Soybean.

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    Bing Wang

    Full Text Available Soybean is one of the most important crops grown across the globe. In the United States, approximately 15% of the soybean yield is suppressed due to various pathogen and pests attack. Sudden death syndrome (SDS is an emerging fungal disease caused by Fusarium virguliforme. Although growing SDS resistant soybean cultivars has been the main method of controlling this disease, SDS resistance is partial and controlled by a large number of quantitative trait loci (QTL. A proteinacious toxin, FvTox1, produced by the pathogen, causes foliar SDS. Earlier, we demonstrated that expression of an anti-FvTox1 single chain variable fragment antibody resulted in reduced foliar SDS development in transgenic soybean plants. Here, we investigated if synthetic FvTox1-interacting peptides, displayed on M13 phage particles, can be identified for enhancing foliar SDS resistance in soybean. We screened three phage-display peptide libraries and discovered four classes of M13 phage clones displaying FvTox1-interacting peptides. In vitro pull-down assays and in vivo interaction assays in yeast were conducted to confirm the interaction of FvTox1 with these four synthetic peptides and their fusion-combinations. One of these peptides was able to partially neutralize the toxic effect of FvTox1 in vitro. Possible application of the synthetic peptides in engineering SDS resistance soybean cultivars is discussed.

  11. Negative Effects of Low Dose Atrazine Exposure on the Development of Effective Immunity to FV3 in Xenopus laevis (United States)

    Sifkarovski, Jason; Grayfer, Leon; De Jesús Andino, Francisco; Lawrence, B. Paige; Robert, Jacques


    The recent dramatic increase of the prevalence and range of amphibian host species and populations infected by ranaviruses such as Frog Virus 3 (FV3) raises concerns about the efficacies of amphibian antiviral immunity. In this context, the potential negative effects of water contaminants such as the herbicide atrazine, at environmentally relevant levels, on host antiviral immunity remains unclear. Here we describe the use of the amphibian Xenopus laevis as an ecotoxiciology platform to elucidate the consequences of exposure to ecologically relevant doses of atrazine on amphibian antiviral immunity. X. laevis were exposed at tadpole and adult stages as well as during metamorphosis to atrazine (range from 0.1 to 10.0 ppb) prior to infection with FV3. Quantitative analysis of gene expression revealed significant changes in the pro-inflammatory cytokine, TNF-α and the antiviral type I IFN gene in response to FV3 infection. This was most marked in tadpoles that were exposed to atrazine at doses as low 0.1 ppb. Furthermore, atrazine exposure significantly compromised tadpole survival following FV3 infections. In contrast, acute atrazine exposure of mature adult frogs did not induce detectable effects on anti-FV3 immunity, but adults that were exposed to atrazine during metamorphosis exhibited pronounced defects in FV3-induced TNF-α gene expression responses and slight diminution in type I IFN gene induction. Thus, even at low doses, atrazine exposure culminates in impaired development of amphibian antiviral defenses. PMID:24984115

  12. The Development of a Recombinant scFv Monoclonal Antibody Targeting Canine CD20 for Use in Comparative Medicine.

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    Saurabh Jain

    Full Text Available Monoclonal antibodies are leading agents for therapeutic treatment of human diseases, but are limited in use by the paucity of clinically relevant models for validation. Sporadic canine tumours mimic the features of some human equivalents. Developing canine immunotherapeutics can be an approach for modeling human disease responses. Rituximab is a pioneering agent used to treat human hematological malignancies. Biologic mimics that target canine CD20 are just being developed by the biotechnology industry. Towards a comparative canine-human model system, we have developed a novel anti-CD20 monoclonal antibody (NCD1.2 that binds both human and canine CD20. NCD1.2 has a sub-nanomolar Kd as defined by an octet red binding assay. Using FACS, NCD1.2 binds to clinically derived canine cells including B-cells in peripheral blood and in different histotypes of B-cell lymphoma. Immunohistochemical staining of canine tissues indicates that the NCD1.2 binds to membrane localized cells in Diffuse Large B-cell lymphoma, Marginal Zone Lymphoma, and other canine B-cell lymphomas. We cloned the heavy and light chains of NCD1.2 from hybridomas to determine whether active scaffolds can be acquired as future biologics tools. The VH and VL genes from the hybridomas were cloned using degenerate primers and packaged as single chains (scFv into a phage-display library. Surprisingly, we identified two scFv (scFv-3 and scFv-7 isolated from the hybridoma with bioactivity towards CD20. The two scFv had identical VH genes but different VL genes and identical CDR3s, indicating that at least two light chain mRNAs are encoded by NCD1.2 hybridoma cells. Both scFv-3 and scFv-7 were cloned into mammalian vectors for secretion in CHO cells and the antibodies were bioactive towards recombinant CD20 protein or peptide. The scFv-3 and scFv-7 were cloned into an ADEPT-CPG2 bioconjugate vector where bioactivity was retained when expressed in bacterial systems. These data identify a

  13. The Development of a Recombinant scFv Monoclonal Antibody Targeting Canine CD20 for Use in Comparative Medicine. (United States)

    Jain, Saurabh; Aresu, Luca; Comazzi, Stefano; Shi, Jianguo; Worrall, Erin; Clayton, John; Humphries, William; Hemmington, Sandra; Davis, Paul; Murray, Euan; Limeneh, Asmare A; Ball, Kathryn; Ruckova, Eva; Muller, Petr; Vojtesek, Borek; Fahraeus, Robin; Argyle, David; Hupp, Ted R


    Monoclonal antibodies are leading agents for therapeutic treatment of human diseases, but are limited in use by the paucity of clinically relevant models for validation. Sporadic canine tumours mimic the features of some human equivalents. Developing canine immunotherapeutics can be an approach for modeling human disease responses. Rituximab is a pioneering agent used to treat human hematological malignancies. Biologic mimics that target canine CD20 are just being developed by the biotechnology industry. Towards a comparative canine-human model system, we have developed a novel anti-CD20 monoclonal antibody (NCD1.2) that binds both human and canine CD20. NCD1.2 has a sub-nanomolar Kd as defined by an octet red binding assay. Using FACS, NCD1.2 binds to clinically derived canine cells including B-cells in peripheral blood and in different histotypes of B-cell lymphoma. Immunohistochemical staining of canine tissues indicates that the NCD1.2 binds to membrane localized cells in Diffuse Large B-cell lymphoma, Marginal Zone Lymphoma, and other canine B-cell lymphomas. We cloned the heavy and light chains of NCD1.2 from hybridomas to determine whether active scaffolds can be acquired as future biologics tools. The VH and VL genes from the hybridomas were cloned using degenerate primers and packaged as single chains (scFv) into a phage-display library. Surprisingly, we identified two scFv (scFv-3 and scFv-7) isolated from the hybridoma with bioactivity towards CD20. The two scFv had identical VH genes but different VL genes and identical CDR3s, indicating that at least two light chain mRNAs are encoded by NCD1.2 hybridoma cells. Both scFv-3 and scFv-7 were cloned into mammalian vectors for secretion in CHO cells and the antibodies were bioactive towards recombinant CD20 protein or peptide. The scFv-3 and scFv-7 were cloned into an ADEPT-CPG2 bioconjugate vector where bioactivity was retained when expressed in bacterial systems. These data identify a recombinant anti-CD20

  14. Negative effects of a disulfide bond mismatch in anti-rabies G protein single-chain antibody variable fragment FV57. (United States)

    Duan, Ye; Gu, Tiejun; Zhang, Xizhen; Jiang, Chunlai; Yuan, Ruosen; Li, Zhuang; Wang, Dandan; Chen, Xiaoxu; Wu, Chunlai; Chen, Yan; Wu, Yongge; Kong, Wei


    Rabies virus (RV) causes a fatal infectious disease requiring efficient post-exposure prophylaxis (PEP), which includes a rabies vaccine and rabies immunoglobulin (RIG). The single-chain antibody variable fragment (scFv), a small engineered antibody fragment derived from an antibody variable heavy chain and light chain, has the potential to replace the current application of RIG. In previous studies, we constructed and evaluated an anti-rabies virus G protein scFv (FV57) based on the monoclonal antibody CR57. Of the five cysteines in FV57, four are linked in intra-chain disulfide bonds (Cys-VH28/Cys-VH98 and Cys-VL16/Cys-VL84), and one is free (Cys-VL85). However, the thiol in Cys-VL85 neighboring Cys-VL84 in the CDR3 of the light chain is likely to mismatch with the thiol in Cys-VL16 during the renaturing process. In order to study effects of the mismatched disulfide bond, Cys-VL85 and Cys-VL84 of FV57 were mutated to serine to construct mutants FV57(VL85S) and FV57(VL84S). Furthermore, the disulfide bonds in the light chain of FV57, FV57(VL85S) and FV57(VL84S) were deleted by mutating Cys-VL16 to serine. All mutants were prepared and evaluated along with the original FV57. The results indicated that the mismatched disulfide bond of FV57 linking the light chain FR1 and CDR3 would confer deleterious negative effects on its activity against RV, likely due to spatial hindrance in the light chain CDR3. Moreover, avoidance of the disulfide bond mismatch provided an additional 30% protective efficacy against RV infection in the mouse RV challenge model. Thus, modifications of FV57 to eliminate the disulfide bond mismatch may provide a candidate therapeutic agent for effective PEP against rabies.

  15. Rapid Selection of Phage Se-scFv with GPX Activity via Combination of Phage Display Antibody Library with Chemical Modification

    Institute of Scientific and Technical Information of China (English)


    Glutathione peroxidase (GPX) plays an important role in scavenging reactive oxygen species. A series of catalytic antibodies with GPX activity have been generated by the authors of this study. To obtain humanized catalytic antibodies, the phage-displayed human antibody library was used to select novel antibodies by repetitive screening. Phage antibodies, scFv-B8 and scFv-H6 with the GSH-binding site, were obtained from the library by enzyme-linked immunosorbent assay(ELISA) analysis with 4 rounds of selection against their respective haptens, S-2,4-dinitriphenyl t-butyl ester(GSH-s-DNP-Bu) and S-2,4-dinitriphenyl t-hexyl ester(GSH-s-DNP-He). Nevertheless, several studies need to be conducted to determine whether scFv-B8 and scFv-H6 possess GPX activity. To enhance the speed of the selection, selenocysteine(Sec, the catalytic group of GPX) was incorporated directly into the phages, scFv-B8 and scFv-H6, by chemical mutation to form the phages Se-scFv-B8 and Se-scFv-H6. The GPX activities were found to be 3012 units/μmol and 2102 units/μmol, respectively. To improve the GPX activity of the phage Se-scFv-B8, DNA shuffling was used to construct a secondary library and another positive phage antibody scFv-B9 was screened out by another panning against GSH-s-DNP-Bu. When Sec was incorporated via chemical mutation into the phage antibody scFv-B9, its GPX activity reached 3560 units/μmol, which is 1.17-fold higher than the phage antibody Se-scFv-B8and almost approached the order of magnitude of native GPX. The rapid selection is the prerequisite for generating humanized Se-scFv with GPX activity.

  16. Humoral immune responses induced by anti-idiotypic antibody fusion protein of 6B11scFv/hGM-CSF in BALB/c mice

    Institute of Scientific and Technical Information of China (English)


    Background We have previously developed and characterized a monoclonal anti-idiotype antibody, designated 6B11, which mimics an ovarian carcinoma associated antigen OC166-9 and whose corresponding monoclonal antibody is COC166-9 (Ab1). In this study, we evaluate the humoral immune responses induced by the fusion protein 6B11 single-chain variable fragment (scFv)/human granulocyte macrophage colony-stimulating factor (hGM-CSF) and 6B11scFv in BALB/c mice. Methods The fusion protein 6B11scFv/hGM-CSF was constructed by fusing a recombinant single-chain variable fragment of 6B11scFv to GM-CSF. BALB/c mice were administrated by 6B11scFv/hGM-CSF and 6B11scFv, respectively. Results The fusion protein 6B11scFv/hGM-CSF retained binding to the anti-mouse F(ab)2' and was also biologically active as measured by proliferation of human GM-CSF dependent cell TF1 in vitro. After immunization with the 6B11scFv/hGM-CSF and 6B11ScFv, BALB/c mice showed significantly enhanced Ab3 antibody responses to 6B11scFv/hGM-CSF compared with the 6B11scFv alone. The level of Ab3 was the highest after the first week and maintained for five weeks after the last immunization. Another booster was given when the Ab3 titer descended, and it would reach to the high level in a week. Conclusion The fusion protein 6B11scFv/hGM-CSF can induce humoral immunity against ovarian carcinoma in vivo. We also provide the theoretical foundation for the application of the fusion protein 6B11scFv/hGM-CSF for active immunotherapy of ovarian cancer.

  17. CXCR4 and CXCL12 (SDF-1) in prostate cancer: inhibitory effects of human single chain Fv antibodies. (United States)

    Vaday, Gayle G; Hua, Shao-Bing; Peehl, Donna M; Pauling, Michelle H; Lin, Yu-Huei; Zhu, Li; Lawrence, Diana M; Foda, Hussein D; Zucker, Stanley


    Metastasis is a major cause of morbidity in prostate cancer (PCa). Several studies have shown that the chemokine receptor CXCR4 and its ligand, CXCL12 (stromal cell-derived factor-1), regulate tumor cell metastasis to specific organs. Recently, it was demonstrated that CXCL12 enhances PCa cell adhesion, migration, and invasion, implicating CXCR4 in PCa metastasis. In this study, we examined the inhibitory effects of anti-CXCR4 antibodies on CXCL12-mediated PCa cell activities. We developed fully human single chain Fv antibodies (scFv), Ab124 and Ab125, against CXCR4 using the yeast two-hybrid system. We performed immunofluorescent staining, flow cytometry, and ELISA-binding assays to measure scFv binding to PCa cells. We also examined the effects of scFv on CXCL12-mediated calcium mobilization, cell migration, and invasion. Our results confirmed that PCa cell lines express cell-surface CXCR4. Real-time quantitative reverse transcription-PCR and immunohistochemical staining also verified that CXCR4 is expressed in primary cultures of prostate epithelial cells from adenocarcinomas and in human prostate tissues. Ab124 and Ab125 demonstrated specific binding to PCa cell lines by flow cytometry and in binding assays. Preincubation with scFv resulted in significant reduction of CXCL12-induced calcium mobilization in PC-3 and LNCaP cells. Ab124 and Ab125 also inhibited PCa cell migration toward CXCL12, as well as invasion through extracellular matrix gels. Ab124 and Ab125 inhibit CXCL12-mediated cellular activities by binding the receptor CXCR4. Recombinant scFv are an efficient mode of targeting tumor antigens. Considering the high incidence of PCa, the development of fully human scFv may be a useful therapeutic approach in the prevention and treatment of PCa metastasis.

  18. Characterization of Fv-1 host range strains of murine retroviruses by titration and p30 protein characteristics

    Energy Technology Data Exchange (ETDEWEB)

    Tennant, R.W. (Oak Ridge National Lab., TN); Otten, J.A.; Brown, A.; Yang, W.K.; Kennel, S.J.


    A standardized, direct XC plaque assay was used to determine the titration hitness patterns of Fv-1 host range murine retroviruses obtained from various laboratories. The N- or B-tropic viruses were tested on a variety of cells with Fv-1/sup nn/, Fv-1/sup bb/, or Fv-1/sup nb/ genotypes, and, with one exception, a discrete two-hit pattern was obtained on cells with the restrictive genotype (i.e., N-tropic virus on BALB/c, SIM.R, and B6C3F/sub 1/ cells, and B-tropic virus on SME, SIM, and B6C3F/sub 1/ cells). The single exception to the two-hit titration effect was a strain (TOR-B) which is poorly infectious for all cells, and which does not show a clear N- or B-tropism. In addition, it was possible to convert the two-hit pattern of another B-tropic virus (OR-B) to a one-hit curve by coinfection with an XC-negative N-tropic virus. Relative to SC-1 (Fv-1/sup - -/) cells, it was possible to define three components of restriction of ecotropic virus infection of mouse cells: The first two components, the two-hit kinetics and 10/sup 2/- to 10/sup 3/-fold reduction in titer on restrictive cells, are Fv-1 determined; the third a decreased infectivity on all cells relative to SC-1 cells, is independent of Fv-1 genotype.

  19. Selection of affinity-improved neutralizing human scFv against HBV PreS1 from CDR3 VH/VL mutant library. (United States)

    Chen, YanMin; Bai, Yin; Guo, XiaoChen; Wang, WenFei; Zheng, Qi; Wang, FuXiang; Sun, Dejun; Li, DeShan; Ren, GuiPing; Yin, JieChao


    A CDR3 mutant library was constructed from a previously isolated anti-HBV neutralizing Homo sapiens scFv-31 template by random mutant primers PCR. Then the library was displayed on the inner membrane surface in Escherichia coli periplasmic space. Seven scFv clones were isolated from the mutant library through three rounds of screening by flow cytometry. Competition ELISA assay indicates that isolated scFv fragments show more efficient binding ability to HBV PreS1 compared with parental scFv-31. HBV neutralization assay indicated that two clones (scFv-3 and 59) show higher neutralizing activity by blocking the HBV infection to Chang liver cells. Our method provides a new strategy for rapid screening of mutant antibody library for affinity-enhanced scFv clones and the neutralizing scFvs obtained from this study provide a potential alternative of Hepatitis B immune globulin.

  20. Effect of radiochemical modification on biodistribution of scFvD2B antibody fragment recognising prostate specific membrane antigen. (United States)

    Frigerio, Barbara; Benigni, Fabio; Luison, Elena; Seregni, Ettore; Pascali, Claudio; Fracasso, Giulio; Morlino, Sara; Valdagni, Riccardo; Mezzanzanica, Delia; Canevari, Silvana; Figini, Mariangela


    Antibody-based reagents represent a promising strategy as clinical diagnostic tools. Prostate cancer (PCa) is the second-leading cause of death in males in the Western population. There is a presently unmet need for accurate diagnostic tool to localize and define the extent of both primary PCa and occult recurrent disease. One of the most suitable targets for PCa is the prostate-specific membrane antigen (PSMA) recognised by the monoclonal antibody D2B that we re-shaped into the single chain Fv (scFv format). Aim of this study was to evaluate in preclinical in vivo models the target specificity of scFvD2B after labelling with different radionuclides. (111)In radiolabelling was performed via the chelator Bz-NOTA, and (131)I radioiodination was performed using iodogen. The potential for molecular imaging and the biological behaviour of the radiolabelled scFvD2B were evaluated in mice bearing two subcutaneous PCa isogenic cell lines that differed only in PSMA expression. Biodistribution studies were performed at 3, 9, 15 and 24h after injection to determine the optimal imaging time point. A significant kidney accumulation, as percentage of injected dose of tissue (%ID/g), was observed for (111)In-scFvD2B at 3h after injection (45%ID/g) and it was maintained up to 24h (26%ID/g). By contrast, kidney accumulation of (131)I-scFvD2B was only marginally (0.3%ID/g at 24h). At the optimal time point defined between 15h and 24h, regardless of the radionuclide used, the scFvD2B was able to localize significantly better in the PSMA expressing tumours compared to the negative control; with (131)I-scFvD2B yielding a significantly better target/background ratio compared to (111)In-scFvD2B. These data suggest that, besides antigen specificity, chemical modification may affect antibody fragment biodistribution.

  1. FV520B钢十字焊接接头的疲劳性能%Fatigue performance of cruciform welded joints of FV520B steel

    Institute of Scientific and Technical Information of China (English)

    樊俊铃; 郭杏林; 吴承伟; 邓德伟


    Microstructure of base metal, weld seam and heat-affected zone of welded joints of FV520B steel were examined by optical microscopy, SEM and TEM. The results show that the mierostructure of the base metal eonsists of fine tempered martensite and dispersively precipitated phase particles with high-density dislocations, whereas the microstructure of the weld seam is composed of coarse lath martensite and few of precipitated phase particles, indicating the better mechanical properties of the base metal. Fatigue tests were carried out to obtain the fatigue strength and the S-N curve of the welded joints subjected to a high mean stress. The observation of the fatigue fracture surface shows that fatigue failure occurs at the weld toe and welding defects due to serious stress concentration of these locations accelerating fatigue crack initiation.%用金相显微镜和扫描电镜观察了FV520B钢焊接接头母材、焊缝及热影响区的微观组织;并通过透射电镜给出了母材和焊缝的微观形貌。结果表明:母材组织为细小均匀的回火马氏体、弥散分布的析出相及高密度位错,而焊缝为粗大的板条马氏体及少量的析出相,说明母材力学性能优于焊缝。利用疲劳实验获得了高平均拉应力下接头的疲劳强度及S-N曲线等。通过对疲劳断口的观察,发现疲劳断裂主要有两种形式:破坏于焊趾处和焊接缺陷处,由于这里严重的应力集中,加速了疲劳裂纹的萌生。

  2. scFv from Antibody That Mimics gp43 Modulates the Cellular and Humoral Immune Responses during Experimental Paracoccidioidomycosis. (United States)

    Jannuzzi, Grasielle Pereira; Tavares, Aldo Henrique F P; Kaihami, Gilberto Hideo; de Almeida, José Roberto Fogaça; de Almeida, Sandro Rogério; Ferreira, Karen Spadari


    Paracoccidioidomycosis (PCM), caused by Paracoccidioides species is a prevalent systemic and progressive mycosis that occurs in Latin America. It is caused by Paracoccidioides species. Immunization with dendritic cells transfected with a plasmid encoding the scFv (pMAC/PS-scFv) that mimics the main antigen of P. brasiliensis (gp43) confers protection in experimental PCM. DCs link innate and adaptive immunity by recognizing invading pathogens and selecting the type of effector T cell to mediate the immune response. Here, we showed that DC-pMAC/PS-scFv induces the activation of CD4+ and CD8+ T cells. Moreover, our results demonstrated that BALB/c mice infected with P. brasiliensis and treated with DC-pMAC/PS-scFv showed the induction of specific IgG production against gp43 and IFN-γ, IL-12 and IL-4 cytokines. Analysis of regional lymph nodes revealed increases in the expression of clec7a, myd88, tlr2, gata3 and tbx21, which are involved in the immune response. Taken together, our results indicate that the scFv modulates the humoral and cellular immune responses and presents epitopes to CD4+ and CD8+ T cells.

  3. scFv from Antibody That Mimics gp43 Modulates the Cellular and Humoral Immune Responses during Experimental Paracoccidioidomycosis.

    Directory of Open Access Journals (Sweden)

    Grasielle Pereira Jannuzzi

    Full Text Available Paracoccidioidomycosis (PCM, caused by Paracoccidioides species is a prevalent systemic and progressive mycosis that occurs in Latin America. It is caused by Paracoccidioides species. Immunization with dendritic cells transfected with a plasmid encoding the scFv (pMAC/PS-scFv that mimics the main antigen of P. brasiliensis (gp43 confers protection in experimental PCM. DCs link innate and adaptive immunity by recognizing invading pathogens and selecting the type of effector T cell to mediate the immune response. Here, we showed that DC-pMAC/PS-scFv induces the activation of CD4+ and CD8+ T cells. Moreover, our results demonstrated that BALB/c mice infected with P. brasiliensis and treated with DC-pMAC/PS-scFv showed the induction of specific IgG production against gp43 and IFN-γ, IL-12 and IL-4 cytokines. Analysis of regional lymph nodes revealed increases in the expression of clec7a, myd88, tlr2, gata3 and tbx21, which are involved in the immune response. Taken together, our results indicate that the scFv modulates the humoral and cellular immune responses and presents epitopes to CD4+ and CD8+ T cells.

  4. Construction and sequencing analysis of scFv antibody fragment derived from monoclonal antibody against norfloxacin (Nor155

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    J. Mala


    Full Text Available Norfloxacin belongs to the group of fluoroquinolone antibiotics which has been approved for treatment in animals. However, its residues in animal products can pose adverse side effects to consumer. Therefore, detection of the residue in different food matrices must be concerned. In this study, a single chain variable fragment (scFv that recognizes norfloxacin antibiotic was constructed. The cDNA was synthesized from total RNA of hybridoma cells against norfloxacin. Genes encoding VH and VL regions of monoclonal antibody against norfloxacin (Nor155 were amplified and size of VH and VL fragments was 402 bp and 363 bp, respectively. The scFv of Nor155 was constructed by an addition of (Gly4Ser3 as a linker between VH and VL regions and subcloned into pPICZαA, an expression vector of Pichia pastoris. The sequence of scFv Nor155 (GenBank No. AJG06891.1 was confirmed by sequencing analysis. The complementarity determining regions (CDR I, II, and III of VH and VL were specified by Kabat method. The obtained recombinant plasmid will be useful for production of scFv antibody against norfloxacin in P. pastoris and further engineer scFv antibody against fluoroquinolone antibiotics.

  5. A Novel Human scFv Library with Non-Combinatorial Synthetic CDR Diversity. (United States)

    Bai, Xuelian; Kim, Jihye; Kang, Seungmin; Kim, Wankyu; Shim, Hyunbo


    The present work describes the construction and validation of a human scFv library with a novel design approach to synthetic complementarity determining region (CDR) diversification. The advantage of synthetic antibody libraries includes the possibility of exerting fine control over factors like framework sequences, amino acid and codon usage, and CDR diversity. However, random combinatorial synthesis of oligonucleotides for CDR sequence diversity also produces many clones with unnatural sequences and/or undesirable modification motifs. To alleviate these issues, we designed and constructed a novel semi-synthetic human scFv library with non-combinatorial, pre-designed CDR diversity and a single native human framework each for heavy, kappa, and lambda chain variable domains. Next-generation sequencing analysis indicated that the library consists of antibody clones with highly nature-like CDR sequences and the occurrence of the post-translational modification motifs is minimized. Multiple unique clones with nanomolar affinity could be isolated from the library against a number of target antigens, validating the library design strategy. The results demonstrate that it is possible to construct a functional antibody library using low, non-combinatorial synthetic CDR diversity, and provides a new strategy for the design of antibody libraries suitable for demanding applications.

  6. Quantum Dot- Conjugated Anti-GRP78 scFv Inhibits Cancer Growth in Mice

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    David Hornby


    Full Text Available Semiconductor quantum dots (Qdots have recently been shown to offer significant advantages over conventional fluorescent probes to image and study biological processes. The stability and low toxicity of QDs are well suited for biological applications. Despite this, the potential of Qdots remains limited owing to the inefficiency of existing delivery methods. By conjugating Qdots with small antibody fragments targeting membrane-bound proteins, such as GRP78, we demonstrate here that the Quantum dot- Anti-GRP78 scFv (Qdot-GRP78 retains its immunospecificity and its distribution can be monitored by visualization of multi-color fluorescence imaging both in vitro and in vivo. Moreover we demonstrate here for the first time that Qdot-GRP78 scFv bioconjugates can be efficiently internalized by cancer cells, thus upregulate phophosphate-AKT-ser473 and possess biological anti-tumour activity as shown by inhibition of breast cancer growth in a xenograft model. This suggests that nanocarrier-conjugated scFvs can be used as a therapeutic antibody for cancer treatment.

  7. FV10: an efficient single-layer approach to HDR coding, with backward compatibility options (United States)

    Topiwala, Pankaj; Dai, Wei; Krishnan, Madhu


    High Dynamic Range and Wide Color Gamut (HDR/WCG) video is now at the forefront of modern broadcast and other video delivery systems. The efficient transmission and display of such video over diverse networks and systems is an important problem. This paper presents a novel, state of the art approach in HDR/WCG video coding (called FV10) which uses a new, fully automatic video data adaptive regrading process, which converts HDR to Standard Dynamic Range (SDR). Our method differs from one developed recently in standards committees (the Joint Collaborative Team on Video Coding, or JCT-VC, of ITU|ISO/IEC), based on the HEVC Main10 Profile as the core codec, which is an HDR10 compliant system ("anchor"). FV10 also works entirely within the framework of HEVC Main10 Profile, but makes greater use of existing SEI messages. Reconstructed video using our methods show a subjective visual quality superior to the output of an example HDR10 anchor. Moreover, a usable backwards compatible SDR video is obtained as a byproduct in the processing chain, allowing service efficiencies. Representative objective results for the system include: results for RGB-PSNR, DE100, MD100, tOSNR-XYZ were -46.0%, -21.6%, -29.6%, 16.2% respectively.

  8. Involvement of FvSet1 in Fumonisin B1 Biosynthesis, Vegetative Growth, Fungal Virulence, and Environmental Stress Responses in Fusarium verticillioides

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    Qin Gu


    Full Text Available Fusarium verticillioides (teleomorph, Gibberella moniliformis is an important plant pathogen that causes seedling blight, stalk rot, and ear rot in maize (Zea mays. During infection, F. verticillioides produce fumonsins B1 (FB1 that pose a serious threat to human and animal health. Recent studies showed that Set1, a methyltransferase of H3K4, was responsible for toxin biosynthesis in filamentous fungi. However, to date, the regulation of FvSet1 on FB1 biosynthesis remains unclear. In the current study, we identified only one Set1 ortholog in F. verticillioides (FvSet1 and found that the deletion of FvSET1 led to various defects in fungal growth and pathogenicity. More interestingly, the FvSET1 deletion mutant (ΔFvSet1 showed a significant defect in FB1 biosynthesis and lower expression levels of FUM genes. FvSet1 was also found to play an important role in the responses of F. verticillioides to multiple environmental stresses via regulating the phosphorylation of FvMgv1 and FvHog1. Taken together, these results indicate that FvSet1 plays essential roles in the regulation of FB1 biosynthesis, fungal growth and virulence, as well as various stress responses in F. verticillioides.

  9. 金针菇FV908菌株液体培养工艺的研究%CULTURE TECHNOLOGY OF Flammulina velutipes FV908 IN LIQUID MEDIUM

    Institute of Scientific and Technical Information of China (English)



    通过单因子试验统计分析,优化筛选了适于金针菇(Flammulina velutipes)FV908的适宜培养基和摇瓶培养条件,结果表明,其适宜的液体培养基组成为玉米粉 5.0 %,麸皮 2.0 %,KH2PO4 0.1 %,MgSO4*7H2O 0.05 %,10 μg VB1/100 mL,50 μg VB2/100 mL;适宜的摇瓶培养条件为:培养基的起始pH 6.0~7.0,500 mL摇瓶装量为 150 mL,接种量为 10 %,培养温度 25 ℃,摇床转速为 120 r/min,菌丝干收率 39 g/L.

  10. Functional characterization of an scFv-Fc antibody that immunotherapeutically targets the common cancer cell surface proteoglycan CSPG4. (United States)

    Wang, Xinhui; Katayama, Akihiro; Wang, Yangyang; Yu, Ling; Favoino, Elvira; Sakakura, Koichi; Favole, Alessandra; Tsuchikawa, Takahiro; Silver, Susan; Watkins, Simon C; Kageshita, Toshiro; Ferrone, Soldano


    Cell surface chondroitin sulfate proteoglycan 4 (CSPG4) is an attractive target for antibody-based cancer immunotherapy because of its role in tumor cell biology, its high expression on malignant cells including cancer-initiating cells, and its restricted distribution in normal tissues. The clinical use of CSPG4 has been hampered by the lack of a CSPG4-specific chimeric, humanized, or fully human monoclonal antibody. To overcome this limitation, we generated a CSPG4-specific fully human single-chain antibody termed scFv-FcC21 and characterized its specificity and antitumor activity. Viable CSPG4(+) melanoma cells were used in a screen of a human scFv phage display library that included CDR3 engineered to optimize antibody binding sites. The scFv antibody isolated was then recombinantly engineered with a human immunoglobulin G1 Fc region to construct the fully human antibody scFv-FcC21, which recognized tumors of neuroectodermal origin, various types of carcinomas, mesotheliomas, and sarcomas as well as myeloid leukemias. scFv-FcC21 inhibited in vitro growth and migration of tumor cells and in vivo growth of human tumor xenografts. These effects were mediated by inhibition of the activation of extracellular signal-regulated kinase and focal adhesion kinase signaling pathways that are critical for tumor cell growth and migration, respectively. Our findings define the CSPG4-specific fully human scFv-FcC21 antibody as a candidate therapeutic agent to target the many types of tumors that express CSPG4.

  11. Generation of functional scFv intrabody to abate the expression of CD147 surface molecule of 293A cells

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    Mai Sabine


    Full Text Available Abstract Background Expression of intracellular antibodies (intrabodies has become a broadly applicable technology for generation of phenotypic knockouts in vivo. The method uses surface depletion of cellular membrane proteins to examine their biological function. In this study, we used this strategy to block the transport of cell surface molecule CD147 to the cell membrane. Phage display technology was introduced to generate the functional antibody fragment to CD147, and we subsequently constructed a CD147-specific scFv that was expressed intracellularly and retained in the endoplasmic reticulum by adenoviral gene transfer. Results The recombinant antibody fragments, Fab and scFv, of the murine monoclonal antibody (clone M6-1B9 reacted specifically to CD147 by indirect enzyme-linked immunosorbent assays (ELISA using a recombinant CD147-BCCP as a target. This indicated that the Fab- and scFv-M6-1B9 displaying on phage surfaces were correctly folded and functionally active. We subsequently constructed a CD147-specific scFv, scFv-M6-1B9-intrabody, in 293A cells. The expression of CD147 on 293A cell surface was monitored at 36 h after transduction by flow cytometry and demonstrated remarkable reduction. Colocalization of scFv-M6-1B9 intrabody with CD147 in the ER network was depicted using a 3D deconvolution microscopy system. Conclusion The results suggest that our approach can generate antibody fragments suitable for decreasing the expression of CD147 on 293A cells. This study represents a step toward understanding the role of the cell surface protein, CD147.

  12. Screening of Human Antibody Fab Fragment against HBsAg and the Construction of its dsFv Form

    Directory of Open Access Journals (Sweden)

    Leili Jia, Jiyun Yu, Hongbin Song, Xuelin Liu, Weina Ma, Yuanyong Xu, Chuanfu Zhang, Shicun Dong, Qiao Li


    Full Text Available The objective of this study was to pursue the techniques involving the screening of the human antibody Fab fragment against hepatitis B virus surface antigen (HBsAg and the construction of its disulfide-stabilized Fv fragment (dsFv. The phage antibody Fab fragments against HBsAg were screened from the human combinatorial immunoglobulin library. Sequence analysis revealed that its heavy chain gene was complete, but the light chain gene was lost. To improve the affinity of the antibody by chain shuffling, a human antibody light chain gene repertoire was generated by reverse transcriptase-polymerase chain reaction (RT-PCR from the human peripheral blood lymphocytes. A phage antibody sub-library was then constructed by inserting the light chain gene repertoire into the phagmid that contained the Fd gene. Five clones with appreciably higher absorbance than that of the original clone were obtained, which indicated that the affinity of the light chain-shuffled phage antibodies was improved. Then, the mutated genes of dsFv against HBsAg were constructed by using PCR-based point mutagenesis method. Purified VH and VL proteins were folded into a 25-kDa protein, designated as anti-HBsAg dsFv. ELISA and competition ELISA revealed that the dsFv maintained the specificity of the Fab by binding to HBsAg, even through with a lower binding activity. These results have facilitated the undertaking of further functional analyses of the constructed dsFv, and may therefore provide an improved technique for the production and application of dsFvs against HBsAg.

  13. Comparative response of platelet fV and plasma fV to activated protein C and relevance to a model of acute traumatic coagulopathy.

    Directory of Open Access Journals (Sweden)

    James E Campbell

    Full Text Available BACKGROUND: Acute traumatic coagulopathy (ATC has been linked to an increase in activated protein C (aPC from 40 pM in healthy individuals to 175 pM. aPC exerts its activity primarily through cleavage of active coagulation factor Va (fVa. Platelets reportedly possess fVa which is more resistant to aPC cleavage than plasma fVa; this work examines the hypothesis that normal platelets are sufficient to maintain coagulation in the presence of elevated aPC. METHODS: Coagulation responses of normal plasma, fV deficient plasma (fVdp, and isolated normal platelets in fVdp were conducted: prothrombin (PT tests, turbidimetry, and thromboelastography (TEG, including the dose response of aPC on the samples. RESULTS: PT and turbidimetric assays demonstrate that normal plasma is resistant to aPC at doses much higher than those found in ATC. Additionally, an average physiological number of washed normal platelets (200,000 platelets/mm3 was sufficient to eliminate the anti-coagulant effects of aPC up to 10 nM, nearly two orders of magnitude above the ATC concentration and even the steady-state pharmacological concentration of human recombinant aPC, as measured by TEG. aPC also demonstrated no significant effect on clot lysis in normal plasma samples with or without platelets. CONCLUSIONS: Although platelet fVa shows slightly superior resistance to aPC's effects compared to plasma fVa in static models, neither fVa is sufficiently cleaved in simulations of ATC or pharmacologically-delivered aPC to diminish coagulation parameters. aPC is likely a correlative indicator of ATC or may play a cooperative role with other activity altering products generated in ATC.

  14. Preconception Screening for Gene Polymorphisms Associated with Thrombophilia and Hyperhomocysteinemia Risk in Healthy Young Women

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    Elena Yu. Glotova


    Full Text Available The frequency characteristics of the gene polymorphisms (FVL G1691A, FII G20210A, MTHFR C677T, MTHFR A1298C, MTRR A66G associated with thrombophilia, hyperhomocysteinemia risk and different perinatal or pregnancy complications were studied. This examination was conducted among 130 planned-pregnancy healthy young women aged between 19 and 29 years. A gene mutation analysis was performed using a real-time polymerase chain reaction (real-time PCR. Factor V Leiden (FVL G1691A and prothrombin gene (FII G20210A mutations were not identified in the women surveyed. The frequency of the occurrence of the heterozygous FVL 1691G/A genotype associated with the risk of thrombosis during pregnancy was very low in these women (0.8%. The frequency of the MTHFR (methylenetetrahydrofolate reductase 1298C/С mutant genotype was 11.5%, MTHFR 677T/Т – 5.4%, and MTRR (methionine synthase reductase 66G/G – 31.5%. A combination of the MTHFR 677TT/1298CC and MTHFR 677TТ/MTRR 66GG mutant genotypes, which significantly increased the risk of pregnancy loss and neural tube defects, were found to occur in 0.8% of the cases.We concluded that selective thrombophilia screening (FVL G1691A and FII G20210A based on prior personal and/or family history of venous thromboembolism was more cost-effective than a universal preconception screening in all planning pregnancy women. However, in order to decrease the risk of congenital anomalies and pregnancy complications associated with folate dependent homocysteine metabolism, preconception care should include folate supplementation

  15. Hydrographic and Impairment Statistics Database: FIIS (United States)

    National Park Service, Department of the Interior — Hydrographic and Impairment Statistics (HIS) is a National Park Service (NPS) Water Resources Division (WRD) project established to track certain goals created in...

  16. The use of scFv-displaying yeast in mammalian cell surface selections. (United States)

    Wang, Xin Xiang; Shusta, Eric V


    Yeast surface display has proven to be a powerful tool for the directed evolution of immunological proteins when soluble ligands are available (Cho, B.K., Kieke, M.C., Boder, E.T., Wittrup, K.D., Kranz, D.M., 1998. A yeast surface display system for the discovery of ligands that trigger cell activation. J. Immunol. Methods 220, 179; Boder, E.T., Midelfort, K.S., Wittrup, K.D., 2000. Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity. Proc. Natl. Acad. Sci. U. S. A. 97, 10701; Shusta, E.V., Holler, P.D., Kieke, M.C., Kranz, D.M., Wittrup, K.D., 2000. Directed evolution of a stable scaffold for T-cell receptor engineering. Nat. Biotechnol. 18, 754; Esteban, O., Zhao, H., 2004. Directed evolution of soluble single-chain human class II MHC molecules. J. Mol. Biol. 340, 81). This investigation extends the utility of this display platform by demonstrating its capacity for use in cell panning selections. This was accomplished by employing a model single-chain antibody (scFv)-hapten system that allowed for detailed investigation of the factors governing panning success. Yeast displaying anti-fluorescein scFv (4-4-20) exhibited specific interactions with the fluoresceinated endothelial cells and could be recovered from large backgrounds of irrelevant yeast in just three rounds. Successful selections required as few as 1700 fluorescein ligands per cell, and a three-round enrichment ratio of 10(6) was possible. These results indicate that yeast surface display is a viable option for use in cell or tissue-based selections.

  17. Expression, production, and renaturation of a functional single-chain variable antibody fragment (scFv against human ICAM-1

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    H. Sun


    Full Text Available Intercellular adhesion molecule-1 (ICAM-1 is an important factor in the progression of inflammatory responses in vivo. To develop a new anti-inflammatory drug to block the biological activity of ICAM-1, we produced a monoclonal antibody (Ka=4.19×10−8 M against human ICAM-1. The anti-ICAM-1 single-chain variable antibody fragment (scFv was expressed at a high level as inclusion bodies in Escherichia coli. We refolded the scFv (Ka=2.35×10−7 M by ion-exchange chromatography, dialysis, and dilution. The results showed that column chromatography refolding by high-performance Q Sepharose had remarkable advantages over conventional dilution and dialysis methods. Furthermore, the anti-ICAM-1 scFv yield of about 60 mg/L was higher with this method. The purity of the final product was greater than 90%, as shown by denaturing gel electrophoresis. Enzyme-linked immunosorbent assay, cell culture, and animal experiments were used to assess the immunological properties and biological activities of the renatured scFv.

  18. Recombinant scFv Antibodies against E Protein and N Protein of Severe Acute Respiratory Syndrome Virus

    Institute of Scientific and Technical Information of China (English)

    Hui LIU; Yan-Li DING; Wei HAN; Mei-Yun LIU; Rui-Yang TIAN; Sheng-Li YANG; Yi GONG


    Three single chain antibodies(scFv)against the proteins of severe acute respiratory syndrome coronavirus(SARS-CoV)were isolated by phage display from an scFv antibody library.Bio-panning was carried out against immobilized purified envelope(E)and nucleocapsid(N)proteins of SARS-CoV.Their binding activity and specificity to E or N protein of SARS-CoV were characterized by phage-ELISA.Two of them,B 10 and C20,could recognize non-overlapping epitopes of the E protein according to the two-site binding test result.Clone A 17 could recognize N protein.The sequence of the epitope or overlapping epitope of scFv antibody A17 was PTDSTDNNQNGGRNGARPKQRRPQ.The affinity(equilibrium dissociation constant,Kd)of SARS-CoV E protein was 5.7×10-8 M for B10 and 8.9×10-8 M for C20.The affinity of A17for N protein was 2.1 x 10-6 M.All three scFv antibodies were purified with affinity chromatography and determined by Western blot.

  19. Construction of bifunctional molecules specific to antigen and antibody’s Fc-fragment by fusion of scFv-antibodies with staphylococcal protein A

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    Kolibo D. V.


    Full Text Available Aim. To develop approach for detection of scFv and their complexes with antigens. Methods. The fusion proteins, which include sequences of scFv and staphylococcal protein A, were constructed and the obtained bifunctional molecules were immunochemically analysed. Results. It was shown, that scFv fused with protein A and their complexes with antigens are effectively recognized by labelled immunoglobulins with unrestricted antigenic specificity. Conclusions. The fusion of scFv with protein A fragment is a perspective approach to increase the efficiency of application in ELISA. The obtained scFv, fused with protein A, could be used for development of test-systems for the detection of diphtheria toxin.

  20. Thrombophilia genetic testing in Romanian young women with acute thrombotic events: role of Factor V Leiden, Prothrombin G20210A, MTHFR C677T and A1298C polymorphisms / Evaluarea genetică a trombofiliilor la femei tinere din România cu evenimente acute trombotice: rolul Factorului V Leiden, Protrombinei G20210A, polimorfismelor MTHFR C677T și A1298C

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    Daraban Ana Maria


    Full Text Available Objective: The present case-control study aimed at evaluating the contribution of thrombophilic polymorphisms to acute venous (VTE as well as arterial thrombotic events (ATE in a population of young women with few traditional thrombotic factors (CVRF.

  1. CF750-A33scFv-Fc-Based Optical Imaging of Subcutaneous and Orthotopic Xenografts of GPA33-Positive Colorectal Cancer in Mice

    Directory of Open Access Journals (Sweden)

    Danfeng Wei


    Full Text Available Antibody-based imaging agents are attractive as adjuvant diagnostic tools for solid tumors. GPA33 is highly expressed in most human colorectal cancers and has been verified as a diagnostic and therapeutic target. Here, we built an A33scFv-Fc antibody against GPA33 by fusing A33scFv to the Fc fragment of human IgG1 antibodies. The A33scFv-Fc specifically binds GPA33-positive colorectal cancer cells and tumor tissues. After the intravenous injection of mice bearing subcutaneous GPA33-positive LS174T tumor grafts with near-infrared fluorescence probe CF750-labeled A33scFv-Fc (CF750-A33scFv-Fc, high contrast images of the tumor grafts could be kinetically documented within 24 h using an optical imaging system. However, GPA33-negative SMMC7721 tumor grafts could not be visualized by injecting the same amount of CF750-A33scFv-Fc. Moreover, in subcutaneous LS174T tumor-bearing mice, tissue scanning revealed that the CF750-A33scFv-Fc accumulated in the tumor grafts, other than the kidney and liver. In mice with orthotopic tumor transplantations, excrescent LS174T tumor tissues in the colon were successfully removed under guidance by CF750-A33scFv-Fc-based optical imaging. These results indicate that CF750-A33scFv-Fc can target GPA33, suggesting the potential of CF750-A33scFv-Fc as an imaging agent for the diagnosis of colorectal cancer.

  2. Effect of Heat Treatments on Fatigue Properties of FV520B Steel Using Infrared Thermography%热处理对FV520B钢疲劳性能的影响

    Institute of Scientific and Technical Information of China (English)

    樊俊铃; 郭杏林; 吴承伟; 邓德伟


    The effects of heat treatments on microstructures and mechanical properties of FV520B stainless steel were investigated. The infrared thermographic method (TM) was used to monitor the external temperature of specimens subjected to fatigue loading in order to fast determine fatigue strengths of the virgin and heat-treated FV520B steels, The relationship between the surface temperature and internal microstructures are closely linked to analyze the damage status for safety evaluation. The metallographic investigation shows that the improvement of mechanical properties of FV520B steel can be attributed to the formation of tempered martensite and secondary phase particles uniformly distributed in the matrix. The fatigue microcracks initiate from the favorably oriented grains at the corner of the specimen surface.%用红外热像法监测交变载荷下试件表面的温升变化规律,快速确定FV520B原材料和热处理材料的疲劳强度,研究了热处理对FV520B不锈钢组织和力学性能的影响。将疲劳试验过程中的试件表面的温度变化与材料内部微观结构的演化相联系,以分析疲劳损伤状态,进行安全性评估。金相观测发现,FV520B钢力学性能的提高是回火马氏体和基体中均匀弥散分布的第二相颗粒共同作用的结果。扫描电子显微镜对疲劳断口形貌特征的观察发现,疲劳裂纹萌生于试件表面棱角上位向有利的晶粒处。

  3. An efficient method for variable region assembly in the construction of scFv phage display libraries using independent strand amplification. (United States)

    Sotelo, Pablo; Collazo, Noberto; Zuñiga, Roberto; Gutiérrez-González, Matías; Catalán, Diego; Ribeiro, Carolina Hager; Aguillón, Juan Carlos; Molina, María Carmen


    Phage display library technology is a common method to produce human antibodies. In this technique, the immunoglobulin variable regions are displayed in a bacteriophage in a way that each filamentous virus displays the product of a single antibody gene on its surface. From the collection of different phages, it is possible to isolate the virus that recognizes specific targets. The most common form in which to display antibody variable regions in the phage is the single chain variable fragment format (scFv), which requires assembly of the heavy and light immunoglobulin variable regions in a single gene. In this work, we describe a simple and efficient method for the assembly of immunoglobulin heavy and light chain variable regions in a scFv format. This procedure involves a two-step reaction: (1) DNA amplification to produce the single strand form of the heavy or light chain gene required for the fusion; and (2) mixture of both single strand products followed by an assembly reaction to construct a complete scFv gene. Using this method, we produced 6-fold more scFv encoding DNA than the commonly used splicing by overlap extension PCR (SOE-PCR) approach. The scFv gene produced by this method also proved to be efficient in generating a diverse scFv phage display library. From this scFv library, we obtained phages that bound several non-related antigens, including recombinant proteins and rotavirus particles.

  4. Cloning of scFv from hybridomas using a rational strategy: Application as a receptor to sensitive detection microcystin-LR in water. (United States)

    Zhang, Xiuyuan; He, Kuo; Zhao, Ruiping; Wang, Lixia; Jin, Yandan


    Single chain variable fragment (scFv), containing of heavy and light chains (VH and VL) joined by a short peptide linker, has been used widely for immunodetection. Nevertheless, cloning functional variable genes is still a bottle neck for the scFv generation technology. Here, a rational strategy for cloning and selecting variable region genes from an anti-microcystin-LR hybridoma was devised, then the functional VH and VL genes were recloned and assembled to scFv using splicing overlap extension PCR. The resulting scFv gene was recombinantly expressed as a soluble scFv-alkaline phosphatase fusion protein (scFv-AP) by vector PLIP6/GN. Then an indirect competitive chemiluminescent enzyme immunoassay (ic-CLEIA) for detection of microcystin-LR was developed. The half-maximum inhibition concentrations (IC50) and limits of detection (LODs, IC15) were 0.81 ± 0.04 μgL(-1) and 0.13 ± 0.03 μgL(-1), respectively. With the mean coefficient of variation lowing 8%, the mean recovery in intra-assay and inter-assay were 100.06% and 96.46%, The proposed strategy should be useful for generation scFv in a rapid and simple way.


    Institute of Scientific and Technical Information of China (English)

    杨章民; 司履生; 王一理; 来宝长


    Objective To design and construct the eukaryotic expression vector which expresses Anti-CD3 ScFv-B7.1 fusion molecules and predict the biological characteristics, the rationality and feasibility of the spacer. Methods To analyze the flexibility, Hoop & Woods hydrophilicity and the epitope of Anti-CD3 ScFv-B7.1 fusion molecule at secondary structure level by computer simulation utilizing the GoldKey software. Results By comparing with Anti-CD3 ScFv and B7.1 respectively, it shows that Anti-CD3 ScFv-B7.1 fusion molecules can form correct secondary structure with the linking of the spacer, the fusion does not change the original hydrophilicity and epitopes of both Anti-CD3 ScFv and B7.1, no new epitopes emerge; The spacer is flexible and shows low antigenicity. Conclusion The design of Anti-CD3 ScFv-B7.1 fusion molecule are rational and feasible, the expressed fusion protein could retain the maximum biological activity and the function of both Anti-CD3 ScFv and B7.1.

  6. Generation and Characterization of C305, a Murine Neutralizing scFv Antibody That Can Inhibit BLyS Binding to Its Receptor BCMA

    Institute of Scientific and Technical Information of China (English)

    Mei-Yun LIU; Wei HAN; Yan-Li DING; Tian-Hong ZHOU; Rui-Yang TIAN; Sheng-Li YANG; Hui LIU; Yi GONG


    B-lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor (TNF) family and a key regulator of B cell response. Neutralizing single-chain fragment variable (scFv) antibody against BLyS binding to its receptor BCMA has the potential to play a prominent role in autoimmune disease therapy. A phage display scFv library constructed on pIII protein of M13 filamentous phage was screened using BLyS.After five rounds of panning, their binding activity was characterized by phage-ELISA. Nucleotide sequencing revealed that at least two different scFv gene fragments (C305 and D416) were obtained. The two different scFv gene fragments were expressed to obtain the soluble scFv antibodies, then the soluble scFv antibodies were characterized by means of competitive ELISA and in vitro neutralization assay. The results indicated that C305 is the neutralizing scFv antibody that can inhibit BLyS binding to its receptor BCMA.

  7. Antiepidermal growth factor variant III scFv fragment: effect of radioiodination method on tumor targeting and normal tissue clearance

    Energy Technology Data Exchange (ETDEWEB)

    Shankar, Sriram [Department of Radiology, Duke University Medical Center, Durham, NC 27710 (United States); Vaidyanathan, Ganesan [Department of Radiology, Duke University Medical Center, Durham, NC 27710 (United States); Kuan, C.-T. [Department of Pathology, Duke University Medical Center, Durham, NC 27710 (United States); Bigner, Darell D. [Department of Pathology, Duke University Medical Center, Durham, NC 27710 (United States); Zalutsky, Michael R. [Department of Radiology, Duke University Medical Center, Durham, NC 27710 (United States) and Department of Pathology, Duke University Medical Center, Durham, NC 27710 (United States) and Department of Biomedical Engineering, Duke University, Durham, NC 27708 (United States)]. E-mail:


    Introduction: MR1-1 is a single-chain Fv (scFv) fragment that binds with high affinity to epidermal growth factor receptor variant III, which is overexpressed on gliomas and other tumors but is not present on normal tissues. The objective of this study was to evaluate four different methods for labeling MR1-1 scFv that had been previously investigated for the radioiodinating of an intact anti-epidermal growth factor receptor variant III (anti-EGFRvIII) monoclonal antibody (mAb) L8A4. Methods: The MR1-1 scFv was labeled with {sup 125}I/{sup 131}I using the Iodogen method, and was also radiohalogenated with acylation agents bearing substituents that were positively charged-N-succinimidyl-3-[*I]iodo-5-pyridine carboxylate and N-succinimidyl-4-guanidinomethyl-3-[*I]iodobenzoate ([*I]SGMIB)-and negatively charged-N-succinimidyl-3-[*I]iodo-4-phosphonomethylbenzoate ([*I]SIPMB). In vitro internalization assays were performed with the U87MG{delta}EGFR cell line, and the tissue distribution of the radioiodinated scFv fragments was evaluated in athymic mice bearing subcutaneous U87MG{delta}EGFR xenografts. Results and Conclusion: As seen previously with the anti-EGFRvIII IgG mAb, retention of radioiodine activity in U87MG{delta}EGFR cells in the internalization assay was labeling method dependent, with SGMIB and SIPMB yielding the most prolonged retention. However, unlike the case with the intact mAb, the results of the internalization assays were not predictive of in vivo tumor localization capacity of the labeled scFv. Renal activity was dependent on the nature of the labeling method. With MR1-1 labeled using SIPMB, kidney uptake was highest and most prolonged; catabolism studies indicated that this uptake primarily was in the form of {epsilon}-N-3-[*I]iodo-4-phosphonomethylbenzoyl lysine.

  8. Cloning approach and functional analysis of anti-intimin single-chain variable fragment (scFv

    Directory of Open Access Journals (Sweden)

    Elias Waldir P


    Full Text Available Abstract Background Intimin is an important virulence factor involved in the pathogenesis of enteropathogenic Escherichia coli (EPEC and enterohemorrhagic Escherichia coli (EHEC. Both pathogens are still important causes of diarrhea in children and adults in many developing and industrialized countries. Considering the fact that antibodies are important tools in the detection of various pathogens, an anti-intimin IgG2b monoclonal antibody was previously raised in immunized mice with the conserved sequence of the intimin molecule (int388-667. In immunoblotting assays, this monoclonal antibody showed excellent specificity. Despite good performance, the monoclonal antibody failed to detect some EPEC and EHEC isolates harboring variant amino acids within the 338-667 regions of intimin molecules. Consequently, motivated by its use for diagnosis purposes, in this study we aimed to the cloning and expression of the single-chain variable fragment from this monoclonal antibody (scFv. Findings Anti-intimin hybridoma mRNA was extracted and reversely transcripted to cDNA, and the light and heavy chains of the variable fragment of the antibody were amplified using commercial primers. The amplified chains were cloned into pGEM-T Easy vector. Specific primers were designed and used in an amplification and chain linkage strategy, obtaining the scFv, which in turn was cloned into pAE vector. E. coli BL21(DE3pLys strain was transformed with pAE scFv-intimin plasmid and subjected to induction of protein expression. Anti-intimin scFv, expressed as inclusion bodies (insoluble fraction, was denatured, purified and submitted to refolding. The protein yield was 1 mg protein per 100 mL of bacterial culture. To test the functionality of the scFv, ELISA and immunofluorescence assays were performed, showing that 275 ng of scFv reacted with 2 mg of purified intimin, resulting in an absorbance of 0.75 at 492 nm. The immunofluorescence assay showed a strong reactivity with

  9. 利用高光谱植被指数监测紧凑型玉米叶绿素荧光参数Fv/Fm%Monitoring the Chlorophyll Fluorescence Parameter Fv/Fm in Compact Corn Based on Different Hyperspectral Vegetation Indices

    Institute of Scientific and Technical Information of China (English)

    谭昌伟; 黄文江; 金秀良; 王君婵; 童璐; 王纪华; 郭文善


    为进一步评价遥感监测紧凑型玉米叶绿素荧光参数Fv/Fm的可行性,通过开展小区紧凑型玉米试验,分析紧凑型玉米整个生育期Fv/Fm与高光谱植被指数的相关关系,建立紧凑型玉米Fv/Fm高光谱监测模型.结果表明,紧凑型玉米Fv/Fm与选取的高光谱植被指数均呈极显著正相关,其中结构敏感色素指数(SIPI)与Fv/Fm的相关性最好,相关系数(r)为0.88.用SIPI建立紧凑型玉米Fv/Fm的监测模型,其决定系数(R2)为0.812 6,均方根误差(RMSE)为0.082.研究表明,利用高光谱植被指数可以有效地监测紧凑型玉米整个生育期的Fv/Fm.%In order to further assess the feasibility of monitoring the chlorophyll fluorescence parameter Fv/Fm in compact corn by hyperspectral remote sensing data, in the present study, hyperspectral vegetation indices from in-situ remote sensing measurements were utilized to monitor the chlorophyll fluorescence parameter Fv/Fm measured in the compact corn experiment. The relationships were analyzed between hyperspectral vegetation indices and Fv/Fm and the monitoring models were established for Fv/Fm in the whole growth stages of compact corn. The results indicated that Fv/Fm was significantly correlated to the hyperspectral vegetation indices. Among them, structure-sensitive pigment index (SIPI) was the most sensitive remote sensing variable for monitoring Fv/Fm with correlation coefficient (r) of 0. 88. The monitoring model of Fv/Fm was established on the base of SIPI, and the determination coefficients (r2) and the root mean square errors (RMSE) were 0. 812 6 and 0. 082 respectively. The overall results suggest that hyperspectral vegetation indices can be potential indicators to monitor Fv/Fm during growth stages of compact corn.

  10. 基于FV-OWA算子的不确定多属性决策方法%Uncertain multiple attribute decision making method based on FV-OWA operator

    Institute of Scientific and Technical Information of China (English)

    汪新凡; 杨小娟



  11. Cloning single-chain antibody fragments (ScFv) from hyrbidoma cells. (United States)

    Toleikis, Lars; Frenzel, André


    Despite the rising impact of the generation of antibodies by phage display and other technologies, hybridoma technology still provides a valuable tool for the generation of high-affinity binders against different targets. But there exist several limitations of using hybridoma-derived antibodies. The source of the hybridoma clones are mostly rat or mouse B-lymphocytes. Therefore a human-anti-mouse or human-anti-rat antibody response may result in immunogenicity of these antibodies. This leads to the necessity of humanization of these antibodies where the knowledge of the amino acid sequence of the proteins is inalienable. Furthermore, additional in vitro modifications, e.g., affinity maturation or fusion to other proteins, are dependent on cloning of the antigen-binding domains.Here we describe the isolation of RNA from hybridoma cells and the primers that can be used for the amplification of VL and VH as well as the cloning of the antibody in scFv format and its expression in Escherichia coli.

  12. Clinical Applications of Phage-Derived sFvs and sFv Fusion Proteins

    Directory of Open Access Journals (Sweden)

    K. A. Chester


    Full Text Available Single chain Fv antibodies (sFvs have been produced from filamentous bacteriophage libraries obtained from immunised mice. MFE-23, the most characterised of these sFvs, is reactive with carcinoembryonic antigen (CEA, a glycoprotein that is highly expressed in colorectal adenocarcinomas. MFE-23 has been expressed in bacteria and purified in our laboratory for two clinical trials; a gamma camera imaging trial using 123I-MFE-23 and a radioimmunoguided surgery trial using 125I-MFE-23, where tumour deposits are detected by a hand-held probe during surgery. Both these trials show MFE-23 is safe and effective in localising tumour deposits in patients with cancer. We are now developing fusion proteins which use MFE-23 to deliver a therapeutic moiety; MFE-23::CPG2 targets the enzyme carboxypeptidase G2 (CPG2 for use in the ADEPT (antibody directed enzyme prodrug therapy system and MFE::TNFα aims to reduce sequestration and increase tumor concentrations of systemically administered TNFα.

  13. Isolation of BNYVV coat protein-specific single chain Fv from a mouse phage library antibody. (United States)

    Jahromi, Zahra Moghaddassi; Salmanian, Ali Hatef; Rastgoo, Nasrin; Arbabi, Mehdi


    Beet necrotic yellow vein virus (BNYVV) infects sugar beet plants worldwide and is responsible for the rhizomania disease and severe economic losses. Disease severity and lack of naturally occurring resistant plants make it very difficult to control the virus, both from epidemiological and economic standpoints. Therefore, early detection is vital to impose hygiene restrictions and prevent further spread of the virus in the field. Immunoassays are one of the most popular methodologies for the primary identification of plant pathogens including BNYVV since they are robust, sensitive, fast, and inexpensive. In this study, the major coat protein (CP21) of BNYVV was cloned and expressed in Escherichia coli. Thereafter, mice were immunized with purified CP21 and a phage antibody library was constructed from their PCR-amplified immunoglobulin repertoire. Following filamentous phage rescue of the library and four rounds of panning against recombinant CP21 antigen, several specific single chain Fv fragments were isolated and characterized. This approach may pave the way to develop novel immunoassays for a rapid detection of viral infection. Moreover, it will likely provide essential tools to establish antibody-mediated resistant transgenic technology in sugar beet plants.

  14. Antimalarial activity of granzyme B and its targeted delivery by a granzyme B-single-chain Fv fusion protein. (United States)

    Kapelski, Stephanie; de Almeida, Melanie; Fischer, Rainer; Barth, Stefan; Fendel, Rolf


    We present here the first evidence that granzyme B acts against Plasmodium falciparum (50% inhibitory concentration [IC50], 1,590 nM; 95% confidence interval [95% CI], 1,197 to 2,112 nM). We created a novel antimalarial fusion protein consisting of granzyme B fused to a merozoite surface protein 4 (MSP4)-specific single-chain Fv protein (scFv), which targets the enzyme to infected erythrocytes, with up to an 8-fold reduction in the IC50 (176 nM; 95% CI, 154 to 202 nM). This study confirms the therapeutic efficacies of recombinant antibody-mediated antimalarial immunotherapeutics based on granzyme B.

  15. Validation of glypican-3-specific scFv isolated from paired display/secretory yeast display library

    Directory of Open Access Journals (Sweden)

    Li Yonghai


    Full Text Available Abstract Background Glypican-3 (GPC3 is a heparan-sulfate proteoglycan frequently expressed on the cell membrane of malignant hepatocytes in hepatocellular carcinoma. The capacity for screening potential antibodies in vitro using human hepatocellular lines is critical to ensure binding to this highly post-translationally modified glycophosphatidylinositiol-linked protein. We hypothesized that we could utilize a recently described paired display/secretory yeast library to isolate human-derived scFv against glypican-3 for potential diagnostic and/or therapeutic application. Results Using two different biotinylated antigen targets, a synthesized 29mer fragment GPC3550-558 and a truncated GPC3368-548 fused with glutathione S-transferase (GST we enriched the yeast display library to greater than 30% target-specific yeast with both positive selection and depletion of streptavidin- and GST-specific clones. After cloning of scFv cDNA from the enriched sub-library, scFv specificity was validated by ELISA for binding to recombinant protein from prokaryotic and eukaryotic sources and ultimately naturally presented human protein on the cell membrane of human hepatocellular cell lines. Specificity was confirmed using non-expressing cell lines and shRNA knockdown. Ultimately, five unique scFv with affinity EC50 ranging from 5.0-110.9nM were identified. Conclusions Using a paired display/secretory yeast library, five novel and unique scFvs for potential humoral or chimeric therapeutic development in human hepatocellular carcinoma were isolated and characterized.

  16. Construction of a human functional single-chain variable fragment (scFv) antibody recognizing the malaria parasite Plasmodium falciparum. (United States)

    Wajanarogana, Sumet; Prasomrothanakul, Teerawat; Udomsangpetch, Rachanee; Tungpradabkul, Sumalee


    Falciparum malaria is one of the most deadly and profound human health problems around the tropical world. Antimalarial drugs are now considered to be a powerful treatment; however, there are drugs currently being used that are resistant to Plasmodium falciparum parasites spreading in different parts of the world. Although the protective immune response against intraerythrocytic stages of the falciparum malaria parasite is still not fully understood, immune antibodies have been shown to be associated with reduced parasite prevalence. Therefore antibodies of the right specificity present in adequate concentrations and affinity are reasonably effective in providing protection. In the present study, VH (variable domain of heavy chain) and VL (variable domain of light chain) were isolated from human blood lymphocytes of P. falciparum in one person who had high serum titre to RESA (ring-infected erythrocyte surface antigen). Equal amounts of VH and VL were assembled together with universal linker (G4S)3 to generate scFvs (single-chain variable fragments). The scFv antibodies were expressed with a phage system for the selection process. Exclusively, an expressed scFv against asynchronous culture of P. falciparum-infected erythrocytes was selected and characterized. Sequence analysis of selected scFv revealed that this clone could be classified into a VH family-derived germline gene (VH1) and Vkappa family segment (Vkappa1). Using an indirect immunofluorescence assay, we could show that soluble expressed scFv reacted with falciparum-infected erythrocytes. The results encourage the further study of scFvs for development as a potential immunotherapeutic agent.

  17. Development of single-chain variable fragments (scFv) against influenza virus targeting hemagglutinin subunit 2 (HA2). (United States)

    Li, Tai-Wei; Cheng, Shu-Fang; Tseng, Yen-Tzu; Yang, Yu-Chih; Liu, Wen-Chun; Wang, Sheng-Cyuan; Chou, Mei-Ju; Lin, Yu-Jen; Wang, Yueh; Hsiao, Pei-Wen; Wu, Suh-Chin; Chang, Ding-Kwo


    Influenza A viruses (IAV) are widespread in birds and domestic poultry, occasionally causing severe epidemics in humans and posing health threats. Hence, the need to develop a strategy for prophylaxis or therapy, such as a broadly neutralizing antibody against IAV, is urgent. In this study, single-chain variable fragment (scFv) phage display technology was used to select scFv fragments recognizing influenza envelope proteins. The Tomlinson I and J scFv phage display libraries were screened against the recombinant HA2 protein (rHA2) for three rounds. Only the third-round elution sample of the Tomlinson J library showed high binding affinity to rHA2, from which three clones (3JA18, 3JA62, and 3JA78) were chosen for preparative-scale production as soluble antibody by E. coli. The clone 3JA18 was selected for further tests due to its broad affinity for influenza H1N1, H3N2 and H5N1. Simulations of the scFv 3JA18-HA trimer complex revealed that the complementarity-determining region of the variable heavy chain (VH-CDR2) bound the stem region of HA. Neutralization assays using a peptide derived from VH-CDR2 also supported the simulation model. Both the selected antibody and its derived peptide were shown to suppress infection with H5N1 and H1N1 viruses, but not H3N2 viruses. The results also suggested that the scFvs selected from rHA2 could have neutralizing activity by interfering with the function of the HA stem region during virus entry into target cells.

  18. Development of single chain variable fragment (scFv) antibodies against Xylella fastidiosa subsp. pauca by phage display. (United States)

    Yuan, Qing; Jordan, Ramon; Brlansky, Ronald H; Istomina, Olga; Hartung, John


    Xylella fastidiosa is a member of the gamma proteobacteria. It is fastidious, insect-vectored and xylem-limited and causes a variety of diseases, some severe, on a wide range of economically important perennial crops, including grape and citrus. Antibody based detection assays are commercially available for X. fastidiosa, and are effective at the species, but not at the subspecies level. We have made a library of scFv antibody fragments directed against X. fastidiosa subsp. pauca strain 9a5c (citrus) by using phage display technology. Antibody gene repertoires were PCR-amplified using 23 primers for the heavy chain variable region (V(H)) and 21 primers for the light chain variable region (V(L)). The V(H) and V(L) were joined by overlap extension PCR, and then the genes of the scFv library were ligated into the phage vector pKM19. The library contained 1.2×10(7) independent clones with full-length scFv inserts. In each of 3cycles of affinity-selection with 9a5c, about 1.0×10(12) phage were used for panning with 4.1×10(6), 7.1×10(6), 2.1×10(7) phage recovered after the first, second and third cycles, respectively. Sixty-six percent of clones from the final library bound X. fastidiosa 9a5c in an ELISA. Some of these scFv antibodies recognized strain 9a5c and did not recognize X. fastidiosa strains that cause Pierce's disease of grapevine.

  19. Primary structure and functional scFv antibody expression of an antibody against the human protooncogen c-myc. (United States)

    Fuchs, P; Breitling, F; Little, M; Dübel, S


    The immunoglobulin heavy- and light-chain variable region (Vh and Vl) genes were isolated from Myc1-9E10 hybridoma cells, which secreted monoclonal antibody against human oncogen c-myc. The expression vector pOPE52-c-myc was constructed for the recombinant production in E. coli. A 30 kDa single chain fragment (scFv) expression product was found in the periplasmic space by SDS-PAGE and immunoblotting. A significant fraction was processed correctly as demonstrated with an antiserum recognizing the processed aminoterminus only. The specific binding of the scFv fragment to the peptide epitope of the maternal monoclonal antibody was demonstrated and the primary sequence of the variable regions was determined. Sequence comparison with previously published partial Vh and Vl sequences from this hybridoma cell line revealed a genetic heterogeneity for the light chain variable region. The potential use of this scFv as a new tool for detection and purification of tagged proteins, for adding costimulatory signals to the surface of cancer cells as well as for analyzing c-myc function in the living cell by cytoplasmic expression is discussed.

  20. Engineering an Anti-Transferrin Receptor ScFv for pH-Sensitive Binding Leads to Increased Intracellular Accumulation.

    Directory of Open Access Journals (Sweden)

    Benjamin J Tillotson

    Full Text Available The equilibrium binding affinity of receptor-ligand or antibody-antigen pairs may be modulated by protonation of histidine side-chains, and such pH-dependent mechanisms play important roles in biological systems, affecting molecular uptake and trafficking. Here, we aimed to manipulate cellular transport of single-chain antibodies (scFvs against the transferrin receptor (TfR by engineering pH-dependent antigen binding. An anti-TfR scFv was subjected to histidine saturation mutagenesis of a single CDR. By employing yeast surface display with a pH-dependent screening pressure, scFvs having markedly increased dissociation from TfR at pH 5.5 were identified. The pH-sensitivity generally resulted from a central cluster of histidine residues in CDRH1. When soluble, pH-sensitive, scFv clone M16 was dosed onto live cells, the internalized fraction was 2.6-fold greater than scFvs that lacked pH-sensitive binding and the increase was dependent on endosomal acidification. Differences in the intracellular distribution of M16 were also observed consistent with an intracellular decoupling of the scFv M16-TfR complex. Engineered pH-sensitive TfR binding could prove important for increasing the effectiveness of TfR-targeted antibodies seeking to exploit endocytosis or transcytosis for drug delivery purposes.

  1. Selection and Characterization of Human Single Chain Fv (scFv)against Interleukin-21%全人源抗白介素21单链抗体的筛选与鉴定

    Institute of Scientific and Technical Information of China (English)

    吴沁航; 王彤; 张弢; 张娟; 王旻


    Interleukin-21 ( IL-21) is a potent immunomodulatory four-a-helical-bundle type I cytokine, mainly produced by CD4 + T cells and has pleiotropic effects on both innate and adaptive immune responses. Abnormal expression of IL-21 is involved in the pathogenesis of inflammation and autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus and inflammatory bowel disease, suggesting that blocking the IL-21 signal might ameliorate autoimmune symptoms. Antibodies are some of the most powerful tools in therapy and diagnostics and are currently one of the fastest growing classes of therapeutic molecules. Single-chain variable fragments ( scFvs) are becoming popular therapeutic alternatives to full length monoclonal antibodies since they are smaller, possessing different properties that are advantageous in certain medical applications. In this study,scFv antibodies against IL-21 were screened from Griffin 1 human phage display library. After four rounds of panning and selection on IL-21 absorbed immunotubes, individual clones were isolated, sequenced and characterized by Enzyme-Linked Immunosorbent Assay ( ELISA). 65% of the selected phage antibodies showed significant binding to IL-21, and a high affinity scFv FG8 was selected. The scFv FG8 was expressed in soluble form in Escherichia coli HB2151 .purified by IMAC,the soluble 30 kDa FG8 scFv-antibody was verified in SDS-PAGE and Western-blot. The binding specificity of scFv FG8 was confirmed by quantitative ELISA, and a dose-dependent manner was obtained. The obtained scFv FG8 are fully human in nature, and have a higher therapeutic value compared with mouse and humanized antibodies, since it has a lower potential to cause adverse immunological effect. This study demonstrated that phage display libraries provide a valuable system for the rapid generation of specific antibody fragments, and the scFvs antibodies can be easily produced using bacterial protein synthesis systems. The scFv FG8 obtained can

  2. Construction and Analysis of Three-dimensional Graphic Model of Single-chain Fv Derived from an Anti-human Placental Acidic Isoferritin Monoclonal Antibody by Computer

    Institute of Scientific and Technical Information of China (English)

    ZHOU Chun; SHEN Guanxin; ZHU Huifen; YANG Jing; ZHANG Yue; FENG Jiannan; SHEN Beifen


    A three-dimensional (3D) graphic model of a single-chain Fv (scFv) which was derived from an anti-human placental acidic isoferritin (PAF) monoclonal antibody (Mab) was constructed by a homologous protein-predicting computer algorithm on Silicon graphic computer station.The structure, surface static electricity and hydrophobicity of scFv were investigated. Computer graphic modelling indicated that all regions of scFv including the linker, variable regions of the heavy (VH) and light (VL) chains were suitable. The VH region and the VL region were involved in composing the "hydrophobic pocket". The linker was drifted away VH and VL regions. The complementarity determining regions (CDRs) of VH and VL regions surrounded the "hydrophobic pocket". This study provides a theory basis for improving antibody affinity, investigating antibody structure and analyzing the functions of VH and VL regions in antibody activity.

  3. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library. (United States)

    Wang, Han; Yu, Rui; Fang, Ting; Yu, Ting; Chi, Xiangyang; Zhang, Xiaopeng; Liu, Shuling; Fu, Ling; Yu, Changming; Chen, Wei


    Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective.

  4. A novel human scFv fragment against TNF-alpha from de novo design method. (United States)

    Chang, Hong; Qin, Weisong; Li, Yan; Zhang, Jiyan; Lin, Zhou; Lv, Ming; Sun, Yingxun; Feng, Jiannan; Shen, Beifen


    Anti-TNF antibody has been an effective therapeutic strategy for the diseases related to aberrant production of TNF-alpha, such as rheumatoid arthritis (RA) and Crohn's disease. The limitations of large molecule inhibitors in the therapy of these diseases prompted the search for other potent novel TNF-alpha antagonists. Antagonistic peptides, derived directly or designed rationally from complementarity-determining regions (CDRs) of neutralizing antibodies against TNF-alpha, have been demonstrated for their ability of inhibiting TNF-alpha. However, their activity is very low. In this study, to increase the affinity and bioactivity, human antibody variable region was used as scaffold to display antagonistic peptides, which were designed on the interaction between TNF-alpha and its neutralizing monoclonal antibody (mAb Z12). Based on the previously designed domain antibody (framework V(H)5), framework V(kappa)1 was used as light chain scaffold. On the basis of computer-guided molecular design method, a novel human scFv fragment (named as TSA1) was designed. Theoretical analysis showed that TSA1 could bind to TNF-alpha with more hydrogen bonds and lower binding free energy than the designed domain antibody. The biological experiments demonstrated that TSA1 could directly bind with TNF-alpha, competitively inhibit the binding of mAb Z12 to TNF-alpha and block the binding of TNF-alpha to TNFR I and TNFR II. TSA1 could also inhibit TNF-induced cytotoxicity on L929 cells and TNF-mediated NF-kappaB activation on HEK-293T cells. The bioactivity of TSA1 was significantly increased over the domain antibody. This study indicated that the framework of antibody variable region could serve as an ideal scaffold for displaying the peptides and provides a novel strategy to design TNF-alpha inhibitors with the ability to block the deleterious biological effects of TNF-alpha.

  5. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library (United States)

    Wang, Han; Yu, Rui; Fang, Ting; Yu, Ting; Chi, Xiangyang; Zhang, Xiaopeng; Liu, Shuling; Fu, Ling; Yu, Changming; Chen, Wei


    Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective. PMID:27626445

  6. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library

    Directory of Open Access Journals (Sweden)

    Han Wang


    Full Text Available Tetanus neurotoxin (TeNT produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective.




    Introduction Autoantibody-induced complement activation, which causes disruption of the postsynaptic membrane, is recognized as a key pathogenic factor in myasthenia gravis (MG). Therefore, specific targeting of complement inhibitors to the site of complement activation is a potential therapeutic strategy for treatment of MG. Methods We assessed expression of single-chain antibody fragment–decay accelerating factor (scFv-DAF), comprising a single-chain fragment scFv1956 based on the rat complement inhibitor DAF in prokaryotic systems, and studied its inhibitory effect on complement deposition in vitro. Results The recombinant conjugate scFv-DAF completely retained the wild-type binding activity of scFv1956 to AChR and inhibited complement activation of DAF in vitro. Conclusions We found that scFv-DAF could bind specifically to TE671 cells, and it is significantly more potent at inhibiting complement deposition than the untargeted parent molecule DAF. scFv-DAF may be a candidate for in vivo protection of the AChR in MG. PMID:22499093

  8. Dendritic cells transfected with scFv from Mab 7.B12 mimicking original antigen gp43 induces protection against experimental Paracoccidioidomycosis.

    Directory of Open Access Journals (Sweden)

    Karen S Ferreira

    Full Text Available Paracoccidioidomycosis (PCM, endemic in Latin America, is a progressive systemic mycosis caused by Paracoccidioides brasiliensis (P. brasiliensis, which primarily attacks lung tissue. Dendritic cells (DCs are able to initiate a response in naïve T cells, and they also participate in Th-cell education. Furthermore, these cells have been used for therapy in several disease models. Here we transfected DCs with a plasmid (pMAC/PS-scFv encoding a single chain variable fragment (scFv of an anti-Id antibody that is capable of mimicking gp43, the main antigenic component of P. brasiliensis. First, Balb/c mice were immunized subcutaneously with pMAC/PS-scFv and, after seven days, scFv protein was presented to the regional lymph nodes cells. Moreover, we showed that the DCs transfected with scFv were capable of efficiently activating proliferation of total lymph node cells and inducing a decrease in lung infection. Therefore, our results suggested that the use of scFv-transfected DCs may be a promising therapy in the paracoccidioidomycosis (PCM model.

  9. Dendritic cells transfected with scFv from Mab 7.B12 mimicking original antigen gp43 induces protection against experimental Paracoccidioidomycosis. (United States)

    Ferreira, Karen S; Maranhão, Andrea Q; Garcia, Maria C C; Brígido, Marcelo M; Santos, Suelen S; Lopes, José D; Almeida, Sandro R


    Paracoccidioidomycosis (PCM), endemic in Latin America, is a progressive systemic mycosis caused by Paracoccidioides brasiliensis (P. brasiliensis), which primarily attacks lung tissue. Dendritic cells (DCs) are able to initiate a response in naïve T cells, and they also participate in Th-cell education. Furthermore, these cells have been used for therapy in several disease models. Here we transfected DCs with a plasmid (pMAC/PS-scFv) encoding a single chain variable fragment (scFv) of an anti-Id antibody that is capable of mimicking gp43, the main antigenic component of P. brasiliensis. First, Balb/c mice were immunized subcutaneously with pMAC/PS-scFv and, after seven days, scFv protein was presented to the regional lymph nodes cells. Moreover, we showed that the DCs transfected with scFv were capable of efficiently activating proliferation of total lymph node cells and inducing a decrease in lung infection. Therefore, our results suggested that the use of scFv-transfected DCs may be a promising therapy in the paracoccidioidomycosis (PCM) model.

  10. Exceptionally Potent Anti-Tumor Bystander Activity of an scFv:sTRAIL Fusion Protein with Specificity for EGP2 Toward Target Antigen-Negative Tumor Cells

    Directory of Open Access Journals (Sweden)

    Edwin Bremer


    Full Text Available Previously, we reported on the target cell-restricted fratricide apoptotic activity of scFvC54:sTRAIL, a fusion protein comprising human-soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL genetically linked to the antibody fragment scFvC54 specific for the cell surface target antigen EGP2. In the present study, we report that the selective binding of scFvC54:sTRAIL to EGP2-positive target cells conveys an exceptionally potent pro-apoptotic effect toward neighboring tumor cells that are devoid of EGP2 expression (bystander cells. The anti-tumor bystander activity of scFvC54:sTRAIL was detectable at target-tobystander cell ratios as low as 1:100. Treatment in the presence of EGP2-blocking or TRAIL-neutralizing antibody strongly inhibited apoptosis in both target and bystander tumor cells. In the absence of target cells, bystander cell apoptosis induction was abrogated. The bystander apoptosis activity of scFvC54:sTRAIL did not require internalization, enzymatic conversion, diffusion, or communication (gap junctional intracellular communication between target and bystander cells. Furthermore, scFvC54:sTRAIL showed no detectable signs of innocent bystander activity toward freshly isolated blood cells. Further development of this new principle is warranted for approaches where cancer cells can escape from antibody-based therapy due to partial loss of target antigen expression.

  11. Synthesis and application of a N-1' fluorescent biotinyl derivative inducing the specific carboxy-terminal dual labeling of a novel RhoB-selective scFv. (United States)

    Chaisemartin, L; Chinestra, P; Favre, G; Blonski, C; Faye, J C


    The fluorescent site-specific labeling of protein would provide a new, easy-to-use alternative to biochemical and immunochemical methods. We used an intein-mediated strategy for covalent labeling of the carboxy-terminal amino acid of a RhoB-selective scFv previously isolated from a phage display library (a human synthetic V(H) + V(L) scFv phage library). The scFv fused to the Mxe intein was produced in E. coli and purified and was then labeled with a newly synthesized fluorescent biotinyl cysteine derivative capable of inducing scFv-Mxe intein splicing. In this study, we investigated the splicing and labeling properties of various amino acids in the hinge domain between scFv and Mxe under thiol activation. In this dual labeling system, the fluorescein is used for antibody detection and biotin is used for purification, resulting in a high specific activity for fluorescence. We then checked that the purified biotinylated fluorescent scFv retained its selectivity for RhoB without modification of its affinity.

  12. Inhibitory effect of pIRES-scFvCD44-TRAIL on breast cancer cells%pIRES-scFvCD 44-TRAIL 对人乳腺癌细胞株抑制作用的研究

    Institute of Scientific and Technical Information of China (English)

    魏杨辉; 黄耀; 张卫星


    目的:探讨CD44单链抗体scFvCD44、肿瘤坏死因子相关凋亡诱导配体( TRAIL)双基因共表达真核载体(pIRES-scFvCD44-TRAIL)对人乳腺癌MDA-MB-231细胞株的抑制作用。方法①细胞实验:采用RT-PCR二步法构建pIRES-scFvCD44-TRAIL并转染MDA-MB-231细胞(转染组);设转染pIRES空质粒的空白组及不作任何处理的阴性组。转染24 h后采用流式细胞仪检测各组细胞凋亡情况,计算细胞凋亡率;行划痕试验比较各组细胞迁移情况,计算划痕愈合率。②移植瘤实验:建立乳腺癌荷瘤裸鼠模型8只并随机分为实验组及对照组。实验组皮下注射pIRES-scFvCD44-TRAIL 100μL/只,隔日1次,共5次;对照组注射等量生理盐水。观察两组移植瘤生长情况,计算抑瘤率;采用免疫组化法检测两组瘤组织中Ki67的表达水平。结果①细胞实验:转染组细胞凋亡率明显高于、划痕愈合率明显低于空白组和阴性组。②移植瘤实验:观察组抑瘤率为79.8%;观察组Ki67的阳性细胞率明显低于对照组,P<0.05。结论 pIRES-scFvCD44-TRAIL可抑制人乳腺癌细胞及其移植肿瘤的生长及转移。%Objective To investigate the inhibitory effects of CD 44 single-chain antibody scFvCD 44 and tumor necrosis factor-related apoptosis inducing ligand eukaryotic expression vector ( pIRES-scFvCD44-TRAIL ) on breast cancer cells MDA-MB-231.Methods ①Cellexperiments:TheplasmidDNAofpIRES-scFvCD44-TRAIL,whosegeneswereampli-fied by reverse transcriptase-polymerase chain reaction ( RT-PCR) , was transfected into breast cancer cells MDA-MB-231 ( the transfected group ) .The blank group was transfected with the blank pIRES plasmid and the negative group was not treated.After 24 hours, the apoptosis rate was calculated by flow cytometry .The scratch test was performed to observe the cell motility assay as well as to calculate the scratch healing rate .② Graft model experiments

  13. Preventive DNA vaccination against CEA-expressing tumors with anti-idiotypic scFv6.C4 DNA in CEA-expressing transgenic mice. (United States)

    Denapoli, Priscila M A; Zanetti, Bianca F; Dos Santos, Adara A; de Moraes, Jane Z; Han, Sang W


    Carcinoembryonic antigen (CEA) is expressed during embryonic life and in low level during adult life. Consequently, the CEA is recognized by the immune system as a self-antigen and thus CEA-expressing tumors are tolerated. Previously, we constructed a single chain variable fragment using the 6.C4 (scFv6.C4) hybridoma cell line, which gave rise to antibodies able to recognize CEA when C57/Bl6 mice were immunized. Here, the scFv6.C4 ability to prevent the CEA-expressing tumor growth was assessed in CEA-expressing transgenic mice CEA2682. CEA2682 mice immunized with the scFv6.C4 expressing plasmid vector (uP/PS-scFv6.C4) by electroporation gave rise to the CEA-specific AB3 antibody after the third immunization. Sera from immunized mice reacted with CEA-expressing human colorectal cell lines CO112, HCT-8, and LISP-1, as well as with murine melanoma B16F10 cells expressing CEA (B16F10-CEA). Cytotoxic T lymphocytes (CTL) from uP/PS-scFv6.C4 immunized mice lysed B16F10-CEA (56.7%) and B16F10 expressing scFv6.C4 (B16F10-scFv6.C4) (46.7%) cells, against CTL from uP-immunized mice (10%). After the last immunization, 5 × 10(5) B16F10-CEA cells were injected into the left flank. All mice immunized with the uP empty vector died within 40 days, but uP/PS-scFv6.C4 vaccinated mice (40%) remained free of tumor for more than 100 days. Splenocytes obtained from uP/PS-scFv6.C4 vaccinated mice showed higher T-cell proliferative activity than those from uP vaccinated mice. Collectively, DNA vaccination with the uP-PS/scFv6.C4 plasmid vector was able to give rise to specific humoral and cellular responses, which were sufficient to retard growth and/or eliminate the injected B16F10-CEA cells.

  14. Diagnostic potential of recombinant scFv antibodies generated against hemagglutinin protein of influenza A virus

    Directory of Open Access Journals (Sweden)

    Roopali eRajput


    Full Text Available Human influenza A viruses have been the cause of enormous socio-economic losses worldwide. In order to combat such a notorious pathogen, hemagglutinin protein (HA has been a preferred target for generation of neutralizing-antibodies, as potent therapeutic/ diagnostic agents. In the present study, recombinant anti-HA single chain variable fragment (scFv antibodies were constructed using the phage display technology to aid in diagnosis and treatment of human influenza A virus infections. Spleen cells of mice hyper-immunized with A/New Caledonia/20/99 (H1N1 virus were used as the source for recombinant antibody (rAb production. The antigen-binding phages were quantified after 6 rounds of bio-panning against A/New Caledonia/20/99 (H1N1, A/California/07/2009 (H1N1-like, or A/Udorn/307/72(H3N2 viruses. The phage yield was maximum for the A/New Caledonia/20/99 (H1N1, however, considerable cross-reactivity was observed for the other virus strains as well. The HA-specific polyclonal rAb preparation was subjected to selection of single clones for identification of high reactive relatively conserved epitopes. The high affinity rAbs were tested against certain known conserved HA epitopes by peptide ELISA. Three recombinant mAbs showed reactivity with both the H1N1 strains and one (C5 showed binding with all the three viral strains. The C5 antibody was thus used for development of an ELISA test for diagnosis of influenza virus infection. Based on the sample size in the current analysis, the ELISA test demonstrated 83.9% sensitivity and 100% specificity. Thus, the ELISA, developed in our study, may prove as a cheaper alternative to the presently used real time RT-PCR test for detection of human influenza A viruses in clinical specimens, which will be beneficial, especially in the developing countries. Since, the two antibodies identified in this study are reactive to conserved HA epitopes; these may prove as potential therapeutic agents as well.

  15. Generation and characterization of a human single-chain fragment variable (scFv antibody against cytosine deaminase from Yeast

    Directory of Open Access Journals (Sweden)

    Tombesi Marina


    Full Text Available Abstract Background The ability of cytosine deaminase (CD to convert the antifungal agent 5-fluorocytosine (5-FC into one of the most potent and largely used anticancer compound such as 5-fluorouracil (5-FU raised considerable interest in this enzyme to model gene or antibody – directed enzyme-prodrug therapy (GDEPT/ADEPT aiming to improve the therapeutic ratio (benefit versus toxic side-effects of cancer chemotherapy. The selection and characterization of a human monoclonal antibody in single chain fragment (scFv format represents a powerful reagent to allow in in vitro and in vivo detection of CD expression in GDEPT/ADEPT studies. Results An enzymatic active recombinant CD from yeast (yCD was expressed in E. coli system and used as antigen for biopanning approach of the large semi-synthetic ETH-2 antibody phage library. Several scFvs were isolated and specificity towards yCD was confirmed by Western blot and ELISA. Further, biochemical and functional investigations demonstrated that the binding of specific scFv with yCD did not interfere with the activity of the enzyme in converting 5-FC into 5-FU. Conclusion The construction of libraries of recombinant antibody fragments that are displayed on the surface of filamentous phage, and the selection of phage antibodies against target antigens, have become an important biotechnological tool in generating new monoclonal antibodies for research and clinical applications. The scFvH5 generated by this method is the first human antibody which is able to detect yCD in routinary laboratory techniques without interfering with its enzymatic function.

  16. Whole-body imaging of HER2/neu-overexpressing tumors using scFv-antibody conjugated quantum dots (United States)

    Balalaeva, Irina V.; Zdobnova, Tatiana A.; Brilkina, Anna A.; Krutova, Irina M.; Stremovskiy, Oleg A.; Lebedenko, Elena N.; Vodeneev, Vladimir V.; Turchin, Ilya V.; Deyev, Sergey M.


    Semiconductor quantum dots (QDs) are widely used in different fields of bioscience and biotechnology due to their unique optical properties. QDs can be used as fluorescent markers for optical detection and monitoring of deeply located tumors in vivo after specific labeling achieved by conjugating of QDs with targeting molecules. In this work the possibilities of intravital tumor labeling with QDs and subsequent in vivo tumor imaging were estimated. The experiments were run on immunodeficient nu/nu mice bearing human breast carcinoma SKBR-3, overexpressing surface protein HER2/neu. We used quantum dots Qdot 705 ITK (Invitrogen, USA) linked to anti-HER2/neu 4D5 scFv antibody. Antibody scFv fragments as a targeting agent for directed delivery of fluorophores possess significant advantages over full-size antibodies due to their small size, lower cross-reactivity and immunogenicity. QDs were bound to 4D5 scFv by barnase-barstar system (bn-bst) analogous to the streptavidin-biotidin system. Whole-body images were obtained using diffuse fluorescence tomography (DFT) setup with low-frequency modulation and transilluminative configuration of scanning, created at the Institute of Applied Physics of RAS, Russia). DFT-results were confirmed ex vivo by confocal microscopy. We report the results of in vivo whole-body tumor imaging with QDs complexes as contrasting agents. Intravital images of QDs-labeled tumors were obtained using specific tumor cells targeting and fluorescence transilluminative imaging method, while "passive" QD-labeling failed to mark effectively the tumor.

  17. The effects of N-terminal insertion into VSV-G of an scFv peptide

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    Piechaczyk Marc


    Full Text Available Abstract Recombinant retroviruses, including lentiviruses, are the most widely used vectors for both in vitro and in vivo stable gene transfer. However, the inability to selectively deliver transgenes into cells of interest limits the use of this technology. Due to its wide tropism, stability and ability to pseudotype a range of viral vectors, vesicular stomatitis virus G protein (VSV-G is the most commonly used pseudotyping protein. Here, we attempted to engineer this protein for targeting purposes. Chimaeric VSV-G proteins were constructed by linking a cell-directing single-chain antibody (scFv to its N-terminal. We show that the chimaeric VSV-G molecules can integrate into retroviral and lentiviral particles. HIV-1 particles pseudotyped with VSV-G linked to an scFv against human Major Histocompatibility Complex class I (MHC-I bind strongly and specifically to human cells. Also, this novel molecule preferentially drives lentiviral transduction of human cells, although the titre is considerably lower that viruses pseudotyped with VSV-G. This is likely due to the inefficient fusion activity of the modified protein. To our knowledge, this is the first report where VSV-G was successfully engineered to include a large (253 amino acids exogenous peptide and where attempts were made to change the infection profile of VSV-G pseudotyped vectors.

  18. Binding Activity Difference of Anti-CD20 scFv-Fc Fusion Protein Derived from Variable Domain Exchange

    Institute of Scientific and Technical Information of China (English)

    Shusheng Geng; Beifen Shen; Jiannan Feng; Yan Li; Yingxun Sun; Xin Gu; Ying Huang; Yugang Wang; Xianjiang Kang; Hong Chang


    Two novel engineered antibody fragments binding to antigen CD20 were generated by fusing a murine IgM-type anti-CD20 single-chain Fv fragment (scFv) to the human IgG1 CH2 (I.e., Cγ2) and CH3 (I.e., Cγ3) domains with the human IgG1 hinge (I.e. Hγ). Given the relationship between structure and function of protein, the 3-D structures of the two engineered antibody fragments were modeled using computer-aided homology modeling method.Furthermore, the relationship between 3-D conformation and their binding activity was evaluated theoretically.Due to the change of active pocket formed by CDRs, the HL23 (VH-Linker-VL-Hγ-Cγ2-Cγ3) remained its activity because of its preserved conformation, while the binding activity of the LH23 (VL-Linker-VH-Hγ-Cγ2-Cγ3) was impaired severely. Experimental studies by flow cytometry and fluorescence microscopy showed that HL23 possessed significantly superior binding activity to CD20-expressing target cells than LH23. That is to say, the order of variable regions could influence the binding activity of the fusion protein to CD20+ cell lines, which was in accordance with the theoretical results. The study highlights the potential relationship between the antibody binding activity and their 3-D conformation, which appears to be worthwhile in providing direction for future antibody design of recombinant antibody.

  19. Re-engineering of the PAM1 phage display monoclonal antibody to produce a soluble, versatile anti-homogalacturonan scFv

    DEFF Research Database (Denmark)

    Manfield, I. W.; Bernal Giraldo, Adriana Jimena; Møller, I.


    Antibody phage display is an increasingly important alternative method for the production of monoclonal antibodies (mAbs) and involves the expression of antibody fragments (scFvs) at the surface of bacteriophage particles. We have previously used this technique to generate a phage mAb (PAM1phage...... of the PAM1 mAb, we describe here the production of a phage-free, soluble scFv version of the PAM1 mAb (PAM1scFv). Using the new PAM1scFv probe, the occurrence of the HG epitope recognized can now be localized with high resolution within micro-domains of plant cell walls....

  20. Optimisation of production of a domoic acid-binding scFv antibody fragment in Escherichia coli using molecular chaperones and functional immobilisation on a mesoporous silicate support. (United States)

    Hu, Xuejun; O'Hara, Liam; White, Simon; Magner, Edmond; Kane, Marian; Wall, J Gerard


    Domoic acid is a potent neurotoxin that can lead to amnesic shellfish poisoning in humans through ingestion of contaminated shellfish. We have produced and purified an anti-domoic acid single-chain Fragment variable (scFv) antibody fragment from the Escherichia coli periplasm. Yields of functional protein were increased by up to 100-fold upon co-production of E. coli DnaKJE molecular chaperones but co-overproduction of GroESL led to a reduction in solubility of the scFv. Co-production of the peptidyl-prolyl isomerase trigger factor resulted in accumulation of unprocessed scFv in the E. coli cytoplasm. This was due to an apparent bottleneck in translocation of the cytoplasmic membrane by the recombinant polypeptide. Co-expression of the E. coli disulfide bond isomerase dsbC increased scFv yields by delaying lysis of the host bacterial cells though this effect was not synergistic with molecular chaperone co-production. Meanwhile, use of a cold-shock promoter for protein production led to accumulation of greater amounts of scFv polypeptide which was predominantly in insoluble form and could not be rescued by chaperones. Purification of the scFv was achieved using an optimised metal affinity chromatography procedure and the purified protein bound domoic acid when immobilised on a mesoporous silicate support. The work outlines the potential benefit of applying a molecular chaperone/folding catalyst screening approach to improve antibody fragment production for applications such as sensor development.

  1. Preparation of EGFRVⅢ specific-chain Fv and its targeting activity%EGFRvⅢ特异性单链抗体的制备及其靶向性能

    Institute of Scientific and Technical Information of China (English)

    张庆丽; 石必枝; 蒋华; 周敏; 王海; 孔娟; 谢海龙


    Objective: To screen for EGFRvⅢ specific single-chain Fv (scFv) by phage display library and to examine its targeting activity. Methods: EGFRv Ⅲ specific scFv phage library was constructed, and the positive EGFRv Ⅲ-scFv clone was screened by ELISA. After cloned into pCANTAB-Thrombin-His vector, EGFRv Ⅲ-scFv plasmid was transformed into E. coli HB2151, and soluble EGFRv Ⅲ-scFv was induced by IPTG. The specific binding activity of EGFRv Ⅲ-scFv with EGFRv Ⅲ was studied by indirect immunofluorescence and in vivo imaging. Results: An EGFRv Ⅲ-scFv phage library was successfully constructed and 16 EGFRv Ⅲ-scFv positive clones were identified by ELISA. One clone named EGFRv Ⅲ-scFv-2A1 was re-cloned into pCANTAB-Thrombin-His vector and soluble EGFRv Ⅲ-scFv-2Al was successfully obtained. EGFRv Ⅲ-scFv-2A1 could specifically bind with HuH7-EGFRv Ⅲ and HuH7 hepatoma cells, but not with HuH7-EGFR and HuH7 cells in vitro. In vivo, fluorescence-labeled EGFRv Ⅲ-scFv-2A1 could only bind with U87MG-EGFRv Ⅲ glioma cells implanted tumor tissues, but not with that of U87MG cells implanted ones. Conclusion: The prepared EGFRv Ⅲ-scFv-2A1 can specifically bind with EGFRv Ⅲ, and it might be used for diagnosis and targeted therapy of tumors.%目的:应用噬菌体展示技术筛选出能与EGFRvⅢ特异性结合的单链抗体(single-chain Fv,scFv),并研究其靶向性能.方法:构建EGFRvⅢ特异性scFv噬菌体库,ELISA筛选阳性克隆,阳性EGFRvⅢ-scFv质粒重新克隆人pCANTAB-Throm-bin-His载体,转化E.coli HB2151,IPTG诱导可溶性EGFRvⅢ-scFv表达.间接免疫荧光及裸鼠活体成像技术鉴定EGFRv Ⅲ-scFv与EGFRvⅢ的特异性结合.结果:成功构建了EGFRvⅢ-scFv噬菌体库,ELISA筛选得到16个EGFRvⅢ-scFv克隆,取一克隆命名为EGFRvⅢ-scFv-2Al.EGFRvⅢ-scFv-2Al质粒重新克隆人pCANTAB-Thrombin-His载体,成功表达可溶性EGFRvⅢ-scFv-2Al.EGFRvⅢ-seFv-2Al在体外可特异性结合HuH7-EGFRvⅢ

  2. 轮枝镰孢菌FvST12基因敲除载体的构建及功能分析%Gene Knockout Vector Construction and Function Assay of FvST12 in Fusarium verticillioides

    Institute of Scientific and Technical Information of China (English)

    张岳平; 瞿华香


    [目的]鉴定并分析轮枝镰孢菌中 STE12 类转录因子的基因功能.[方法]以尖孢镰孢菌转录因子Fost12蛋白为靶序列,在轮枝镰孢菌基因组数据库中进行BlastP搜索,发现一个与Fost12蛋白同源性高达98%的基因,命名为FvST12.基于Double-Joint PCR技术,构建该基因的敲除载体,通过真菌原生质体转化及筛选获得基因敲除突变体,并对突变体的表型及致病性进行系统分析,以明确该基因的功能.[结果l突变体在生长速率、菌落形态,产孢量以及高渗和氧化等逆境胁迫反应上与野生型无显著差异;致病性分析表明,突变体致病力明显下降.[结论]轮枝镰孢菌FvST12对致病性起重要调控作用,但不参与营养生长、产孢和高渗和氧化胁迫等逆境反应.%[Objective]The objective of this study is to identify and analyze the STE12 homolog transcription factor gene in Fusarium verticillioides.[Method]The F.oxysporum Fostl2 transcription factor was used as a target sequence to search its homolog by BlastP in F.verticillioides genome sequence.FVEG_05267.3, a predicted gene in F.verticillioides, was identified and it showed 98% similarity to Fostl2.It was called FvST12 in this study.Based on the Double-Joint PCR, the knockout vector was constructed and null knockout mutants were generated through fungal protoplast transformation.The gene knockout mutants were confirmed by PCR and Southern blot.Furthermore, the function of the gene in F.vertieillioides was systematically characterized [Result]Compared with the wild type, Fvstl2 mutant has normal growth rate, condiation, colony morphology, and stress responses (osmotic and oxidative stress) tested in this study.However, the mutant reduced the virulence on maize ear and stalk.[Conclusion]The transcription factor FvST12 is an important virulence factor but it isn't involved in the vegetative growth, conidiation, osmotic and oxidative stress responses in F.verticillioides.

  3. Studies on soil to grass transfer factor (Fv) and grass to milk transfer coefficient (Fm) for cesium in Kaiga region. (United States)

    Karunakara, N; Ujwal, P; Yashodhara, I; Rao, Chetan; Sudeep Kumara, K; Dileep, B N; Ravi, P M


    Detailed studies were carried out to establish site-specific soil to grass transfer factors (Fv) and grass to cow milk transfer coefficients (Fm) for radioactive cesium ((137)Cs) and stable cesium (Cs) for Kaiga region, where a nuclear power station has been in operation for more than 10 years. The study included adopted cows, cows of local farmers, and cows from the dairy farm. A grass field was developed specifically for the study and 2 local breed cows were adopted and allowed to graze in this grass field. The soil and grass samples were collected regularly from this field and analyzed for the concentrations of (137)Cs and stable Cs to evaluate the soil to grass Fv values. The milk samples from the adopted cows were analyzed for the (137)Cs and stable Cs concentrations to evaluate Fm values. For comparison, studies were also carried out in dominant grazing areas in different villages around the nuclear power plant and the cows of local farmers which graze in these areas were identified and milk samples were collected and analyzed regularly. The geometric mean values of Fv were found to be 1.1 × 10(-1) and 1.8 × 10(-1) for (137)Cs and stable Cs, respectively. The Fm of (137)Cs had geometric mean values of 1.9 × 10(-2) d L(-1) and 4.6 × 10(-2) d L(-1), respectively, for adopted Cows 1 and 2; 1.7 × 10(-2) d L(-1) for the cows of local farmers, and 4.0 × 10(-3) d L(-1) for the dairy farm cows. The geometric mean values of Fm for stable Cs were similar to those of (137)Cs. The Fm value for the dairy farm cows was an order of magnitude lower than those for local breed cows. The Fm values observed for the local breed cows were also an order of magnitude higher when compared to the many values reported in the literature and in the IAEA publication. Possible reasons for this higher Fm values were identified. The correlation between Fv and Fm values for (137)Cs and stable Cs and their dependence on the potassium content ((40)K and stable K) in

  4. Remote monitoring of solar PV system for rural areas using GSM, V-F & F-V converters (United States)

    Tejwani, R.; Kumar, G.; Solanki, C. S.


    The Small capacity photovoltaic (PV) systems like solar lantern and home lighting systems installed in remote rural area often fail without any prior warning due to lack of monitoring and maintenance. This paper describes implementation of remote monitoring for small capacity solar PV system that uses GSM voice channel for communication. Through GSM analog signal of sine wave with frequency range 300-3500 Hz and amplitude range 2.5-4 V is transmitted. Receiver is designed to work in the same frequency range. The voltage from solar PV system in range of 2 to 7.5 V can be converted to frequency directly at the transmitting end. The frequency range from 300-6000 Hz can be sensed and directly converted to voltage signal at receiving end. Testing of transmission and reception of analog signal through GSM voice channel is done for voltage to frequency (V-F) and frequency to voltage (F-V) conversions.

  5. 重组免疫毒素scFv-psm-ETA对人前列腺癌细胞生长抑制作用的研究%Construction of scFv-psm-ETA recombinant immunotoxin and its antitumor effect on human prostate cancer cells in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    陈凌武; 王晓波; 陈炜; 徐鸿绪; 梅骅


    目的探讨重组免疫毒素scFv-psm-ETA对人前列腺癌细胞生长的抑制作用. 方法制备具有良好器官特异性的免疫毒素scFv-psm-ETA,从抗前列腺特异膜抗原(PSM)单抗杂交瘤细胞中克隆抗PSM单抗重、轻链可变区基因,与切去细胞结合区的ETA基因相连,构建成表达scFv-psm-ETA的质粒pSW200-psm,转化E.coli株CC118,表达有生物活性的融合蛋白scFv-psm-ETA,经M1 FLAG柱纯化,检测其体外对前列腺癌细胞的细胞毒杀伤活性和在荷瘤裸鼠体内的抑瘤作用. 结果制备的 scFv-psm-ETA免疫毒素能特异性地与PSM高表达的LNCaP细胞结合,在浓度为100 ng/ml时对80%的LNCaP细胞有体外细胞毒杀伤作用;荷LNCaP前列腺癌裸鼠治疗组及对照组肿瘤大小分别为153 mm3及272 mm3(P<0.01),显示scFv-psm-ETA对荷瘤裸鼠有抑制肿瘤生长作用. 结论重组免疫毒素scFv-psm-ETA对PSM高表达的前列腺癌细胞LNCaP有体外细胞毒杀伤及体内抑瘤作用.

  6. Exceptionally potent anti-tumor bystander activity of an scFv : sTRAIL fusion protein with specificity for EGP2 toward target antigen-negative tumor cells

    NARCIS (Netherlands)

    Bremer, E; Samplonius, D; Kroesen, BJ; van Genne, L; de Leij, L; Helfrich, W


    Previously, we reported on the target cell-restricted fratricide apoptotic activity of scFvC54:sTRAIL, a fusion protein comprising human-soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) genetically linked to the antibody fragment scFvC54 specific for the cell surface target

  7. Evaluation of a high throughput method for the detection of mutations associated with thrombosis and hereditary hemochromatosis in Brazilian blood donors.

    Directory of Open Access Journals (Sweden)

    Vivian Dionisio Tavares Niewiadonski

    Full Text Available The aim of this study was to evaluate the OpenArray platform for genetic testing of blood donors and to assess the genotype frequencies of nucleotide-polymorphisms (SNPs associated with venous thrombosis (G1691A and G20210A, hyperhomocysteinemia (C677T, A1298C, and hereditary hemochromatosis (C282Y, H63D and S65C in blood donors from Sao Paulo, Brazil.We examined 400 blood donor samples collected from October to November 2011. The SNPs were detected using OpenArray technology. The blood samples were also examined using a real-time PCR-FRET system to compare the results and determine the accuracy of the OpenArray method.We observed 100% agreement in all assays tested, except HFE C282Y, which showed 99.75% agreement. The HFE C282Y assay was further confirmed through direct sequencing, and the results showed that OpenArray analysis was accurate. The calculated frequencies of each SNP were FV G1691A 98.8% (G/G, 1.2% (G/A; FII G2021A 99.5% (G/G, 0.5% (G/A; MTHFR C677T 45.5% (C/C, 44.8% (C/T, 9.8% (T/T; MTHFR A1298C 60.3% (A/A, 33.6% (A/C, 6.1% (C/C; HFE C282Y 96%(G/G, 4%(G/A, HFE H63D 78.1%(C/C, 20.3% (C/G, 1.6% (G/G; and HFE S65C 98.1% (A/A, 1.9% (A/T.Taken together, these results describe the frequencies of SNPs associated with diseases and are important to enhance our current knowledge of the genetic profiles of Brazilian blood donors, although a larger study is needed for a more accurate determination of the frequency of the alleles. Furthermore, the OpenArray platform showed a high concordance rate with standard FRET RT-PCR.

  8. The change of the scFv into the Fab format improves the stability and in vivo toxin neutralization capacity of recombinant antibodies. (United States)

    Quintero-Hernández, Veronica; Juárez-González, Victor R; Ortíz-León, Mauricio; Sánchez, Rosalba; Possani, Lourival D; Becerril, Baltazar


    The antigen-binding fragment (Fab) has been considered a more functionally stable version of recombinant antibodies than single chain antibody fragments (scFvs), however this intuitive consideration has not been sufficiently proven in vivo. This communication shows that three out of four specific scFvs against a scorpion toxin, with different affinities and stabilities, become neutralizing in vivo when expressed as Fabs, despite the fact that they are not neutralizing in the scFv format. A scFv fragment previously obtained from a neutralizing mouse antibody (BCF2) was used to produce three derived scFvs by directed evolution. Only one of them was neutralizing, however when expressed as Fab, all of them became neutralizing fragments in vivo. The initial scFvBCF2 (earlier used for directed evolution) was not neutralizing in the scFv format. After expressing it as Fab did not become a neutralizing fragment, but did reduce the intoxication symptoms of experimental mice. The stability of the four Fabs derived from their respective scFvs was improved when tested in the presence of guanidinium chloride. The in vitro stability of the Fab format has been shown earlier, but the physiological consequences of this stability are shown in this communication. The present results indicate that improved functional stability conferred by the Fab format can replace additional maturation steps, when the affinity and stability are close to the minimum necessary to be neutralizing.

  9. Affinity Purification of Tumor Necrosis Factor-α Expressed in Raji Cells by Produced scFv Antibody Coupled CNBr-Activated Sepharose

    Directory of Open Access Journals (Sweden)

    Safar Farajnia


    Full Text Available Purpose: Recombinant tumor necrosis factor-alpha (TNF-α has been utilized as an antineoplastic agent for the treatment of patients with melanoma and sarcoma. It targets tumor cell antigens by impressing tumor-associated vessels. Protein purification with affinity chromatography has been widely used in the downstream processing of pharmaceutical-grade proteins. Methods: In this study, we examined the potential of our produced anti-TNF-scFv fragments for purification of TNF-α produced by Raji cells. he Raji cells were induced by lipopolysaccharides (LPS to express TNF-α. Western blotting and Fluorescence-activated cell sorting (FACS flow cytometry analyses were used to evaluate the TNF-α expression. The anti-TNF-α scFv selected from antibody phage display library was coupled to CNBr-activated sepharose 4B beads used for affinity purification of expressed TNF-α and the purity of the protein was assessed by SDS-PAGE. Results: Western blot and FACS flow cytometry analyses showed the successful expression of TNF-α with Raji cells. SDS-PAGE analysis showed the performance of scFv for purification of TNF-α protein with purity over 95%. Conclusion: These findings confirm not only the potential of the produced scFv antibody fragments but also this highly pure recombinant TNF-α protein can be applied for various in vitro and in vivo applications.

  10. Crystal structures of mono- and bi-specific diabodies and reduction of their structural flexibility by introduction of disulfide bridges at the Fv interface (United States)

    Kim, Jin Hong; Song, Dong Hyun; Youn, Suk-Jun; Kim, Ji Won; Cho, Geunyoung; Kim, Sun Chang; Lee, Hayyoung; Jin, Mi Sun; Lee, Jie-Oh


    Building a sophisticated protein nano-assembly requires a method for linking protein components in a predictable and stable structure. Diabodies are engineered antibody fragments that are composed of two Fv domains connected by short peptide linkers. They are attractive candidates for mediators in assembling protein nano-structures because they can simultaneously bind to two different proteins and are rigid enough to be crystallized. However, comparison of previous crystal structures demonstrates that there is substantial structural diversity in the Fv interface region of diabodies and, therefore, reliable prediction of its structure is not trivial. Here, we present the crystal structures of ten mono- and bi-specific diabodies. We found that changing an arginine residue in the Fv interface to threonine greatly reduced the structural diversity of diabodies. We also found that one of the bispecific diabodies underwent an unexpected process of chain swapping yielding a non-functional monospecific diabody. In order to further reduce structural flexibility and prevent chain shuffling, we introduced disulfide bridges in the Fv interface regions. The disulfide-bridged diabodies have rigid and predictable structures and may have applications in crystallizing proteins, analyzing cryo-electron microscopic images and building protein nano-assemblies. PMID:27682821

  11. Overproduction of anti-Tn antibody MLS128 single-chain Fv fragment in Escherichia coli cytoplasm using a novel pCold-PDI vector. (United States)

    Subedi, Ganesh P; Satoh, Tadashi; Hanashima, Shinya; Ikeda, Akemi; Nakada, Hiroshi; Sato, Reiko; Mizuno, Mamoru; Yuasa, Noriyuki; Fujita-Yamaguchi, Yoko; Yamaguchi, Yoshiki


    Overproduction of recombinant proteins in Escherichia coli is often hampered by their failure to fold correctly, leading to their accumulation within inclusion bodies. To overcome the problem, a variety of techniques aimed at soluble expression have been developed including low temperature expression and/or fusion of soluble tags and chaperones. However, a general protocol for bacterial expression of disulfide bond-containing proteins has hitherto not been established. Single chain Fv fragments (scFvs) are disulfide bond-containing proteins often difficult to express in soluble forms in E. coli. We here examine in detail the E. coli expression of a scFv originating from an anti-carbohydrate MLS128 antibody as a model system. We combine three techniques: (1) tagging scFv with thioredoxin, DsbC and protein disulfide isomerase (PDI), (2) expressing the proteins at low temperature using the pCold vector system, and (3) using Origami E. coli strains with mutations in the thioredoxin reductase and glutathione reductase genes. We observed a high expression level of soluble MLS128-scFv in the Origami strain only when PDI is used as a tag. The recombinant protein retains full binding activity towards synthetic carbohydrate antigens. The developed "pCold-PDI" vector has potential for overproduction of other scFvs and disulfide-containing proteins in the Origami strains.

  12. FvBck1, a Component of Cell Wall Integrity MAP Kinase Pathway, is Required for Virulence and Oxidative Stress Response in Sugarcane Pokkah Boeng Pathogen

    Directory of Open Access Journals (Sweden)

    Chengkang eZhang


    Full Text Available Fusarium verticillioides (formerly F. moniliforme is suggested as one of the causal agents of Pokkah Boeng, a serious disease of sugarcane worldwide. Currently, detailed molecular and physiological mechanism of pathogenesis is unknown. In this study, we focused on cell wall integrity MAPK pathway as one of the potential signaling mechanisms associated with Pokkah Boeng pathogenesis. We identified FvBCK1 gene that encodes a MAP kinase kinase kinase homolog and determined that it is not only required for growth, micro- and macro-conidia production, and cell wall integrity but also for response to osmotic and oxidative stresses. The deletion of FvBCK1 caused a significant reduction in virulence and FB1 production, a carcinogenic mycotoxin produced by the fungus. Moreover, we found the expression levels of three genes, which are known to be involved in superoxide scavenging, were down regulated in the mutant. We hypothesized that the loss of superoxide scavenging capacity was one of the reasons for reduced virulence, but overexpression of catalase or peroxidase gene failed to restore the virulence defect in the deletion mutant. When we introduced Magnaporthe oryzae MCK1 into the FvBck1 deletion mutant, while certain phenotypes were restored, the complemented strain failed to gain full virulence. In summary, FvBck1 plays a diverse role in F. verticillioides, and detailed investigation of downstream signaling pathways will lead to a better understanding of how this MAPK pathway regulates Pokkah Boeng on sugarcane.

  13. Cloning and expression in Escherichia coli of a human gelatinase B-inhibitory single-chain immunoglobulin variable fragment (scFv). (United States)

    Zhou, N; Paemen, L; Opdenakker, G; Froyen, G


    The murine monoclonal antibody REGA-3G12 selectively and specifically inhibits the activity of human gelatinase B. The cDNA fragments which encode the variable regions of the light and heavy chains were isolated by PCR-mediated cloning and sequenced. Single-chain Fv expression constructs for Escherichia coli were generated in which c-myc tag sequences were encoded. Inducible expression of the scFv and secretion to the periplasm were obtained with higher yields when the c-myc tag sequence was positioned at the amino-terminal side. The inhibitory activity of purified scFv on neutrophil gelatinase B was tested in a gelatin degradation assay and it was found to possess a similar specific activity as that of the intact monoclonal antibody and of the pepsin-clipped F(ab')2 derivative. This shows for the first time that inhibition of soluble enzymes with scFv is possible and opens new perspectives for the treatment of diseases with excessive and detrimental enzyme production in the host.

  14. Wheat cultivars selected for high Fv/Fm under heat stress maintain high photosynthesis, total chlorophyll, stomatal conductance, transpiration and dry matter

    DEFF Research Database (Denmark)

    Sharma, Dew Kumari; Andersen, Sven Bode; Ottosen, Carl-Otto


    ) than the low group, accompanied by higher stomatal conductance (gs), transpiration rate (E) and evaporative cooling of the leaf (ΔT). The difference in PN between the groups was not caused by differences in PSII capacity or gs as the variation in Fv/Fm and intracellular CO2 (Ci) was non...

  15. Evaluation of anti-HER2 scFv-conjugated PLGA-PEG nanoparticles on 3D tumor spheroids of BT474 and HCT116 cancer cells (United States)

    Thuy Duong Le, Thi; Pham, Thu Hong; Nghia Nguyen, Trong; Giang Ngo, Thi Hong; Nhung Hoang, Thi My; Huan Le, Quang


    Three-dimensional culture cells (spheroids) are one of the multicellular culture models that can be applied to anticancer chemotherapeutic development. Multicellular spheroids more closely mimic in vivo tumor-like patterns of physiologic environment and morphology. In previous research, we designed docetaxel-loaded pegylated poly(D, L-lactide-co-glycolide) nanoparticles conjugated with anti-HER2 single chain antibodies (scFv-Doc-PLGA-PEG) and evaluated them in 2D cell culture. In this study, we continuously evaluate the cellular uptake and cytotoxic effect of scFv-Doc-PLGA-PEG on a 3D tumor spheroid model of BT474 (HER2-overexpressing) and HCT116 (HER2-underexpressing) cancer cells. The results showed that the nanoparticle formulation conjugated with scFv had a significant internalization effect on the spheroids of HER2-overexpressing cancer cells as compared to the spheroids of HER2-underexpressing cancer cells. Therefore, cytotoxic effects of targeted nanoparticles decreased the size and increased necrotic score of HER2-overexpressing tumor spheroids. Thus, these scFv-Doc-PLGA-PEG nanoparticles have potential for active targeting for HER2-overexpressing cancer therapy. In addition, BT474 and HCT116 spheroids can be used as a tumor model for evaluation of targeting therapies.

  16. Generation of anti-idiotype scFv for pharmacokinetic measurement in lymphoma patients treated with chimera anti-CD22 antibody SM03.

    Directory of Open Access Journals (Sweden)

    Qi Zhao

    Full Text Available Pre-clinical and clinical studies of therapeutic antibodies require highly specific reagents to examine their immune responses, bio-distributions, immunogenicity, and pharmacodynamics in patients. Selective antigen-mimicking anti-idiotype antibody facilitates the assessment of therapeutic antibody in the detection, quantitation and characterization of antibody immune responses. Using mouse specific degenerate primer pairs and splenocytic RNA, we generated an idiotype antibody-immunized phage-displayed scFv library in which an anti-idiotype antibody against the therapeutic chimera anti-CD22 antibody SM03 was isolated. The anti-idiotype scFv recognized the idiotype of anti-CD22 antibody and inhibited binding of SM03 to CD22 on Raji cell surface. The anti-idiotype scFv was subsequently classified as Ab2γ type. Moreover, our results also demonstrated firstly that the anti-idiotype scFv could be used for pharmacokinetic measurement of circulating residual antibody in lymphoma patients treated with chimera anti-CD22 monoclonal antibody SM03. Of important, the present approach could be easily adopted to generate anti-idiotype antibodies for therapeutic antibodies targeting membrane proteins, saving the cost and time for producing a soluble antigen.

  17. Ligation-based assembly for constructing mouse synthetic scFv libraries by chain shuffling with in vivo-amplified VH and VL fragments. (United States)

    Nishi, Michiru; Jian, Nan; Yamamoto, Keiko; Seto, Haruyo; Nishida, Yuichi; Tonoyama, Yasuhiro; Shimizu, Nobuyoshi; Nishi, Yoshisuke


    In vitro assembly of two or three PCR fragments using primers is a common method of constructing scFv fragments for display on the surface of phage. However, mismatch annealing often occurs during in this step, leading to cloning and display of incomplete Fab or scFv fragments. To overcome this limitation, we developed a ligation-based two-fragment assembly (LTFA) protocol that involved separately cloning VH and Vκ fragments into the high-copy-number plasmid pUC18. The VH and Vκ fragments had randomized complementarity-determining region 3 (CDR3) and were joined with a peptidyl linker composed of (G4S)3. Using this approach, complete sequences of scFv fragments were successfully constructed, and the sequencing of 83 scFv clones revealed that none of the sequences, including the linker region, contained deletions or mutations. In contrast, linker sequences generated using a conventional two-fragment PCR assembly (TFPA) protocol often contained sequence anomalies, including large truncations. Using the LTFA protocol, a final library size of 1.0×10(8)cfu was achieved. Examination of the amino acid profiles of the generated scFv fragments within the randomized regions introduced using degenerate codons did not detect any bias from that expected based on stochastic distribution. After several cycles of panning with this library, antigen-specific scFvs against two reference antigens, hen egg lysozyme and streptavidin were detected. In addition, scFvs with specificity against peptidyl antigens in the loop region of the Medaka ortholog of human C6orf89, which encodes a histone deacetylase enhancer that interacts with the bombesin receptor, were also obtained. The LTFA protocol developed here is robust and allows for the easy construction of integral scFv fragments compared with conventional TFPA. Utilizing LTFA, other CDRs can be readily combined. This approach also allows for the in vitro maturation of scFv fragments by separately introducing randomization in CDRs or

  18. Glycosylphosphatidylinositol-Anchored Anti-HIV scFv Efficiently Protects CD4 T Cells from HIV-1 Infection and Deletion in hu-PBL Mice. (United States)

    Ye, Chaobaihui; Wang, Weiming; Cheng, Liang; Li, Guangming; Wen, Michael; Wang, Qi; Zhang, Qing; Li, Dan; Zhou, Paul; Su, Lishan


    Despite success in viral inhibition and CD4 T cell recovery by highly active antiretroviral treatment (HAART), HIV-1 is still not curable due to the persistence of the HIV-1 reservoir during treatment. One patient with acute myeloid leukemia who received allogeneic hematopoietic stem cell transplantation from a homozygous CCR5 Δ32 donor has had no detectable viremia for 9 years after HAART cessation. This case has inspired a field of HIV-1 cure research focusing on engineering HIV-1 resistance in permissive cells. Here, we employed a glycosylphosphatidylinositol (GPI)-scFv X5 approach to confer resistance of human primary CD4 T cells to HIV-1. We showed that primary CD4 T cells expressing GPI-scFv X5 were resistant to CCR5 (R5)-, CXCR4 (X4)-, and dual-tropic HIV-1 and had a survival advantage compared to control cells ex vivo In a hu-PBL mouse study, GPI-scFv X5-transduced CD4 T cells were selected in peripheral blood and lymphoid tissues upon HIV-1 infection. Finally, GPI-scFv X5-transduced CD4 T cells, after being cotransfused with HIV-infected cells, showed significantly reduced viral loads and viral RNA copy numbers relative to CD4 cells in hu-PBL mice compared to mice with GPI-scFv AB65-transduced CD4 T cells. We conclude that GPI-scFv X5-modified CD4 T cells could potentially be used as a genetic intervention against both R5- and X4-tropic HIV-1 infections. Blocking of HIV-1 entry is one of most promising approaches for therapy. Genetic disruption of the HIV-1 coreceptor CCR5 by nucleases in T cells is under 2 clinical trials and leads to reduced viremia in patients. However, the emergence of viruses using the CXCR4 coreceptor is a concern for therapies applying single-coreceptor disruption. Here, we report that HIV-1-permissive CD4 T cells engineered with GPI-scFv X5 are resistant to R5-, X4-, or dual-tropic virus infection ex vivo In a preclinical study using hu-PBL mice, we show that CD4 T cells were protected and that GPI-scFv X5-transduced cells were

  19. Isolation and characterisation of a human-like antibody fragment (scFv that inactivates VEEV in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Torsten Rülker

    Full Text Available Venezuelan equine encephalitis virus (VEEV belongs to the Alphavirus genus and several species of this family are pathogenic to humans. The viruses are classified as potential agents of biological warfare and terrorism and sensitive detection as well as effective prophylaxis and antiviral therapies are required.In this work, we describe the isolation of the anti-VEEV single chain Fragment variable (scFv, ToR67-3B4, from a non-human primate (NHP antibody gene library. We report its recloning into the bivalent scFv-Fc format and further immunological and biochemical characterisation.The scFv-Fc ToR67-3B4 recognised viable as well as formalin and ß-propionolactone (ß-Pl inactivated virus particles and could be applied for immunoblot analysis of VEEV proteins and immuno-histochemistry of VEEV infected cells. It detected specifically the viral E1 envelope protein of VEEV but did not react with reduced viral glycoprotein preparations suggesting that recognition depends upon conformational epitopes. The recombinant antibody was able to detect multiple VEEV subtypes and displayed only marginal cross-reactivity to other Alphavirus species except for EEEV. In addition, the scFv-Fc fusion described here might be of therapeutic use since it successfully inactivated VEEV in a murine disease model. When the recombinant antibody was administered 6 hours post challenge, 80% to 100% of mice survived lethal VEEV IA/B or IE infection. Forty to sixty percent of mice survived when scFv-Fc ToR67-3B4 was applied 6 hours post challenge with VEEV subtypes II and former IIIA. In combination with E2-neutralising antibodies the NHP antibody isolated here could significantly improve passive protection as well as generic therapy of VEE.

  20. Implementation of the NCAR Community Land Model (CLM) in the NASA/NCAR finite-volume Global Climate Model (fvGCM) (United States)

    Radakovich, Jon D.; Wang, Guiling; Chern, Jiundar; Bosilovich, Michael G.; Lin, Shian-Jiann; Nebuda, Sharon; Shen, Bo-Wen


    In this study, the NCAR CLM version 2.0 land-surface model was integrated into the NASA/NCAR fvGCM. The CLM was developed collaboratively by an open interagency/university group of scientists and based on well-proven physical parameterizations and numerical schemes that combine the best features of BATS, NCAR-LSM, and IAP94. The CLM design is a one-dimensional point model with 1 vegetation layer, along with sub-grid scale tiles. The features of the CLM include 10-uneven soil layers with water, ice, and temperature states in each soil layer, and five snow layers, with water flow, refreezing, compaction, and aging allowed. In addition, the CLM utilizes two-stream canopy radiative transfer, the Bonan lake model and topographic enhanced streamflow based on TOPMODEL. The DAO fvGCM uses a genuinely conservative Flux-Form Semi-Lagrangian transport algorithm along with terrain- following Lagrangian control-volume vertical coordinates. The physical parameterizations are based on the NCAR Community Atmosphere Model (CAM-2). For our purposes, the fvGCM was run at 2 deg x 2.5 deg horizontal resolution with 55 vertical levels. The 10-year climate from the fvGCM with CLM2 was intercompared with the climate from fvGCM with LSM, ECMWF and NCEP. We concluded that the incorporation of CLM2 did not significantly impact the fvGCM climate from that of LSM. The most striking difference was the warm bias in the CLM2 surface skin temperature over desert regions. We determined that the warm bias can be partially attributed to the value of the drag coefficient for the soil under the canopy, which was too small resulting in a decoupling between the ground surface and the canopy. We also discovered that the canopy interception was high compared to observations in the Amazon region. A number of experiments were then performed focused on implementing model improvements. In order to correct the warm bias, the drag coefficient for the soil under the canopy was considered a function of LAI (Leaf


    Medecigo, M.; Manoutcharian, K.; Vasilevko, V.; Govezensky, T.; Munguia, M. E.; Becerril, B.; Luz-Madrigal, A.; Vaca, L.; Cribbs, D. H.; Gevorkian, G.


    Anti-amyloid immunotherapy has been proposed as an appropriate therapeutic approach for Alzheimer’s disease (AD). Significant efforts have been made towards the generation and assessment of antibody-based reagents capable of preventing and clearing amyloid aggregates as well as preventing their synaptotoxic effects. In this study, we selected a novel set of human anti-amyloid-beta peptide 1-42 (Aβ1-42) recombinant monoclonal antibodies in a single chain fragment variable (scFv) and a single domain (VH) formats. We demonstrated that these antibody fragments recognize in a specific manner amyloid beta deposits in APP/Tg mouse brains, inhibit toxicity of oligomeric Aβ1-42 in neuroblastoma cell cultures in a concentration-dependently manner and reduced amyloid deposits in APP/Tg2576 mice after intracranial administration. These antibody fragments recognize epitopes in the middle/C-terminus region of Aβ, which makes them strong therapeutic candidates due to the fact that most of the Aβ species found in the brains of AD patients display extensive N-terminus truncations/modifications. PMID:20451261

  2. Generation and functional characterization of the anti-transferrin receptor single-chain antibody-GAL4 (TfRscFv-GAL4 fusion protein

    Directory of Open Access Journals (Sweden)

    Ye Qing


    Full Text Available Abstract Background The development of vectors for cell-specific gene delivery is a major goal of gene therapeutic strategies. Transferrin receptor (TfR is an endocytic receptor and identified as tumor relative specific due to its overexpression on most tumor cells or tissues, and TfR binds and intakes of transferrin-iron complex. We have previously generated an anti-TfR single-chain variable fragments of immunoglobulin (scFv which were cloned from hybridoma cell line producing antibody against TfR linked with a 20 aa-long linker sequence (G4S4. In the present study, the anti-TfR single-chain antibody (TfRscFv was fused to DNA-binding domain of the yeast transcription factor GAL4. The recombinant fusion protein, designated as TfRscFv-GAL4, is expected to mediate the entry of DNA-protein complex into targeted tumor cells. Results Fusion protein TfRscFv-GAL4 was expressed in an E. coli bacterial expression system and was recovered from inclusion bodies with subsequent purification by metal-chelate chromatography. The resulting proteins were predominantly monomeric and, upon refolding, became a soluble biologically active bifunctional protein. In biological assays, the antigen-binding activity of the re-natured protein, TfRscFv-GAL4, was confirmed by specific binding to different cancer cells and tumor tissues. The cell binding rates, as indicated by flow cytometry (FCM analysis, ranged from 54.11% to 8.23% in seven different human carcinoma cell lines. It showed similar affinity and binding potency as those of parent full-length mouse anti-TfR antibody. The positive binding rates to tumor tissues by tissue microarrays (TMA assays were 75.32% and 63.25%, but it showed weakly binding with hepatic tissue in 5 cases, and normal tissues such as heart, spleen, adrenal cortex blood vessel and stomach. In addition, the re-natured fusion protein TfRscFv-GAL4 was used in an ELISA with rabbit anti-GAL4 antibody. The GAL4-DNA functional assay through the GAL4

  3. Application of fusion protein 4D5 scFv-dibarnase:barstar-gold complex for studying P185HER2 receptor distribution in human cancer cells. (United States)

    Ivanova, Julia L; Edelweiss, Evelina F; Leonova, Olga G; Balandin, Taras G; Popenko, Vladimir I; Deyev, Sergey M


    Overexpression of the P185(HER2) protein determines the malignancy and unfavorable prognosis of ovarian and breast tumors. In this work, the distribution of P185(HER2) in human cancer cells was studied by electron microscopy, using a novel approach. It is based on the interaction between barnase (a ribonuclease from Bacillus amyloliquefaciens) and its specific inhibitor barstar. The monoclonal antibody 4D5 scFv to extracellular P185(HER2) domain fused with two molecules of barnase was used as a recognizing agent, and the conjugate of colloidal gold with barstar, as an electron dense label for electron microscopic visualization. For labeling, we used supramolecular complexes 4D5 scFv-dibarnase:barstar-Au. The distribution of P185(HER2) in human ovarian carcinoma cells SKOV-3 and breast carcinoma cells BT-474 was studied at 4 °C and 37 °C. It was shown that at 4 °C the protein P185(HER2) occurs exclusively on the cell surface, mainly on protrusions or close to their bases. At 37 °C, the internalization of P185(HER2) caused by its interaction with 4D5 scFv-dibarnase was observed. Inside the cells, P185(HER2) was located in the coated pits and vesicles, endosomes and multivesicular bodies. The data obtained indicate that the supramolecular 4D5 scFv-dibarnase:barstar-gold complex can be used as a new immunodetection system for exploring the P185(HER2) distribution.

  4. Affinity Purification of Tumor Necrosis Factor-α Expressed in Raji Cells by Produced scFv Antibody Coupled CNBr-Activated Sepharose



    Purpose: Recombinant tumor necrosis factor-alpha (TNF-α) has been utilized as an antineoplastic agent for the treatment of patients with melanoma and sarcoma. It targets tumor cell antigens by impressing tumor-associated vessels. Protein purification with affinity chromatography has been widely used in the downstream processing of pharmaceutical-grade proteins. Methods: In this study, we examined the potential of our produced anti-TNF-scFv fragments for purification of TNF-α produced by Raj...

  5. Recognition of Vipera ammodytes meridionalis neurotoxin vipoxin and its components using phage-displayed scFv and polyclonal antivenom sera. (United States)

    Stoyanova, Vishnya; Aleksandrov, Radoslav; Lukarska, Maria; Duhalov, Deyan; Atanasov, Vasil; Petrova, Svetla


    Vipoxin is a potent postsynaptic heterodimeric neurotoxin isolated from the venom of the Bulgarian snake Vipera ammodytes meridionalis, whose snakebites cause different and strongly manifested pathophysiological effects (neurotoxic, hemolytic, anticoagulant, convulsant, hypotensive, hyperglycemic etc.). The neutralization of snake toxins calls for extensive research through the application of different approaches: antibodies, non-immunologic inhibitors, natural products derived from plants and animals, as well as synthetic drugs. In this study, we applied naive Tomlinson I + J (Cambridge, UK) libraries to obtain recombinant human scFv antibodies against the vipoxin's two subunits--basic and toxic phospholipase A₂ (PLA₂) and acidic, non-toxic component. We found that 33 of more than hundred tested clones were positive and recognized vipoxin and its subunits. Enriched scFv-phage samples (1.2 × 10⁹ pfu/ml) were analyzed for their binding (ELISA) and enzyme-inhibiting abilities. Single chain Fv-phage clones--D₁₂, E₃, F₆, D₁₀ and G₅ exhihest binding affinity for the toxic component. Clones A₁, D₁₂ and C₁₂ recognized preferentially vipoxin's acidic component. Clones E₃, G₅ and H₄ inhibited the enzymatic activity of both vipoxin and its purified and separated toxic subunit to the highest extent. Six of the selected clones (E₃, G₅, H₄, C₁₂, D₁₀ and A₁₁) inhibited direct hemolytic activity of vipoxin and its pure PLA₂ subunit. The obtained specific scFv antibodies will be used for epitope mapping studies required to shed light on the role of the phospholipase A₂ activity for the vipoxin toxicity and its effective neutralization. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. Rapid Purification of a New Humanized Single-chain Fv Antibody/Human Interleukin-2 Fusion Protein Reactive against HER2 Receptor

    Institute of Scientific and Technical Information of China (English)

    Wei-Yun ZHANG; Tak-Chun YIP; Cheuk-Sang KWOK


    Human embryonic kidney 293 cells were transfected with plasmid pcDNA-H520C9scFv-rhIL2 containing a chimeric cDNA encoding the humanized 520C9 scFv/recombinant human IL-2 fusion protein (H520C9scFv-rhIL-2). The transfected cells in plateau growing phase were cultured in serum-free medium for three days. The supernatant was collected, concentrated and purified using an affinity column packed with CNBr-activated Sepharose 4B coupled with anti-rhIL-2 mouse monoclonal antibody. The purified fusion protein was analyzed by ELISA, SDS-PAGE and Western blot. The fusion protein showed only one band in both silver stained electrophoresis gel and Western blot developed by ECL chemiluminescence system.Its molecular weight was confirmed to be about 45 kD. This fusion protein possessed binding specificity against p 185 positive SKOV3 and B 16/neu cells, and it might stimulate IL-2-dependent CTLL-2 cell proliferation as well.

  7. Aptamers, antibody scFv, and antibody Fab' fragments: An overview and comparison of three of the most versatile biosensor biorecognition elements. (United States)

    Crivianu-Gaita, Victor; Thompson, Michael


    The choice of biosensing elements is crucial for the development of the optimal biosensor. Three of the most versatile biosensing elements are antibody single-chain Fv fragments (scFv), antibody fragment-antigen binding (Fab') units, and aptamers. This article provides an overview of these three biorecognition elements with respects to their synthesis/engineering, various immobilization techniques, and examples of their use in biosensors. Furthermore, the final section of the review compares and contrasts their characteristics (time/cost of development, ease and variability of immobilization, affinity, stability) illustrating their advantages and disadvantages. Overall, scFv fragments are found to display the highest customizability (i.e. addition of functional groups, immobilizing peptides, etc.) due to recombinant synthesis techniques. If time and cost are an issue in the development of the biosensor, Fab' fragments should be chosen as they are relatively cheap and can be developed quickly from whole antibodies (several days). However, if there are sufficient funds and time is not a factor, aptamers should be utilized as they display the greatest affinity towards their target analytes and are extremely stable (excellent biosensor regenerability).

  8. The efficient elimination of solid tumor cells by EGFR-specific and HER2-specific scFv-SNAP fusion proteins conjugated to benzylguanine-modified auristatin F. (United States)

    Woitok, Mira; Klose, Diana; Niesen, Judith; Richter, Wolfgang; Abbas, Muhammad; Stein, Christoph; Fendel, Rolf; Bialon, Magdalena; Püttmann, Christiane; Fischer, Rainer; Barth, Stefan; Kolberg, Katharina


    Antibody-drug conjugates (ADCs) combine the potency of cytotoxic drugs with the specificity of monoclonal antibodies (mAbs). Most ADCs are currently generated by the nonspecific conjugation of drug-linker reagents to certain amino acid residues in mAbs, resulting in a heterogeneous product. To overcome this limitation and prepare ADCs with a defined stoichiometry, we use SNAP-tag technology as an alternative conjugation strategy. This allows the site-specific conjugation of O(6)-benzylguanine (BG)-modified small molecules to SNAP-tag fusion proteins. To demonstrate the suitability of this system for the preparation of novel recombinant ADCs, here we conjugated SNAP-tagged single chain antibody fragments (scFvs) to a BG-modified version of auristatin F (AURIF). We used two scFv-SNAP fusion proteins targeting members of the epidermal growth factor receptor (EGFR) family that are frequently overexpressed in breast cancer. The conjugation of BG-AURIF to EGFR-specific 425(scFv)-SNAP and HER2-specific αHER2(scFv)-SNAP resulted in two potent recombinant ADCs that specifically killed breast cancer cell lines by inducing apoptosis when applied at nanomolar concentrations. These data confirm that SNAP-tag technology is a promising tool for the generation of novel recombinant ADCs.

  9. Expression of anti-tumor necrosis factor alpha (TNFα) single-chain variable fragment (scFv) in Spirodela punctata plants transformed with Agrobacterium tumefaciens. (United States)

    Balaji, Parthasarathy; Satheeshkumar, P K; Venkataraman, Krishnan; Vijayalakshmi, M A


    Therapeutic antibodies against tumor necrosis factor alpha (TNFα) have been considered effective for some of the autoimmune diseases such as rheumatoid arthritis, Crohn's diseases, and so on. But associated limitations of the current therapeutics in terms of cost, availability, and immunogenicity have necessitated the need for alternative candidates. Single-chain variable fragment (scFv) can negate the limitations tagged with the anti-TNFα therapeutics to a greater extent. In the present study, Spirodela punctata plants were transformed with anti-TNFα through in planta transformation using Agrobacterium tumefaciens strain, EHA105. Instead of cefotaxime, garlic extract (1 mg/mL) was used to remove the agrobacterial cells after cocultivation. To the best of our knowledge, this report shows for the first time the application of plant extracts in transgenic plant development. 95% of the plants survived screening under hygromycin. ScFv cDNA integration in the plant genomic DNA was confirmed at the molecular level by PCR. The transgenic protein expression was followed up to 10 months. Expression of scFv was confirmed by immunodot blot. Protein expression levels of up to 6.3% of total soluble protein were observed. β-Glucuronidase and green fluorescent protein expressions were also detected in the antibiotic resistant plants. The paper shows the generation of transgenic Spirodela punctuata plants through in planta transformation.

  10. Profile data from CTD casts aboard the F/V Ocean Explorer in the Arctic Ocean and Beaufort Sea from 2008-08-06 to 2008-08-22 (NODC Accession 0001920) (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — This profile data aboard the F/V Ocean Explorer in the Arctic Ocean and Beaufort Sea from August 6, 2008 to August 22, 2008 was supported by the Minerals Management...

  11. Evaluation and characterization of 30 solar home systems (SHS) Ovonic-Unisolar; Caracterizacion y evaluacion de 30 sistemas FV domiciliarios ovonic-unisolar

    Energy Technology Data Exchange (ETDEWEB)

    Flores, J. Roberto; Agredano, Jaime; Munguia, Gonzalo; Lagunas, Javier; Huacuz, Jorge [Instituto de Investigaciones Electricas, Temixco, Morelos (Mexico); Brennan, Steve [Troy, MI (United States)


    In this work the first results of the evaluation and characterization of 30 solar home systems (SHS) Ovonic-Unisolar (16 of 30 W and 14 of 60 W of capacity) are presented. The components of the SHS are: One or two PV modules, a charge controller, a nickel metal-hybride (NiMH) battery with a nominal capacity of 85 Ah rated at 3 hours, two or four 8 W lamps, and a CD/CD converter for connecting a radio or a TV W/B a maximum power of 20W. Of all systems evaluated, 29 were installed in three communities of the Oaxaca State, and the other 2 are installed at the Instituto de Investigaciones Electricas (IIE) the laboratory as prototype systems. Before the systems installation at the rural communities, all of them were tested in laboratory. Eight systems installed in the field are being monitoring with data acquisition systems. The main motivation of this projects is to know the behavior of the NiMH battery in the SHS. [Spanish] En este trabajo se presentan los primeros resultados de la caracterizacion y evaluacion de 30 sistemas fotovoltaicos (FV) domiciliarios Ovonic-Unisolar (16 con capacidad de 30 W y 14 con capacidad de 60 W). Los sistemas estan integrados por uno o dos modulos FV de 30 W (dependiendo de la capacidad del sistema), un controlador de carga con termo-interuptor, una bateria del tipo niquel hidruros metalicos (NiHM) con capacidad de 85 Ah a una razon de descarga de 3 horas, 2 o 4 lamparas compactas de alta eficiencia de 8 W y un convertidor CD/CD que permite a los usuarios utilizar una radiograbadora y/o una television B/N con potencia no mayor a 20 W. De estos 30 sistemas FV, 28 se instalaron en 3 comunidades rurales del Estado de Oaxaca y los dos sistemas restantes se tienen instalados en el laboratorio FV del Instituto de Investigaciones Electricas (IIE) como sistemas testigos. Previo a la instalacion en campo, todos los sistemas fueron evaluados en el laboratorio para garantizar su operacion en las comunidades rurales. De los 28 sistemas instalados en

  12. Anti-HER2 antibody and ScFvEGFR-conjugated antifouling magnetic iron oxide nanoparticles for targeting and magnetic resonance imaging of breast cancer

    Directory of Open Access Journals (Sweden)

    Chen H


    Full Text Available Hongwei Chen,1,* Liya Wang,1,2,* Qiqi Yu,1,2 Weiping Qian,3 Diana Tiwari,1 Hong Yi,4 Andrew Y Wang,5 Jing Huang,1,2 Lily Yang,3 Hui Mao1,2 1Department of Radiology and Imaging Sciences, 2Center for Systems Imaging, 3Department of Surgery, Emory University School of Medicine, 4Robert Apkarian Electron Microscopy Core, Emory University, Atlanta, GA, 5Ocean NanoTech LLC, Springdale, AK, USA *These authors contributed equally to this work Abstract: Antifouling magnetic iron oxide nanoparticles (IONPs coated with block copolymer poly(ethylene oxide-block-poly(γ-methacryloxypropyltrimethoxysilane (PEO-b-PγMPS were investigated for improving cell targeting by reducing nonspecific uptake. Conjugation of a HER2 antibody, Herceptin®, or a single chain fragment (ScFv of antibody against epidermal growth factor receptor (ScFvEGFR to PEO-b-PγMPS-coated IONPs resulted in HER2-targeted or EGFR-targeted IONPs (anti-HER2-IONPs or ScFvEGFR-IONPs. The anti-HER2-IONPs bound specifically to SK-BR-3, a HER2-overexpressing breast cancer cell line, but not to MDA-MB-231, a HER2-underexpressing cell line. On the other hand, the ScFvEGFR-IONPs showed strong reactivity with MDA-MB-231, an EGFR-positive human breast cancer cell line, but not with MDA-MB-453, an EGFR-negative human breast cancer cell line. Transmission electron microscopy revealed internalization of the receptor-targeted nanoparticles by the targeted cancer cells. In addition, both antibody-conjugated and non-antibody-conjugated IONPs showed reduced nonspecific uptake by RAW264.7 mouse macrophages in vitro. The developed IONPs showed a long blood circulation time (serum half-life 11.6 hours in mice and low accumulation in both the liver and spleen. At 24 hours after systemic administration of ScFvEGFR-IONPs into mice bearing EGFR-positive breast cancer 4T1 mouse mammary tumors, magnetic resonance imaging revealed signal reduction in the tumor as a result of the accumulation of the targeted IONPs

  13. Development of single chain variable fragment (scFv) antibodies against surface proteins of 'Ca. Liberibacter asiaticus'. (United States)

    Yuan, Qing; Jordan, Ramon; Brlansky, Ronald H; Minenkova, Olga; Hartung, John


    'Candidatus Liberibacter asiaticus' is the causal agent of citrus huanglongbing, the most serious disease of citrus worldwide. We have developed and applied immunization and affinity screening methods to develop a primary library of recombinant single chain variable fragment (scFv) antibodies in an M13 vector, pKM19. The antibody population is enriched for antibodies that bind antigens of 'Ca. Liberibacter asiaticus'. The primary library has more than 10(7) unique antibodies and the genes that encode them. We have screened this library for antibodies that bind to specifically-chosen proteins that are present on the surface of 'Ca. Liberibacter asiaticus'. These proteins were used as targets for affinity-based selection of scFvs that bind to the major outer membrane protein, OmpA; the polysaccharide capsule protein KpsF; a protein component of the type IV pilus (CapF); and, two flagellar proteins FlhA and FlgI. These scFvs have been used in ELISA and dot blot assays against purified protein antigens and 'Ca. Liberibacter asiaticus' infected plant extracts. We have also recloned many of these scFvs into a plasmid expression vector designed for the production of scFvs. Screening of these scFvs was more efficient when phage-bound, rather than soluble scFvs, were used. We have demonstrated a technology to produce antibodies at will and against any protein target encoded by 'Ca. Liberibacter asiaticus'. Applications could include advanced diagnostic methods for huanglongbing and the development of immune labeling reagents for in planta applications.

  14. IG and TR single chain fragment variable (scFv) sequence analysis: a new advanced functionality of IMGT/V-QUEST and IMGT/HighV-QUEST. (United States)

    Giudicelli, Véronique; Duroux, Patrice; Kossida, Sofia; Lefranc, Marie-Paule


    IMGT®, the international ImMunoGeneTics information system® ( ), was created in 1989 in Montpellier, France (CNRS and Montpellier University) to manage the huge and complex diversity of the antigen receptors, and is at the origin of immunoinformatics, a science at the interface between immunogenetics and bioinformatics. Immunoglobulins (IG) or antibodies and T cell receptors (TR) are managed and described in the IMGT® databases and tools at the level of receptor, chain and domain. The analysis of the IG and TR variable (V) domain rearranged nucleotide sequences is performed by IMGT/V-QUEST (online since 1997, 50 sequences per batch) and, for next generation sequencing (NGS), by IMGT/HighV-QUEST, the high throughput version of IMGT/V-QUEST (portal begun in 2010, 500,000 sequences per batch). In vitro combinatorial libraries of engineered antibody single chain Fragment variable (scFv) which mimic the in vivo natural diversity of the immune adaptive responses are extensively screened for the discovery of novel antigen binding specificities. However the analysis of NGS full length scFv (~850 bp) represents a challenge as they contain two V domains connected by a linker and there is no tool for the analysis of two V domains in a single chain. The functionality "Analyis of single chain Fragment variable (scFv)" has been implemented in IMGT/V-QUEST and, for NGS, in IMGT/HighV-QUEST for the analysis of the two V domains of IG and TR scFv. It proceeds in five steps: search for a first closest V-REGION, full characterization of the first V-(D)-J-REGION, then search for a second V-REGION and full characterization of the second V-(D)-J-REGION, and finally linker delimitation. For each sequence or NGS read, positions of the 5'V-DOMAIN, linker and 3'V-DOMAIN in the scFv are provided in the 'V-orientated' sense. Each V-DOMAIN is fully characterized (gene identification, sequence description, junction analysis, characterization of mutations and amino

  15. Production of monoclonal anti-GP Ⅱ b/Ⅲa scFv antibodies from scFv phage libraries%从scFv噬菌体抗体库中筛选抗GPⅡ b/Ⅲa单链抗体

    Institute of Scientific and Technical Information of China (English)

    夏红利; 谭最; 陈德杰; 乔建国; 邱仁峰


    目的 从scFv噬菌体库中获取人源化特异性Anti-GPⅡb/Ⅲa单克隆scFv抗体.方法 对Tomlinson I+J scFv文库进行3次淘洗,富集特异性的抗GPⅡb/Ⅲa抗体.通过酶联免疫吸附试验(ELISA)和双脱氧终止法基因测序及同源性对比,检测出人源化的抗GPⅡb/Ⅲa单克隆抗体.结果 在3次淘洗后,得到了抗GPⅡb/Ⅲa单克隆噬菌体抗体,阳性克隆的获取率在95.6%以上;ELISA和基因测序筛选出25种不同的全长抗GPⅡb/Ⅲa噬菌体抗体,这些基因序列与人免疫球蛋白可变区基因同源性达到89%以上;分泌性抗体ELISA检测提示这些抗体顺利表达了蛋白并特异性结合GPⅡb/Ⅲa,其中15种scFv对GPⅡb/Ⅲa有更强的阳性反应.结论 人源化的特异性抗-GPⅡb/Ⅲa scFv能通过噬菌体展示技术快速有效地获得.%Objective To screen monoclonal anti-GP Ⅱ b/Ⅲ a antibodies from scFv phage libraries and obtain specific monoclonal anti-GP Ⅱ b/Ⅲ a scFv. Methods Specific anti-GP Ⅱ b/Ⅲ a scFv antibodies were enriched by three rounds of selection from Tomlinson Ⅰ + J libraries. By using polyclonal and monoclonal phage enzyme linked immunosorbent assay ( ELISA) and gene sequencing by bideoxy chain termination, full-length specific monoclonal anti-GP Ⅱ b/Ⅲ a antibodies were picked out and their gene sequences were obtained. Results Twenty-five different full-length monoclonal scFv phage fragments were obtained after three rounds of panning and their gene sequences were identified, and positive rate of monoclones was above 95.6%; homology comparison with variable regions of human immunoglobulin gene showed the similarity was above 89%; the result of soluble scFv ELISA showed that these specific scFv could be expressed smoothly, and 15 full-length monoclonal scFv antibodies were stronger positive than the other in these scFv. Conclusion Antibody phage display was a rapid and effective method to obtain Anti-GP Ⅱ b/Ⅲ a scFv fragements.


    Directory of Open Access Journals (Sweden)



    Full Text Available This article is result of research on the validity of the college entrance test. College entrance test including programs of study must have the characteristics of a good test especially predictive validity. Entrance test study program Master of Arabic Education (PBA FITK UIN Jakarta consists of four types of tests; the Academic Potential Test (TPA, TOAFL, TOEFL and interviews, the four must be able to predict exactly when the students join the program. That is, students who earn higher test scores should be able to follow the course as evidenced by the value of IP and or higher GPA compared with the results of the entry test lower. Similarly in predicting students graduate on time, which requires in addition to success in the lecture was also successful in the final project / Thesis which some students a frightening specter. Its mean that students with higher admission tests should be faster in completing the study compared with the lower value of the entry test. If not, then it must be held meta-evaluation of the instruments and implementation of entrance tests which have already become standards.


    Directory of Open Access Journals (Sweden)

    Raswan Raswan


    Full Text Available This article is result of research on the validity of the college entrance test. College entrance test including programs of study must have the characteristics of a good test especially predictive validity. Entrance test study program Master of Arabic Education (PBA FITK UIN Jakarta consists of four types of tests; the Academic Potential Test (TPA, TOAFL, TOEFL and interviews, the four must be able to predict exactly when the students join the program. That is, students who earn higher test scores should be able to follow the course as evidenced by the value of IP and or higher GPA compared with the results of the entry test lower. Similarly in predicting students graduate on time, which requires in addition to success in the lecture was also successful in the final project / Thesis which some students a frightening specter. Its mean that students with higher admission tests should be faster in completing the study compared with the lower value of the entry test. If not, then it must be held meta-evaluation of the instruments and implementation of entrance tests which have already become standards.DOI: 10.15408/a.v2i1.1300

  18. De zaak-Kronos: spiegelbeeld van de FII-jurisprudentie

    NARCIS (Netherlands)

    de Groot, I.M.


    In deze bijdrage gaat de auteur in op de zaak-Kronos. In deze zaak is in geschil of de voormalige Duitse regelgeving, op basis waarvan (onder voorwaarden) voor binnenlandse dividenden de "full-creditmethode" werd toegepast en voor buitenlandse dividenden de vrijstellingsmethode, in strijd is met de

  19. Efficient production of human bivalent and trivalent anti-MUC1 Fab-scFv antibodies in Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Haustraete Jurgen


    system for the production of recombinant multivalent Fab-scFv antibody derivatives.

  20. In vivo near-infrared fluorescence imaging of FAP-expressing tumors with activatable FAP-targeted, single-chain Fv-immunoliposomes. (United States)

    Rüger, Ronny; Tansi, Felista L; Rabenhold, Markus; Steiniger, Frank; Kontermann, Roland E; Fahr, Alfred; Hilger, Ingrid


    Molecular and cellular changes that precede the invasive growth of solid tumors include the release of proteolytic enzymes and peptides in the tumor stroma, the recruitment of phagocytic and lymphoid infiltrates and alteration of the extracellular matrix. The reactive tumor stroma consists of a large number of myofibroblasts, characterized by high expression of fibroblast activation protein alpha (FAP). FAP, a type-II transmembrane sialoglycoprotein is an attractive target in diagnosis and therapy of several pathologic disorders especially cancer. In the underlying work, a fluorescence-activatable liposome (fluorescence-quenched during circulation and fluorescence activation upon cellular uptake), bearing specific single-chain Fv fragments directed against FAP (scFv'FAP) was developed, and its potential for use in fluorescence diagnostic imaging of FAP-expressing tumor cells was evaluated by whole body fluorescence imaging. The liposomes termed anti-FAP-IL were prepared via post-insertion of ligand-phospholipid-conjugates into preformed DY-676-COOH-containing liposomes. The anti-FAP-IL revealed a homogeneous size distribution and showed specific interaction and binding with FAP-expressing cells in vitro. The high level of fluorescence quenching of the near-infrared fluorescent dye sequestered in the aqueous interior of the liposomes enables fluorescence imaging exclusively upon uptake and degradation by cells, which results in fluorescence activation. Only FAP-expressing cells were able to take up and activate fluorescence of anti-FAP-IL in vitro. Furthermore, anti-FAP-IL accumulated selectively in FAP-expressing xenograft models in vivo, as demonstrated by blocking experiments using free scFv'FAP. The local tumor fluorescence intensities were in agreement with the intrinsic degree of FAP-expression in different xenograft models. Thus, anti-FAP-IL can serve as a suitable in vivo diagnostic tool for pathological disorders accompanied by high FAP-expression.

  1. DNA immunization combined with scFv phage display identifies antagonistic GCGR specific antibodies and reveals new epitopes on the small extracellular loops. (United States)

    van der Woning, Bas; De Boeck, Gitte; Blanchetot, Christophe; Bobkov, Vladimir; Klarenbeek, Alex; Saunders, Michael; Waelbroeck, Magali; Laeremans, Toon; Steyaert, Jan; Hultberg, Anna; De Haard, Hans


    The identification of functional monoclonal antibodies directed against G-protein coupled receptors (GPCRs) is challenging because of the membrane-embedded topology of these molecules. Here, we report the successful combination of llama DNA immunization with scFv-phage display and selections using virus-like particles (VLP) and the recombinant extracellular domain of the GPCR glucagon receptor (GCGR), resulting in glucagon receptor-specific antagonistic antibodies. By immunizing outbred llamas with plasmid DNA containing the human GCGR gene, we sought to provoke their immune system, which generated a high IgG1 response. Phage selections on VLPs allowed the identification of mAbs against the extracellular loop regions (ECL) of GCGR, in addition to multiple VH families interacting with the extracellular domain (ECD) of GCGR. Identifying mAbs binding to the ECL regions of GCGR is challenging because the large ECD covers the small ECLs in the energetically most favorable 'closed conformation' of GCGR. Comparison of Fab with scFv-phage display demonstrated that the multivalent nature of scFv display is essential for the identification of GCGR specific clones by selections on VLPs because of avid interaction. Ten different VH families that bound 5 different epitopes on the ECD of GCGR were derived from only 2 DNA-immunized llamas. Seven VH families demonstrated interference with glucagon-mediated cAMP increase. This combination of technologies proved applicable in identifying multiple functional binders in the class B GPCR context, suggesting it is a robust approach for tackling difficult membrane proteins.

  2. Pharmacological efficacy of anti-IL-1β scFv, Fab and full-length antibodies in treatment of rheumatoid arthritis. (United States)

    Qi, Jianying; Ye, Xianlong; Ren, Guiping; Kan, Fangming; Zhang, Yu; Guo, Mo; Zhang, Zhiyi; Li, Deshan


    Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease that mainly causes the synovial joint inflammation and cartilage destruction. Interleukin-1β (IL-1β) is an important proinflammatory cytokine involved in the pathogenesis of RA. In this study, we constructed and expressed anti-IL-1β-full-length antibody in CHO-K1-SV, anti-IL-1β-Fab and anti-IL-1β-scFv in Rosetta. We compared the therapeutic efficacy of three anti-IL-1β antibodies for CIA mice. Mice with CIA were subcutaneously injected with humanized anti-IL-1β-scFv, anti-IL-1β-Fab or anti-IL-1β-full-length antibody. The effects of treatment were determined by arthritis severity score, autoreactive humoral, cellular immune responses, histological lesion and cytokines production. Compared with anti-IL-1β-scFv treatments, anti-IL-1β-Fab and anti-IL-1β-full-length antibody therapy resulted in more significant effect in alleviating the severity of arthritis by preventing bone damage and cartilage destruction, reducing humoral and cellular immune responses, and down-regulating the expression of IL-1β, IL-6, IL-2, IFN-γ, TNF-α and MMP-3 in inflammatory tissue. The therapeutic effects of anti-IL-1β-Fab and anti-IL-1β-full-length antibodies on CIA mice had no significant difference. However, production of anti-IL-1β-full-length antibody in eukaryotic system is, in general, time-consuming and more expensive than that of anti-IL-1β-Fab in prokaryotic systems. In conclusion, as a small molecule antibody, anti-IL-1β-Fab is an ideal candidate for RA therapy.

  3. Autoconsumo de energía solar fotovoltáica con gestión activa de la demanda:El sistema GEDELOS-FV


    Caamaño Martín, Estefanía; Castillo Cagigal, Manuel; Masa Bote, Daniel; Gutiérrez Martín, Álvaro; Matallanas de Avila, Eduardo; Monasterio-Huelin Maciá, Felix; Jiménez Leube, Francisco Javier


    El sistema GeDELOS-FV es un ejemplo de valor añadido de la energía solar fotovoltaica que combina tecnología fotovoltaica híbrida de última generación con estrategias de Gestión de la Demanda Eléctrica. En este trabajo se ha configurado el sistema con el objetivo de maximizar la cantidad e energía fotovoltaica generada localmente y consumida por las cargas locales (autoconsumo), tanto de manera directa como indirecta mediante el uso de almacenamiento eléctrico. Se han realizado simulacione...

  4. Autoconsumo de Energía Solar Fotovoltaica con Gestión Activa de la Demanda: El Sistema GeDELOS-FV


    Caamaño Martín, Estefanía; Castillo Cagigal, Manuel; Masa Bote, Daniel; Gutiérrez Martín, Álvaro; Matallanas de Avila, Eduardo; Monasterio-Huelin Maciá, Felix; Jiménez Leube, Francisco Javier


    El sistema GeDELOS-FV es un ejemplo de valor añadido de la energía solar fotovoltaica que combina tecnología fotovoltaica híbrida de última generación con estrategias de Gestión de la Demanda Eléctrica. En este trabajo se ha configurado el sistema con el objetivo de maximizar la cantidad e energía fotovoltaica generada localmente y consumida por las cargas locales (autoconsumo), tanto de manera directa como indirecta mediante el uso de almacenamiento eléctrico. Se han realizado simulacion...

  5. DARPA Antibody Technology Program. Standardized Test Bed for Antibody Characterization: Characterization of an MS2 ScFv Antibody Produced by Illumina (United States)


    air bubbles from the samples. Mineral oil (Sigma-Aldrich) was applied to the top of each sample to prevent sample evaporation. The plate was placed...quantitative melting temperature (Tm) for each of the Illumina MS2 scFv proteins. The Tm should predict the results of subsequent ELISA and surface 1× PBS and divided into five tubes. One aliquot was kept on ice for the duration of the experiment and was marked time 0. The remaining four

  6. 提取纯化过程对金针菇FV-19菌株发酵液多糖相对分子质量的影响%Effects of the courses of extraction and purification on the relative molecularmass of fermenting filtrate's polysaccharide of Flatmmulina velutipes (Curt. exFr. ) Sing "FV-19"

    Institute of Scientific and Technical Information of China (English)



    金针菇FV-19菌株深层发酵醪液.经离心、沉淀、透析处理得到发酵液多糖样品,并用凝胶色谱法测定其相对分子质量.探讨了提取纯化过程对发酵液多糖相对分子质量的影响以及贮存期对其相对分子质量的影响.%Polysaccharide samples of fermenting filtrate were isolated by means of centrifugation, sediment,dialysis from the mash of submerged culture of Flammulina velutipes (Curt. ex Fr. ) Sing "FV-19", itsrelative molecular mass was determined by gel permeation chromatography. The effects of the course ofextraction and purification on the relative molecular mass of fermenting filtrate's polysaccharide and theeffects of storage time on its relative molecular mass were explored and discussed.

  7. Expression and optimization of anti-AFB1 scFv in Escherichia coli%抗黄曲霉毒素B1单链抗体在大肠杆菌中的表达及优化

    Institute of Scientific and Technical Information of China (English)

    杨炼; 张艳红; 丁虎生; 王丽云; 陈卫; 张灏


    [Objective] A drawback of the expression of single chain antibody fragment (scFv) in prokaryotic system is the protein accumulation in the cytoplasm as inclusion body. We aimed at high-level production of an anti-aflatoxin Bl scFv in functional form. [Methods] The gene of scFv-H4 was cloned into pET22b vector and transformed into E. coli BL21(DE3) and Origami (DE3), respectively. The amount of functional scFv-H4 was optimized in terms of IPTG concentration and induction temperature. [Results] scFv-H4 could be expressed in both BL21(DE3) and Origami (DE3). Compared with BL21(DE3), Origami(DE3) could express multifunctional scFv-H4 (35 mg/mL) and less in inclusion body (11% of the total expression) . The expression of scFv-H4 was significantly affected by induction temperature rather than IPTG concentration. [Conclusion] The p£T22b could be used for high-level expression of the functional scFv-H4 in Origami (DE3) , which has an oxidative cytoplasm. In addition, the induction at low temperature avoided the formation of inclusion body.%[目的]继杂交瘤技术后,重组抗体技术是新一代的抗体制备技术.然而如何用原核系统中较多地表达具有生物活性的单链抗体,避免包涵体形成仍是一个需要探讨的问题.[方法]将目的基因scFv-H4克隆到载体pET22b上,分别转入大肠杆菌BL21(DE3)和Origami(DE3)中,通过改变诱导温度和IPTG浓度,比较具有生物活性的蛋白量以及包涵体的量.[结果]在BL21(DE3)中,pET22b能产生大量表达scFv-H4,而BL21(DE3)的含有trxA和gor双突变的衍生菌Origami(DE3)表达的scFv-H4的总量较少,但是具有生物活性的蛋白量较多(35 mg/L培养物),具有生物活性的蛋白比例也较BL2l(DE3)高.另外IFrG的浓度对scFv-H4表达没有显著影响,而较高的诱导温度会促使表达的蛋白形成包涵体.[结论]在较低的温度下,pET22b能在Origami(DE3)能较好地表达具有生物活性的scFv-H4,减少包涵体的比例,为后续的抗

  8. Inhibitory effect of humanized anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles conjugate on growth of human hepatocellular carcinoma:in vitro andin vivo studies

    Institute of Scientific and Technical Information of China (English)

    Xiang-Bao Yin; Lin-Quan Wu; Hua-Qun Fu; Ming-Wen Huang; Kai Wang; Fan Zhou; Xin Yu; Kai-Yang Wang


    Objective:To investigate the inhibitory effect of humanized anti-VEGFR-2ScFv-As2O3-stealth nanoparticles conjugate on growth of human hepatocellular carcinoma bothin vitro andin vivo, which may be a potential agents with sensitivity and targeting ability for human hepatocellular cancer.Methods:Humanized anti-VEGFR-2ScFv-As2O3-stealth nanoparticles conjugate was previously constructed using ribosome display technology and antibody conjugate technology. In this combinedin vitro andin vivo study, the inhibitory effects of anti-VEGFR-2ScFv-As2O3-stealth nanoparticles conjugate on tumor growth, invasion, and metastasis was observed with human liver carcinoma cell lineBel7402 and normal cellL02 byMTT assay,Tanswell assay, Hochest33258 staining, andDNA ladder analysis.The anticancer activity and distribution of anti-VEGFR-2ScFv-As2O3-stealth nanoparticles was then verified in a mouse model ofBel7402 xenografts.Results:Anti-VEGFR-2ScFv-As2O3-stealth nanoparticles significantly inhibited the proliferation ofBel7402 in the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay while had almost no effects onL02 cells.And the apoptosis inducing effects were proved byHochest33258 staining andDNA ladder analysis.Transwell assay found that the drug also inhibited the metastasis ability of tumor cells.Furthermore, anti-VEGFR-2ScFv-As2O3-stealth nanoparticles significantly delayed the growth ofBel7402 xenografts after administration(92.9%), followed byAs2O3-stealth nanoparticles, anti-VEGFR-2ScFv, andAs2O3(61.4%,58.8%,20.5%, P<0.05).The concentration ofAs2O3 in anti-VEGFR-2ScFv-As2O3-stealth nanoparticles group was more selectively.Conclusions:Anti-VEGFR-2ScFv-As2O3-stealth nanoparticles is a potent and selective anti-hepatocellular carcinoma agent which could inhibit the growth of liver cancer as a targeting agent bothin vitro andin vivo and also significantly inhibit angiogenesis.

  9. Delineation of BmSXP antibody V-gene usage from a lymphatic filariasis based immune scFv antibody library. (United States)

    Rahumatullah, Anizah; Ahmad, Azimah; Noordin, Rahmah; Lim, Theam Soon


    Phage display technology is an important tool for antibody generation or selection. This study describes the development of a scFv library and the subsequent analysis of identified monoclonal antibodies against BmSXP, a recombinant antigen for lymphatic filariasis. The immune library was generated from blood of lymphatic filariasis infected individuals. A TA based intermediary cloning approach was used to increase cloning efficiency for the library construction process. A diverse immune scFv library of 10(8) was generated. Six unique monoclonal antibodies were identified from the 50 isolated clones against BmSXP. Analysis of the clones showed a bias for the IgHV3 and Vκ1 (45.5%) and IgHV2 and Vκ3 (27.3%) gene family. The most favored J segment for light chain is IgKJ1 (45.5%). The most favored D and J segment for heavy chain are IgHD6-13 (75%) and IgHJ3 (47.7%). The information may suggest a predisposition of certain V genes in antibody responses against lymphatic filariasis.

  10. Two ScFv antibody libraries derived from identical VL-VH framework with different binding site designs display distinct binding profiles. (United States)

    Huovinen, Tuomas; Syrjänpää, Markku; Sanmark, Hanna; Brockmann, Eeva-Christine; Azhayev, Alex; Wang, Qi; Vehniäinen, Markus; Lamminmäki, Urpo


    In directed evolution experiments, a single randomization scheme of an antibody gene does not provide optimal diversity for recognition of all sizes of antigens. In this study, we have expanded the recognition potential of our universal library, termed ScFvP, with a second distinct diversification scheme. In the second library, termed ScFvM, diversity was designed closer to the center of the antigen binding site in the same antibody framework as earlier. Also, the CDR-H3 loop structures were redesigned to be shorter, 5-12 aa and mostly without the canonical salt bridge between Arg106H and Asp116H to increase the flexibility of the loop and to allow more space in the center of the paratope for binding smaller targets. Antibodies were selected from the two libraries against various antigens separately and as a mixture. The origin and characteristics of the retrieved antibodies indicate that complementary diversity results in complementary functionality widening the spectrum of targets amenable for selection.

  11. Screening and identification of human ZnT8-specific single-chain variable fragment (scFv) from type 1 diabetes phage display library. (United States)

    Wu, Qian; Wang, Xiaodong; Gu, Yong; Zhang, Xiao; Qin, Yao; Chen, Heng; Xu, Xinyu; Yang, Tao; Zhang, Mei


    Zinc transporter 8 (ZnT8) is a major autoantigen and a predictive marker in type 1 diabetes (T1D). To investigate ZnT8-specific antibodies, a phage display library from T1D was constructed and single-chain antibodies against ZnT8 were screened and identified. Human T1D single-chain variable fragment (scFv) phage display library consists of approximately 1×10(8) clones. After four rounds of bio-panning, seven unique clones were positive by phage ELISA. Among them, C27 and C22, which demonstrated the highest affinity to ZnT8, were expressed in Escherichia coli Top10F' and then purified by affinity chromatography. C27 and C22 specifically bound ZnT8 N/C fusion protein and ZnT8 C terminal dimer with one Arg325Trp mutation. The specificity to human islet cells of these scFvs were further confirmed by immunohistochemistry. In conclusion, we have successfully constructed a T1D phage display antibody library and identified two ZnT8-specific scFv clones, C27 and C22. These ZnT8-specific scFvs are potential agents in immunodiagnostic and immunotherapy of T1D.

  12. Nano-yeast-scFv probes on screen-printed gold electrodes for detection of Entamoeba histolytica antigens in a biological matrix. (United States)

    Grewal, Yadveer S; Shiddiky, Muhammad J A; Spadafora, Lauren J; Cangelosi, Gerard A; Trau, Matt


    The time and costs associated with monoclonal antibody production limit the potential for portable diagnostic devices to penetrate the market. Replacing the antibody with a low-cost alternate affinity reagent would reduce the costs of diagnostic development and use, and lead to new portable diagnostic devices towards many diseases. Herein, we present low-cost affinity reagents, nano-yeast-scFv, on commercially available, inexpensive, and portable screen-printed electrodes for the label-free electrochemical detection of Entamoeba histolytica cyst antigens. The biosensor was able to detect antigen at concentrations down to 10 pg mL(-1) in buffer with an inter-assay reproducibility of (% RSD, n=3) 4.1%. The applicability of two differently engineered nano-yeast-scFv to each specifically detect their cognant E. histolytica cyst antigens was demonstrated in a biological matrix derived from human stool. Because of the simple, inexpensive, and sensitive nature of this methodology, it may offer a low-cost alternative to immunosensors based on antibody-target recognition.

  13. The factor V leiden , the G20210A mutation of prothrombin gene and their association with deep vein thrombophilia%凝血因子V基因Leiden 突变和凝血酶原基因G20210A突变与下肢深静脉血栓形成关系的探讨

    Institute of Scientific and Technical Information of China (English)

    郭辰虹; 郭琼行; 龚瑶琴; 陈丙玺; 常宏; 周海斌


    目的:探讨凝血因子V 基因G1691A突变(Leiden 突变)和凝血酶原基因G20210A突变与下肢深静脉血栓形成的关系.方法:采用PCR---RFLP技术对86例下肢深静脉血栓形成患者和100例正常对照的健康人群进行凝血因子 V 基因G1691A突变和凝血酶原基因G20210A突变检测.结果:86例下肢深静脉血栓形成患者和100例正常对照组中均未检出凝血因子V基因 G1691A突变和凝血酶原基因G20210A突变.结论:凝血因子V基因G1691A突变和凝血酶原基因G20210A突变可能不是中国汉族人群下肢深静脉血栓形成的主要风险因子.

  14. Association of prothrombin G20210A and coagulation factor Leiden gene mutation with coronary heart disease%凝血酶原基因G20210A和凝血因子V Leiden突变与冠心病的相关性探讨

    Institute of Scientific and Technical Information of China (English)

    金国栋; 徐耕; 傅国胜; 马骥; 施育平; 单江


    目的:探讨凝血酶原基因G20210A和凝血因子VG1691A(Leiden)突变在中国汉族人群中的发生率及与冠心病的关系.方法:用聚合酶链反应和限制性内切酶片段长度多态性技术,对234例冠心病患者和210名正常人的凝血酶原基因G20210A和凝血因子V Leiden突变进行分析.结果:找到了一例正常人的凝血酶原基因G20210A变异,突变率为0.2%.冠心病和正常人中均未发现凝血因子V Leiden突变.结论:汉族人群中存在凝血酶原基因G20210A突变,但凝血酶原基因G20210A和凝血因子V Leiden变异在我国的发生率较低,不足以作为冠心病的遗传性危险因素.

  15. Targeting the active site of the placental isozyme of alkaline phosphatase by phage-displayed scFv antibodies selected by a specific uncompetitive inhibitor

    Directory of Open Access Journals (Sweden)

    Kala Mrinalini


    Full Text Available Abstract Background The isozymes of alkaline phosphatase, the tissue non-specific, intestinal and placental, have similar properties and a high degree of identity. The placental isozyme (PLAP is an oncofetal antigen expressed in several malignancies including choriocarcinoma, seminoma and ovarian carcinoma. We had earlier attempted to isolate PLAP-specific scFv from a synthetic human immunoglobulin library but were unable to do so, presumably because of the similarity between the isozymes. In this work, we have employed a PLAP-specific uncompetitive inhibitor, L-Phe-Gly-Gly, to select isozyme specific scFvs. An uncompetitive inhibitor binds to the enzyme in the presence of substrate and stabilizes the enzyme-substrate complex. Several uncompetitive inhibitors have varying degrees of isozyme specificity for human alkaline phosphatase isozymes. A specific uncompetitive inhibitor would be able to unmask conformational differences between the otherwise very similar molecules. Also, such inhibitors would be directed to regions at/close to the active site of the enzyme. In this work, the library was first incubated with PLAP and the bound clones then eluted by incubation with L-Phe-Gly-Gly along with the substrate, para-nitro phenyl phosphate (pNPP. The scFvs were then studied with regard to the biochemical modulation of their binding, isozyme specificity and effect on enzyme activity. Results Of 13 clones studied initially, the binding of 9 was inhibited by L-Phe-Gly-Gly (with pNPP and 2 clones were inhibited by pNPP alone. Two clones had absolute and 2 clones had partial specificity to PLAP. Two clones were cross-reactive with only one other isozyme. Three scFv clones, having an accessible His6-tag, were purified and studied for their modulation of enzyme activity. All the three scFvs inhibited PLAP activity with the kinetics of competitive inhibition. Cell ELISA could demonstrate binding of the specific scFvs to the cell surface expressed PLAP

  16. Comparison of a mouse and a novel human scFv-SNAP-auristatin F drug conjugate with potent activity against EGFR-overexpressing human solid tumor cells

    Directory of Open Access Journals (Sweden)

    Woitok M


    Full Text Available Mira Woitok,1,2 Diana Klose,1 Stefano Di Fiore,1 Wolfgang Richter,3 Christoph Stein,1 Gerrit Gresch,1 Elena Grieger,1 Stefan Barth,1 Rainer Fischer,1,2 Katharina Kolberg,1,* Judith Niesen1,* 1Fraunhofer Institute for Molecular Biology and Applied Ecology (IME, Aachen, Germany; 2Institute of Molecular Biotechnology (Biology VII, RWTH Aachen University, Aachen, Germany; 3Tube Pharmaceuticals GmbH, Vienna, Austria *These authors contributed equally to this work Abstract: Antibody–drug conjugates (ADCs can deliver toxins to specific targets such as tumor cells. They have shown promise in preclinical/clinical development but feature stoichiometrically undefined chemical linkages, and those based on full-size antibodies achieve only limited tumor penetration. SNAP-tag technology can overcome these challenges by conjugating benzylguanine-modified toxins to single-chain fragment variables (scFvs with 1:1 stoichio­metry while preserving antigen binding. Two (human and mouse scFv-SNAP fusion proteins recognizing the epidermal growth factor receptor (EGFR were expressed in HEK 293T cells. The purified fusion proteins were conjugated to auristatin F (AURIF. Binding activity was confirmed by flow cytometry/immunohistochemistry, and cytotoxic activity was confirmed by cell viability/apoptosis and cell cycle arrest assays, and a novel microtubule dynamics disassembly assay was performed. Both ADCs bound specifically to their target cells in vitro and ex vivo, indicating that the binding activity of the scFv-SNAP fusions was unaffected by conjugation to AURIF. Cytotoxic assays confirmed that the ADCs induced apoptosis and cell cycle arrest at nanomolar concentrations and microtubule disassembly. The SNAP-tag technology provides a platform for the development of novel ADCs with defined conjugation sites and stoichiometry. We achieved the stable and efficient linkage of AURIF to human or murine scFvs using the SNAP-tag technology, offering a strategy to

  17. Factor V Leiden, factor V Cambridge, factor II GA20210, and methylenetetrahydrofolate reductase in cerebral venous and sinus thrombosis: A case-control study. (United States)

    Saadatnia, Mohammad; Salehi, Mansour; Movahedian, Ahmad; Shariat, Seyed Ziaeddin Samsam; Salari, Mehri; Tajmirriahi, Marzieh; Asadimobarakeh, Elham; Salehi, Rasoul; Amini, Gilda; Ebrahimi, Homa; Kheradmand, Ehsan


    Factor V G1691A (FV Leiden), FII GA20210, and methylenetetrahydrofolate reductase (MTHFR) C677T mutations are the most common genetic risk factors for thromboembolism in the Western countries. However, there is rare data in Iran about cerebral venous and sinus thrombosis (CVST) patients. The aim of this study was to evaluate the frequency of common genetic thrombophilic factors in CVST patients. Forty consequently CVST patients from two University Hospital in Isfahan University of Medical Sciences aged more than 15 years from January 2009 to January 2011 were recruited. In parallel, 51 healthy subjects with the same age and race from similar population selected as controls. FV Leiden, FII GA20210, MTHFR C677T, and FV Cambridge gene mutations by polymerase chain reaction technique were evaluated in case and control groups. FV Leiden, FII GA20210, and FV Cambridge gene mutations had very low prevalence in both case (5%, 2%, 0%) and control (2.5%, 0%, 0%) and were not found any significant difference between groups. MTHFR C677T mutations was in 22 (55%) of patients in case group and 18 (35.5%) of control group (P = 0.09). This study showed that the prevalence of FV Leiden, FII GA20210, and FV Cambridge were low. Laboratory investigations of these mutations as a routine test for all patients with CVST may not be cost benefit.

  18. Characterization of an engineered human purine nucleoside phosphorylase fused to an anti-her2/neu single chain Fv for use in ADEPT

    Directory of Open Access Journals (Sweden)

    Wu Anna M


    Full Text Available Abstract Background Antibody Directed Enzyme Prodrug Therapy (ADEPT can be used to generate cytotoxic agents at the tumor site. To date non-human enzymes have mainly been utilized in ADEPT. However, these non-human enzymes are immunogenic limiting the number of times that ADEPT can be administered. To overcome the problem of immunogenicity, a fully human enzyme, capable of converting a non-toxic prodrug to cytotoxic drug was developed and joined to a human tumor specific scFv yielding a fully human targeting agent. Methods A double mutant of human purine nucleoside phosphorylase (hDM was developed which unlike the human enzyme can cleave adenosine-based prodrugs. For tumor-specific targeting, hDM was fused to the human anti-HER2/neu single chain Fv (scFv, C6 MH3B1. Enzymatic activity of hDM with its natural substrates and prodrugs was determined using spectrophotomeric approaches. A cell proliferation assay was used to assess the cytotoxicity generated following conversion of prodrug to drug as a result of enzymatic activity of hDM. Affinity of the targeting scFv, C6 MH3B1 fused to hDM to Her2/neu was confirmed using affinity chromatography, surface plasmon resonance, and flow-cytometry. Results In vitro hDM-C6 MH3B1 binds specifically to HER2/neu expressing tumor cells and localizes hDM to tumor cells, where the enzymatic activity of hDM-C6 MH3B1, but not the wild type enzyme, results in phosphorolysis of the prodrug, 2-fluoro-2'-deoxyadenosine to the cytotoxic drug 2-fluoroadenine (F-Ade causing inhibition of tumor cell proliferation. Significantly, the toxic small drug diffuses through the cell membrane of HER2/neu expressing cells as well as cells that lack the expression of HER2/neu, causing a bystander effect. F-Ade is toxic to cells irrespective of their growth rate; therefore, both the slowly dividing tumor cells and the non-dividing neighboring stromal cells that support tumor growth should be killed. Analysis of potential novel MHCII

  19. The Study of the Targeting Selectivity and Binding the Surface of Breast Cancer Cells with the Fusion Protein Anti-HER2-ScFv-GFP in vitro Experiments%Anti-HER2-ScFv-GFP融合蛋白靶向结合体外乳腺癌细胞表面受体的研究

    Institute of Scientific and Technical Information of China (English)

    高国辉; 黄奇迪; 王金丹; 杨纪锋; 包兵兵; 胡孝渠


    为了研究不同表达系统获得的携带绿色荧光抗HER2单链抗体(Anti-HER2-ScFv-GFP)是否既可靶向结合HER2阳性乳腺癌细胞表面,也可通过观察绿色荧光变化直接判断抗体结合乳腺癌细胞表面后细胞的动态变化,在前期成功构建两种表达系统的基础上,利用Ni2+-NTA亲和层析法纯化来源于真核表达系统pFAST Bac to Bac HTA/Tn-5B1-4和原核表达系统pBAD His B/TOP10的融合蛋白Anti-HER2-ScFv-GFP,设置HER2阳性细胞SKBR3为实验组、HER2阴性细胞MCF7为对照组,分别与之混合24 h后,1×PBS洗脱细胞3次,激光共聚焦显微镜观察到两种不同表达系统获得的融合蛋白在HER2阳性细胞SKBR3 表面分布均有绿色荧光,真核表达的蛋白结合效率明显高于原核表达的蛋白,SKBR3结合高浓度的融合蛋白后细胞表现出皱缩,绿色荧光明显增强,而两种不同来源的融合蛋白与HER2阴性MCF7混合后均易被洗脱.GFP标准品与SKBR3混合后也容易被洗脱.实验表明构建的携带绿色荧光抗HER2单链抗体同时具有靶向结合和报告作用两方面的功能.%The goal of this study was to test the targeting binding efficiency of the fusion protein Anti-HER2-ScFv-GFP on the surface of breast cancer cells. We constructed the eukaryotic expression system pFAST Bac to Bac HT A/Anti-HER2-ScFv-GFP/Tn-5Bl-4 and the prokaryotic system pBAD His B/Anti-HER2-ScFv-GFP/TOP10. And then the fusion protein Anti-HER2-ScFv-GFP was separated to get the purification with Ni2+-NTA argrose from the eukaryotic expression system pFAST Bac to Bac HT A/Tn-5Bl-4 and the prokaryotic expression system pBAD His B/TOP10. Then we incubated SKBR3 (HER2+ cell) and MCF7(HER2-cell) containing the purification of the fusion proteins in 24 h, eluted these cells with 1×PBS three times, examined the targeting binding efficiency of the fusion protein Anti-HER2-ScFv-GFP on the surface of breast cancer cells with laser confocal microscopy system

  20. Co-expression of Dsb proteins enables soluble expression of a single-chain variable fragment (scFv) against human type 1 insulin-like growth factor receptor (IGF-1R) in E. coli. (United States)

    Sun, Xue-Wen; Wang, Xiao-Hua; Yao, Yan-Bing


    Type 1 insulin-like growth factor receptor (IGF-1R) is a promising therapeutic target for cancer treatment. A single-chain variable fragment (scFv) against human IGF-1R forms inclusion body when expressed in periplasmic space of E. coli routinely. Here, we described that co-expression of appropriate disulfide bonds (Dsb) proteins known to catalyze the formation and isomerization of Dsb can markedly recover the soluble expression of target scFv in E. coli. A 50 % recovery in solubility of the scFv was observed upon co-expression of DsbC alone, and a maximum solubility (80 %) was obtained when DsbA and DsbC were co-expressed in combination. Furthermore, the soluble scFv present full antigen-binding activity with IGF-1R, suggesting its correct folding. This study also suggested that the selection of Dsb proteins should be tested case-by-case if the approach of co-expression of Dsb system is adopted to address the problem of insoluble expression of proteins carrying Dsb.

  1. Sequence Analysis of ScFv Gene for Anti-alpha-toxin%两种抗α毒素单链抗体基因的序列分析

    Institute of Scientific and Technical Information of China (English)

    赵宝华; 许崇波


    应用RT-PCR技术,从两株分泌具有中和活性的抗A型产气荚膜梭菌α毒素单克隆抗体(McAb)的杂交瘤细胞株2E3和1A8中,分别扩增出抗体VH和VL基因,用Linker(Gly4Ser)3基因,将VH和VL基因连接成ScFv基因2E3-ScFv和1A8-ScFv,并将其克隆至pGEM-T载体中.经核苷酸序列分析证实,VH和VL基因以及Linker基因拼接正确,2E3-ScFv基因全长为729bp,经计算机分析,VH和VL基因均为新发现的基因序列,符合功能性重排的鼠抗体可变区基因特征.2E3-ScFv的VH和VL基因分别属于鼠免疫球蛋白重链Ⅱ(B)和轻链kⅢ家簇;而1A8-ScFv的VH和VL基因分别属于鼠免疫球蛋白重链Ⅱ(A)和轻链κⅥ家簇.%The VH and VL genes were amplified from two hybridoma cell lines producing mouse McAb against alpha-txoin of Clostridium perfringens type A by RT-PCR. The VH and VL genes were connected through a flexible Linker (Gly4Ser)3,and the VH-Linker-VL(ScFv) fusion gene was cloned into a clone vector pGEM-T. The 2E3-ScFv and 1A8-ScFv gene were sequenced and analyzed by computer. The ScFv genes consist of 726 bp and 729 bp respectively.Both VH and VL genes were functionally rearranged mouse immunoglobulin variable region genes and appeared to be new genes. According to Kabat classed method,the VH gene segment and VL gene segment of 2E3-ScFv belong to the mouse Ig heavy chain subgroup Ⅱ (B) and к chain subgroup Ⅲ respectively,but the VH gene segment and VL gene segment of 1A8 -ScFv belong to the mouse Ig heavy chain subgroup Ⅱ (A) and к chain subgroup Ⅵ respectively.

  2. Stability engineering of anti-EGFR scFv antibodies by rational design of a lambda-to-kappa swap of the VL framework using a structure-guided approach. (United States)

    Lehmann, Andreas; Wixted, Josephine H F; Shapovalov, Maxim V; Roder, Heinrich; Dunbrack, Roland L; Robinson, Matthew K


    Phage-display technology facilitates rapid selection of antigen-specific single-chain variable fragment (scFv) antibodies from large recombinant libraries. ScFv antibodies, composed of a VH and VL domain, are readily engineered into multimeric formats for the development of diagnostics and targeted therapies. However, the recombinant nature of the selection strategy can result in VH and VL domains with sub-optimal biophysical properties, such as reduced thermodynamic stability and enhanced aggregation propensity, which lead to poor production and limited application. We found that the C10 anti-epidermal growth factor receptor (EGFR) scFv, and its affinity mutant, P2224, exhibit weak production from E. coli. Interestingly, these scFv contain a fusion of lambda3 and lambda1 V-region (LV3 and LV1) genes, most likely the result of a PCR aberration during library construction. To enhance the biophysical properties of these scFvs, we utilized a structure-based approach to replace and redesign the pre-existing framework of the VL domain to one that best pairs with the existing VH. We describe a method to exchange lambda sequences with a more stable kappa3 framework (KV3) within the VL domain that incorporates the original lambda DE-loop. The resulting scFvs, C10KV3_LV1DE and P2224KV3_LV1DE, are more thermodynamically stable and easier to produce from bacterial culture. Additionally, C10KV3_LV1DE and P2224KV3_LV1DE retain binding affinity to EGFR, suggesting that such a dramatic framework swap does not significantly affect scFv binding. We provide here a novel strategy for redesigning the light chain of problematic scFvs to enhance their stability and therapeutic applicability.

  3. Preperation and biological functions of anti-hγδTCR-ScFv%抗人γδ TCR单链抗体G5-4ScFv的制备及生物学功能分析

    Institute of Scientific and Technical Information of China (English)

    郑静; 崔莲仙; 何维


    Objective To construct and express the single chain variable fragment (Fv) of a monoclonal antibody against human γδ TCR which can stimulate proliferation of human γδ T cells. Methods The genes of the heavy (VH) and light chains (VL) of antibody were cloned by RT-PCR from a hybfidoma cell line G5-4,which secreted the monoclonal antibody (mAb) against human γδ TCR. Overlapping extension PCR was used to connect the VH and VL genes with a short linker encoding (Gly4Ser)3 to construct the anti-hγδ TCR-ScFv gene G5-4ScFv. Recom-binant plasmid pET22b( + )-G5-4ScFv was constructed by inserting G5-4ScFv fragment into the prokaryotic expression vector pET22b( + ). The anti-hγδTCR-SeFv (G5-4ScFv) was expressed in E. coli TransB(DE3) by induction with IPTG and purified with Nickel-affinity chromatography column. Then G5-4ScFv was analyzed by SDS-PAGE and Western-blot assay. Its binding ability to human γδ T cells was measured by flow cytometry. The purity and biological characteristics including cytokine secretion and cytotoxicity of γδ T cells expanded by G5-4ScFv stimulation, were detected by flow cytometry and LDH methods. Results G5-4ScFv showed a molecular weight of 30 ku in SDS-PAGE. G5-4ScFv binded to 78 T cells and greatly inhibited the binding of a pan-γδTCR antibody with γδ T cells. The purity of γδ T cells expanded by G5-4ScFv stimulation was up to 90% after culture of 2 weeks. These γδ T cells exhibited IFN-γ and TNF-α secretion ability and strong cytotoxicity against Daudi cells. Conclusions anti-h-y&TCR-ScFv with biological functions has been successfully constructed and expressed in prokaryotic expression system, which provides a concrete base for further studying on its potential application in tumor immunotherapy.%目的 构建并应用原核表达体系表达能体外刺激人γδ T细胞增殖的抗人γδ TCR单链抗体.方法 在成功建立稳定分泌鼠抗人γδTCR单克隆抗体杂交瘤细胞系G5-4的基础上,通过RT-PCR

  4. α-半乳糖苷酶A缺乏对凝血因子V Leiden小鼠组织纤维蛋白沉积和血栓形成的影响%Effect α-galaetosidase A deficiency on FV Leiden fibtin deposition and thrombosis in mice

    Institute of Scientific and Technical Information of China (English)

    沈粤春; 何兆初; 蔡茹丽; 潘洁贞; 王晓明; 李君


    Objective To evaluate the effect of q-galaetosidase A(Gla)deficiency on FV Leiden (FVL)associated thrombosis in vivo.Metlaods To generate the mice carrying mutations in Gla and F V L and analyze the tissue fibrin deposition in organs and thrombosis.Results in the presence of F V L,Gla deficiency greatly increased tissue fibrin deposition compared with that in wild-type[Gla-/0 FV Q/Q vs.Gla+/0 FV Q/Q=(0.24±0.07)% vs.(0.086±0.049)%,P<0.0001;Gla-/-FV Q/Q vs.Gla+/+ FV Q/Q=(0.32±0.03)% vs.(0.06±0.005)%,P<0.05].With Gla deficiency,the number of thrombi on organ sectiona in F V L mice was signifieantly increased[(Gla-/-FV Q/Q and Gla-/0 FV Q/Q) vs.(Gla+/+ FV Q/Q and Gla+/0 FV Q/Q)=1.9±0.7 vs.0.3±0.1,P<0.05].Conclusions Gla deficieney could be an important genetic modifier for the enhaneed thrombosis associated with F V L.%目的 评价α-半乳糖苷酶A(Gla)缺乏与凝血因子V(FV)Leiden(FVL)在体内对血栓形成的影响.方法 建立Gla与FVL基因复合突变小鼠,并分析其器官组织纤维蛋白沉积和血栓形成情况.结果 在FVL存在情况下,野生型小鼠组织器官纤维蛋白沉积[Gla+/0 FV Q/Q,(0.08±0.05)%]较Gla缺乏型[Gla-/0 FV Q/Q,(0.24±0.07)%)]显著降低(P<0.01),Gla缺乏型纯合子小鼠组织器官纤维蛋白沉积[Gla-/-FV Q/Q,(0.32±0.03)%)]较Gla野生型[Gla+/+FV Q/Q,(0.06±0.01)%)]显著增加(P<0.05);同时Gla缺乏型小鼠(Gla-/-FV Q/Q和Gla-/0 FV Q/Q)血栓数目[(1.9±0.7)个]亦显著高于Gla野生型小鼠(Gla+/+FV Q/Q和Gla+/0 FV Q/Q)的血栓数目[(0.3±0.1)个](P<0.05).结论 Gla缺乏加重FVL突变小鼠的组织纤维蛋白沉积和血栓形成,提示Cla缺乏可能是重要的增强FVL患者血栓形成的遗传因素,临床上FVL患者出现早发和严重的血栓疾病可能合并其他遗传性血栓相关的基因缺陷.

  5. Construction and identification of anti-ENR phage display scFv libraries%抗恩诺沙星噬菌体单链抗体库的构建及鉴定

    Institute of Scientific and Technical Information of China (English)

    温凯; 沈建忠; 孟辉; 吴聪明; 王战辉; 张素霞


    本研究以兽药恩诺沙星为对象,构建噬菌体抗体库,为低成本、快速制备目的抗体提供新的途径.以恩诺沙星-鸡卵清蛋白(ENR-OVA)为免疫原对Balb/c小鼠进行免疫,取其脾细胞提取总RNA,并分别扩增全套抗体轻、重链基因,通过重叠延伸PCR技术,以编码柔性多肽(Gly3Ser)4的基因为接头,将轻重链基因组装为完整的scFv基因,将之克隆入pCANTAB5E载体,转化大肠杆菌XL1-Blue,以辅助噬菌体M13KO7对其进行超感染,构建噬菌体抗体库并进行富集、筛选和鉴定;构建了库容量约为2.2×106的抗恩诺沙星噬菌体单链抗体库,并筛选出26株阳性克隆,为表达单链抗体、建立免疫检测方法奠定基础.%This study focuses on construction and identification of immunized phage display library from splenocytes of hyperimmunized BALB/C mice for screening and isolation of scFv fragment against ENR, an alternative method to produce antibodies for veterinary drug residues detection. The total RNA was isolated from splenocytes of a BALB/C mouse hyperimmunized with the Enrofloxacin conjugated to chicken OVA. Variable light and heavy domains of the immunoglobulin genes were amplified by PCR and assembled to produce full-length single-chain Fv(scFV) by overlap extension PCR using a linker primer containing flexible polypeptide-(Gly3Ser)4. The scFv DNA fragment was ligated into phagemid vector pCANTAB5E and electroporated into E. coli XL1-Blue cells. The transformed cells were rescued by M13KO7 helper phage and phage libraries were constructed. The size of antibody libraries is 2.2 × 106. Following the construction of phage display scFv libraries, the recombinant phage displaying scFv were enriched and identified. There are 26 clones against ENR generated in this study.

  6. Recovery of active anti TNF-α ScFv through matrix-assisted refolding of bacterial inclusion bodies using CIM monolithic support. (United States)

    Sushma, Krishnan; Bilgimol, Chuvappumkal Joseph; Vijayalakshmi, Mookambeswaran A; Satheeshkumar, Padikara Kutty


    Anti TNF-α molecules are important as therapeutic agents for many of the autoimmune diseases in chronic stage. Here we report the expression and purification of a recombinant single chain variable fragment (ScFv) specific to TNF-α from inclusion bodies. In contrast to the conventional on column refolding using the soft gel supports, an efficient methodology using monolithic matrix has been employed. Nickel (II) coupled to convective interaction media (CIM) support was utilized for this purpose with 6M guanidine hydrochloride (GuHCl) as the chaotropic agent. The protein purified after solubilization and refolding proved to be biologically active with an IC₅₀ value of 15 μg. To the best of our knowledge, this is the first report showing the application of methacrylate based chromatographic supports for matrix-assisted refolding and purification of Escherichia coli inclusion bodies. The results are promising to elaborate the methodology further to exploit the potential positive features of monoliths in protein refolding science.

  7. Molecular characterization of a single-chain antibody variable fragment (scFv) specific for PspA from Streptococcus pneumoniae. (United States)

    Jang, ShinA; Kim, Gyuhee; Oh, Jihye; Lee, Seungyeop; Kim, Dongho; Kim, Kook-Han; Kim, Yong Ho; Rhee, Dong-Kwon; Lee, Sangho


    Streptococcus pneumoniae is a major infectious agent responsible for pneumonia, otitis media, sepsis and meningitis. Pneumococcal surface protein A (PspA) is a well-characterized virulence factor localized on the surface and a target for vaccine development. In this study, we screened a single-chain antibody variable fragment (scFv) using phage display from a human synthetic library to select a clone 2B11. Affinity (Kd) of 2B11 was measured to be 5 nM using biolayer interferometry. 2B11 exhibited a dose-dependent recognition of recombinant PspA with no cross-reactivity towards pneumococcal antigens. The epitope on PspA was defined to residues 231-242 by mutational analysis. Molecular docking analysis supported the experimentally determined epitope, suggesting that the helix spanning residues 231-242 can bind to 2B11 with residues in the CDR-H3 (complementarity determining region 3 in the heavy chain) actively participating in the molecular contacts. Comparison of 2B11 with a commercial PspA antibody revealed that 2B11 exhibited a better specificity towards recombinant PspA antigen. 2B11 was capable of detecting endogenous PspA from pneumococcal lysates with affinity similar to that of the commercial antibody. Our study provides a molecular tool for biosensors detecting pneumococcal diseases.

  8. Evaluation of an anti-p185{sup HER2} (scFv-C{sub H}2-C{sub H}3){sub 2} fragment following radioiodination using two different residualizing labels: SGMIB and IB-Mal-D-GEEEK

    Energy Technology Data Exchange (ETDEWEB)

    Vaidyanathan, Ganesan [Duke University Medical Center, Durham, NC 27710 (United States)], E-mail:; Jestin, Emmanuelle [Duke University Medical Center, Durham, NC 27710 (United States); Olafsen, Tove; Wu, Anna M. [Department of Molecular and Medical Pharmacology, Crump Institute for Molecular Imaging, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, CA 90095 (United States); Zalutsky, Michael R. [Duke University Medical Center, Durham, NC 27710 (United States)


    Introduction: A 105-kDa double mutant single-chain Fv-Fc fragment (scFv-Fc DM) derived from the anti-p185{sup HER2} hu4D5v8 antibody (trastuzumab; Herceptin) has been described recently. The goal of this study was to investigate whether improved tumor targeting could be achieved with this fragment through the use of residualizing radioiodination methods. Methods: The scFv-Fc DM fragment was radioiodinated using N-succinimidyl 4-guanidinomethyl 3-[{sup 131}I]iodobenzoate ([{sup 131}I]SGMIB) and N{sup {epsilon}}-(3-[{sup 131}I]iodobenzoyl)-Lys{sup 5}-N{sup {alpha}}- maleimido-Gly{sup 1}-GEEEK ([{sup 131}I]IB-Mal-D-GEEEK), two residualizing radioiodination agents that have been used successfully with intact antibodies. Paired-label internalization assays of the labeled fragments were performed in vitro using MCF7 human breast cancer cells transfected to express HER2 (MCF7-HER2); comparisons were made to scFv-Fc DM directly radioiodinated using Iodogen. The tissue distribution of the scFv-Fc DM labeled with [{sup 125}I]IB-Mal-D-GEEEK and [{sup 131}I]SGMIB was compared in athymic mice bearing MCF7-HER2 xenografts. Results: The scFv-Fc DM fragment was labeled with [{sup 131}I]SGMIB and [{sup 131}I]IB-Mal-D-GEEEK in conjugation yields of 53% and 25%, respectively, with preservation of immunoreactivity for HER2. Internalization assays indicated that labeling via SGMIB resulted in a 1.6- to 3.5-fold higher (P<.05) retention of radioactivity, compared to that from the directly labeled fragment, in HER2-expressing cells during a 24-h observation period. Likewise, the amount of radioactivity retained in cells from the IB-Mal-D-GEEEK-labeled fragment was 1.4- to 3.3-fold higher (P<.05). Tumor uptake of radioiodine activity in athymic mice bearing MCF7-HER2 xenografts in vivo was significantly higher for the [{sup 125}I]IB-Mal-D-GEEEK-labeled scFv-Fc DM fragment compared with that of the [{sup 131}I]SGMIB-labeled fragment, particularly at later time points. The uptake of {sup

  9. The Evaluation of Serum Copeptin Levels and Some Commonly Seen Thrombophilic Mutation Prevalence in Acute Pulmonary Embolism. (United States)

    Ozturk, Nurinnisa; Baygutalp, Nurcan Kilic; Bayramoglu, Atif; Polat, Harun; Gul, Mehmet Ali; Bakan, Ebubekir; Aslan, Sahin; Gunes, Ozge Nur


    Acute pulmonary embolism (PE) is a common, emergent condition and may affect a large number of patients. Copeptin has been indicated to be a sensitive biomarker of arginine vasopressin release, and has diagnostic and prognostic value in various clinical conditions. Genetic mutations are considerable components of thrombophilic diseases, and factor II gene G20210A, (FII20210A), factor V Leiden (FVL, G1691A) and methylenetetrahydrofolate reductase gene C677T (MTHFR677T) single nucleotide polymorphisms are the most common mutations of thrombophilic diseases. In this study, serum copeptin levels were determined in patients with PE and healthy controls, and the results were discussed. The prevalence of some commonly seen thrombophilic mutations was also evaluated in patients with PE. The study included 32 patients (18 male, 14 female) with PE and 24 (13 male, 11 female) age- and gender-matched healthy controls. A significant difference in serum copeptin levels was determined between the patient and control groups (8.58 ± 4.42 and 4.07 ± 1.02 pmol/L, respectively). Heterozygous mutant genotype for FII20210A and heterozygous mutant genotype for FVL were observed in 3.1 and 9.4% of patients, respectively. Mutant genotype of 49% was determined for MTHFR677T mutations. It was concluded that copeptin may have diagnostic value for PE.

  10. Short-Term Responses in Maximum Quantum Yield of PSII (Fv/Fm to ex situ Temperature Treatment of Populations of Bryophytes Originating from Different Sites in Hokkaido, Northern Japan

    Directory of Open Access Journals (Sweden)

    Annika K. Jägerbrand


    Full Text Available There is limited knowledge available on the thermal acclimation processes for bryophytes, especially when considering variation between populations or sites. This study investigated whether short-term ex situ thermal acclimation of different populations showed patterns of site dependency and whether the maximum quantum yield of PSII (Fv/Fm could be used as an indicator of adaptation or temperature stress in two bryophyte species: Pleurozium schreberi (Willd. ex Brid. Mitt. and Racomitrium lanuginosum (Hedw. Brid. We sought to test the hypothesis that differences in the ability to acclimate to short-term temperature treatment would be revealed as differences in photosystem II maximum yield (Fv/Fm. Thermal treatments were applied to samples from 12 and 11 populations during 12 or 13 days in growth chambers and comprised: (1 10/5 °C; (2 20/10 °C; (3 25/15 °C; (4 30/20 °C (12 hours day/night temperature. In Pleurozium schreberi, there were no significant site-dependent differences before or after the experiment, while site dependencies were clearly shown in Racomitrium lanuginosum throughout the study. Fv/Fm in Pleurozium schreberi decreased at the highest and lowest temperature treatments, which can be interpreted as a stress response, but no similar trends were shown by Racomitrium lanuginosum.

  11. Reversible dimer formation and stability of the anti-tumour single-chain Fv antibody MFE-23 by neutron scattering, analytical ultracentrifugation, and NMR and FT-IR spectroscopy. (United States)

    Lee, Yie Chia; Boehm, Mark K; Chester, Kerry A; Begent, Richard H J; Perkins, Stephen J


    MFE-23 is a single chain Fv (scFv) antibody molecule used to target colorectal cancer through its high affinity for the tumour marker carcinoembryonic antigen (CEA). ScFv molecules are formed from peptide-linked antibody V(H) and V(L) domains, and many of these form dimers. Our recent crystal structure for MFE-23 showed that this formed an unusual symmetric back-to-back association of two monomers that is consistent with a domain-swapped diabody structure. Neutron scattering and modelling fits showed that MFE-23 existed as compact V(H)-V(L)-linked monomers at therapeutically relevant concentrations below 1 mg/ml. Size-exclusion gel chromatography showed that the monomeric and dimeric forms of MFE-23 could be separated, and that the proportions of these two forms depended on the starting MFE-23 concentration. Sedimentation equilibrium experiments by analytical ultracentrifugation at nine concentrations of MFE-23 indicated a reversible monomer-dimer self-association equilibrium with an association constant of 1.9x10(3)-2.2x10(3) M(-1). Sedimentation velocity experiments using the time derivative g(s(*)) method showed that MFE-23-His has a concentration-dependent weight average sedimentation coefficient that increased from 1.8 S for the monomer to about 3-6 S for the dimer. Both values agreed with those calculated from the MFE-23 crystal structure. In relation to the thermal stability of MFE-23, denaturation experiments by (1)H NMR and FT-IR spectroscopy showed that the molecule is stable up to 47 degrees C, after which denaturation was irreversible. MFE-23 dimerisation is discussed in terms of a new model for diabody structures, in which the V(H) and V(L) domains in the monomer are able to dissociate and reassociate to form a dimer, or diabody, but in which symmetric back-to-back contacts between the two monomers are formed. This dimerisation in solution is attributed to the complementary nature of the C-terminal surface of the MFE-23 monomer. Crystal structures for

  12. Crystal structure of the anti-(carcinoembryonic antigen) single-chain Fv antibody MFE-23 and a model for antigen binding based on intermolecular contacts. (United States)

    Boehm, M K; Corper, A L; Wan, T; Sohi, M K; Sutton, B J; Thornton, J D; Keep, P A; Chester, K A; Begent, R H; Perkins, S J


    MFE-23 is the first single-chain Fv antibody molecule to be used in patients and is used to target colorectal cancer through its high affinity for carcinoembryonic antigen (CEA), a cell-surface member of the immunoglobulin superfamily. MFE-23 contains an N-terminal variable heavy-chain domain joined by a (Gly(4)Ser)(3) linker to a variable light-chain (V(L)) domain (kappa chain) with an 11-residue C-terminal Myc-tag. Its crystal structure was determined at 2.4 A resolution by molecular replacement with an R(cryst) of 19.0%. Five of the six antigen-binding loops, L1, L2, L3, H1 and H2, conformed to known canonical structures. The sixth loop, H3, displayed a unique structure, with a beta-hairpin loop and a bifurcated apex characterized by a buried Thr residue. In the crystal lattice, two MFE-23 molecules were associated back-to-back in a manner not seen before. The antigen-binding site displayed a large acidic region located mainly within the H2 loop and a large hydrophobic region within the H3 loop. Even though this structure is unliganded within the crystal, there is an unusually large region of contact between the H1, H2 and H3 loops and the beta-sheet of the V(L) domain of an adjacent molecule (strands DEBA) as a result of intermolecular packing. These interactions exhibited remarkably high surface and electrostatic complementarity. Of seven MFE-23 residues predicted to make contact with antigen, five participated in these lattice contacts, and this model for antigen binding is consistent with previously reported site-specific mutagenesis of MFE-23 and its effect on CEA binding.

  13. Optimization of production of the anti-keratin 8 single-chain Fv TS1-218 in Pichia pastoris using design of experiments

    Directory of Open Access Journals (Sweden)

    Sundström Birgitta E


    Full Text Available Abstract Background Optimization of conditions during recombinant protein production for improved yield is a major goal for protein scientists. Typically this is achieved by changing single crucial factor settings one at a time while other factors are kept fixed through trial-and-error experimentation. This approach may introduce larger bias and fail to identify interactions between the factors resulting in failure of finding the true optimal conditions. Results In this study we have utilized design of experiments in order to identify optimal culture conditions with the aim to improve the final yield of the anti-keratin 8 scFv TS1-218, during expression in P. pastoris in shake flasks. The effect of: pH, temperature and methanol concentration on the yield of TS1-218 using buffered minimal medium was investigated and a predictive model established. The results demonstrated that higher starting pH and lower temperatures during induction significantly increased the yield of TS1-218. Furthermore, the result demonstrated increased biomass accumulation and cell viability at lower temperatures which suggested that the higher yield of TS1-218 could be attributed to lower protease activity in the culture medium. The optimal conditions (pH 7.1, temperature 11°C and methanol concentration 1.2% suggested by the predictive model yielded 21.4 mg TS1-218 which is a 21-fold improvement compared to the yield prior to optimization. Conclusion The results demonstrated that design of experiments can be utilized for a rapid optimization of initial culture conditions and that P. pastoris is highly capable of producing and secreting functional single-chain antibody fragments at temperatures as low as 11°C.

  14. Paternal factor V Leiden and recurrent pregnancy loss: a new concept behind fetal genetics? (United States)

    Udry, S; Aranda, F M; Latino, J O; de Larrañaga, G F


    In up to 50% of couples affected by recurrent pregnancy loss, no identifiable cause is established. Fetal and maternal factors may be equally important in the establishment and maintenance of the placental/maternal arteriovenous anastomoses. Therefore,the inheritance of thrombophilia-related genes may be an important factor in the pathophysiology of recurrent pregnancy loss. Most of the research on recurrent pregnancy loss and thrombophilia has focused on maternal factors, but little is known about the paternal contribution. On that basis, we studied the association between inherited paternal thrombophilias and recurrent pregnancy loss in a narrowly selective group of 42 Argentine males from couples that presented without any known risk factors for recurrent pregnancy loss. The genotypic distributions of factor (F) V Leiden and prothrombin G20210A among cases were compared with those from a reference group composed of 200 Argentine men. We found a significant difference in the distribution of FV Leiden between both groups (16.7% vs. 3.0%), but no difference was found in the distribution of prothrombin G20210A (2.4% vs.2.0%). Those couples with paternal FV Leiden carriage would be six times more likely to experience recurrent pregnancy loss despite no other apparent cause (OR = 6.47; 95% CI, 2.06–20.39). We found evidence of an association between the paternal carriage of FV Leiden and the predisposition to recurrent pregnancy loss, thereby supporting the hypothesis that genetic contributions from both parents are essential factors in the development of this obstetric disorder.

  15. 人源性抗乳腺癌单链抗体库的筛选与初步鉴定%Isolation and identification of single chain Fv antibodies against breast cancer from a human phage display library

    Institute of Scientific and Technical Information of China (English)

    王净; 王慧; 王超; 袁媛; 崔岩; 叶菁; 李青


    [目的]本研究旨在已构建的大容量人源性抗乳腺癌噬菌体单链抗体库的基础上,筛选出高亲和力的特异性单链抗体(scFv)并对抗体基本特性进行初步鉴定.[方法]以人乳腺癌细胞系MCF-7为靶标,经过4轮淘洗,筛选出高亲和力的特异性抗乳腺癌scFv,并对其结构序列进行分析;通过ELISA和Western blot方法,鉴定scFv的亲和力和特异性,以及其蛋白的基本表达情况.[结果]成功构建具有高亲和力的抗乳腺癌单链抗体库,获得scFv的长度约为750 bp,ELISA证实所得抗体对乳腺癌细胞具有良好的亲和力和高度的特异性,IPTG诱导表达及Western blot结果显示,scFv为相对分子质量(Mr)30 000的可溶性蛋白.[结论]本研究在已构建的大容量抗乳腺癌单链抗体库的基础上,筛选获得了高亲和力的抗乳腺癌单链抗体库.研究结果为进一步获得可应用于临床诊断和治疗的乳腺癌靶向性抗体奠定了良好的基础.%AIM: To Isolate and identify single chain Fv (scFv) antibodies against breast cancer from a constructed human phage display library. METHODS: Recombinant phages specific for breast cancer cells were enriched after four-round screening with MCF-7 cells. We selected the antigen-positive ones from the enriched clones by phage ELISA. The positive clones were used to infect E. coli HB2151 to express soluble scFv antibody. The antigen binding activity of the soluble antibodies was detected by West-em blotting. RESULTS; The specific antibodies against MCF-7 cells were enriched after four rounds of affinity selection. SDS-PAGE and Western blotting showed a band at relative molecular mass 30 000 Da, which indicated soluble antibodies were present. ELISA analysis revealed that soluble antibodies had the affinity to a human breast cancer cell line MCF-7 but not to other cancer cell line, which demonstrated scFv could react specifically with breast cancer cells. CONCLUSION; We constructed a scFv phage

  16. 抗胃癌单链抗体免疫毒素重组质粒的构建及表达%Construction of Anti-carcinoma ScFv Immunotoxin Recombina nt Plasmid and its Expression

    Institute of Scientific and Technical Information of China (English)

    韩梅; 戴寿芝; 靳更林; 寿成超


    Objectives To construct the recombinant plasm id that expresses anti-carcinoma ScF v immunotoxin.Methods By recombinant technique,the cDNA coding the anti-carcinoma ScFv antibody was recombined with the cDNA coding Pseudomonas areuginosa exotoxin to form the recombinanr plasmid.The E Coli.BL-21,after bein g transformed with the recombinant plasmid,was induced by IPTG to express the an ti-carcinoma ScFv immumotoxin.Results The recombinantion of pla smid was identifide to be correct by endonuclease restriction analysis and the B L-21 that contain the recombinant plasmid abundantly expressed the immunotoxin b y IPTG inducement.The size of the expressed protein is similar to that anticipat ed in MW. Conclusions The recombinant plasmid that can express the anti -carcinoma ScFv immunotoxin is successfully obtained.%目的 构建能表达抗胃癌单链抗体免疫毒素的重组质粒并进行表达。方法 应用基因工程方法,将抗胃癌的单链抗体基因与绿脓杆菌外毒素基因进行重组,得到的重组质粒转化大肠杆菌BL-21 ,鉴定后的阳性克隆经IPTG诱导表达。结果 重组质粒经酶切鉴定正确,IPTG诱导后可看到明显的表达带,分子大小与预计值相符。结论 得到了能表达抗胃癌单链抗体免疫毒素的重组质粒。

  17. Generation of a rabbit single-chain fragment variable (scFv) antibody for specific detection of Bradyrhizobium sp. DOA9 in both free-living and bacteroid forms. (United States)

    Vu, Nguyen Xuan; Pruksametanan, Natcha; Srila, Witsanu; Yuttavanichakul, Watcharin; Teamtisong, Kamonluck; Teaumroong, Neung; Boonkerd, Nantakorn; Tittabutr, Panlada; Yamabhai, Montarop


    A simple and reliable method for the detection of specific nitrogen-fixing bacteria in both free-living and bacteroid forms is essential for the development and application of biofertilizer. Traditionally, a polyclonal antibody generated from an immunized rabbit was used for detection. However, the disadvantages of using a polyclonal antibody include limited supply and cross-reactivity to related bacterial strains. This is the first report on the application of phage display technology for the generation of a rabbit recombinant monoclonal antibody for specific detection and monitoring of nitrogen-fixing bacteria in both free-living form and in plant nodules. Bradyrhizobium sp. DOA9, a broad host range soil bacteria, originally isolated from the root nodules of Aeschynomene americana in Thailand was used as a model in this study. A recombinant single-chain fragment variable (scFv) antibody library was constructed from the spleen of a rabbit immunized with DOA9. After three rounds of biopanning, one specific phage-displayed scFv antibody, designated bDOA9rb8, was identified. Specific binding of this antibody was confirmed by phage enzyme-linked immunosorbent assay (phage ELISA). The phage antibody could bind specifically to DOA9 in both free-living cells (pure culture) and bacteroids inside plant nodules. In addition to phage ELISA, specific and robust immunofluorescence staining of both free-living and bacteroid forms could also be observed by confocal-immunofluorescence imaging, without cross-reactivity with other tested bradyrhizobial strains. Moreover, specific binding of free scFv to DOA9 was also demonstrated by ELISA. This recombinant antibody can also be used for the study of the molecular mechanism of plant-microbe interactions in the future.

  18. 人源抗恶性疟原虫单链抗体库的制备及鉴定%Preparation and identification of a human single chain Fv antibody against Plasmodium falciparum

    Institute of Scientific and Technical Information of China (English)

    何光志; 俞琦; 王乾宇; 曹峰; 黄高; 韩洁; 蔡琨; 安传伟; 田维毅; 邓树轩; 高英; 李世军; 吴玛莉; 王平; 王文佳; 奚锦


    OBJECTIVE To construct humanize phage antibody library against Plasmodium falciparum and to screen specificity scFv to Plasmodium falciparum. METHODS Total RN A was extracted from fresh blood of patients with Plasmodium falciparum followed RT-PCR. The heavy-chain and light-chain variable region genes (VH and VL) repertoire of immunoglobulin were amplified respectively by PCR. Then they were linked as VH-linker-VL sequence forming scFv by SOE (splicing by overlap extension) PCR. These fragments were cloned into the phagemid pCANTAB5E and the recombinant phagemids were transformed to susceptible Escherichi a coli TGI by roporation. After rescuing with helper phage M13K07, A natural single-chain antibody library for Plasmodium falciparum was successfully constructed. RESULTS 15 antigen binding clones were identified from 20 individual phagemid clones. CONCLUSION This study shows the strategy of phage display technique and of RT-PCR from peripheral blood lymphocytes mRNA may provide an alternative approach.%目的 通过噬菌体展示技术构建人源抗恶性疟原虫单链抗体库(ScFv),从中筛选出高亲和力的抗体基因片段,为进一步单链抗体表达打下基础.方法 收集患者新鲜外周血,进行淋巴细胞分离和总RNA的提取,提取总RNA经RT-PCR扩增出入源抗体重链和轻链抗体库,采用用重叠延伸拼接法将VH与VL以Linker相连组装成单链抗体基因(ScFv),将ScFv基因片段连接至载体pCANTAB5E,转化大肠杆菌TG1感受态细胞,构建的鼠源性抗恶性疟原虫天然噬菌体抗体库.结果 从20个噬菌体克隆中筛选到15个具有弓形虫可溶性抗原结合活性的阳性克隆.结论 从外周血淋巴细胞中获取可变区基因,利用噬菌体抗体库技术制备人源抗恶性疟原虫单链抗体的策略是可行的.

  19. Human umbilical cord-drived mesenchymal stem cells as vehicles of CD20 specific-TRAIL fusion protein against non-Hodgkin’ s lymphoma%脐带间充质干细胞运载scFvCD20:sTRAIL融合蛋白对B-淋巴瘤细胞的生长抑制作用

    Institute of Scientific and Technical Information of China (English)

    范冬梅; 张晓龙; 张晴; 卢杨; 杨圆圆; 袁向飞; 张砚君; 熊冬生


    Objective:To study the therapeutic effect of a novel double-target system,in which human umbilical cord-derived MSCs were used as vehicles to deliver fusion protein scFvCD20:sTRAIL to non-Hodgkin ’ s lymphoma. Methods: The traditional methods in molecular biology were used to construct lentivirus expression vectors pLenR. scFvCD20: sTRAIL and contrast vectors. Human umbilical cord-derived MSCs ( HUMSCs ) were labeled with the copGFP by transducing with pseudo viral particles which had been packaged in 293T cells with four plasmid-lentivirus packaging system. Fusion protein scFvCD20:sTRAIL were secreted from MSC. scFvCD20:sTRAIL after that HUMSCs were infected by pseudo viral particles. CCK8 assay was applied to detect the antigen-restricted cell death induced by scFvCD20:sTRAIL in CD20-positive BJAB and Raji cells as well as CD20-negtive Jurkat cells and human normal peripheral blood mononuclear cells (PBMCs). To evaluate the therapeutic effect of MSC. scFvCD20:sTRAIL in vivo,ge-netically modified HUMSCs were intravenously injected into tumor-bearing mice with BJAB cells. The volume of tumor was measured every three days, and the inhibition ratio of tumor was calculated according to tumor volume. Results: Lentivirus expression vectors pLenR. scFvCD20:sTRAIL, pLenR. ISZ:sTRAIL, pLenR. scFvCD20 and pLenR. CopGFP were successfully constructed and these constructs could be expressed stably in HUMSCs by lentivirus transduction. scFvCD20:sTRAIL fusion protein produced a potent inhibition of cell proliferation in CD20-positive BJAB cells,moderate inhibition of the growth of Raji cells,and weak inhibition in CD20-negtive Jurkat cells when compared with ISZ-sTRAIL treatment,and it had no effect on normal human peripheral blood mononuclear cells (PBMCs). The MSC. scFvCD20:sTRAIL treatment significantly inhibited the tumor growth when compared with those treated with MSC. ISZ-sTRAIL. Conclusion: A double-target therapeutic system is well established, in which HUMSCs

  20. 抗黄曲霉毒素B1单链抗体在Sf9昆虫细胞中的表达与性质分析%Expression and Characterization of Anti-AFB1 scFv Expressed in Sf9 Cell

    Institute of Scientific and Technical Information of China (English)

    刘爱平; 李诚; 刘书亮; 王小红; 陈福生


    目的:在原核表达抗黄曲霉毒素B1(aflatoxin B1,AFB1)单链抗体(single chain Fv fragment,scFv)研究的基础上,为进一步了解和提高抗AFB1 scFv的活性,利用Sf9昆虫细胞表达抗AFB1scFv,并对其活性进行探索研究.方法:构建pFastBac 1-scFv2E6VHVL重组质粒,将重组质粒转化Escherichia coli(E.coli)DH10Bac细胞,进行蓝白斑筛选,挑取阳性克隆.提取相应的重组杆状病毒穿梭载体Bacmid侵染Sf9昆虫细胞,表达scFv,利用镍亲和层析法纯化scFv,并以ELISA检测scFv活性.结果:蓝白斑筛选后,经菌落PCR和测序验证挑取的白斑阳性单克隆含有正确的单链抗体基因.提取相应的重组杆状病毒穿梭载体Bacmid侵染Sf9昆虫细胞,通过Westem blot检测得知抗AFB1 scFv在Sf9昆虫细胞中成功表达.AFB1对scFv的抑制中浓度(IC50)为30μg/ml.结论:与E.coli BL21 (DE3)表达系统相比,scFv灵敏度转好,但仍有较大提升空间.

  1. Estudio de factibilidad para la implementación de un taller cautivo (desarrollo de productos) de clase mundial, para la empresa manufacturera FV Área Andina S.A. en la división griferia, Sangolquí, Ecuador.


    Castaneda Naranjo, Jacqueline Elizabeth


    El presente trabajo consiste en una propuesta para integrar y organizar un conjunto de departamentos de la vicepresidencia industrial de FV Área andina en una sola unidad de negocio de la empresa bajo la figura de un Taller Cautivo que además apalanque eficientemente los objetivos estratégicos de la empresa. En general se ha planteado estructurar al Taller Cautivo de Clase mundial para que sea autosustentable en el sentido de que aunque no sea fuente directa de ingresos para FV...

  2. 抗镰刀菌单链抗体在大肠杆菌中可溶性表达条件的研究%A Study on the Optimization of Condition for Soluble Expression of Fusarium-specific scFv Antibody in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    胡祖权; 李和平; 吴平; 廖玉才; 张静柏


    The objectives of the work is to optimize the conditions for inducing expression, and obtain the soluble and high-yield expression of a Fusarium-specific single-chain variable fragment(scFv)antibody in the periplasmic space of Escherichia coli XL1-Blue. The recombinant plasmid containing a Fusarium-specific scFv antibody FvSG7 was transferred into E. coli XL1-Blue. After the culture medium for inducing selected, the soluble expression level and activity of FvSG7 antibody were analyzed by Western blot and ELISA detection for studying the influence of temperature, IPTG concentration and induction time on expression level. The maximum productivity of soluble FvSG7 antibody was obtained after induction at 25℃for 2 h, with a final concentration of 0.1 mmol/L β-D-thiogalactopyranoside(IPTG)and the cultured bacteria growing to the OD600 value of 0.5. In conclusion, the soluble expression of FvSG7 antibody in the periplasm of E. coli XL1-Blue increased significantly by optimizing the expression’s conditions of induction temperature, IPTG concentration and induction time.%通过优化诱导表达条件,实现抗镰刀菌单链抗体在大肠杆菌周质中高效可溶性表达。将抗镰刀菌单链抗体FvSG7转化到大肠杆菌XL1-Blue中,确定诱导表达培养基,诱导温度、诱导剂IPTG的浓度和诱导时间条件下培养重组大肠杆菌,通过Western杂交和ELISA检测分析单链抗体的可溶性表达情况以及抗体的活性。重组大肠杆菌培养至OD600nm为0.5时加入终浓度为0.1 mmol/L IPTG,25℃诱导表达2 h可获得最大量的可溶性单链抗体。通过对诱导温度、IPTG浓度和诱导时间等表达条件的优化,可以显著提高FvSG7抗体在大肠杆菌XL1-Blue周质中的表达量。

  3. Antitumor mechanism and efficacy of the fusion protein consisting of lidamycin and anti-gelatinases single-chain Fv antibody%抗明胶酶单链抗体与力达霉素融合蛋白抗肿瘤作用机制及疗效研究

    Institute of Scientific and Technical Information of China (English)

    高瑞娟; 尚伯杨; 盛唯瑾; 赵春燕; 李电东; 甄永苏


    目的:探讨抗明胶酶单链抗体 Fv 与力达霉素(LDM)融合蛋白的抗肿瘤作用机制及疗效。方法采用三联径向加压 C4柱制备 LDM 发色团 AE,体外分子重建将其装入融合蛋白 Fv-LDP 中,Superdex 75层析获得 Fv-LDP-AE。反相 HPLC 测定纯度或相对含量。流式细胞术测定 DNA 含量;采用 SA-β-gal 染色、Western blot 和细胞伤口愈合实验分别检测药物对细胞衰老、蛋白表达和迁移的影响;通过动物模型评价药物疗效。结果 Fv-LDP-AE 承袭了 LDM 诱导肿瘤细胞周期阻滞、凋亡和裂亡的作用特点,并显示出更强的诱导衰老和抗迁移作用。Fv-LDP-AE 诱导凋亡和抑制迁移依次与 caspases 通路激活和 MMP-2降调节有关。Fv-LDP-AE 可抑制小鼠和裸鼠移植瘤生长,抑制率分别可达到86.6%和82.5%。结论 Fv-LDP-AE 对小鼠和裸鼠移植瘤有效,可能与其对细胞周期阻滞、凋亡、裂亡和衰老的诱导以及迁移的抑制有关。%Objective To investigate the antitumor mechanism and efficacy of the fusion protein Fv-LDP-AE composed of lidamycin (LDM) and anti-gelatinases single-chain Fv antibody. Methods Free AE (the active enediyne chromophore of LDM) prepared using the triple radial pressure C4 column was reassembled into fusion protein Fv-LDP by in vitro molecular reconstitution. The rebuilt fusion protein Fv-LDP-AE was obtained by Superdex 75 chromatography to remove the free AE. Reverse phase HPLC was used to determine the purity or the relative content of the drug components. DNA content was measured by flow cytometry. SA-β-gal staining, Western blot and cell wound healing assay were exploited to detect the effect of tested drugs on cell senescence, protein expression and cell migration, respectively. Antitumor efficacies of tested drugs were evaluated by different animal models. Results Fv-LDP-AE inherited the action characteristics derived from LDM including the induction of cell cycle

  4. Screening and immunological identification of the human ScFv antibody against PSMA%全人源抗PSMA单链抗体的筛选及免疫活性鉴定

    Institute of Scientific and Technical Information of China (English)

    张才田; 刘金霞


    Objective To screen and identify a human single-chain variable fragment(scFv) antibody against prostate specific membrane antigen(PSMA) from a human scFv antibody library.Methods Using a synthetic PSMA peptide as the coating antigen, the antibody library was screened by five rounds of combining-eluting-amplification.The phage antibody against PSMA with high specificity was screened out from the human scFv antibody library and its binding ability to the antigen was tested by ELISA.The soluble antibody was produced by plasmids extracted from highly specific clones, whose binding ability to PSMA was further identified by Western blot and immunohistochemistry.The affinity constant of the soluble antibody was measured by non-competitive ELISA.Results The screened phage antibody was specific for PSMA by ELISA.The soluble antibody was also specific for PSMA, its molecular weight was about 30 kD by SDS-PAGE and its affinity constant was about 5.077 × 106 L/mol.Conclusions The screened scFv antibody is specific and has low immunogenicity.It can be further used in the target treatment of malignant tumors.%目的 从全人源单链噬菌体抗体库中筛选出抗前列腺特异性膜抗原(PSMA)特异性单链抗体并进行免疫活性鉴定.方法 以合成的PSMA多肽为抗原,经过五轮吸附-洗脱-扩增,从单链噬菌体抗体库中筛选出特异性抗PSMA噬菌体抗体,ELISA检测其抗原结合能力,并对特异性较强的克隆提取质粒,表达可溶性抗体.Western Blotting和免疫组织化学检测其抗原结合性,非竞争ELSIA法检测其亲和常数.结果 从单链噬菌体抗体库中筛选出的噬菌体抗体,经ELISA鉴定为抗PSMA的特异性噬菌体抗体.抗PSMA可溶性抗体相对分子质量约为3.0×104,与PSMA特异性结合,其亲和常数约为5.077×106L/mol.结论 所得全人源抗PSMA单链抗体保留完整抗体分子结合抗原的特异性,免疫原性弱,是肿瘤导向治疗的理想栽体.

  5. Functional characterization of two scFv-Fc antibodies from an HIV controller selected on soluble HIV-1 Env complexes: a neutralizing V3- and a trimer-specific gp41 antibody.

    Directory of Open Access Journals (Sweden)

    Maria Trott

    Full Text Available HIV neutralizing antibodies (nAbs represent an important tool in view of prophylactic and therapeutic applications for HIV-1 infection. Patients chronically infected by HIV-1 represent a valuable source for nAbs. HIV controllers, including long-term non-progressors (LTNP and elite controllers (EC, represent an interesting subgroup in this regard, as here nAbs can develop over time in a rather healthy immune system and in the absence of any therapeutic selection pressure. In this study, we characterized two particular antibodies that were selected as scFv antibody fragments from a phage immune library generated from an LTNP with HIV neutralizing antibodies in his plasma. The phage library was screened on recombinant soluble gp140 envelope (Env proteins. Sequencing the selected peptide inserts revealed two major classes of antibody sequences. Binding analysis of the corresponding scFv-Fc derivatives to various trimeric and monomeric Env constructs as well as to peptide arrays showed that one class, represented by monoclonal antibody (mAb A2, specifically recognizes an epitope localized in the pocket binding domain of the C heptad repeat (CHR in the ectodomain of gp41, but only in the trimeric context. Thus, this antibody represents an interesting tool for trimer identification. MAb A7, representing the second class, binds to structural elements of the third variable loop V3 and neutralizes tier 1 and tier 2 HIV-1 isolates of different subtypes with matching critical amino acids in the linear epitope sequence. In conclusion, HIV controllers are a valuable source for the selection of functionally interesting antibodies that can be selected on soluble gp140 proteins with properties from the native envelope spike.

  6. Preparation and Identification of a Human Single Chain Fv Antibody Against Ascaris lumbricoides%抗蛔虫人源单链抗体库的构建及鉴定

    Institute of Scientific and Technical Information of China (English)

    何光志; 田维毅; 高英; 王平; 王文佳; 奚锦; 俞琦; 王乾宇; 黄高


    Objective To construct humanize phage antibody library against Ascaris lumbricoides and to screen specificity scFv to Ascaris lumbricoides. Methods Total RNA was abstracted from peripheral blood lympho-cytes of 20 persons, and was used to amplify VH and VL gene by RT-PCR. The amplified VH and VL genes were spliced to form scFv gene which was cloned into pCANTAB-SE phagemid, and the constructed recombinant phage-mid was transformed to E. Coli TC1 to construct human natural single-chain antibody library from which positive clones were screened. Results A primary library of 1.5 × 106 and a second library of 1.2 × 106 were constructed. Conclusion This study was to provide us the basis for radionuclide imaging and therapy for ascariasis.%目的:构建抗蛔虫人源单链抗体库,从中筛选建抗蛔虫人源特异性单链抗体.方法:分离10个患蛔虫病人的淋巴细胞,提取总RNA反转录为cDNA,PCR扩增人抗体重链(VH)和轻链(VL)可变区基因,采用SOE-PCR法将VH和VL片段随机拼接成scFv片段,并克隆入噬菌粒载体pCANTAB5E中,构建噬菌体单链抗体库.结果:初级库库容量为1.8×106,在大肠杆菌TG1中重组后得到1.6×106的次级抗体库.结论:本研究成功构建抗蛔虫人源单链抗体库,拟在为蛔虫病的预防、诊断、治疗奠定基础.

  7. 肿瘤干细胞表面标志物CD133单链抗体基因的分离与鉴定%Identification and characterization of single chain antibody of cancer stem cell surface marker CD133 scFv CD133

    Institute of Scientific and Technical Information of China (English)

    韩瑜; 周炳喜; 张延瑞; 韩双印


    Objective To identify,construct and express scFv CD133,verify its biological function.Methods VL and VH were isolated from hybridoma of mAb CD133 by using antibody engineering technology.Its DNA sequencing and CDR were determined.scFv CD133 was then cloned into pET32a,transformed into Origami,induced by IPTG,purified by Ni2+-NTA His resin.Its affinity and specificity were tested by NH4SCN elution and ELISA.Results The size of VL and VH of scFv CD133 was 339 bp and 342 bp,which coded 113 and 114 amino acid separately.Its VL belonged to mouse Igκ chain and VH belonged to mouse IgG heavy chain subtype I.The molecular weight of scFv CD133 was about 27 × 103 which was testified by SDSPAGE and Western blot.Its affinity and specificity were also verified.Conclusion scFv CD133 has been successfully constructed and expressed in Origami,which could supply basis for target therapy of CD+133 cancer stem cell.%目的 分离、构建和表达抗人CD133单链抗体(scFvCD133),测定其生物学活性.方法 用抗体工程技术从抗人CD133单克隆抗体(mAb)杂交瘤细胞中分离抗体可变区基因(VL和VH),测定DNA序列并确定抗体互补决定区(CDR);将scFv CD133基因克隆至pET32a中,转化Origami菌株,IPTG诱导,Ni2+-NTA His树脂纯化单链抗体,梯度硫氰酸盐洗脱法和酶联免疫吸附(ELISA)法检测其亲和性和特异性.结果 scFvCD133基因VL和VH长度分别为339 bp和342 bp,各编码113和114个氨基酸,其中VL隶属于小鼠Ig κ轻链,VH隶属于小鼠Ig重链Ⅰ亚类.scFvCD133经SDS-PAGE和Western blot 分析证明相对分子质量为27 × 103,体外实验具有一定的亲和性和特异性.结论 获得scFv CD133基因,为CD+133肿瘤干细胞的靶向治疗奠定了基础.

  8. [Preparation of heat shock protein 70 (Hsp70) and idiotypic determinant single-chain antibody (Id-ScFv) in a patient with B-cell chronic lymphatic leukemia (B-CLL) and antitumor effect of peptide complex Hsp70-Id]. (United States)

    Ye, Qin; Wang, Zhi-Hua; Qin, Shu-Kui


    Idiotypic determinant (Id) of malignant B lymphocyte surface membrane immunoglobulin (SmIg) is not only a specific marker for chronic B-cell lymphatic leukemia (B-CLL) but also a specific antigen as an attractive target for active immunotherapy. However, as a small self antigen, it has poor immunogenicity. As an important molecule for antigen presentation, heat shock protein 70 (Hsp70) can efficiently strengthen the immunogenicity of antigen. In this study, we prepared Hsp70 and idiotypic determinant single-chain antibody (Id-ScFv) fragment against SmIg in patient with B-CLL and explored the in vitro antitumor effect of peptide complex Hsp70-Id and the possible immune mechanism. Id-ScFv and human Hsp70 proteins were expressed in E.coli and identified by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and enzyme-linked immunosorbent assay (ELISA), and purified by metal chelate chromatography and DEAE ion-exchange chromatography. The purified Hsp70 was combined with the prepared Id-ScFv to produce peptide complex Hsp70-Id in vitro. Peripheral blood mononuclear cells (PBMCs) from healthy human were stimulated with Id-ScFv, HSP70 and HSP70-Id, separately. The proliferation rate of PBMCs was determined by MTT assay; the changes of T lymphocyte subsets were assessed by flow cytometry (FCM)û the levels of interleukin-12 (IL-12) and tumor necrosis factor-alpha (TNF-alpha) in supernatant were detected by ELISA. Killing efficiency of activated PBMCs to chronic B-cell leukemia cell line Daudi, chronic myelogenous leukemia cell line K562 and hepatoma carcinoma cell line HepG2 were tested by cell counting. Id-ScFv and Hsp70 were expressed successfully in E.coli. The proliferation rate of PBMCs and secreting levels of IL-12 and TNF-alpha of lymphocytes in Hsp70-Id group were significantly higher than those in Id-ScFv and HSP70 groups (P<0.05), which were significantly higher than that in negative control group (P<0.05). The proportion of CD8+ T cells was

  9. Coastal Vulnerability Index (CVI) for Fire Island National Seashore (fiis_shore) (United States)

    U.S. Geological Survey, Department of the Interior — A coastal vulnerability index (CVI) was used to map the relative vulnerability of the coast to future sea-level rise within Fire Island National Seashore in New...

  10. Investigating causal relationship between stock return with respect to exchange rate and FII: evidence from India


    Kumar, Sundaram


    The purpose of this paper is to investigate the relationship between macroeconomic parameters like Exchange rate and foreign institutional investment with stock returns in India, in particular at National Stock Exchange. I find that both stock returns and exchange rate are integrated of order one. The Engle–Granger Cointegration test is then performed, suggesting that there is not a long-run equilibrium relationship between stock returns and exchange rates at 5% significance level. Moreover, ...

  11. FII 2 and the applicable freedoms of movement in third country situations

    NARCIS (Netherlands)

    E. Nijkeuter (Erwin); M.F. de Wilde (Maarten)


    markdownabstractIn this article, the authors discuss the approach that the Court of Justice of the European Union (CJEU) adopts in deciding which of the European treaty freedoms apply to third country situations. On 13 November 2012 the CJEU delivered a landmark ruling on this matter in the Test Cla

  12. Coastal Vulnerability Index (CVI) for Fire Island National Seashore (fiis_shore) (United States)

    U.S. Geological Survey, Department of the Interior — A coastal vulnerability index (CVI) was used to map the relative vulnerability of the coast to future sea-level rise within Fire Island National Seashore in New...

  13. 全人源抗鼻咽癌噬菌体单链抗体的筛选与鉴定%Screening and characterization of human scFv antibodies against nasopharyngeal carcinoma

    Institute of Scientific and Technical Information of China (English)

    李艳东; 谢平丽; 王甲甲; 李跃辉; 胡锦跃; 李官成


    目的 从全人源抗鼻咽癌噬菌体抗体库中筛选特异性单链抗体(ScFv),并对其特异性进行鉴定.方法 通过噬菌体表面展示技术把ScFv表达在噬菌体表面,以鼻咽癌细胞作为抗原,用抗原递减法,通过"吸附-洗脱-扩增"过程筛选并富集特异性抗体,及ELISA筛选,获得特异阳性克隆进行免疫组化鉴定并测序.结果 通过对抗体库进行三轮正负淘洗和富集后,随机挑选4212个克隆进行ELISA,发现3个克隆对CNE2呈强阳性反应,而与人正常细胞系HUVEC等呈弱阳性反应或不反应.对克隆HNSAO33进一步进行免疫细胞化学验证,结果与ELISA反应一致;免疫组织化学鉴定表明克隆HNSAO33与鼻咽癌组织和鼻咽组织阳性率的差别有统计学意义.结论 通过淘选富集、ELISA和免疫化学鉴定获得特异性较强的噬菌体克隆,为鼻咽癌发病机制的研究和临床诊断以及治疗奠定了基础.%Objective To screen the anti-nasopharyngeal carcinoma scFv from a human anti-nasopharyngeal carcinoma single-chain phage antibody library, and identify its characteristics. Methods The single-chain phage antibody library was subjected to three rounds of positive and negative cell panning and enrichment, and then it was selected by ELISA. The binding specificity of phage antibodies with naso-pharyngeal carcinoma cells was confirmed by immunohistochemistry. Results After panning, enrichment and testing by ELISA, 3 phage an-tibody clones reacting with CNE2 more strongly than HUVEC and NP69 were picked out from 4212 clones. One clone, HNSAO33, was fur-ther analyzed after DNA sequencing. The results of immunohistochemistry with cultured cells were similar to those of ELISA. HNSAO33 spe-cifically reacted to nasopharyngeal carcinoma cells in most human nasopharyngeal carcinoma tissue sections except a few human normal naso-pharyngeal tissue sections. The distinction of positive rates was of a great statistical significance. Conclusion ELISA

  14. Prothrombin polymorphism A19911G, factor V HR2 haplotype A4070G, and plasminogen activator-inhibitor-1 polymorphism 4G/5G and the risk of retinal vein occlusion. (United States)

    Kuhli-Hattenbach, Claudia; Hellstern, Peter; Nägler, Dorit Karin; Kohnen, Thomas; Hattenbach, Lars-Olof


    Thus far, no data has become available to evaluate systematically the prevalences of prothrombin polymorphism A19911G (PT A19911G), factor V HR2 haplotype A4070G (FV A4070G), or plasminogen activator-inhibitor-1 polymorphism 4G/5G (PAI-1 4G/5G) in patients who develop retinal vein occlusion (RVO) without cardiovascular risk factors. We retrospectively evaluated comprehensive thrombophilia data from 42 preselected RVO patients without cardiovascular risk factors. The prevalences of different gene mutations and polymorphisms including factor V Leiden mutation G1691A (FVL), FV A4070G, prothrombin mutation G20210A, PT A19911G, and PAI-1 4G/5G were compared with 241 healthy controls matched for age and sex. A total of 20 patients (47.7%) were found to carry thrombophilic gene polymorphisms including FVL, FV A4070G, and homozygous PT A19911G compared with 72 of 241 controls (29.9%; p = 0.03). Subgroup analysis of patients with a significant personal or family history of thromboembolism revealed a high prevalence of FVL, FV A4070G, and homozygous PT A19911G (p = 0.005). FV A4070G was found to be significantly associated with at least two other heterozygous or one homozygous gene polymorphisms (p = 0.02). Multivariate analysis revealed the presence of FVL (p = 0.0017) and homozygous PT A19911G (p = 0.03) polymorphism as independent risk factors for the development of RVO. Our results indicate that in selected RVO patients screening for thrombophilic gene polymorphisms including FVL, FV A4070G and homozygous PT G19911A may be helpful in a high percentage of cases. Our findings suggest that hereditary thrombophilia associated with RVO is more likely to be multigenic than caused by any single risk factor.

  15. Recurrence of superficial vein thrombosis in patients with varicose veins. (United States)

    Karathanos, Christos; Spanos, Konstantinos; Saleptsis, Vassileios; Tsezou, Aspasia; Kyriakou, Despina; Giannoukas, Athanasios D


    To investigate which factors other than history of superficial vein thrombosis (SVT) are associated with recurrent spontaneous SVT episodes in patients with varicose veins (VVs). Patients with a history of spontaneous SVT and VVs were followed up for a mean period of 55 months. Demographics, comorbidities, and thrombophilia screening test were analyzed. Patients were grouped according to the clinical-etiology-anatomy-pathophysiology classification. A multiple logistic regression analysis with the forward likelihood ratio method was undertaken. Thirteen patients out of 97 had a recurrence SVT episode during the follow-up period. All those patients were identified to have a thrombophilia defect. Protein C and S, antithrombin, and plasminogen deficiencies were more frequently present in patients without recurrence. Gene mutations were present in 38% in the nonrecurrence group and 77% in the recurrence group. After logistic regression analysis, patients with dislipidemia and mutation in prothrombin G20210A (FII) had an increased risk for recurrence by 5.4-fold and 4.6-fold, respectively. No deep vein thrombosis or pulmonary embolism occurred. Dislipidemia and gene mutations of F II are associated with SVT recurrence in patients with VVs. A selection of patients may benefit from anticoagulation in the short term and from VVs intervention in the long term. © The Author(s) 2015.

  16. Thrombophilia risk is not increased in children after perinatal stroke. (United States)

    Curtis, Colleen; Mineyko, Aleksandra; Massicotte, Patricia; Leaker, Michael; Jiang, Xiu Yan; Floer, Amalia; Kirton, Adam


    Perinatal stroke causes cerebral palsy and lifelong disability. Specific diseases are definable but mechanisms are poorly understood. Evidence suggests possible associations between arterial perinatal stroke and prothrombotic disorders but population-based, controlled, disease-specific studies are limited. Understanding thrombophilia in perinatal stroke informs pathogenesis models and clinical management. We conducted a population-based, prospective, case-control study to determine the association of specific perinatal stroke diseases with known thrombophilias. Children with idiopathic, MRI-classified neonatal arterial ischemic stroke (NAIS), arterial presumed perinatal ischemic stroke (APPIS), or fetal periventricular venous infarction (PVI) were recruited. Standardized thrombophilia evaluations were performed after 12 months of age on stroke cases and controls including quantified protein C and S, antithrombin, factors VIII/IX/XI, fibrinogen, lipoprotein(a), homocysteine, lupus anticoagulant, anticardiolipin antibodies and genotyping of factor V Leiden (FVL), factor II G20210A (FII), and MTHFR C677T. A total of 212 children were studied: 46 NAIS, 34 APPIS, 55 PVI, and 77 controls (53% male, median 4.8 years). Of 14 parameters, no differences were observed in 12 including all common thrombophilias. Mean prothrombin time was shorter in arterial strokes (p<0.001). Rates of antiphospholipid antibodies were low, comparable to controls, and resolved on repeat testing. FVL and FII rates were comparable to population norms. Total number of possible abnormalities did not differ between cases and controls. Our prospective, population-based, controlled, disease-specific study suggests minimal association between perinatal stroke and thrombophilia. This does not exclude the possibility of disordered coagulation at the time of stroke but suggests testing in childhood is not indicated.

  17. 一种特异性识别斑马鱼卵黄蛋白原的噬菌体展示单链抗体的可溶性表达方案%Strategy for soluble expression of phage-displayed scFv antibody specific for zebrafish vitellogenin

    Institute of Scientific and Technical Information of China (English)

    饶瑜; 钟利桥; 张凤; 张晓华; 戴和平


    We developed a new method for soluble expression of phage-displayed scFv antibody specific for zebrafish vitellogenin. The scFv antibody F5 could bind zebrafish vitellogenin specifically in phage-displayed form but not soluble form. The gene of scFv antibody F5 was cloned into vector pET 32a and transferred into Escherichia coli ori DE3. With inducible expression, soluble scFv antibody 32a-F5 was obtained successfully and could also specifically bind to zebrafish vitellogenin. The insoluble expression of phage-displayed scFv antibody was a common problem in the practical use of phage display. This study offered a feasible way to express soluble scFv antibodies with biological activity.%为了实现特异性识别斑马鱼卵黄蛋白原的噬菌体展示单链抗体的可溶性表达,将不能以可溶性蛋白形式表达的、只能以噬菌体展示形式特异性识别斑马鱼卵黄蛋白原的单链抗体F5的基因,克隆到pET 32a载体并转化入大肠杆菌ori DE3中.结果表明,通过诱导表达,可获得可溶性的并且仍特异性识别斑马鱼卵黄蛋白原的单链抗体32a-F5.噬菌体展示单链抗体不能可溶性表达是噬菌体展示技术应用中常见的问题,该方法提供了一种表达可溶性单链抗体的可行性方案.

  18. Construction, expression and identification of scFv against microcystin-LR%抗微囊藻毒素单链抗体基因的构建、表达及初步鉴定

    Institute of Scientific and Technical Information of China (English)

    宋丽敏; 张维; 陈明; 林敏; 潘家荣


    利用RT-PCR方法从抗微囊藻毒素-LR(MC-LR)单克隆抗体的杂交瘤细胞中扩增出抗体的V_H和V_L基因,构建了抗MC-LR分子的单链抗体(scFv)基因.SDS-PAGE和Western blot分析结果显示,该单链抗体基因在大肠杆菌Origami 2中特异性表达出分子量约为30kD的融合蛋白.通过Ni-NTA金属亲和层析法对可溶性表达产物进行纯化,获得的目的蛋白浓度为0.115mg/mL.ELISA反应结果表明该单链抗体能与MC-LR特异性结合.此研究为制备多价高亲和力抗MC-LR抗体奠定了基础.

  19. 绿脓杆菌外毒素A单链抗体基因的制备及其克隆的研究%Preparation of the Anti-PEA Monoclone Antibody and the Cloning of Its ScFv

    Institute of Scientific and Technical Information of China (English)

    崔淑芳; 汤球; 郝光荣; 曹平



  20. Specific single chain variable fragment (ScFv) antibodies to angiotensin II AT(2) receptor: evaluation of the angiotensin II receptor expression in normal and tumor-bearing mouse lung. (United States)

    Tamura, Masaaki; Yan, Heping; Zegarra-Moro, Ofelia; Edl, Jennifer; Oursler, Stephanie; Chard-Bergstrom, Cindy; Andrews, Gordon; Kanehira, Tsutomu; Takekoshi, Susumu; Mernaugh, Ray


    To gain insight into the mechanism by which angiotensin II type 2 receptor (AT(2)) regulates carcinogen-induced lung tumorigenesis, we have newly developed anti-AT(2) single chain variable fragment (ScFv) antibodies using a rodent phage-displayed recombinant antibody library with various peptide fragments of the receptor protein, and investigated the expression of the AT(2) receptor protein. The specificity of the antibodies was verified using AT(2) over-expressing COS-7 cells and AT(2) naturally expressing PC12W cells. In control wild type mouse lung, a stronger immunoreactivity was observed in bronchial epithelial cells. A moderate immunoreactivity was detected in pulmonary vascular walls and vascular endothelial cells. In the lungs possessing tobacco-specific nitrosamine (NNK)-induced tumors, significantly increased AT(2) and AT(1 )immunostaining was observed in adenomatous lesions. These data suggest that the increase in both receptors' expression in the alveolar epithelial cells may be accompanied with the onset of NNK-induced tumorigenesis and hence play important roles in lung tumorigenesis.

  1. 人源抗创伤弧菌溶血素蛋白单链抗体的筛选及初步鉴定%Screening and initial identification of human anti-vibrio vulnificus haemolysin ScFv

    Institute of Scientific and Technical Information of China (English)

    谢学渊; 付凯飞; 王欲晓; 吴成林; 周丽君


    目的 从天然单链(scFv)大容量噬菌体抗体库中筛选特异性抗创伤弧菌溶血素蛋白(vibrio vulnificus haemolysin,VVH)的scFv并进行初步鉴定.方法 以表达纯化后的VVH为靶抗原,采用酸洗脱的方法从天然大容量噬菌体抗体库中筛选人源抗VVH噬菌体单链抗体,并将噬菌体抗体的上清梯度稀释后感染HB2151菌,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达后获得抗VVH蛋白可溶性单链抗体,ELISA检测其与抗原结合活性,DNA序列测定鉴定抗体序列.结果 经过4轮筛选,共筛选到22株可以与VVH结合的噬菌体阳性克隆,其中8株获得可溶性表达,DNA序列分析表明获得3株可变区基因型不同的单链抗体.结论 利用大容量噬菌体抗体库筛选技术获得抗创伤弧菌溶血素蛋白的可溶性抗体,为创伤弧菌感染的治疗奠定了基础.%Objective To screen specific human antibodies against Vibrio vulnificus hemolysin (VVH ) from natural single stranded large capacity phage antibody library and to perform initial identification of the antibodies,for the treatment of vibrio vulnificus infection.Methods The specific antibody against purified VVH was screened from large natural phage antibody library by 4 rounds of " adsorption-elution-amplification" screening process.Soluble ScFvs were prepared through infecting E coli.HB2151 and inducing expression with IPTG.The antigen binding activity and DNA sequence of the antibodies against VVH were identified by ELISA and DNA fingerprint analysis.Results After 4 rounds of panning,22 positive clones with specific binding ability to VVH were obtained,of which 8 obtained soluble expressions.DNA sequence analyses showed that the variable region genes of the clones belonged to different subgroups of immunoglobulin gene family.Conclusions The acquirement of human anti-VVH ScFv from large phage antibody library has laid a good foundation for the treatment of vibrio vulnificus infection.

  2. Soluble expression and charaterization of anti-carbofuran single chain Fv%抗克百威单链抗体的可溶性表达载体的构建及鉴定

    Institute of Scientific and Technical Information of China (English)

    邓龙; 王弘; 周思; 冼燕萍; 郭新东


    为构建克百威(carbofuran,CBF)单链抗体(scFv)可溶性表达载体,实现其在大肠杆菌中的表达.以pCANTAB5E-scFv质粒为模板扩增CBF的重链(VH)及轻链(VL)片段,通过引物设计引入由15个氨基酸组成的连接肽(Gly4Ser)3,经重叠延伸拼接重链及轻链片段,并通过PCR扩增得到scFv基因,再与载体pLIP6/GN连接,转化大肠杆菌BI21,阳性克隆质粒经PCR鉴定并测序.重组菌经0.6 μmol/L异丙基硫代半乳糖苷(IPTG)及低温诱导表达重组单链抗体,并通过SDS-PAGE和Western blotting对其鉴定.采用ELISA法检测重组单链抗体的抗原结合活性.结果表明重组质粒pLIP6/GN-scFv含有插入片段,scFv与碱性磷酸酶(AP)相连得到重组蛋白的分子量接近78 ku,可被游离的CBF竞争性抑制,IC50值为26.80 ng/mL.这说明成功构建了重组质粒pLIP6/GN-scFv并实现了其在大肠杆菌中的可溶性表达,为研究其在免疫分析方法中的应用奠定了基础.

  3. Thermodynamic Stability Analysis of Tolbutamide Polymorphs and Solubility in Organic Solvents. (United States)

    Svärd, Michael; Valavi, Masood; Khamar, Dikshitkumar; Kuhs, Manuel; Rasmuson, Åke C


    Melting temperatures and enthalpies of fusion have been determined by differential scanning calorimetry (DSC) for 2 polymorphs of the drug tolbutamide: FI(H) and FV. Heat capacities have been determined by temperature-modulated DSC for 4 polymorphs: FI(L), FI(H), FII, FV, and for the supercooled melt. The enthalpy of fusion of FII at its melting point has been estimated from the enthalpy of transition of FII into FI(H) through a thermodynamic cycle. Calorimetric data have been used to derive a quantitative polymorphic stability relationship between these 4 polymorphs, showing that FII is the stable polymorph below approximately 333 K, above which temperature FI(H) is the stable form up to its melting point. The relative stability of FV is well below the other polymorphs. The previously reported kinetic reversibility of the transformation between FI(L) and FI(H) has been verified using in situ Raman spectroscopy. The solid-liquid solubility of FII has been gravimetrically determined in 5 pure organic solvents (methanol, 1-propanol, ethyl acetate, acetonitrile, and toluene) over the temperature range 278 to 323 K. The ideal solubility has been estimated from calorimetric data, and solution activity coefficients at saturation in the 5 solvents determined. All solutions show positive deviation from Raoult's law, and all van't Hoff plots of solubility data are nonlinear. The solubility in toluene is well below that observed in the other investigated solvents. Solubility data have been correlated and extrapolated to the melting point using a semiempirical regression model.

  4. Acquired Activated Protein C Resistance, Thrombophilia and Adverse Pregnancy Outcomes: A Study Performed in an Irish Cohort of Pregnant Women

    Directory of Open Access Journals (Sweden)

    Sara Sedano-Balbás


    Full Text Available The combination of thrombophilia and pregnancy increases the risk of thrombosis and the potential for adverse outcomes during pregnancy. The most significant common inherited risk factor for thrombophilia is activated protein C resistance (APCR, a poor anticoagulant response of APC in haemostasis, which is mainly caused by an inherited single-nucleotide polymorphism (SNP, factor V G1691A (FV Leiden (FVL, referred as inherited APCR. Changes in the levels of coagulation factors: FV, FVIII, and FIX, and anticoagulant factors: protein S (PS and protein C (PC can alter APC function causing acquired APCR. Prothrombin G20210A and methylenetetrahydrofolate reductase (MTHFR C677T are prothrombotic SNPs which in association with APCR can also increase the risk of thrombosis amongst Caucasians. In this study, a correlation between an acquired APCR phenotype and increased levels of factors V, VIII, and IX was demonstrated. Thrombophilic mutations amongst our acquired APCR pregnant women cohort are relatively common but do not appear to exert a severe undue adverse effect on pregnancy.

  5. Genetic polymorphisms and cerebrovascular disease in children with sickle cell anemia from Rio de Janeiro, Brazil

    Directory of Open Access Journals (Sweden)

    Isaac Lima da Silva Filho


    Full Text Available The aim of the present work was to examine possible genetic risk factors related to the occurrence of cerebrovascular disease (CVD in Brazilian population, the frequency of βS-globin gene haplotypes and co-inheritance with α-thalassemia (-α3.7kb and single nucleotide polymorphism of methylenetetrahydrofolate reductase (MTHFR-C677T, Factor V Leiden (FV-G1691A and prothrombin (PT-G20210A genes in children from Rio de Janeiro. Ninety four children with sickle cell anemia (SCA were included, 24 patients with cerebrovascular involvement and 70 patients without CVD as control group. The mean age of children at the time of the cerebrovascular event was similar to the control group. The frequency of -α3.7kb thalassemia was similar in both groups (p=0.751. Children with Bantu/Atypical βS-globin gene haplotype presented 15 times more chance (OR=15.4 CI 95% 2.9-81.6 of CVD than the other βS-globin gene haplotypes. The C677T polymorphism of MTHFR gene was similar in both groups (p=0.085. No mutation in the FV Leiden or PT genes was found. A large study seems necessary to establish the role of these genetic polymorphisms in Brazilian miscegenated population.

  6. 去唾液酸糖蛋白受体特异性单链抗体的优化表达及亲和常数的测定%Optimized expression of a single-chain Fv antibody against human asialoglycoprotein receptor and determination of its affinity constant

    Institute of Scientific and Technical Information of China (English)

    王炜煜; 易继林; 邓云华; 司进; 王从俊; 李翔; 曹利民


    目的: 表达及纯化抗去唾液酸糖蛋白受体(ASGPR)的单链抗体的可溶性,并测定其亲和常数.方法: 用噬菌体C1克隆感染E.coli HB2151,挑取单个菌落接种于2×TY培养基中,于37℃震荡培养过夜.将培养物作1∶ 100稀释并转种后,用终浓度为0.25、0.5、1.0 mmol/L的IPTG,分别在37℃、25℃和20℃下诱导表达过夜.取其培养上清,用饱和硫酸铵沉淀后,以120 g/L SDS-PAGE分析.另外,将饱和硫酸铵沉淀物用30 mL PBS重新溶解、透析除盐后,用Ni2+螯合柱进行纯化,再以120 g/L SDS-PAGE鉴定纯化scFv C1的纯度.用非竞争酶免疫法测定scFv的亲和常数.结果: 用0.5 mmol/L IPTG在25℃诱导过夜,表达的scFv C1的量较多,其相对分子质量(Mr)约为28 000,以可溶性的形式存在于培养基中.通过Ni2+亲和柱纯化后scFv C1的纯度在95%以上,产量约为0.8 mg/L.scFv的亲和常数为(2.31±0.36)×10-7 mol/L.结论: 以筛选的C1噬菌体感染E.coli HB2151后可表达低亲和力的可溶性scFv,对肝癌的基因治疗具有潜在的应用价值.

  7. 鼠源性人乳腺癌单链抗体库的构建与乳腺癌相关抗体的筛选%Construction of scFv Library with Human Breast Cancer from Immunised Mice and Screening Antibodies Binding to Breast Cancer

    Institute of Scientific and Technical Information of China (English)

    袁翔; 胡宝成


    目的:获得乳腺癌的噬菌体呈现型单链抗体(scFv)库,筛选与乳腺癌细胞特异结合的抗体,为乳腺癌的诊断和治疗奠定基础.方法:用乳腺癌细胞系MCF-7、T47D、MDA-MB-435免疫BALB/c小鼠,取脾脏提取总RNA,用RT-PCR分别扩增抗体重、轻链可变区(VH和VL)基因,经Linker连接形成scFv基因片段.将scFv基因片段与噬菌粒载体pCANTAB 5E的连接产物转化大肠杆菌TG1.用辅助噬菌体M13KO7进行超感染,获得重组噬菌体抗体.选用乳腺癌细胞系MCF-7和人正常肝细胞系HL02做正负差异的筛选细胞,通过5轮筛选,随机挑取克隆,经phage-ELISA筛选特异性结合MCF-7细胞的scFv.结果:构建了1个库容为1.3×106的单链抗体库.筛选到2株与MCF-7细胞有较高结合活性的噬菌体-单链抗体scFv-873和scFv-874.数据库搜索表明这2株单链抗体基因是与以往抗体序列不同的新基因.用Western blot检测了这2株单链抗体在琥珀密码子非抑制型菌株TOP10中的表达情况.结论:筛选到2个与乳腺癌细胞结合特异性较好的单链抗体,为乳腺癌的诊断和治疗研究奠定了基础.

  8. 抗人宫颈癌单链抗体的基因构建、空间结构和进化分析%Construction, structure prediction and phylogenetic analysis of murine scFv gene against human cervical cancer

    Institute of Scientific and Technical Information of China (English)

    王莹; 李旭; 陈葳


    目的: 构建抗人宫颈癌单链抗体(scFv)基因CSAs-1,并进行二级结构和三级结构预测、理化性质分析及进化树构建.方法: 采用重叠延伸PCR方法, 以能特异性分泌抗人宫颈癌mAb的杂交瘤细胞株CSA125为原料,构建抗人宫颈癌scFv基因CSAs-1, 进行测序; 并通过Internet对其进行结构预测和进化树构建.结果: 抗人宫颈癌scFv基因CSAs-1有834 bp,对应278个氨基酸的多肽, 等电点预测值7.215,为碱性蛋白质; PHDsec二级结构显示CSAs-1属于α+β蛋白, V H和V L区均有多个蛋白激酶C磷酸化位点、酪蛋白激酶II磷酸化位点; CSAs-1三级结构建模显示V L和V H形成一个疏水的“口袋”, Linker游离于此结构之外.以上结果表明: CSAs-1符合scFv的结构特点,明显具有抗原结合位点的空间构象, 理论上应该具有良好的抗原结合活性.进化分析发现脊椎动物V基因形成三个进化群, CSAs-1 V区分布于进化树的A群中.结论: 构建一个鼠源性抗人宫颈癌scFv基因CSAs-1,为该基因的进一步原核和真核表达打下了基础; 应用信息学技术所获得的预测和分析结果为CSAs-1表达、纯化和活性研究提供了大量的信息; 为进一步分析抗体V H、V L在抗体功能中的作用、改造抗体、实现抗体的人源化、提高抗体的活性等方面提供了一定的依据.

  9. 细胞表达流感病毒抗体CR6261VH与scFv对流感病毒感染的影响%Effect of antibody CR6261 VH and scFv expression on influenza virus infection

    Institute of Scientific and Technical Information of China (English)

    李燕; 李秀岚; 严景华


    The emerging CR6261 antibody could neutralize several subtype influenza virus with high affinity, whose VH domain binds to the HA protein conserved domain. Therefore, it has drawn much attention as a potential broad-spectrum therapeutic antibody against influenza virus. In this study, we constructed the eukaryotic expression vectors pCR6261VH, pCR6261VH-GFP and pCR6261scFv and screened the monoclonal cell lines that could stably express CR6261VH, CR6261VH-GFP and CR6261scFv on the cell membrane. After influenza virus infecting the stable cell lines, the titers of viruses were tested by hemagglutination inhibition test. The result shows that the titers of viruses in CR6261scFv and CR6261VH-GFP stable expression cell lines decreased and there was no obvious discrimination between the CR6261VH expression cell line and the negative control, suggesting that CR6261VH and CR6261scFv expressing on the cell membrane could partly inhibit the virus infection. Though the effective inhibition strategy is undergoing, our research will provide new clues for the breeding of anti influenza transgenic animals.%由于新发现的流感病毒抗体CR6261可以中和多种亚型的流感病毒,而且是其VH区结合于流感病毒蛋白HA的保守区,因此该抗体有望成为一种广谱的流感治疗性抗体而备受关注.通过构建真核表达栽体pCR6261VH、pCR6261VH-GFP、pCR6261 scFv,成功地筛选到在细胞膜上稳定表达CR6261 VH、CR6261VH-GFP与CR6261 scFv的单克隆细胞系.用流感病毒去感染稳定细胞系,间隔12 h取上清检测病毒血凝效价.结果表明:稳定表达CR6261 scFv和CR6261VH-GFP细胞系能够降低病毒的血凝效价,而表达CR6261VH细胞系的血凝效价与对照无显著差异.说明CR6261VH与CR6261scFv在细胞膜上表达能够部分抑制病毒侵染细胞.尽管更有效的抑制策略还在研究中,但本研究将为抗流感转基因动物的育种提供新的思路.

  10. Screening scFv Specific to Vcam-1 by Phage Display Library and Its Activity Evaluation%噬菌体展示库筛选构建血管细胞黏附分子-1单链抗体及其效价检测

    Institute of Scientific and Technical Information of China (English)

    刘纯宝; 宋夷龄; 张永学


    目的:从噬菌体重组抗体库中筛选获得靶向血管细胞黏附分子‐1(Vcam‐1)的单链抗体,纯化浓缩后进行亲和性鉴定,并与单克隆抗体效价进行比较。方法扩增Vcam‐1基因克隆质粒并转入真核细胞中表达获得Vcam‐1抗原蛋白,纯化后包被免疫管,通过4轮压力逐渐增大的“吸附‐洗脱‐扩增”过程筛选获得阳性克隆。对阳性克隆进行ELISA检测,选取效价高的克隆送予测序并转入大肠埃希菌进行表达,认定高表达的样品为最终的阳性克隆。将该阳性克隆转染入感受态细胞表达单链抗体,纯化后经ELISA检测并评价其抗原亲和性。结果真核细胞表达的Vcam‐1抗原蛋白的分子量为85~90 kD。以Vcam‐1抗原蛋白为免疫原筛选4轮所得的阳性克隆进行单噬菌体ELISA检测,从检测结果中选取9个效价高的克隆经基因测序共获得3个序列,其中1个序列对应的克隆高表达。表达的单链抗体分子量约30 kD ,ELISA检测其对Vcam‐1抗原蛋白有较高亲和性,效价较单克隆抗体低。结论利用噬菌体展示技术获得了靶向Vcam‐1的单链抗体,为随后的诊断和治疗应用奠定了基础。%Objective To screen out single chain variable fragment antibody (scFv)specific to vascular cell adhesion mole‐cule 1(Vcam‐1)from phage recombinant antibody library ,and to evaluate its activity and compare its activity with full‐length monoclonal antibody.Methods Amplification of Vcam‐1 was performed by PCR and Vcam‐1 gene plasmid was transferred into eukaryotic cells to express Vcam‐1 antigen protein.Immune cuvette was coated with purified Vcam‐1 antigen ,and the positive clones were screened out by 4 rounds of “adhesion‐elution‐proliferation” process with gradually increasing pressure.The posi‐tive clones were tested by ELISA method and high titer clones were chosen for gene sequencing.Then the high‐titer clones

  11. Generating the human asialoglycoprotein receptor specific scFv by capture phage ELISA%捕获噬菌体酶联免疫吸附法筛选抗去唾液酸糖蛋白受体单链抗体的价值

    Institute of Scientific and Technical Information of China (English)

    王炜煜; 易继林; 邓云华; 曹利民


    目的评价捕获噬菌体酶免疫吸附试验法(ELISA)在筛选抗去唾液酸糖蛋白受体(ASGPR)单链抗体(scFv)中的价值.方法以含ASGPR基因的质粒(CRDH1/pET3b)为模板,通过聚合酶链反应扩增获得ASGPR H1亚单位糖识别区基因(CRDH1),定向克隆至原核表达载体pET-32c,并经IPTG诱导表达,其表达产物用Ni2+螯合柱亲和纯化;然后,采用捕获噬菌体ELISA对人源噬菌体抗体库进行筛选和鉴定,同时对筛选的抗CRDH1单链抗体进行DNA测序、表达、纯化和免疫印记鉴定.然后,与间接噬菌体ELISA比较.结果 CRDH1/pET-32c在原核系统中经诱导表达出分子量约35 000的融合蛋白,且以包涵体形式存在;通过Ni2+亲和柱纯化获得CRDH1融合蛋白后,采用捕获噬菌体ELISA进行四轮筛选,在60个噬菌体克隆中有45株与CRDH1特异性结合,其中仅1株与重组蛋白标签有交叉结合反应.DNA序列分析表明,有9株DNA序列各异,9株可分泌性表达与CRDH1特异性结合的scFv,纯化的scFv也可特异性识别rCRDH1.计算该筛选方法的假阳性率为2%,低于间接噬菌体ELISA筛选的假阳性率(58%).结论捕获噬菌体ELISA筛选CRDH1特异性scFv可有效降低硫氧还蛋白标签(Trx·Tag)引起的假阳性,提高筛选阳性率,并成功筛选出9株具有rCRDH1特异性的scFv.

  12. Binding of HIV-1 gp41-directed neutralizing and non-neutralizing fragment antibody binding domain (Fab and single chain variable fragment (ScFv antibodies to the ectodomain of gp41 in the pre-hairpin and six-helix bundle conformations.

    Directory of Open Access Journals (Sweden)

    John M Louis

    Full Text Available We previously reported a series of antibodies, in fragment antigen binding domain (Fab formats, selected from a human non-immune phage library, directed against the internal trimeric coiled-coil of the N-heptad repeat (N-HR of HIV-1 gp41. Broadly neutralizing antibodies from that series bind to both the fully exposed N-HR trimer, representing the pre-hairpin intermediate state of gp41, and to partially-exposed N-HR helices within the context of the gp41 six-helix bundle. While the affinities of the Fabs for pre-hairpin intermediate mimetics vary by only 2 to 20-fold between neutralizing and non-neutralizing antibodies, differences in inhibition of viral entry exceed three orders of magnitude. Here we compare the binding of neutralizing (8066 and non-neutralizing (8062 antibodies, differing in only four positions within the CDR-H2 binding loop, in Fab and single chain variable fragment (ScFv formats, to several pre-hairpin intermediate and six-helix bundle constructs of gp41. Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity. There is a large differential (≥ 150-fold in binding affinity between neutralizing and non-neutralizing antibodies to the six-helix bundle of gp41 and binding to the six-helix bundle does not involve displacement of the outer C-terminal helices of the bundle. The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs. We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.

  13. Fluobodies : green fluorescent single-chain Fv fusion proteins

    NARCIS (Netherlands)

    Griep, R.A.; Twisk, van C.; Wolf, van der J.M.; Schots, A.


    An expression system (pSKGFP), which permits the expression of single-chain variable fragments as fusion proteins with modified green fluorescent proteins, was designed. This expression system is comparable to frequently used phage display vectors and allows single-step characterization of the selec

  14. Economical analysis of FV power plants according installed performance

    Directory of Open Access Journals (Sweden)

    Peter Tauš


    Full Text Available According prognosis of future development of power capacities in Slovakia till 2030 there is assumed increasing of the electricalenergy volume, produced from renewable energy sources from present 260 MW approximately to 2100 MW (Petrovič, 2008, thatpresents almost 800 % increasing! In Slovakia position of photovoltaic in this sector was due to the high investment cost and on the lastplace due to the low system efficiency. Only possible way for implementation of the photovoltaic to the energetic system of the stateis 0donation. Slovakia will go this way also.

  15. FIIS_Breach_Shorelines.shp - Fire Island National Seashore Wilderness Breach Shoreline Data Collected from Fire Island, New York, October 2014 to September 2016 (United States)

    U.S. Geological Survey, Department of the Interior — Hurricane Sandy made U.S. landfall, coincident with astronomical high tides, near Atlantic City, New Jersey, on October 29, 2012. The storm, the largest on...

  16. Anti-ASGPR scFv-mediated multi-function targeting molecule construction and characterization%靶向去唾液酸糖蛋白受体多功能分子的构建及抗肝癌效应

    Institute of Scientific and Technical Information of China (English)

    王炜煜; 曹利民; 罗剑; 曾建平; 徐立宁; 易继林; 董家鸿


    目的 构建具有靶向去唾液酸糖蛋白受体、溶酶体逃逸、DNA结合的多功能融合蛋白,并对表达产物进行功能鉴定.方法 合成编码Melittin的两条寡核苷酸单链(GenBank:X02007),退火形成寡核苷酸双链;双酶切含抗去唾液酸糖蛋白单链抗体( ASGPR scFv) C1基因的载体C1/pIT2,0.7%低融点琼脂糖凝胶回收C1;以pSW50-Gal4为模板,通过聚合酶链反应(PCR)扩增Gal4基因;采用分子克隆技术将其定向克隆至载体pGC4C26H中,获得重组质粒C1 MG/pGC4C26H;双酶切载体C1MG/pGG4C26H,胶回收片段C1MG定向克隆至载体pET-32c中,将含C1MG/pET-32c的BL21单菌落1∶100接种至1000 ml LB肉汤培养基中培养,并通过IPTG诱导表达.表达产物用Ni2+螯合柱亲和纯化,免疫印迹技术( Western blot)和免疫组织化学分析重组蛋白C1 MG的抗原结合能力,溶血实验分析C1MG的生物学活性,肿瘤细胞生长抑制实验分析C1MG的DNA结合能力.结果 成功构建原核表达质粒C1MG/pET-32c,并经测序证实:在大肠杆菌BL21中有效表达重组融合蛋白C1MG;表达产物以包涵体形式存在;纯化的C1 MG大小为64.1 kDa,浓度为0.6g/L;Western blot结果说明C1MG能有效识别重组ASGPR,免疫组织化学结果证实C1 MG能结合到鼠肝细胞表面;溶血实验显示ClMG具有裂解红细胞膜功能;肿瘤细胞生长抑制实验证实C1 MG将pEBAF/tk-GAL4rec质粒有效地导入表达ASGPR的细胞中并表达TK基因,氯喹对肿瘤细胞生长抑制无明显影响.结论 在大肠杆菌中成功表达和纯化得到单链抗体-蜂毒肽-酵母转录因子(C1MG)的融合蛋白,该融合蛋白至少具有以下功能:ASGPR靶向识别能力、溶酶体膜裂解功能、以及DNA特异性结合功能,提示对肝癌的靶向治疗有潜在的应用价值.%Objective To construct a multi-function fusion protein with the target desasialoglycoprotein receptor ( ASGPR ),endosome escape and DNA-binding ability,and identify

  17. Polymorphism of clotting factors in Hungarian patients with Raynaud's phenomenon. (United States)

    Shemirani, Amir-Houshang; Szomják, Edit; Balogh, Emese; András, Csilla; Kovács, Dóra; Acs, Judit; Csiki, Zoltán


    Patients with primary Raynaud's phenomenon may have a genetically determined risk for clotting factors that predispose them to aberrant microvascular thrombosis. We investigated the prevalence of factor V substitution of G to A at position 1691 (FVLeiden), prothrombin G20210A, and methyltetrahydrofolate reductase C677T mutations in these patients. Two hundred (158 women, 42 men, mean age of 42.4 ± 13.7 years) consecutive patients with primary Raynaud's phenomenon and 200 age-sex-matched healthy controls of Hungarian origin were included in a case-control study. The prevalence of methyltetrahydrofolate reductase C677T homozygous among patients was significantly lower than in the control group (odds ratio 0.4, 95% confidence interval 0.2-0.9, P Raynaud's phenomenon and control subjects. FVLeiden, prothrombin G20210A, and polymorphism, prothrombin G20210A mutations have no apparent effect on the etiology of primary Raynaud's phenomenon.

  18. Risk of placenta-mediated pregnancy complications or pregnancy-related VTE in VTE-asymptomatic families of probands with VTE and heterozygosity for factor V Leiden or G20210 prothrombin mutation. (United States)

    Cordoba, Iris; Pegenaute, Carlota; González-López, Tomás José; Chillon, Carmen; Sarasquete, Maria Eugenia; Martin-Herrero, Francisco; Guerrero, Carmen; Cabrero, Mónica; Garcia Sanchez, Maria Helena; Pabon, Pedro; Lozano, Francisco Santiago; Gonzalez, Marcos; Alberca, Ignacio; González-Porras, José Ramón


    Few studies have evaluated the risk of pregnancy-related adverse events in asymptomatic relatives of probands for VTE and factor V Leiden or the G20210A variant. The antepartum management of this population ranges from antepartum anticoagulation therapy to clinical surveillance. To evaluate the risk of placenta-mediated pregnancy complications and pregnancy-related VTE in VTE-asymptomatic families of probands with VTE and who are heterozygous carriers of either factor V Leiden or PT-G20210A mutation. One hundred and fifty-eight relatives, who had 415 pregnancies, were retrospectively evaluated. Odds ratios and 95% confidence intervals were calculated to compare pregnancy outcomes between women with and without thrombophilia. In the factor V Leiden group, 22 placenta-mediated pregnancy events of 152 pregnancies (14.4%) were reported, compared with 25 adverse events of 172 pregnancies in the G20210A prothrombin group (14.5%) and 13 adverse events of 91 pregnancies in the non-carrier group (14.2%). Carriers of factor V Leiden or G20210A prothrombin were not associated with a higher risk of pregnancy-adverse outcomes compared with non-carriers: OR 1.02 (95% CI, 0.40-2.25) and 1.25 (95% CI, 0.48-3.24), respectively. Four episodes of pregnancy-associated VTE of 415 pregnancies (0.96%) were recorded. Two episodes of VTE in the G20210A group, one in the factor V Leiden group, and one episode in the non-carrier group were noted. In VTE-asymptomatic relatives of probands with VTE, the presence of factor V Leiden or the G20210A prothrombin mutation in heterozygosis should not lead to a decision to instigate antepartum prophylaxis. © 2012 John Wiley & Sons A/S.

  19. Comparative Response of Platelet fV and Plasma fV to Activated Protein C and Relevance to a Model of Acute Traumatic Coagulopathy (United States)


    Valle P, et al. (1998) Plasma levels of endothelial cell protein C receptor are elevated in patients with sepsis and systemic lupus erythematosus : lack of... lupus erythematosus [40]. However, over the range of 0 200 nM sEPCR, no effect on fibrin crosslinking was observed beyond that generated by aPC alone...Report Documentation Page Form ApprovedOMB No. 0704-0188 Public reporting burden for the collection of information is estimated to average 1 hour

  20. Risk of venous thromboembolism associated with single and combined effects of Factor V Leiden, Prothrombin 20210A and Methylenetethraydrofolate reductase C677T

    DEFF Research Database (Denmark)

    Simone, Benedetto; De Stefano, Valerio; Leoncini, Emanuele


    Genetic and environmental factors interact in determining the risk of venous thromboembolism (VTE). The risk associated with the polymorphic variants G1691A of factor V (Factor V Leiden, FVL), G20210A of prothrombin (PT20210A) and C677T of methylentetrahydrofolate reductase (C677T MTHFR) genes ha...

  1. Establishment of reference intervals for von Willebrand factor antigen and eight coagulation factors in a Korean population following the Clinical and Laboratory Standards Institute guidelines. (United States)

    Jang, Ja-Hyun; Seo, Ja-Young; Bang, Sung-Hwan; Park, In-Ae; Kim, Hee-Jin; Kim, Sun-Hee


    Establishment of reference intervals for coagulation molecules is important but is costly and sometimes not feasible. Since reference intervals from manufacturers or the literature are mostly out of date or involved Western populations, the authors determined reference intervals for VWF: Ag and eight factors in a Korean population. VWF: Ag, factor VIII (FVIII), FII, FV, FVII, FIX, FX, FXI, and FXII were determined in Korean individuals visiting for routine checkup following the CLSI (Clinical and Laboratory Standards Institute) guidelines. Reagents by Diagnostica Stago were used on the STA Compact Analyzer (Diagnostica Stago). Exclusion criteria were medical history or laboratory findings that could affect the factor levels. Influence of demographic factors was analyzed. Mean +/- 2 x SD or central 95 percentile was used, as appropriate. We obtained data from 266 adults for VWF: Ag, 371 adults for FVIII, and minimum 136 adults for the rest. Reference interval for VWF was 51-176% (52-155% in blood group O and 71-186% for non-O). Reference interval for FVIII was 64-197% (55-150% in O and 77-205% in non-O). Reference interval for FII was 77-121%, FV 81-160%, FVII 68-149%, FIX 67-154%, FX 69-126%, FXI 59-138%, and FXII 48-177%. The medians of VWF: Ag, FVIII, and FIX were significantly higher in the elderly group (> or =60 years). We established local reference intervals for VWF: Ag and eight coagulation factors in a Korean population according to the CLSI guidelines. Significantly, different reference intervals were obtained in blood group O vs. non-O for VWF: Ag and FVIII. The reference intervals obtained in this study could be adopted in other clinical laboratories after appropriate validation.

  2. The frequencies of six important thrombophilic mutations in a population of the Czech Republic. (United States)

    Kvasnička, T; Hájková, J; Bobčíková, P; Cverhová, V; Malíková, I; Ulrych, J; Bříza, J; Dušková, D; Poletínová, S; Kieferová, V; Kvasnička, J


    The primary aim was to determine frequencies of mutations related to risk of venous thrombosis in healthy Caucasians in Central Bohemia. In a cohort of 1527 healthy individuals the frequency of risk alleles for the mutations FV Leiden and FII 20210G>A was 4.5% and 1.3%, respectively. Frequency of 4G PAI-1 allele was 55.5%. Genotype frequencies were: GG 91.03%, GA 8.91%, and AA 0.07% for FV Leiden; GG 97.45%, GA 2.49%, and AA 0.07% for FII 20210G>A; 4G/4G 30.26%, 4G/5G 50.56%, and 5G/5G 19.19% for PAI-1. Frequency of the risk allele A in polymorphism SERPINC1 (IVS +141G >A) was 11.3%, and frequencies of genotypes were as follows: GG 78.36%, GA 20.66%, and AA 0.98%. Frequency of the risk allele T for polymorphism GP6 13254T>C was 87.7%, and frequencies of genotypes were as follows: TT 77.14%, TC 21.15%, and CC 1.70%. Frequency of the risk allele A in polymorphism CYP4V2 (Lys259Gln) was 65.2%, and frequencies of genotypes were: CC 12.25%, CA 45.12 %, and AA 42.63%. All observed genotypes and alleles frequencies were without gender differences. Their occurrences confirm a relatively high prevalence of hereditary thrombophilia predisposition in the Czech Republic.

  3. The Effect of Carbon Dioxide Insufflation Applied at Different Pressures and Periods on Thrombotic Factors. (United States)

    Sen, Mehmet Celal; Turkyilmaz, Zafer; Sonmez, Kaan; Karabulut, Ramazan; Kaya, Zuhre; Yenicesu, Idil; Gursel, Turkiz; Basaklar, Abdullah Can


    The aim of this experimantal study which is applied on rats, is to determine the differences on the clotting factors over the application of low and high intraabdominal pressure (IAP) values in different periods of time in carbon dioxide (CO2) pneumoperitoneum. Thirty rats were randomized into five groups (n = 6): a control group (Group K) and 1 h and 6 mm Hg IAP (Group A), 2 h and 6 mm Hg IAP (Group B), 1 h and 12 mm Hg IAP (Group C) and 2 h and 12 mm Hg IAP were created with CO2 pneumoperitoneum (Group D). At the end of the experiment, plasma samples taken from subjects and fibrinogen, FII (prothrombin), FV, FVII, FVIII, FIX, FX, FXI, FXII, von willebrand's factor (vWF), ristocetin cofactor, protein C, protein S, antithrombin III (AT III) levels are studied. There were statistically significant differences in the mean levels of FII, FV, FVII, FVIII, FIX, FX, FXI, FXII, and protein S between the groups. A hypercoagulable state occurred with the following: increase in the coagulation parameters compared to the control group; increase in FVII in the group only Group C; decrease in AT III in all groups compared to the control group; decrease in protein C in the group only XII Group D compared to control group; decrease in protein S in all groups except group D compared to control group. CO2 insufflation predisposes to thromboembolic events both by inducing coagulation factors and by suppressing the fibrinolytic system contrary to the controversies in the literature.

  4. Thrombotic gene polymorphisms and postoperative outcome after coronary artery bypass graft surgery

    Directory of Open Access Journals (Sweden)

    Zaim Cagin


    Full Text Available Abstract Background Emerging perioperative genomics may influence the direction of risk assessment and surgical strategies in cardiac surgery. The aim of this study was to investigate whether single nucleotide polymorphisms (SNP affect the clinical presentation and predispose to increased risk for postoperative adverse events in patients undergoing coronary artery bypass grafting surgery (CABG. Methods A total of 220 patients undergoing first-time CABG between January 2005 and May 2008 were screened for factor V gene G1691A (FVL, prothrombin/factor II G20210A (PT G20210A, angiotensin I-converting enzyme insertion/deletion (ACE-ins/del polymorphisms by PCR and Real Time PCR. End points were defined as death, myocardial infarction, stroke, postoperative bleeding, respiratory and renal insufficiency and event-free survival. Patients were compared to assess for any independent association between genotypes for thrombosis and postoperative phenotypes. Results Among 220 patients, the prevalence of the heterozygous FVL mutation was 10.9% (n = 24, and 3.6% (n = 8 were heterozygous carriers of the PT G20210A mutation. Genotype distribution of ACE-ins/del was 16.6%, 51.9%, and 31.5% in genotypes I/I, I/D, and D/D, respectively. FVL and PT G20210A mutations were associated with higher prevalence of totally occluded coronary arteries (p p = 0.002. ACE D/D genotype was associated with hypertension (p = 0.004, peripheral vascular disease (p = 0.006, and previous myocardial infarction (p = 0.007. Conclusions FVL and PT G20210A genotypes had a higher prevalence of totally occluded vessels potentially as a result of atherothrombotic events. However, none of the genotypes investigated were independently associated with mortality.

  5. Functional expression of an scFv on bacterial magnetic particles by in vitro docking

    Energy Technology Data Exchange (ETDEWEB)

    Sugamata, Yasuhiro; Tanaka, Tsuyoshi; Matsunaga, Tadashi; Yoshino, Tomoko, E-mail:


    Highlights: • We present a novel expression system called “in vitro docking” on bacterial magnetic particles. • An scFv–Fc was functionally expressed on bacterial magnetic particles of magnetotactic bacteria. • Our novel expression system on BacMPs will be effective for disulfide-bonded proteins. - Abstract: A Gram-negative, magnetotactic bacterium, Magnetospirillum magneticum AMB-1 produces nano-sized magnetic particles (BacMPs) in the cytoplasm. Although various applications of genetically engineered BacMPs have been demonstrated, such as immunoassay, ligand–receptor interaction or cell separation, by expressing a target protein on BacMPs, it has been difficult to express disulfide-bonded proteins on BacMPs due to lack of disulfide-bond formation in the cytoplasm. Here, we propose a novel dual expression system, called in vitro docking, of a disulfide-bonded protein on BacMPs by directing an immunoglobulin Fc-fused target protein to the periplasm and its docking protein ZZ on BacMPs. By in vitro docking, an scFv–Fc fusion protein was functionally expressed on BacMPs in the dimeric or trimeric form. Our novel disulfide-bonded protein expression system on BacMPs will be useful for efficient screening of potential ligands or drugs, analyzing ligand–receptor interactions or as a magnetic carrier for affinity purification.


    Institute of Scientific and Technical Information of China (English)

    WANG Enrong; YE Biaoming; MA Xiaoqing; SU Chunyi; RAKHEJA Subhash


    An asymmetric damping force generation algorithm is originally proposed to yield the asymmetric force-velocity characteristics for the symmetric magneto-rheological (MR) dampers. The command current is formulated in an asymmetric manner to excite the symmetric MR dampers by employing the "on-off" control law in response to the direction of velocity, and a smooth modulation function is developed without phase shift to suppress strong transients in the damping forces caused by the current-switching discontinuity. The effectiveness of the proposed algorithm is evaluated by analyzing the dynamic responses of a quarter-vehicle suspension system with a symmetric MR-damper by modulating the command current into the asymmetric manner. The simulation results show that the proposed algorithm could achieve a better compromise between the conflicting requirements of the asymmetric damping force ratio and the force-velocity curve smoothness, and the asymmetric damping MR-suspension design can ideally improve the road holding and ride performances of vehicle motion. The proposed algorithm can be generally incorporated with a controller synthesis to realize an intelligent vehicle suspension design with the symmetric MR dampers.

  7. AFSC/ABL: Origins of salmon seized from the F/V Petropavlovsk (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — On 25 July 2001, the Russian factory trawler Petropavlovsk was seized in the Bering Sea seven miles into the U. S. EEZ by the U. S. Coast Guard. There was...

  8. [Toxicity, tolerance and blood concentrations of iron and tylosin with the use of preparation FV-82]. (United States)

    Dilov, P; Minkov, E; Bogdanova, S; Dilova, V


    A pharmacologic evaluation was made of a technologic model of a liquid drug form (code name phi B-82), having the following composition: tylosine tartrate 3500000 UI, cyanocobalamine 0.008 g, pyridoxin hydrochloride 0.500 g, tartaric acid 0.100 g, and feridextran (dextrofer-100) up to 100 cm3; pH from 5.5 to 6.5, and Fe3+ 100 mg/cm3. It was found that phi B-82 at i/m application to rabbits, subcutaneous injection to albino mice, and intra-abdominal introduction to albino rats and mice at rates that were equal to ED100 and 3 to 5 times higher than those used with pigs did not lead to local and total lack of tolerance. The acute toxicity (LD50) of phi B-82 at intra-abdominal application to 18-20 g albino mice was 29.2 cm3/kg. The single muscular application to guinea pigs at 2 cm3 per kg of body mass showed good absorption of the preparation - it did not differ essentially from those of dextrofer-100 and aquaous solution of tylosine tartrate used in equivalent amounts. The bacteriostatic concentrations of tylosine were maintained for 24 hours. It was shown that the optimal effect would be produced by a combined preparation having the qualities of the feridextrane complexes with a rapid absorption and those of the erythropoietic vitamins of the B12 group and B6 along with the participation of tylosine as an antibiotic.

  9. Retargeting T cells for HER2-positive tumor killing by a bispecific Fv-Fc antibody.

    Directory of Open Access Journals (Sweden)

    Lei Wang

    Full Text Available To exploit the biological and pharmacological properties of immunoglobulin constant domain Fc fragment and increase the killing efficacy of T cells, a single chain variable fragment specific to CD3 was fused with Fcab (Fc antigen binding, a mutant Fc fragment with specificity against Human epidermal growth factor receptor 2 (HER2 developed by F-star. The bispecific fusion named as FcabCD3 was expressed by transient transfection in HEK-293T cells and purified by affinity chromatography. Specific cytolytic activity of retargeted T cells to kill HER2 positive SKBR3 cell line was evaluated in vitro. FcabCD3 was able to retarget T cells to kill both Herceptin insensitive Colo205-luc cell line and HER2 low expression MDA-MB-231-luc cell line. Furthermore, FcabCD3 was effective in eliminating the Colo205 tumor established on BALB/c nu/nu mice.

  10. Fruit and vegetable waste (FV) from the market places: A potential source for animal feeding?

    Energy Technology Data Exchange (ETDEWEB)

    Angulo, J.; Yepes, S. A.; Yepes, A. M.; Bustamante, G.; Mahecha, L.


    The generation of organic solid waste and its inappropriate management is considered one of the main environmental problems in the world associated with emissions of methane from landfill sites, with emission of dusts, odors and hazardous gases, and with contamination of water. There are different sources for the generation of solid wastes; the market places are considered one of then on a global scale. (Author)

  11. Influence of soil structure on the "Fv approach" applied to (238)U and (226)Ra. (United States)

    Blanco Rodríguez, P; Vera Tomé, F; Lozano, J C


    The soil-to-plant transfer factors were determined in a granitic area for the two long-lived uranium series radionuclides (238)U and (226)Ra. With the aim to identify a physical fraction of soil whose concentration correlates linearly with the plant concentration, the soil compartment was analyzed in various stages. An initial study identified the soil compartments as being either bulk soil or its labile fraction. The bulk soil was subsequently divided into three granulometric fractions consisting of: coarse sand, fine sand, and silt and clay. The soil-to-plant transfer of radionuclides for each of these three texture fractions was analyzed. Lastly, the labile fraction was extracted from each textural part, and the activity concentration of the radionuclides (238)U and (226)Ra was measured. In order to assess the influence of soil texture on the soil-to-plant transfer process, we sought to identify possible correlations between the activity concentration in the plant compartment and those found in the different fractions within each soil compartment. The results showed that the soil-to-plant transfer process for uranium and radium depends on soil grain size, where the results for uranium showed a linear relationship between the activity concentration of uranium in the plant and the fine soil fraction. In contrast, a linear relation between the activity concentration of radium in the plant and the soil coarse-sand fraction was observed. Additionally, the presence of phosphate and calcium in the soil of all of the compartments studied affected the soil-to-plant transfer of uranium and radium, respectively.

  12. Antibiotic-resistant Klebsiella pneumoniae and Escherichia coli high-risk clones and an IncFII(k) mosaic plasmid hosting Tn1 (blaTEM-4) in isolates from 1990 to 2004. (United States)

    Rodríguez, Irene; Novais, Ângela; Lira, Felipe; Valverde, Aránzazu; Curião, Tânia; Martínez, José Luis; Baquero, Fernando; Cantón, Rafael; Coque, Teresa M


    We describe the genetic background of bla(TEM-4) and the complete sequence of pRYC11::bla(TEM-4), a mosaic plasmid that is highly similar to pKpQIL-like variants, predominant among TEM-4 producers in a Spanish hospital (1990 to 2004), which belong to Klebsiella pneumoniae and Escherichia coli high-risk clones responsible for the current spread of different antibiotic resistance genes. Predominant populations of plasmids and host adapted clonal lineages seem to have greatly contributed to the spread of resistance to extended-spectrum cephalosporins.

  13. 基于Hadoop的海量统计小文件存取优化方案%Accessing Optimization of Massive SmaII StatisticaI FiIes based on Hadoop

    Institute of Scientific and Technical Information of China (English)

    付红阁; 姜华; 张怀锋


    Hadoop作为一个开源的并行计算框架,它提供了一个分布式的文件存储系统 HDFS 。然而,当处理海量小文件时会产生NameNode内存使用率较高、存取性能并不理想的问题,导致NameNode成为系统瓶颈,从而制约了文件系统的可扩展性。本文结合统计工作,提出了一种小文件存储的优化策略,在 HDFS之上增加小文件预处理模块将文件进行分类,合并成M apfile ,并建立全局索引,另外该策略引入了索引预取机制和缓存机制。实验证明,该方法能有效提高大批量小文件的存取性能。%As an open‐source parallel computing framework , Hadoop provides a distributed file storage system HDFS .However ,when dealing with small files ,it will cause NameNode consumes too much memory storage and the accessing performance not ideal ,so NameNode become a bottleneck ,w hich restrictes the file system scalability .Based on the statistical work ,we put forward the optimization strat‐egy for small files ,adding mall file preprocessing module on HDFS will classify the files and merge them into MapFile ,we also establish the global index ,in addition ,introduces index prefetching mechanism and caching mechanism .Experiments show that this method can effectively improve the performance of accessing mass small files .


    Directory of Open Access Journals (Sweden)

    Azis Ikhsanudin


    Full Text Available The essential oil of Ginger rhizome is a natural subtance which has been proved to have a repellent effect to the mosquito. The aim of this study was to know the effect of this subtance/these substances in vanishing cream dosage forms as repellant to the female Aedes aegypti mosquito. The essential oil of Ginger rhizome was isolated with aqueous vapor destillation. Then the result was determined for its physical properties, these were: viscosity, adhesivity, spreadibility and its repellant effect to mosquito as well. The vanishing cream formulation, containing variation of the essential oil concentration were prepared, these were: FI (formulation I, FII, FIII, FIV and FV, containing 6,25%, 12,5%, 15%, 20% and 25% of the essential oil respectivity. Each formulas were tested for their physical properties were viscosity, adhesivity, spreadibility and its repellant effect to mosquito as well. The datas were analyzed. Statistically using SPSS version 16 with Kolmogorov-Smirnov and continued with the t-test (Mann-Whitney (=0,05. The result showed that the duration of the adhesivity of FI was the longest than the others (1,37±0,15 minutes; the spreadibility of the FI was widest (71,69±1,76 mm2 , and the viscosity was the largert than others (27±0 cps. The repellant capasity of the FV was the longest than the others (47,3±1,80 minutes.

  15. Risk of venous thromboembolism and myocardial infarction associated with factor V Leiden and prothrombin mutations and blood type. (United States)

    Sode, Birgitte F; Allin, Kristine H; Dahl, Morten; Gyntelberg, Finn; Nordestgaard, Børge G


    ABO blood type locus has been reported to be an important genetic determinant of venous and arterial thrombosis in genome-wide association studies. We tested the hypothesis that ABO blood type alone and in combination with mutations in factor V Leiden R506Q and prothrombin G20210A is associated with the risk of venous thromboembolism and myocardial infarction in the general population. We used data from 2 Danish studies that followed members of the general public from 1977 through 2010. We obtained the genotype of 66 001 white participants for ABO blood type, factor V Leiden R506Q and prothrombin G20210A. We calculated hazard ratios (HRs) and population attributable risk. Our main outcome measures were venous thromboembolism and myocardial infarction. The multivariable adjusted HR for venous thromboembolism was 1.4 (95% confidence interval [CI] 1.3-1.5) for non-O blood type (v. O blood type). For the factor V Leiden R506Q mutation, the adjusted HR was 2.2 (95% CI 2.0-2.5) for heterozygous participants and 7.0 (95%CI 4.8-10) for homozygous participants (v. participants without the mutation). For prothrombin G20210A, the adjusted HR was 1.5 (95%CI 1.2-1.9) for heterozygous participants and 11 (95% CI 2.8-44) for homozygous participants (v. participants without the mutation). When we combined ABO blood type and factor V Leiden R506Q or prothrombin G20210A genotype, there was a stepwise increase in the risk of venous thromboembolism (trend, pfactor V Leiden R506Q and 1% for prothrombin G20210A. Multivariable adjusted HRs for myocardial infarction by genotypes did not differ from 1.0. ABO blood type had an additive effect on the risk of venous thromboembolism when combined with factor V Leiden R506Q and prothrombin G20210A mutations; blood type was the most important risk factor for venous thromboembolism in the general population.

  16. Determinants of Foreign Institutional Investors’ Investment in India

    Directory of Open Access Journals (Sweden)

    Manjinder KAUR


    Full Text Available The present study aims at exploring the determinants of Foreign Institutional Investors’ (FIIs investment in India. Returns on Indian stock market have positive impact whereas US stock market returns have no significant influence on FIIs investment to India. Stock market risk has negative influence on FIIs inflows to India. Market capitalization and stock market turnover of India have significant positive influence only in short-run. Among macroeconomic determinants, economic growth of India has positive impact on FIIs investment both in long-run and shortrun. But all other macroeconomic factors have significant influence only in long-run like inflation in US has positive influence whereas inflation in India has negative influence on FIIs investment. Further, US interest rate has adverse impact on FIIs investment while liberalization policies of India exhibited significant contribution to FIIs inflows. Study concludes that FIIs inflows in India are determined by both stock market characteristics and macroeconomic factors.


    Directory of Open Access Journals (Sweden)



    Full Text Available Обсуждаются возможные изосимметрийные и деформационные модификации детерминистических модулярных структур из фракталов Вичека FV, канторова множества F(CM(1/3 и итерационной последовательности точек F(IC(1/2 в 2D пространстве на квадратной сетке.

  18. Dicty_cDB: Contig-U14356-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available IDQLDAEGS*kwfklvtlnstmmlkrenakavaligletyqqmgiclwcfclnf f*iiyiiknliiykkkkkkkkkiciyslmnyknkkkkkntlnqfq*fkkkket...GGFAFVNPDASCYIN TIEAVPIDQLDAEGS*kwfklvtlnstmmlkrenakavaligletyqqmgiclwcfclnf f*ii

  19. Prothrombin and risk of venous thromboembolism, ischemic heart disease and ischemic cerebrovascular disease in the general population

    DEFF Research Database (Denmark)

    Weischer, Maren; Juul, Klaus; Zacho, Jeppe


    OBJECTIVE: We tested the hypotheses that Prothrombin G20210A heterozygosity associate with increased risk of venous thromboembolism (VTE), ischemic heart disease (IHD), and ischemic cerebrovascular disease (ICVD) in the general population and re-tested risk of IHD and ICVD in two case......-control studies. METHODS: 9231 individuals from the Danish general population were followed for VTE (VTE=DVT+PE), deep venous thrombosis (DVT), pulmonary embolism (PE), IHD, myocardial infarction (MI), ICVD, and ischemic stroke (IS) for a median of 24 years. Case-control studies included 2461 IHD cases and 867.......0(1.0-3.8) for MI, 1.4(0.7-3.1) for ICVD, and 2.1(0.8-5.4) for IS. CONCLUSION: Prothrombin G20210A heterozygosity alone and in combination with Factor V Leiden R506Q heterozygosity predicts 1.5 and 6.0 fold risk of IHD compared to non-carriers....

  20. Risk of venous thromboembolism and myocardial infarction associated with factor V Leiden and prothrombin mutations and blood type

    DEFF Research Database (Denmark)

    Sode, Birgitte F; Allin, Kristine H; Dahl, Morten


    ABO blood type locus has been reported to be an important genetic determinant of venous and arterial thrombosis in genome-wide association studies. We tested the hypothesis that ABO blood type alone and in combination with mutations in factor V Leiden R506Q and prothrombin G20210A is associated...... with the risk of venous thromboembolism and myocardial infarction in the general population....

  1. Genetic risk factors for placental abruption: a HuGE review and meta-analysis. (United States)

    Zdoukopoulos, Nikos; Zintzaras, Elias


    Although the precise pathophysiology that leads to placental abruption is unknown, there is evidence supporting a genetic etiology. We searched PubMed and systematically reviewed all case-control studies that investigated the association between genetic variants and placental abruption. Pooled genetic risks were estimated using fixed and random effects odds ratios. Twenty-two articles, examining a total of 14 gene polymorphisms were identified. Seven polymorphisms (F5 Arg506Gln, F5 Met385Thr, F2 G20210A, MTHFR A1298C, MTHFD1 Arg653Gln, NOS3 Glu298Asp, AGT Met235Thr) show significant association in individual studies. Six of the 7 (all except F5Met385Thr) were studied more than once and we therefore included them in our meta-analyses. A positive association under the dominant model was found for the F5 Arg506Gln and F2 G20210A polymorphisms. The random-effects odds ratio for the F5 Arg506Gln polymorphism was 3.4 (95% confidence interval = 1.4-8.3) and the fixed-effects odds ratio for the F2 G20210A polymorphism was 6.7 (3.2-13). Considering the multifactorial etiology of abruption and the relatively small numbers of studies and participants, this review provides only the first clues of possible genetic causes. Larger case-control studies that include gene-gene and gene-environment interactions may help to elucidate the genetics of placental abruption further.

  2. Cerebral venous and sinus thrombosis and thrombophilic mutations in Western Iran: association with factor V Leiden. (United States)

    Rahimi, Zohreh; Mozafari, Hadi; Bigvand, Amir Hossein Amiri; Doulabi, Reza Mohammad; Vaisi-Raygani, Asad; Afshari, Dariush; Razazian, Nazanin; Rezaei, Mansour


    The present study aimed at investigating the prevalence of factor V Leiden G1691A, prothrombin G20210A, and MTHFR C677T in cerebral venous and sinus thrombosis (CVST) patients and their possible association with CVST in Western Iran. A total of 24 CVST patients with the mean age of 37.1 +/- 11.7 years and 100 sex- and age-matched healthy individuals from Kermanshah Province of Iran with ethnic background of Kurd were studied for factor V Leiden G1691A, prothrombin G20210A and MTHFR C677T by PCR-RFLP method using Mnl I, Hind III, and Hinf I restriction enzymes, respectively. Prevalence of factor V Leiden was 16.7% in patients and 2% in control group. A significant association was found between factor V Leiden mutation and CVST with odds ratio (OR) of 9.8 (95% confidence intervals [CI] 1.68-57.2, P = .01). No prothrombin G20210A was found among patients. In patients, MTHFR C677T tended to be higher (58.3%) compared to control (44%), OR of 1.8 (95% CI 0.73-4.5, P = .2). Our study for the first time has determined the prevalence of inherited thrombophilia in a homogenous ethnic group of CVST patients and suggests that factor V Leiden, and not the prothrombin gene mutation is a risk factor for CVST in Western Iran.

  3. Frequency of Factor V Leiden and Prothrombin Polymorphism in South of Iran

    Directory of Open Access Journals (Sweden)

    Javad Dehbozorgian


    Full Text Available Normal hemostasis requires balanced regulation of prothromboticand antithrombotic factors. Inherited alteration of factor Vand prothrombin gene, the G20210A mutation, increases the resistanceof factor V to degradation and booster production ofprothrombin respectively. These alterations can increase hypercoagulabilityleading to thrombotic consequences. We aimed toassess the frequencies of these mutations in a group of the populationof southern Iran. In total, 198 healthy volunteers with theage range of 1-64 years were selected and screened for factor VLeiden and prothrombin mutations using polymerase chain reactionand restriction fragment length polymorphism techniques.The carrier frequencies for factor V Leiden and prothrombin mutationin the studied cohort were 4.1% and 3.07%, respectively.In the studied area, the allele frequency of factor V ishigher than the prothrombin G20210A mutation (0.0204 v0.0153. According to the data and Hardy-Weinberger equation,the total risk of thrombosis caused by homozygosity andheterozygosity of factor V Leiden, prothrombin G20210A mutationand compound heterozygosity of these mutations areabout 1 in 500 individuals.

  4. Evaluation of molecular genetic variation associated with deep venous thrombosis in lower limb fractures in traumatic patients

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    Ali Tabrizi


    Full Text Available Background: Genetic variation in multiple genes associated with hemostasis and thrombosis is well documented to impact the rates of future venous thromboembolism; in addition, trauma and orthopedic surgery in lower limb and immobilization are important factors in increasing the incidence of thrombosis. Gene mutation can be predisposing factor for thrombosis in traumatic patients under anti-coagulant agent prophylaxis. The aim of this study is to evaluate the different gene mutations in these patients. Materials and Methods: In this cross-sectional descriptive study, the sample consisted of 53 patients with deep venous thrombosis (DVT and 32 traumatic patients without thrombosis as the control group. Two groups matched together according to sex, age, weight, and medications. DNA analysis for mutation of multivariate of genes in thrombosis was studied. Results: Regarding gene variations, there was statistically significant difference only in Prothrombin (Factor II, G20210A between the patients with thrombosis and control group (P = 0.01. But, there was no difference between two groups considering other gene mutations. Mutation of Prothrombin gene (G20210A was a predictive factor for thrombosis with odds ratio of 1.1 (CI 0.3-1.9. Conclusion: According to the outcomes resulted from this study, genetic mutation in Prothrombin (Factor II [G20210A] is one of the most important genetic variations involved in traumatic patients with DVT despite prophylaxis. Genetic mutation in Prothrombin appears to be predisposing factor for thrombosis associated with trauma.

  5. Polymorphisms in the genes for coagulation factor II,V,VII in patients undergoing coronary angiography

    Institute of Scientific and Technical Information of China (English)

    徐耕; 金国栋; 傅国胜; 马骥; 单江; 王建安


    Objective: To determine whether polymorphisms in the genes for coagulation factor II,V, VII could predispose an individual to increase risk for coronary artery disease (CAD) and/or myocardial infarction (MI) in Chinese. Methods: We screened coagulation factor II(G20210A),V(G1691A),VII (R353Q and HVR4) genotype in 374 patients undergoing coronary angiography by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) assay. Results: The R353Q and HVR4 genotype of the factor VII distribution was in accordance with Hardy-Weinberg equilibrium. The frequencies of FVII genotype or allele did not show statistically significant differences between CAD group and controls or between male and female. The frequencies of the Q allele and (RQ+QQ) genotype were significantly higher among the CAD patients without myocardial infarction (MI) history than among those with MI history (P<0.05). However, HVR4 polymorphism was not significantly different within groups. We only find one normal control of factorII(G20210A) mutation. No coagulation factor V(G1691A) mutation was found in the CAD patients and controls. Conclusion: The factor II(G20210A),V(G1691A) mutation is absent and may not be a major genetic factor for CAD and/or MI; the Q allele of the R353Q polymorphism of the factor VII gene may be a protective genetic factor against myocardial infarction in Chinese.

  6. Polymorphisms in the genes for coagulation factor Ⅱ,Ⅴ,Ⅶ in patients undergoing coronary angiography

    Institute of Scientific and Technical Information of China (English)

    徐耕; 金国栋; 傅国胜; 马骥; 单江; 王建安


    Objective: To determine whether polymorphisms in the genes for coagulation factor Ⅱ,Ⅴ,Ⅶ could predispose an individual to increase risk for coronary artery disease (CAD) and/or myocardial infarction (MI) in Chinese. Methods: We screened coagulation factor Ⅱ( G20210A),Ⅴ( G1691A),Ⅶ( R353Q and HVR4) genotype in 374 patients undergoing coronary angiography by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) assay. Results: The R353Q and HVR4 genotype of the factor Ⅶ distribution was in accordance with Hardy-Weinberg equilibrium. The frequencies of FVⅡ genotype or allele did not show statistically significant differences between CAD group and controls or between male and female.The frequencies of the Q allele and ( RQ + QQ) genotype were significantly higher among the CAD patients without myocardial infarction (MI) history than among those with MI history (P < 0.05). However, HVR4 polymorphism was not significantly different within groups. We only find one normal control of factorⅡ(G20210A) mutation. No coagulation factor Ⅴ(G1691A) mutation was found in the CAD patients and con-trois. Conclusion: The factor Ⅱ(G20210A),Ⅴ(G1691A) mutation is absent and may not be a major genetic factor for CAD and/or MI; the Q allele of the R353Q polymorphism of the factor Ⅶ gene may be a protective genetic factor against myocardial infarction in Chinese.

  7. Interpretation of an E-W seismic cross section in Los Humeros geothermal field, Puebla, Mexico; Interpretacion geofisica de una seccion sismica de reflexion con direccion E-W en el campo geotermico de Los Humeros, Puebla, Mexico

    Energy Technology Data Exchange (ETDEWEB)

    Arredondo Fragoso, Jesus [Comision Federal de Electricidad, Morelia, Michoacan (Mexico)


    On the basis of reflection seismic studies conducted at Los Humeros, Puebla, geothermal field. There were identified several reflection horizons correlated with the following lithology features: the basis of theignimbrites, the Teziutlan andesites, and the top of the calcareous basement and the faults affecting them at depth in the well H-27 sector. It was observed that Los Humeros fault ends at the buried fault F-II that affects the calcareous basement in the well H-8 area. The presence of steaming grounds at the surface suggests that there are fluid transports between them. In the sector corresponding to the location of faults F-II to F-V, at the top of the basement depth, there are bending of the faults traces related to recent magnetic intrusive that metamorphosed the sedimentary basement to produce skarn. These intrusive act as the heat source of the hydrothermal system. It is considered that the best sites to drill the wells to develop the field are those in which the faults bends were observed, because near the fault traces, the best conditions for the flow of fluids are expected. [Spanish] Por medio de estudio de sismica de reflexion realizados en Los Humeros, Puebla, se determinaron reflectores que se correlacionaron con la base de las ignimbritas, el espesor de las andesitas Teziutlan, la cima del basamento calcareo y estructuras que lo desplazan a mayor profundidad en la zona del pozo H-27. Se observo que la falla de los Humeros termina sobre la falla sepultada F-11, la cual desplaza al basamento a mayor profundidad en la zona del pozo H-8. La presencia de manifestaciones de vapor en la superficie sugiere que existe transporte de fluidos entre ellas. En la zona donde se ubican las fallas de la F-11 a la F-V, a la profundidad de la cima del basamento sedimentario, se presentan arqueamientos sobre las trazas de las fallas relacionados con intrusiones magmaticas recientes que metamorfizaron al basamento sedimentario (skarn) y que sirven actualmente de

  8. Superior activity of fusion protein scFvRit : sFasL over cotreatment with rituximab and Fas agonists

    NARCIS (Netherlands)

    Bremer, Edwin; ten Cate, Bram; Samplonius, Douwe F.; Mueller, Nicole; Wajant, Harald; Stel, Aja J.; Chamuleau, Martine; de Loosdrecht, Arjan A. van; Stieglmaier, Julia; Fey, Georg H.; Helfrich, Wijnand


    The clinical efficacy of the CD20-specific chimeric monoclonal antibody rituximab is significantly hampered by intrinsic or acquired resistance to therapy. Rituximab activates antibody-dependent cellular cytotoxicity/complement-dependent cytotoxicity-dependent lysis but also induces apoptosis by cro

  9. FV-projektet - model til Forebyggende Virksomhedsstrategier i SMV’er (også kaldet Produktivitet og Trivsel - projektet)

    DEFF Research Database (Denmark)

    Ipsen, Christine; Poulsen, Signe; Gish, Liv

    Baggrund : Mange små og mellemstore virksomheder (SMV’er) oplever i disse år udfordringer, der knytter sig til det psykiske arbejdsmiljø. Virksomheder, der beskæftiger sig med eksempelvis industriel service, reparation og vedligehold kommer ud for, at deres medarbejdere bliver belastet af forhold...... organisatoriske design. Mange SMV’er har adgang til virkemidler, så de kan udvikle og gennemføre forebyggende strategier i forhold til det psykiske arbejdsmiljø. Men de mangler procesværktøjer til stresshåndtering og står usikre overfor opgaven. Typisk har virksomhederne heller ikke adgang til interne...... spørgsmål: 1. Hvilke arbejdsmæssige og ledelsesmæssige forhold er nødvendige for at SMV’er bliver i stand til at gennemføre en kollektiv proces, hvor virksomhederne selv udvikler, gennemfører og forankrer nye forebyggende strategier og indsatser, der skal forbedre det psykiske arbejdsmiljø? Derudover er der...

  10. A novel approach for immunization, screening and characterization of selected scFv libraries using membrane fractions of tumor cells. (United States)

    Tur, Mehmet Kemal; Rothe, Achim; Huhn, Michael; Goerres, Ute; Klimka, Alexander; Stöcker, Michael; Engert, Andreas; Fischer, Rainerr; Finner, Ricarda; Barth, Stefan


    Isolation of cell-surface specific antibodies prerequisites the functional expressing of antigens on intact cells, which are maintained routinely by cell culturing. However, long-term culturing of tumor cells could alter their antigen expression patterns and stable fixation of whole cells is not guaranteed on plastic surfaces during stringent screening procedures. We prepared functional breast cancer cell-membrane fractions that express surface molecules in their native conformation. Specific binding phages were isolated from phage antibody libraries constructed from the spleen messenger RNA of mice immunized with breast cancer cell-membrane fractions. After negative selection on non-mammary carcinoma cells and four rounds of positive selection on breast carcinoma cell lines, phage antibodies were enriched that bound specifically to breast cancer cell lines as confirmed by phage enzyme linked immunosorbent assay (ELISA) using 96-well plates coated with breast cancer cell membranes. The isolated phage antibodies were highly specific for the breast cancer cell line 8701-BC but not on other carcinoma such as the Hodgkin-derived cell line L540Cy as demonstrated by ELISA and flow cytometry. This report describes a rapid and more versatile method for isolating antibody fragments compared to whole cell screening procedures. One single membrane preparation can be stored for at least 15 months at -80 degrees C and used to immunize mice or for screening of antibody libraries. The selection and screening strategy used should be generally applicable to identify novel cell-surface antigens and their corresponding antibodies.

  11. A large semi-synthetic single-chain Fv phage display library based on chicken immunoglobulin genes

    Directory of Open Access Journals (Sweden)

    Jordaan Frances


    Full Text Available Abstract Background Antibody fragments selected from large combinatorial libraries have numerous applications in diagnosis and therapy. Most existing antibody repertoires are derived from human immunoglobulin genes. Genes from other species can, however, also be used. Because of the way in which gene conversion introduces diversity, the naïve antibody repertoire of the chicken can easily be accessed using only two sets of primers. Results With in vitro diagnostic applications in mind, we have constructed a large library of recombinant filamentous bacteriophages displaying single chain antibody fragments derived from combinatorial pairings of chicken variable heavy and light chains. Synthetically randomised complementarity determining regions are included in some of the heavy chains. Single chain antibody fragments that recognise haptens, proteins and virus particles were selected from this repertoire. Affinities of three different antibody fragments were determined using surface plasmon resonance. Two were in the low nanomolar and one in the subnanomolar range. To illustrate the practical value of antibodies from the library, phage displayed single chain fragments were incorporated into ELISAs aimed at detecting African horsesickness and bluetongue virus particles. Virus antibodies were detected in a competitive ELISA. Conclusion The chicken-derived phage library described here is expected to be a versatile source of recombinant antibody fragments directed against a wide variety of antigens. It has the potential to provide monoclonal reagents with applications in research and diagnostics. For in vitro applications, naïve phage libraries based on avian donors may prove to be useful adjuncts to the selectable antibody repertoires that already exist.

  12. [Comparative studies on the resorption, anti-anemic action and growth effect of FV-82 and dextrofer-100 on pigs]. (United States)

    Dilov, P; Chakŭrov, R; Iotsev, M; Iordanov, S


    Comparative investigations were carried out on the absorption, antianemic action, and growth effect with newborn pigs with FB-82 and dextrofer-100 (an iron dextran complex with 100 mg Fe3+ per cm3) injected i/m. The FB-82 is a combined preparation of 3500000 IU Tylosine tartrate, 0.008 g cyanocobalamin, 0.5 g pyridoxine hydrochloride, 0.1 g tartaric acid, and iron dextran up to 100 cm3 (= 100 mg Fe3+/cm3). Dextrofer-100 enriched with 50 mg Zn, 0.5 mg Co, 200 micrograms cyanocobalamin, and 100 mg pyridoxine hydrochloride for 100 cm3 under the compound name of fericin was also used to compare the anti-anemic effect of FB-82 and dextrofer-100. It was found that FB-82 applied to newborn pigs in a single dose of 2 cm3, i/m, was well absorbed; it developed high concentrations of tylosine in the plasma, and its sideremia and antianemic action were similar to those induced by an equivalent (with regard to iron) amount of dextrofer-100, whereas the result concerning the hematocrit value was better. Compared to dextrofer-100 the FB-82 preparation have better protection to pigs (lowered the mortality rate) and induced better development of the animals. Fericin did not differ essentially from FB-82 in terms of its effect on the red blood picture, however, the percent of protected pigs was lower.

  13. Functional Characterization of an scFv-Fc Antibody that Immunotherapeutically Targets the Common Cancer Cell Surface Proteoglycan CSPG4


    Wang, Xinhui; Katayama, Akihiro; Wang, Yangyang; YU Ling; Favoino, Elvira; Sakakura, Koichi; Favole, Alessandra; Tsuchikawa, Takahiro; Silver, Susan; Watkins, Simon C.; Kageshita, Toshiro; Ferrone, Soldano


    Cell surface chondroitin sulfate proteoglycan 4 (CSPG4) is an attractive target for antibody-based cancer immunotherapy because of its role in tumor cell biology, its high expression on malignant cells including cancer-initiating cells, and its restricted distribution in normal tissues. The clinical use of CSPG4 has been hampered by the lack of a CSPG4-specific chimeric, humanized, or fully human monoclonal antibody. To overcome this limitation, we generated a CSPG4-specific fully human singl...

  14. Evaluation of fruit vegetable wastes (FV) from a market place as a alternative deed stuffs in dairy cattle diets

    Energy Technology Data Exchange (ETDEWEB)

    Angulo, J.; Yepes, S. A.; Yepes, A. M.; Bustamante, G.; Mahecha, L.


    Solid food residues from market places represent about 10% to 20% of the total wastes of a city. A big proportion comes from the overproduction of fruits and vegetables in some production steps, its low price and abundance, turning them into potential contaminants, particularly since not always there is an appropriate knowledge and experience in its alternative use. (Author)

  15. DARPA Antibody Technology Program Standardized Test Bed for Antibody Characterization: Characterization of an MS2 ScFv Antibody (United States)


    position 83 in a construct engineered by DNA2.0 (Menlo Park, CA). Upon receipt, the lyophilized plasmid was rehydrated in ultrapure water and...Applied Biosystems ; Foster City, CA) and was washed with 10 column volumes of 20 mM imidazole in PBS (pH 7.4). MS2CP was eluted with 300 mM imidazole

  16. Fre-2, a locus closely linked to Fv-2, is rearranged in some erythroleukemias induced by Friend murine leukemia virus. (United States)

    Eisel, D; Veit, M; Friedrich, U; Pass, M; Sels, F T; Friedrich, R W


    Friend murine leukemia virus (F-MuLV) induces leukemia by integration into the cellular genome, thereby changing the structure of expression of cellular oncogenes. Here we describe a new F-MuLV integration site Fre-2 isolated from splenic DNA of an erythroleukemic animal. This site has been found rearranged in 5 out of 63 additional tumors; however, no F-MuLV proviruses could be detected in the vicinity of the rearrangement sites in these 5 cases. The rearrangements represented closely clustered chromosomal breakpoints, presumably chromosomal translocations. Exons transcribed into differentially spliced mRNAs of 1.9 and 3.7 kb have been found near the breakpoint. No sequences that are homologous to Fre-2 could be found in databases.

  17. Single chain FV constructs of anti-ganglioside GD2 antibodies for radioimaging and radioimmumotheraphy. Progress report

    Energy Technology Data Exchange (ETDEWEB)

    Cheung, N.K.V.; Larson, S.M.


    For the past several years, we have studied the anti-G{sub D2} murine monoclonal antibody, 3F8, in radiolabeled form, for diagnosis and therapy of neuroblastoma. The targeting properties of this antibody/antigen system are exceptional, with uptakes consistently in the highest range of reported results for in vivo human studies. The radioiodinated antibody 3F8 is now used by us as our criteria for diagnosis and staging of advanced neuroblastoma. This antibody is showing considerable promise also in our Phase I trials in Stage 4 neuroblastoma, and major responses are being seen at current dose level, with manageable marrow toxicity, but no limiting organ toxicity.

  18. AFSC/ABL: Origins of sockeye and chum salmon seized from the F/V Ying Fa (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Samples of chum (Oncorhynchus keta) and sockeye (O. nerka) salmon seized from the stateless fishing vessel Ying Fa were analyzed to determine their region of origin...

  19. Maternal genetic polymorphisms and unexplained recurrent miscarriage: a systematic review and meta-analysis. (United States)

    Shi, X; Xie, X; Jia, Y; Li, S


    The roles of genetic polymorphisms in the pathogenesis of recurrent miscarriage (RM) have been intensively studied. However, the results of these studies were inconsistent, especially when conducted in different populations. Therefore, we performed the current study to systematically review the broad spectrum of genetic polymorphisms that were suspected to be involved in RM, and discussed potential genetic biomarkers of RM. Eligible articles were identified in PubMed, Medline, Embase and CNKI. Odd ratios (ORs) and 95% confidence intervals (CIs) were used to describe the strength of association, and a probability value (p value) of 0.05 or less was considered as statistically significant. A total of 425 eligible articles were included in this systematic review and 369 articles evaluating 124 polymorphisms of 73 genes were meta-analyzed. Significant associations were found between RM and 53 genetic polymorphisms of 37 genes. Our findings suggest that genetic variants of HLA-G, IFNG, TNF, IL-6, IL-10, FII, FV, FXIII, ITGB3, MTR, MTHFR, PAI-1, NOS3, KDR, TP53, VEGFA, CYP17, CYP1A1, CYP2D6, ANXA5, and XCI may serve as biological markers of RM. This study indicates that over-active immunological responses, thrombophilia, abnormal placental function, and disturbance of metabolic regulation may be implicated in the pathogenesis of RM. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  20. Effect of elevated CO2 concentration on photosynthetic characteristics of hyperaccumulator Sedum alfredii under cadmium stress

    Institute of Scientific and Technical Information of China (English)

    Tingqiang Li; Qi Tao; Zhenzhen Di; Fan Lu; Xiaoe Yang


    The combined effects of elevated CO2 and cadmi-um (Cd) on photosynthetic rate, chlorophyl fluorescence and Cd accumulation in hyperaccumulator Sedum alfredi Hance were investigated to predict plant growth under Cd stress with rising atmospheric CO2 concentration. Both pot and hydroponic experiments were conducted and the plants were grown under ambient (350 mL L?1) or elevated (800 mL L?1) CO2. Elevated CO2 significantly (P<0.05) increased Pn (105%–149%), Pnmax (38.8%–63.0%) and AQY (20.0%–34.8%) of S. alfredii in al the Cd treatments, but reduced chlorophyl concentra-tion, dark respiration and photorespiration. After 10 days growth in medium with 50 mM Cd under elevated CO2, PSII activities were significantly enhanced (P<0.05) with Pm, Fv/Fm, F(II) and qP increased by 66.1%, 7.5%, 19.5% and 16.4%, respectively, as compared with ambient-grown plants. Total Cd uptake in shoot of S. alfredi grown under elevated CO2 was increased by 44.1%–48.5%, which was positively correlated with the increase in Pn. These results indicate that elevated CO2 promoted the growth of S. alfredi due to increased photosynthetic carbon uptake rate and photosynthetic light-use efficiency, and showed great potential to improve the phytoextraction of Cd by S. alfredi .

  1. Can inherited thrombophilia modulate the clinical phenotype of patients with antiphospholipid syndrome? (United States)

    Berman, Horacio; Ugarte-Gil, Manuel F; Espinosa, Gerard; Tàssies, Dolors; Monteagudo, Joan; Reverter, Joan Carles; Cervera, Ricard


    The current case-control study was aimed to determine the prevalence and the clinical significance of inherited thrombophilia - factor V Leiden and G20210A prothrombin polymorphisms - in patients with antiphospholipid syndrome (APS). 100 patients with APS (77 with primary APS and 23 with systemic lupus erythematosus [SLE]-APS), and 100 patients with first lower extremity deep venous thrombosis (DVT), and 200 healthy individuals as a control groups were analysed. Patients and control group were tested for factor V Leiden and prothrombin G20210A gene polymorphism. Factor V Leiden variant was found in 1% of APS patients, in 3% of healthy individuals (p=0.49), and 16% of patients with first DVT (p<0.0005). Prothrombin gene polymorphism was found in 6% of APS patients and in 2.5% of healthy subjects (p=0.21), and 13% of patients with DVT (p=0.14). In primary APS patients, factor V Leiden was present in 1.3% (1/77) and prothrombin gene polymorphism in 6.5% (5/77). No patient with SLE-APS had factor V Leiden and prothrombin gene variant was present in only one patient (4.3%). Patients with prothrombin polymorphism had higher prevalence of venous thrombosis, with no statistical significance (80% vs. 47.9%, p=0.35). There were no differences in the prevalence of recurrent thrombosis before or after APS diagnosis in patients with or without prothrombin gene polymorphism. Factor V Leiden and G20210A prothrombin variant seem to play no role in either the development of APS or in the type of involved vessel, with no increased risk of re-thrombosis during follow-up.

  2. The clinical impact of MTHFR polymorphism on the vascular complications of sickle cell disease

    Directory of Open Access Journals (Sweden)

    F. Moreira Neto


    Full Text Available Sickle cell disease (SCD is one of the most common inherited diseases in the world and the patients present notorious clinical heterogeneity. It is known that patients with SCD present activation of the blood coagulation and fibrinolytic systems, especially during vaso-occlusive crises, but also during the steady state of the disease. We determined if the presence of the factor V gene G1691A mutation (factor V Leiden, the prothrombin gene G20210A variant, and methylenetetrahydrofolate reductase (MTHFR C677T polymorphism may be risk factors for vascular complications in individuals with SCD. We studied 53 patients with SCD (60% being women, 29 with SS (sickle cell anemia; 28 years, range: 13-52 years and 24 with SC (sickle-hemoglobin C disease; 38.5 years, range: 17-72 years hemoglobinopathy. Factor V Leiden, MTHFR C677T polymorphism, and prothrombin G20210A variant were identified by PCR followed by further digestion of the PCR product with specific endonucleases. The following vascular complications were recorded: stroke, retinopathy, acute thoracic syndrome, and X-ray-documented avascular necrosis. Only one patient was heterozygous for factor V Leiden (1.8% and there was no prothrombin G20210A variant. MTHFR 677TT polymorphism was detected in 1 patient (1.8% and the heterozygous form 677TC was observed in 18 patients (34%, 9 with SS and 9 with SC disease, a prevalence similar to that reported by others. No association was detected between the presence of the MTHFR 677T allele and other genetic modulation factors, such as alpha-thalassemia, ß-globin gene haplotype and fetal hemoglobin. The presence of the MTHFR 677T allele was associated with the occurrence of vascular complications in SCD, although this association was not significant when each complication was considered separately. In conclusion, MTHFR C677T polymorphism might be a risk factor for vascular complications in SCD.

  3. Life-Threatening Contraceptive-Related Pulmonary Embolism in a 14-Year-Old Girl with Hereditary Thrombophilia

    DEFF Research Database (Denmark)

    Hellfritzsch, Maja; Grove, Erik Lerkevang


    in patients with thrombophilia. CASE REPORT: A 14-year-old girl presented with acute onset of chest pain and dyspnea followed by syncope. She was hypoxic and hemodynamically compromised at admission. Computed tomography pulmonary angiography revealed a large central "saddle" pulmonary embolism causing nearly...... found heterozygote for the prothrombin G20210A mutation, and 9 months before admission she had initiated use of second-generation COCs. CONCLUSIONS: Hereditary thrombophilia and a family history of early-onset venous thromboembolism (VTE) each pose an increased risk of VTE and should be considered...

  4. An Unexpectedly High Rate of Thrombophilia Disorders in Patients with Superficial Vein Thrombosis of the Lower Extremities

    DEFF Research Database (Denmark)

    Sobreira, Marcone Lima; Rogatto, Silvia Regina; Dos Santos, Rodrigo Mattos


    of major hereditary and acquired thrombophilic factors in patients with SVT. METHODS: Sixty-six patients presenting with primary SVT underwent evaluation for thrombophilia: molecular testing for the factor V Leiden and factor II G20210 A (prothrombin) mutations, protein C, protein S, antithrombin...... that 34.2% of patients were heterozygous for factor V Leiden, 23.6% were heterozygous for the factor II mutation, 7.8% had antithrombin deficiency, 2.6% had protein S deficiency, and 2.1% had protein C deficiency. CONCLUSIONS: Our study showed that hereditary and acquired thrombophilias are higher than...

  5. Hypercoagulable States: What the Oral Surgeon Needs to Know. (United States)

    Bona, Robert


    Thrombophilia or hypercoagulable conditions can be thought of as either inherited or acquired. The inherited disorders include deficiencies of antithrombin, protein C, or protein S or the common disorders of factor V Leiden and prothrombin G20210A gene mutation. All these disorders are inherited as autosomal dominant and predispose individuals primarily to venous thrombosis. Acquired thrombophilic conditions are seen in individuals with cancer, phospholipid antibodies, and a whole host of other conditions that alter endothelial function, change blood levels of coagulant or anticoagulant proteins, activate platelets, or have other effects on coagulation proteins, platelet function, or the endothelium. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Identification of Damage in Hysteretic Structures. (United States)



  7. Fabricated or Induced Illness in Children: A Narrative Review of the Literature (United States)

    Lazenbatt, Anne


    Although child maltreatment due to abuse or neglect is pervasive within our society, less is known about fabricated or induced illness by carers (FII), which is considered to be a rare form of child abuse. FII occurs when a caregiver (in 93% of cases, the mother) misrepresents the child as ill either by fabricating, or much more rarely, producing…

  8. Influence of state of charge in lead-acid batteries operating in PV systems; Comportamiento no repetitivo de las baterias de plomo-acido operando en sistemas FV.

    Energy Technology Data Exchange (ETDEWEB)

    Vela, N.; Chenlo, F.


    Correct determination of the overcharge cut-off voltage is a key point for both the optimal operation and maximum life-time of batteries in photovoltaic (PV) systems. This work presents the results of analysing the influence on charge voltage of different operation conditions, mainly current rate, temperature and state of charge (SOC). From the results obtained we have observed that voltage evolution during a charge process depends on its activation degree of the battery. The battery activation is reached when battery was previously fully charged. So, we can conclude that variation of the charge voltage with time as function of starting point (fully charged or fully discharged) together with current rate and temperature should be taking into account in the battery SOC determination and in the design of charge controllers. (Author)

  9. Screening for a human single chain Fv antibody against epitope on amyloid-beta 1-40 from a human phage display library

    Institute of Scientific and Technical Information of China (English)

    ZHAO Zhen-fu; GAO Guo-quan; LIU Shu; ZOU Jun-tao; XIE Yao; YUAN Qun-fang; WANG Hua-qiao; YAO Zhi-bin


    @@ Amyloid-beta peptides (Aβ) are believed to be responsible for the mental decline in patients with Alzheimer's disease (AD). In 1999, Schenk et al1 reported that immunization with Aβ attenuated AD-like pathology in the PDAPP mouse, and developed a new vaccination approach to AD.

  10. CD40-targeted adenoviral gene transfer to dendritic cells through the use of a novel bispecific single-chain Fv antibody enhances cytotoxic T cell activation

    NARCIS (Netherlands)

    Brandao, JG; Scheper, RJ; Lougheed, SM; Curiel, DT; Tillman, BW; Gerritsen, WR; van den Eertwegh, AJM; Pinedo, HM; Haisma, HJ; de Gruijl, TD


    Adenoviral (Ad) transduction of dendritic cells (DC) is a promising vaccination strategy. However, clinical applicability of Ad vectors is hampered by the necessity to use high titers of infectious Ad particles for efficient DC transduction. Here, we report on the production of a bacterially express

  11. Convergence Analysis of a FV-FE Scheme for Partially Miscible Two-Phase Flow in Anisotropic Porous Media

    KAUST Repository

    Saad, Bilal Mohammed


    We study the convergence of a combined finite volume nonconforming finite element scheme on general meshes for a partially miscible two-phase flow model in anisotropic porous media. This model includes capillary effects and exchange between the phase. The diffusion term,which can be anisotropic and heterogeneous, is discretized by piecewise linear nonconforming triangular finite elements. The other terms are discretized by means of a cell-centered finite volume scheme on a dual mesh. The relative permeability of each phase is decentred according the sign of the velocity at the dual interface. The convergence of the scheme is proved thanks to an estimate on the two pressures which allows to show estimates on the discrete time and compactness results in the case of degenerate relative permeabilities. A key point in the scheme is to use particular averaging formula for the dissolution function arising in the diffusion term. We show also a simulation of CO2 injection in a water saturated reservoir and nuclear waste management. Numerical results are obtained by in-house numerical code. © Springer International Publishing Switzerland 2014.

  12. One-Step Recovery of scFv Clones from High-Throughput Sequencing-Based Screening of Phage Display Libraries Challenged to Cells Expressing Native Claudin-1

    Directory of Open Access Journals (Sweden)

    Emanuele Sasso


    Full Text Available Expanding the availability of monoclonal antibodies interfering with hepatitis C virus infection of hepatocytes is an active field of investigation within medical biotechnologies, to prevent graft reinfection in patients subjected to liver transplantation and to overcome resistances elicited by novel antiviral drugs. In this paper, we describe a complete pipeline for screening of phage display libraries of human scFvs against native Claudin-1, a tight-junction protein involved in hepatitis C virus infection, expressed on the cell surface of human hepatocytes. To this aim, we implemented a high-throughput sequencing approach for library screening, followed by a simple and effective strategy to recover active binder clones from enriched sublibraries. The recovered clones were successfully converted to active immunoglobulins, thus demonstrating the effectiveness of the whole procedure. This novel approach can guarantee rapid and cheap isolation of antibodies for virtually any native antigen involved in human diseases, for therapeutic and/or diagnostic applications.

  13. Labeling of Anti-MUC-1 Binding Single Chain Fv Fragments to Surface Modified Upconversion Nanoparticles for an Initial in Vivo Molecular Imaging Proof of Principle Approach

    Directory of Open Access Journals (Sweden)

    Markus Haase


    Full Text Available In vivo optical Imaging is an inexpensive and highly sensitive modality to investigate and follow up diseases like breast cancer. However, fluorescence labels and specific tracers are still works in progress to bring this promising modality into the clinical day-to-day use. In this study an anti-MUC-1 binding single-chain antibody fragment was screened, produced and afterwards labeled with newly designed and surface modified NaYF4:Yb,Er upconversion nanoparticles as fluorescence reporter constructs. The MUC-1 binding of the conjugate was examined in vitro and in vivo using modified state-of-the-art small animal Imaging equipment. Binding of the newly generated upconversion nanoparticle based probe to MUC-1 positive cells was clearly shown via laser scanning microscopy and in an initial proof of principal small animal optical imaging approach.

  14. The recombinant anti-EGF receptor immunotoxin 425(scFv)-ETA' suppresses growth of a highly metastatic pancreatic carcinoma cell line

    NARCIS (Netherlands)

    Bruell, D; Stocker, M; Huhn, M; Redding, N; Kupper, M; Schumacher, P; Paetz, A; Bruns, CJ; Haisma, HJ; Fischer, R; Finnern, R; Barth, S


    Pancreatic carcinoma still has the highest mortality rate in comparison to any other malignancy. Major reasons are late detection of disease, highly aggressive tumor growth and the early formation of metastases. Thus, novel effective therapies are urgently needed to improve the outcome of the patien

  15. Isolation of ScFv antibodies of rP27Kip1 from phage display libraries constructed from immunized and non-immunized repertoires

    Institute of Scientific and Technical Information of China (English)

    曹跃琼; 乔守怡; 袁有忠; 黄建生; 赵寿元


    Through mRNA extract, RT and a series of PCR, phage antibody libraries were constructed from rP27Kiplimmunized and non-immunized mice. After only one round of selection with rP27Kipl, clones from each library were chosen randomly and digested by Taq I and Hinf I. 11 of 64 clones from the immunized animal had consistent restriction pattern, while none of the 64 clones from the non-immunized animal had, except that one had the same fragments pattern as that of the 11 clones. The 12 fragments were expressed in E. coli BL21(DE3)/pET-28b(+) system. ELISA showed that some of the fragments could bind to rP27Kipl specifically. All these results implied that specific antibody can be obtained by genetic engineering without hybridoma or many rounds of growth and panning selection.

  16. Identification of the curve of maximum power of photovoltaic modules using simulation software; Identificacao da curva de maxima potencia de modulos FV utilizando softwares de simulacao

    Energy Technology Data Exchange (ETDEWEB)

    Moreira, Andre Pimentel; Ramalho, Geraldo Luis Bezerra; Dias, Samuel Vieira [Centro Federal de Educacao Tecnologica do Ceara (CEFETCE), Fortaleza, CE (Brazil)], emails:,,; Carvalho, Paulo Cesar Marques de [Universidade Federal do Ceara (PPGEE/UFC), Fortaleza, CE (Brazil). Programa de Pos Graduacao em Engenharia Eletrica], e-mail:; Borges Neto, Manuel Rangel [Centro Federal de Educacao Tecnologica de Petrolina (CEFETPet), Petrolina, PE (Brazil)], email:


    This article is presented the study and compared the behaviour of real and simulated a photovoltaic system, through the design and simulation software, Electronic Workbench (MultiSIM 9), AIM-Spice and identifying the point of maximum power (MPP), with the help of modeling software from Matlab. The results of the simulated model were very close to data collected from a real.photovoltaic system. (author)

  17. Large-Scale Purification of r28M: A Bispecific scFv Antibody Targeting Human Melanoma Produced in Transgenic Cattle.

    Directory of Open Access Journals (Sweden)

    Katrin Spiesberger

    Full Text Available 30 years ago, the potential of bispecific antibodies to engage cytotoxic T cells for the lysis of cancer cells was discovered. Today a variety of bispecific antibodies against diverse cell surface structures have been developed, the majority of them produced in mammalian cell culture systems. Beside the r28M, described here, no such bispecific antibody is known to be expressed by transgenic livestock, although various biologicals for medical needs are already harvested-mostly from the milk-of these transgenics. In this study we investigated the large-scale purification and biological activity of the bispecific antibody r28M, expressed in the blood of transgenic cattle. This tandem single-chain variable fragment antibody is designed to target human CD28 and the melanoma/glioblastoma-associated cell surface chondroitin sulfate proteoglycan 4 (CSPG4.With the described optimized purification protocol an average yield of 30 mg enriched r28M fraction out of 2 liters bovine plasma could be obtained. Separation of this enriched fraction by size exclusion chromatography into monomers, dimers and aggregates and further testing regarding the biological activity revealed the monomer fraction as being the most appropriate one to continue working with. The detailed characterization of the antibody's activity confirmed its high specificity to induce the killing of CSPG4 positive cells. In addition, first insights into tumor cell death pathways mediated by r28M-activated peripheral blood mononuclear cells were gained. In consideration of possible applications in vivo we also tested the effect of the addition of different excipients to r28M.Summing up, we managed to purify monomeric r28M from bovine plasma in a large-scale preparation and could prove that its biological activity is unaffected and still highly specific and thus, might be applicable for the treatment of melanoma.

  18. Oral contraception in women with mild thrombophilia: what have we learned recently? (United States)

    Blanco-Molina, Ángeles


    Mild thrombophilias include heterozygous forms of Factor V Leiden and prothrombin G20210A mutation and high levels of factor VIII. These defects are responsible for only a moderate increase of venous thromboembolism (VTE) risk compared with strong thrombophilias (antithrombin III, protein C and protein S deficiencies and homozygous forms of factor V Leiden and G20210A prothrombin mutation). Combined oral contraceptives increase the VTE risk in women in a fertile age, and in a far more important way in thrombophilia carrier women. Universal screening of thrombophilia test before the use of combined oral contraceptives is not recommended. However, selective VTE-history based screening is associated with a better cost-effectiveness, especially in asymptomatic relatives of carriers of strong thrombophilia. Currently, combined oral contraceptives are discouraged in women with any thrombophilic defects. The VTE risk is higher during pregnancy and postpartum period compared to hormonal contraceptive use period in women with mil thrombophilia. For this reason, a careful election of an alternative birth control method is necessary in these women.

  19. Cerebral Vein Thrombosis:Screening of Acquired and Hereditary Thrombophilic Risk Factors

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    Sarraf Payam


    Full Text Available Cerebral vein thrombosis (CVT is an infrequent condition with a large variety of causes that can lead to serious disabilities. However, in 20% to 35% of cases, no cause is found. In this study we evaluated the hereditary (P & C Proteins, antithrombin, mutation of prothrombin G20210A and factor V Leiden, other risk factors (hyperhomocycteinemia, factor VIII, ACL-ab, APL-ab, and OCP and clinical manifestations among a population of Iranian patients with CVT. 18 women and 10 men aged 16 to 50 years with CVT were screened for inherited and acquired coagulation risk factors. No one had an abnormal ACL-ab, APL-ab or antithrombin III deficiency. One had prothrombin G20210A mutation (heterozygot (3.6%. Hyperhomocycteinemia was observed in 5 patients (17.9%. APC-R was decreased in 3 (10.7%. 2 had positive factor V Leiden mutation (heterozygot (7.1%. 17 had an increased of factor VIII (60.7. PS and PC deficiencies were each detected in two cases (7.1%. Conclusion: Our study suggests that screening for inherited thrombophilia may be an integral part in the diagnostic workup and duration of treatment in patients with CVT.

  20. Etiopathogenesis of Sheehan’s Syndrome: Roles of Coagulation Factors and TNF-Alpha

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    Halit Diri


    Full Text Available Sheehan’s Syndrome (SS is defined as pituitary hormone deficiency due to ischemic infarction of the pituitary gland as a result of massive postpartum uterine hemorrhage. Herein, we aimed to investigate the roles of Factor II (G20210A, Factor V (G1691A, MTHFR (C677T and A1298C, PAI-1 4G/5G, and TNF-α (-308  G>A gene polymorphisms in the etiopathogenesis of SS. Venous blood samples were obtained from 53 cases with SS and 43 healthy women. Standard methods were used to extract the genomic DNAs. Factor II (G20210A, Factor V (G1691A, and MTHFR (C677T and A1298C polymorphisms were identified by real-time PCR. PAI-1 4G/5G and TNF-α (-308  G>A gene polymorphisms were detected with polymerase chain reaction (PCR and restriction fragment length polymorphism (RFLP methods. According to statistical analysis, none of the polymorphisms were found to be significantly higher in the SS group compared to the control group. Hence, we suggest that genetic factors other than Factor II, Factor V, MTHFR, PAI-1, and TNF-α gene polymorphisms should be researched in the etiopathogenesis of SS.

  1. Preterm infant with a catastrophic hemorrhagic-thromboembolic incident. (United States)

    Abdelhamid, Adel E


    Thrombotic disease of the newborn is uncommon but usually associated with serious morbidity and mortality. Although the operating mechanisms of coagulation and fibrinolysis are the same in all age groups, plasma concentrations of the two systems' components are significantly different in neonates compared to children and adults. This places neonates at greater risk for thrombosis that may rise considerably if a predisposing factor is present or a genetic or medical condition predisposing to thrombosis coexists. While marginal, the possibility of abnormal bleeding secondary to congenital prothrombotic disorders has been described. A significant association between thromboembolic/hemorrhagic disease in newborns and each of factor V(Leiden) and prothrombin G20210A mutations has been reported. Although not a frequent occurrence in adults and children, congenital 'multigenic' thrombophilia is well known. However, the combined heterozygote state of both mutations is perhaps underreported in preterm infants. We present a severely intrauterine growth-restricted preterm baby born to consanguineous parents. He had stroke as part of a generalized bleeding-thromboembolic incident caused by combined heterozygote mutation of factor V(Leiden) and prothrombin G20210A, each of which was then found in a heterozygote form in each of the 2 parents.

  2. Etiopathogenesis of Sheehan's Syndrome: Roles of Coagulation Factors and TNF-Alpha. (United States)

    Diri, Halit; Sener, Elif Funda; Bayram, Fahri; Tascioglu, Nazife; Simsek, Yasin; Dundar, Munis


    Sheehan's Syndrome (SS) is defined as pituitary hormone deficiency due to ischemic infarction of the pituitary gland as a result of massive postpartum uterine hemorrhage. Herein, we aimed to investigate the roles of Factor II (G20210A), Factor V (G1691A), MTHFR (C677T and A1298C), PAI-1 4G/5G, and TNF- α (-308  G > A) gene polymorphisms in the etiopathogenesis of SS. Venous blood samples were obtained from 53 cases with SS and 43 healthy women. Standard methods were used to extract the genomic DNAs. Factor II (G20210A), Factor V (G1691A), and MTHFR (C677T and A1298C) polymorphisms were identified by real-time PCR. PAI-1 4G/5G and TNF- α (-308  G > A) gene polymorphisms were detected with polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) methods. According to statistical analysis, none of the polymorphisms were found to be significantly higher in the SS group compared to the control group. Hence, we suggest that genetic factors other than Factor II, Factor V, MTHFR, PAI-1, and TNF- α gene polymorphisms should be researched in the etiopathogenesis of SS.

  3. The Frequency of Some Thrombophilic Mutations in Eastern Turkey. (United States)

    Ozturk, Nurinnisa; Bakan, Ebubekir; Gul, Mehmet Ali; Bakan, Nuri; Sebin, Engin; Kiziltunc, Ahmet


    Factor V / Factor II / Methylenetetrahydrofolate reductase, gene polymorphisms are closely associated with thrombophilia. Regional frequencies of these mutations may show a characteristic state. The aim of our study was to evaluate the frequency of commonly seen Factor V / Factor II / Methylenetetrahydrofolate reductase gene polymorphisms in Eastern Turkey. In 433 patients sent to the laboratory with the suspicion of thrombophilia, using whole blood samples, an automated Nucleic Acid Test was used for mutation determinations in Verigene System. The kit module was designed to detect the Factor V G1691A / Factor II G20210A / Methylenetetrahydrofolate reductase gene C677T single nucleotide polymorphisms. In 433 patients, 8.7% for Factor V G1691A polymorphisms were heterozygous genotype, 3.9% for Factor II G20210A polymorphisms were heterozygous genotype, and 43.9% for methylenetetrahydrofolate reductase 677C>T polymorphisms were heterozygous genotype and 3.0% homozygous mutation genotype. Detection of these commonly seen Factor V / Factor II / Methylenetetrahydrofolate reductase single nucleotide polymorphisms can help to identify patients in high risk group and to evaluate the interaction of genetic and acquired risk factors. Our findings suggest that commonly seen thrombophilic allele mutation frequency in our region is the same as the data reported in the literature.

  4. Puerarin Attenuates Ovalbumin-Induced Lung Inflammation and Hemostatic Unbalance in Rat Asthma Model

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    Feng Dong


    Full Text Available Aim. We aimed to investigate and evaluate the preventive activity of puerarin on the ovalbumin-induced asthma rat model. Materials and Methods. Male Wistar rats were sensitized intraperitoneally on days 0, 7, and 14 and challenged to ovalbumin intratracheally on day 21. Groups of sensitized rats were treated randomly either with placebo, puerarin, dexamethasone, or puerarin combined with dexamethasone, from days 15 to 20. Inflammatory markers, including cell counts in bronchoalveolar lavage fluid (BALF, inflammatory cytokines, histopathology, and coagulation parameters, such as coagulation tests and the activity of coagulation factors, were analyzed. Results. Puerarin significantly inhibited the recruitment of inflammatory cells in BALF and lung tissue. At the same time, the release of IL-4, IL-10, and IFN-γ in serum and the expression of mRNAs in lung tissue homogenate were changed by puerarin. Administration of puerarin also effectively rectified the coagulation disorder in asthmatic rats, such as prothrombin time (PT (P<0.01, thrombin time (TT (P<0.05, fibrinogen (FIB (P<0.01,the activity of factor II (FII (P<0.01, the activity of factor V (FV (P<0.05, the activity of factor VII (FVII (P<0.05, the activity of factor X (FX (P<0.05, the activity of factor VIII (FVIII (P<0.01, the activity of factor IX (FIX (P<0.05, and the activity of factor XII (FXII (P<0.05. Conclusions. Our results provide a clue that puerarin was useful for the preventive of allergic airway disease in rodents.

  5. Molecular analysis of G202010A mutation in factor II of blood coagulation and its relationship with polymorphism rs5030737 of MBL gene in recurrent pregnancy loss

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    Neda Mohammad Rafiee


    Full Text Available Purpose: Miscarriage means ending a pregnancy at any stage of the fetus. Recurrent pregnancy loss is defined as two or more loss of pregnancy to be detected continuous or discontinuous before the twentieth week of pregnancy.Mutations in the gene for coagulation factor IIand MBL gene can be involved in miscarriage. Hence, according to importance of this issue, the purpose of this study is to investigate G20210A mutation of coagulation factor IIand its relationship with polymorphism rs5030737 of MBL gene to evaluate on-time diagnosis and treatment of miscarriage. Method: in order to conduct the study, 41 patients with history of miscarriage and 48 healthy women with successful delivery were selected. A questionnaire was fulfilled by them to insert comprehensive information including history of miscarriage, history of miscarriage among relatives, age, weight, blood type, type of marriage and smoking. Then, blood sample of every one was taken. The blood samples were transferred to the laboratory and after extraction of DNA from each of samples, G20210A mutation in coagulation factor IIandtype of polymorphism rs5030737 in MBL gene was determined using PCR method. Finally, analysis of the results and assessment of other important and effective factors in them was done using Epi Info software and using chi square (X2 test. Results: among the patients, frequency of patients with one miscarriage was determined to 29.25%; frequency of patients with two miscarriages to 58.85% and frequency of patients with 3 miscarriages was obtained to 4.9%. In regard with assessing G20210A mutation in coagulation factor II, frequency percent ofheterozygous or carriers were equal to 7.3% among patients and to 2.1% for healthy individuals. Among them, frequency of available genotypes included GG: 92.6%; GA: 7.3%, AA: 0 in patient group and GG: 97.9%, GA: 2.1% and AA: 0 in healthy individuals. On the other hand, frequency of types of polymorphism of MBL included BB: 17%; AB

  6. Hybrid Fv - Biogas/Diesel System Measuring With Homer. Case of Application: Farm Pozo Verde, Jamundi Municipality, Cauca Valley, Colombia; Dimensionado de un Sistema Hibrido Fv - Biogas/Diesel mediante el Empleo de la Herramienta Homer. Caso de Aplicacion: Granja Pozo Verde, Municipio de Jamundi, Valle del Cauca, Colombia

    Energy Technology Data Exchange (ETDEWEB)

    Dominguez, J.; Manrique, P. A.; Pinedo, I.


    This report completes the previous one, by adding the characterization of renewable energy systems based on optimization and sizing tools. A case study has been carried out in the Valley of Cauca (Colombia) using HOMER, once the solar energy potential was defined. This work is about the production of electricity using hybrid power systems (HPS). The HPS chosen for this study is based on two of the most common energy resources in most of Colombian rural areas: solar energy and biomass waste. The biomass waste is obtained by animal manure from livestock farms, particularly from pigs, which is highly valued in terms of energy production. The breeding of pigs has a huge economic, environmental and social importance in this Colombian region. In this report, the HPS technology and the energy contribution of the biogas produced from animal manure are described. A technical and economic simulation of a HPS has been performed using the optimization tool HOMER. These reports are the result of a collaboration established between Universidad del Valle (Colombia) and CIEMAT (Spain), and have been done by the author while his staying at our center included on his PhD program. Keywords: renewable energies, hybrid systems, biogas, technology sizing, rural electrification, Latin America. (Author) 8 refs.

  7. A Cassette Vector System for the Rapid Cloning and Production of Bispecific Tetravalent Antibodies

    Directory of Open Access Journals (Sweden)

    Stefanie Claudia Pohl


    Full Text Available Bivalent single chain (scFv-Fc antibodies have been used for years as recombinant alternatives of natural immunoglobulins. We have extended this approach to the scFv-Fc-scFv antibody format to obtain tetravalent antigen binding and the possibility to generate bispecific antibodies. We developed a mammalian expression vector system to construct tetravalent scFv-Fc-scFv antibodies with two NcoI+NotI compatible cloning sites flanking the Fc gene fragment. We demonstrated direct cloning from single chain antibody gene libraries and tested various scFv combinations. Transient production of scFv-Fc-scFv antibodies in human embryonic kidney (HEK 293T cells achieved volumetric yields of up to 10 mg/L. However, expression levels were strongly dependent on the carboxyterminal scFv and the scFv combination. All scFv-Fc-scFv antibodies exclusively formed disulfide-linked homodimers. Antigen binding studies revealed dual specificity for all scFv-Fc-scFv employing different scFv fragments. Comparison of C-reactive protein (CRP specific monovalent scFv LA13-IIE3, bivalent scFv-Fc and Fc-scFv LA13-IIE3, and tetravalent scFv-Fc-scFv (scFv LA13-IIE3 in combination with scFvs LA13-IIE3, TOB4-B11, or TOB5-D4 revealed an up to 500-fold increased antigen binding. This novel scFv-Fc-scFv antibody expression system allows simple and fast testing of various scFv combinations.

  8. Dicty_cDB: AFO328 [Dicty_cDB

    Lifescience Database Archive (English)

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  9. Dicty_cDB: VHM682 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available *shc*wcrfmvkinssr*ksfsllynysi*ywffn*ll*f gfqcslrmak*wcc***aystrrwcifk*l*rlvsl*wyaltttnyky*i*qrnefqlcw *hfvyf...ffstk*knr Frame B: in*ikekeneiiiiinfnnnn*f*lwcint*kfsr*se*iwfi*ysksngcstk***fii w

  10. 12 CFR 19.231 - Order to dismiss a director or senior executive officer. (United States)


    ... dismiss from office any director or senior executive officer under section 38(f)(2)(F)(ii) of the FDI Act... based on supervisory criteria other than capital, pursuant to section 38(g) of the FDI Act....

  11. Molecular cloning of Taenia taeniaeformis oncosphere antigen genes. (United States)

    Cougle, W G; Lightowlers, M W; Bogh, H O; Rickard, M D; Johnson, K S


    Infection of mice with the cestode Taenia taeniaeformis exhibits several important features common to other cestode infections, including the ability to vaccinate with crude antigen mixtures. Partial purification of the protective oncosphere antigens has been reported with a cutout from deoxycholate (DOC) acrylamide gels; this cutout was called fraction II (FII), and comprises approximately 10% of total DOC-soluble oncosphere antigen. Western blots of DOC gels probed with anti-FII antisera revealed a series of 3-5 discrete bands within the FII region. Further fractionation of the FII antigens on DOC gels was impractical due to limitations in supply of oncospheres, so a cDNA library was constructed from 150 ng of oncosphere mRNA and screened with alpha-FII antisera. Two distinct clone families were identified, oncA and oncB. Antibodies affinity-purified on either of two representative members, oncA1 and oncB1, recognised all the FII bands. Individual FII bands excised from a DOC gel resolved into an overlapping series of molecules when re-run on SDS-PAGE, indicating that each FII band consisted of several polypeptides of differing molecular weight. Immunoprecipitates resolved on SDS-PAGE revealed that alpha-FII recognised 3 major oncosphere antigens, of 62, 34 and 25 kDa; antisera against oncB precipitated both the 34- and 25-kDa antigens, whereas alpha-oncA antisera precipitated the 62-kDa antigen. We conclude that oncA and oncB encode the major antigens in the FII complex. The 62-kDa antigen encoded by oncA1 was the only common antigen precipitated by anti-FII and two other antisera raised against different protective extracts, suggesting that it may be a protective component in all three. Southern blot results indicate that oncA and oncB are distinct genes present at low copy number in the genome. Evidence is also presented suggesting that some cestode mRNAs, including oncA, may use variant polyadenylation signals.

  12. Uncertain thrombophilia markers. (United States)

    Franchini, Massimo; Martinelli, Ida; Mannucci, Pier Mannuccio


    The development of venous thromboembolism (VTE), which includes deep-vein thrombosis and pulmonary embolism, may be associated with inherited or acquired risk factors that can be measured in plasma or DNA testing. The main inherited thrombophilias include the plasma deficiencies of the natural anticoagulants antithrombin, protein C and S; the gain-of-function mutations factor V Leiden and prothrombin G20210A; some dysfibrinogenaemias and high plasma levels of coagulation factor VIII. Besides these established biomarkers, which usually represent the first-level laboratory tests for thrombophilia screening, a number of additional abnormalities have been less consistently associated with an increased VTE risk. These uncertain causes of thrombophilias will be discussed in this narrative review, focusing on their clinical impact and the underlying pathogenetic mechanisms. Currently, there is insufficient ground to recommend their inclusion within the framework of conventional thrombophilia testing.

  13. Thrombosis and inflammatory bowel disease-the role of genetic risk factors

    Institute of Scientific and Technical Information of China (English)

    Georgia Tsiolakidou; Ioannis E Koutroubakis


    Thromboembolism is a significant cause of morbidity and mortality in patients with inflammatory bowel dis-ease (IBD). Recent data suggest thromboembolism as a disease-specific extraintestinal manifestation of IBD,which is developed as the result of multiple interac-tions between acquired and genetic risk factors. There is evidence indicating an imbalance of procoagulant,anticoagulant and fibrinolitic factors predisposing in thrombosis in patients with IBD. The genetic factors that have been suggested to interfere in the thrombotic manifestations of IBD include factor V Leiden, factor Ⅱ (prothrombin, G20210A), methylenetetrahydrofolate reductase gene mutation (MTHFR, 6777T), plasminogen activator inhibitor type 1 (PAI-1) gene mutation and fac-tor ⅩⅢ (va1341eu). In this article we review the current data and future prospects on the role of genetic risk factors in the development of thromboembolism in IBD.

  14. Venous thromboembolism in children

    Directory of Open Access Journals (Sweden)

    Helena Pereira


    Full Text Available Introduction: Venous thromboembolism is rare among children and has a multi-factorial aetiology. It’s important to establish the diagnosis and evaluate the functional prognosis. Case report: A previously healthy 11 year old adolescent was observed in the emergency room with clinical signs of superficial venous thrombosis of the upper limb. Laboratorial evaluation showed a sedimentation rate, coagulation study, autoimmune antibodies and homocysteine with normal values. Testing for heritable thrombophilia revealed prothrombin mutation (G20210A, heterozigosity and mutation of PAI-1 (4G e -844A, both of which are associated with hypercoagulable state and indication to do prophylaxis with low molecular weight heparin in higher risk situations. Discussion/Conclusion: The rarity of thromboembolic events at this age and the atypical localization lead to an exhaustive laboratorial evaluation. Thrombophilia mutations may clinically become evident in adolescence, and its detection is important because of children’s lifestyle and the need of prophylactic treatment in some situations.

  15. Recurrent pregnancy loss in a subject with heterozygote factor V Leiden mutation; a case report (United States)

    Ebrahimzadeh-Vesal, Reza; Azam, Roza; Ghazarian, Arvin; Hajesmaeili, Mogge; Ranji, Najmeh; Ezzati, Mohammad Reza; Sadri, Mehrdad; Mohammadi, Mohammad Ali; Khavandi, Siamak


    Recurrent pregnancy loss is usually defined as the loss of two or more consecutive pregnancies before 20 weeks of gestation, which occurs in approximately 5% of reproductive-aged women. It has been suggested that women with thrombophilia have an increased risk of pregnancy loss and other adverse pregnancy outcomes. Thrombophilia is an important predisposition to blood clot formation and is considered as a significant risk factor for recurrent pregnancy loss. The inherited predisposition to thrombophilia is most often associated with factor V Leiden mutation, prothrombin G20210A mutation, and methylenetetrahydrofolate reductase C677T and A1298C gene variants. The net effect is an increased cleavage of prothrombin to thrombin and excessive blood coagulation. PMID:26989729

  16. Testosterone, anastrozole, factor V Leiden heterozygosity and osteonecrosis of the jaws. (United States)

    Pandit, Ramesh S; Glueck, Charles J


    Our specific aim is to describe the development of thrombotic osteonecrosis of the jaws after testosterone-anastrozole therapy in a 55-year-old white man subsequently found to have previously undiagnosed factor V Leiden heterozygosity. Before the diagnosis of V Leiden heterozygosity, he was given testosterone gel, 50 mg/day, and on testosterone, serum testosterone (963 ng/dl) and estradiol were high (50 pg/ml). Anastrozole was started, and testosterone was continued. Six months later, osteonecrosis of the jaws was diagnosed. Exogenous testosterone is aromatized to estradiol and estradiol-induced thrombophilia, when superimposed on underlying familial thrombophilia, as in this case, may lead to thrombosis and osteonecrosis. We recommend that before giving testosterone, at a minimum, screening for the factor V Leiden and G20210A mutations, and factor VIII and XI activity be carried out, to avoid unanticipated thrombosis.

  17. Nonarteritic Anterior Ischemic Optic Neuropathy and Double Thrombophilic Defect: A New Observation

    Directory of Open Access Journals (Sweden)

    Eleni Papageorgiou


    Full Text Available We report the first case of nonarteritic anterior ischemic neuropathy (NAION associated with double thrombophilia: protein S deficiency and prothrombin G20210A mutation. A 58-year-old man is presented including the clinical and laboratory findings, cardiovascular profile and thrombophilia screening. The patient presented with 3/10 vision and an inferior altitudinal defect in the right eye. Funduscopic examination of the right eye revealed a hyperemic optic disk with blurred superior optic disk border and sectoral nerve fiber layer edema. Complete blood count, erythrocyte sedimentation rate and C-reactive protein were normal, suggesting a NAION. A workup of cardiovascular risk factors revealed hyperlipidemia, arterial hypertension and high-risk asymptomatic coronary artery disease. Due to the family history of deep vein thrombosis in the patient’s daughter, a thrombophilia screening was additionally performed. The results revealed a double thrombophilic defect, namely congenital protein S deficiency and heterozygosity for prothrombin G20210A mutation, which were also identified in the patient’s daughter. Anticoagulant warfarin therapy was initiated and the patient underwent a triple bypass surgery. At three-month follow-up, the right optic disk edema had resolved, leaving a pale superior optic nerve head. Visual acuity in the right eye had slightly improved to 4/10; however, the dense inferior altitudinal field defect had remained unchanged. The patient is currently treated with warfarin, atorvastatin, irbesartan and metoprolol. This case suggests that the first line of investigation in all patients with NAION involves assessment of cardiovascular risk factors. However, careful history taking will identify NAION patients who are eligible for additional thrombophilia screening: young patients without vasculopathic risk factors, bilateral or recurrent NAION, idiopathic or recurrent venous thromboembolism (VTE, positive family history of VTE

  18. Trombofilias en pacientes con pérdida gestacional recurrente (PGR

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    Gabriel Bedoya


    Full Text Available

    Una de las posibles causas de la PGR es la trombofilia, la
    cual se define como una tendencia a desarrollar trombosis con
    variabilidad en las manifestaciones clínicas dependiente de la
    región vascular afectada por la ausencia del flujo sanguíneo. La
    trombosis es una enfermedad multifactorial, en la cual
    contribuyen factores genéticos y ambientales al riesgo del
    desarrollo de la enfermedad (1. Las trombofilias heredadas están asociadas a polimorfismos en los genes del factor V Leiden
    G1691A, protrombina G20210A y ilentetrahidrofolatoreductasa
    (MTHFR C677T; también se asocia a las deficiencias de los niveles de antitrombina III (AT-III, proteína C (PC y proteína
    S (PS en el sistema vascular y a la resistencia a la proteína C
    activada (RPCa. Varios estudios han encontrado asociación de
    las trombofilias heredadas con PGR y trombosis venosa asociada
    al embarazo (2, aunque los mecanismos fisiopatológicos responsables de las patologías vasculares placentarias en mujeres con trombofilia aun no han sido esclarecidos (3.
    El objetivo de este estudio es establecer la asociación de las
    trombofilias heredadas G1691A, G20210A, C677T, las
    deficiencias de AT-III, PC. PS y la RPCa, con la pérdida gestacional recurrente.



  19. Nonarteritic anterior ischemic optic neuropathy and double thrombophilic defect: a new observation. (United States)

    Papageorgiou, Eleni; Karamagkiolis, Spyridon; Dimera, Vasiliki


    WE REPORT THE FIRST CASE OF NONARTERITIC ANTERIOR ISCHEMIC NEUROPATHY (NAION) ASSOCIATED WITH DOUBLE THROMBOPHILIA: protein S deficiency and prothrombin G20210A mutation. A 58-year-old man is presented including the clinical and laboratory findings, cardiovascular profile and thrombophilia screening. The patient presented with 3/10 vision and an inferior altitudinal defect in the right eye. Funduscopic examination of the right eye revealed a hyperemic optic disk with blurred superior optic disk border and sectoral nerve fiber layer edema. Complete blood count, erythrocyte sedimentation rate and C-reactive protein were normal, suggesting a NAION. A workup of cardiovascular risk factors revealed hyperlipidemia, arterial hypertension and high-risk asymptomatic coronary artery disease. Due to the family history of deep vein thrombosis in the patient's daughter, a thrombophilia screening was additionally performed. The results revealed a double thrombophilic defect, namely congenital protein S deficiency and heterozygosity for prothrombin G20210A mutation, which were also identified in the patient's daughter. Anticoagulant warfarin therapy was initiated and the patient underwent a triple bypass surgery. At three-month follow-up, the right optic disk edema had resolved, leaving a pale superior optic nerve head. Visual acuity in the right eye had slightly improved to 4/10; however, the dense inferior altitudinal field defect had remained unchanged. The patient is currently treated with warfarin, atorvastatin, irbesartan and metoprolol. This case suggests that the first line of investigation in all patients with NAION involves assessment of cardiovascular risk factors. However, careful history taking will identify NAION patients who are eligible for additional thrombophilia screening: young patients without vasculopathic risk factors, bilateral or recurrent NAION, idiopathic or recurrent venous thromboembolism (VTE), positive family history of VTE, and VTE in young age or

  20. Factor Xa generation by computational modeling: an additional discriminator to thrombin generation evaluation.

    Directory of Open Access Journals (Sweden)

    Kathleen E Brummel-Ziedins

    Full Text Available Factor (fXa is a critical enzyme in blood coagulation that is responsible for the initiation and propagation of thrombin generation. Previously we have shown that analysis of computationally generated thrombin profiles is a tool to investigate hemostasis in various populations. In this study, we evaluate the potential of computationally derived time courses of fXa generation as another approach for investigating thrombotic risk. Utilizing the case (n = 473 and control (n = 426 population from the Leiden Thrombophilia Study and each individual's plasma protein factor composition for fII, fV, fVII, fVIII, fIX, fX, antithrombin and tissue factor pathway inhibitor, tissue factor-initiated total active fXa generation was assessed using a mathematical model. FXa generation was evaluated by the area under the curve (AUC, the maximum rate (MaxR and level (MaxL and the time to reach these, TMaxR and TMaxL, respectively. FXa generation was analyzed in the entire populations and in defined subgroups (by sex, age, body mass index, oral contraceptive use. The maximum rates and levels of fXa generation occur over a 10- to 12- fold range in both cases and controls. This variation is larger than that observed with thrombin (3-6 fold in the same population. The greatest risk association was obtained using either MaxR or MaxL of fXa generation; with an ∼2.2 fold increased risk for individuals exceeding the 90(th percentile. This risk was similar to that of thrombin generation(MaxR OR 2.6. Grouping defined by oral contraceptive (OC use in the control population showed the biggest differences in fXa generation; a >60% increase in the MaxR upon OC use. FXa generation can distinguish between a subset of individuals characterized by overlapping thrombin generation profiles. Analysis of fXa generation is a phenotypic characteristic which may prove to be a more sensitive discriminator than thrombin generation among all individuals.

  1. Fruit and Vegetable Attitudes, Norms, and Intake in Low-Income Youth (United States)

    Di Noia, Jennifer; Cullen, Karen Weber


    Fruit and vegetable (FV) attitudes and norms have been shown to influence intake in youth; yet research with low-income youth and studies supplementing self-report with objective measures of intake are lacking. Cross-sectional survey data on self-rated FV intake, FV attitudes, and FV norms were collected in a sample of 116 youth attending a…

  2. The effectiveness of asking behaviors among 9-11 year-old children in increasing home availability and children's intake of fruit and vegetables: Results from the Squire's Quest II self-regulation game intervention (United States)

    Home environment has an important influence on children's fruit and vegetable (FV) consumption, but children may in turn also impact their home FV environment, e.g. by asking for FV. The Squire's Quest II serious game intervention aimed to increase asking behaviors to improve home FV availability an...

  3. Theranostic pretargeted radioimmunotherapy of colorectal cancer xenografts in mice using picomolar affinity {sup 86}Y- or {sup 177}Lu-DOTA-Bn binding scFv C825/GPA33 IgG bispecific immunoconjugates

    Energy Technology Data Exchange (ETDEWEB)

    Cheal, Sarah M.; Lee, Sang-gyu; Punzalan, Blesida; Larson, Steven M. [Memorial Sloan Kettering Cancer Center, Department of Radiology, New York, NY (United States); Memorial Sloan Kettering Cancer Center, Molecular Pharmacology and Chemistry Program, New York, NY (United States); Xu, Hong; Guo, Hong-fen [Memorial Sloan Kettering Cancer Center, Department of Pediatrics, New York, NY (United States); Chalasani, Sandhya; Carrasquillo, Jorge A. [Memorial Sloan Kettering Cancer Center, Department of Radiology, New York, NY (United States); Fung, Edward K. [Memorial Sloan Kettering Cancer Center, Molecular Pharmacology and Chemistry Program, New York, NY (United States); Memorial Sloan Kettering Cancer Center, Department of Medical Physics, New York, NY (United States); Jungbluth, Achim [Memorial Sloan Kettering Cancer Center, Department of Pathology, New York, NY (United States); Zanzonico, Pat B.; O' Donoghue, Joseph [Memorial Sloan Kettering Cancer Center, Department of Medical Physics, New York, NY (United States); Smith-Jones, Peter M. [Stony Brook University, Department of Psychiatry and Behavioral Science, Stony Brook, NY (United States); Stony Brook University, Department of Radiology, Stony Brook, NY (United States); Wittrup, K.D. [Massachusetts Institute of Technology, Department of Chemical Engineering, Cambridge, MA (United States); Massachusetts Institute of Technology, Department of Biological Engineering, Cambridge, MA (United States); Massachusetts Institute of Technology, Koch Institute for Integrative Cancer Research, Cambridge, MA (United States); Cheung, Nai-Kong V. [Memorial Sloan Kettering Cancer Center, Molecular Pharmacology and Chemistry Program, New York, NY (United States); Memorial Sloan Kettering Cancer Center, Department of Pediatrics, New York, NY (United States)


    GPA33 is a colorectal cancer (CRC) antigen with unique retention properties after huA33-mediated tumor targeting. We tested a pretargeted radioimmunotherapy (PRIT) approach for CRC using a tetravalent bispecific antibody with dual specificity for GPA33 tumor antigen and DOTA-Bn-(radiolanthanide metal) complex. PRIT was optimized in vivo by titrating sequential intravenous doses of huA33-C825, the dextran-based clearing agent, and the C825 haptens {sup 177}Lu-or {sup 86}Y-DOTA-Bn in mice bearing the SW1222 subcutaneous (s.c.) CRC xenograft model. Using optimized PRIT, therapeutic indices (TIs) for tumor radiation-absorbed dose of 73 (tumor/blood) and 12 (tumor/kidney) were achieved. Estimated absorbed doses (cGy/MBq) to tumor, blood, liver, spleen, and kidney for single-cycle PRIT were 65.8, 0.9 (TI 73), 6.3 (TI 10), 6.6 (TI 10), and 5.3 (TI 12), respectively. Two cycles of PRIT (66.6 or 111 MBq {sup 177}Lu-DOTA-Bn) were safe and effective, with a complete response of established s.c. tumors (100 - 700 mm{sup 3}) in nine of nine mice, with two mice alive without recurrence at >140 days. Tumor log kill in this model was estimated to be 2.1 - 3.0 based on time to 500-mm{sup 3} tumor recurrence. In addition, PRIT dosimetry/diagnosis was performed by PET imaging of the positron-emitting DOTA hapten {sup 86}Y-DOTA-Bn. We have developed anti-GPA33 PRIT as a triple-step theranostic strategy for preclinical detection, dosimetry, and safe targeted radiotherapy of established human colorectal mouse xenografts. (orig.)

  4. 人源抗A型肉毒毒素单链抗体的体外亲和筛选%Affinity Selection of Human ScFv Against Botulinum Neurotoxin Serotype A

    Institute of Scientific and Technical Information of China (English)

    王慧; 荫俊


    以重组制备的A型肉毒毒素保护性抗原为配体,对人源噬菌体免疫抗体文库进行体外定向亲和筛选,获得特异结合子,其中与抗原高亲和力结合的抗体克隆B17基因全长750 bp,可编码250个氨基酸,抗体可变区基因同源分析表明,分属VH4和κ chainⅡ家族,是一株人源特异单链抗体基因.人源单链抗体B17在大肠杆菌中获得了重组表达,表达产物可以竞争特异肉毒抗毒素马血清与抗原的结合,是国内首次获得的抗A型肉毒毒素保护性抗原的人源单链抗体,可以在肉毒毒素检测和治疗研究中发挥作用.

  5. Exchange of diesel generator's retrofitting by PV-diesel hybrid plants in the south of Morocco; Sustitucion de grupos electrogenos por pequenas centrales hibridas FV-diesel en el sur de Marruecos

    Energy Technology Data Exchange (ETDEWEB)

    Munoz Cano, J.; Narvarte Fernandez, L.; Lorenzo Pigueiras, E.


    This paper describes a pilot experience of diesel generator's retrofitting by PV-diesel hybrid plants in two rural villages in the south of Morocco. After two operating years, we are ready to present the obtained results, especially, those that can be useful for future electrification projects based on this technology, such as the introduction of the energy service, the energy consumption of users and the managerial aspects that contribute to the long-term sustainability of these systems. (Author)

  6. Compton scattering in a unitary approach with causality constraints 11.55.Fv; 13.40.Gp; 13.60.Fz; Nucleon-photon vertex; Off-shell form factors; K-matrix formalism; Compton scattering; Dispersion relations

    CERN Document Server

    Kondratyuk, S


    Pion-loop corrections for Compton scattering are calculated in a novel approach based on the use of dispersion relations in a formalism obeying unitarity. The basic framework is presented, including an application to Compton scattering. In the approach the effects of the non-pole contribution arising from pion dressing are expressed in terms of (half-off-shell) form factors and the nucleon self-energy. These quantities are constructed through the application of dispersion integrals to the pole contribution of loop diagrams, the same as those included in the calculation of the amplitudes through a K-matrix formalism. The prescription of minimal substitution is used to restore gauge invariance. The resulting relativistic-covariant model combines constraints from unitarity, causality, and crossing symmetry.

  7. Targeted elimination of activated hepatic stellate cells by an anti-epidermal growth factor-receptor single chain fragment variable antibody-tumor necrosis factor-related apoptosis-inducing ligand (scFv425-sTRAIL)

    NARCIS (Netherlands)

    Arabpour, Mohammad; Poelstra, Klaas; Helfrich, Wijnand; Bremer, Edwin; Haisma, Hidde J.


    BackgroundProgressive liver fibrosis is the result of chronic liver injury and is characterized by the excessive accumulation of extracellular matrix that may result in liver failure. Activated hepatic stellate cells are known to play a central role in this process and their elimination is a crucial

  8. The therapeutically anti-prion active antibody-fragment scFv-W226: paramagnetic relaxation-enhanced NMR spectroscopy aided structure elucidation of the paratope-epitope interface. (United States)

    Mangels, Christian; Kellner, Ruth; Einsiedel, Jürgen; Weiglmeier, Philipp R; Rosch, Paul; Gmeiner, Peter; Schwarzinger, Stephan


    Antibodies have become indispensable reagents with numerous applications in biological and biotechnical analysis, in diagnostics as well as in therapy. In all cases, selective interaction with an epitope is crucial and depends on the conformation of the paratope. While epitopes are routinely mapped at high throughput, methods revealing structural insights on a rather short timescale are rare. We here demonstrate paramagnetic relaxation-enhanced (PRE) NMR spectroscopy to be a powerful tool unraveling structural information about epitope-orientation in a groove spanned by the complementary determining regions. In particular, we utilize the spin label TOAC, which is fused to the peptidic epitope using standard solid-phase chemistry and which is characterized by a reduced mobility compared to, e.g., spin labels attached to the side-chain functionalities of cysteine or lysine residues. We apply the method to determine the orientation of helix 1 of the prion protein, which is the epitope for the therapeutically anti-prion active scF(v) fragment W226.

  9. Physical profile data collected aboard the F/V Columbia in the Gulf of Alaska during the Lower Cook Inlet (Alaska) seasonal hydrographic surveys, January 8 - 10, 2005 (NCEI Accession 0040248) (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — This is the CTD data from one of sixteen cruises in lower Cook Inlet. Four CTDs were used, but most also included fluorescence, transmissometer, and oxygen...

  10. Community-based interventions for enhancing access to or consumption of fruit and vegetables among five to 18-year olds: a scoping review



    Abstract Background Low fruit and vegetable ( FV) consumption is a key risk factor for morbidity and mortality. Consumption of FV is limited by a lack of access to FV. Enhanced understanding of interventions and their impact on both access to and consumption of FV can provide guidance to public health decision-makers. The purpose of this scoping review is to identify and map literature that has evaluated effects of community-based interventions designed to increase FV access or consumption am...

  11. Expression of a single-chain variable-fragment antibody against a Fusarium virguliforme toxin peptide enhances tolerance to sudden death syndrome in transgenic soybean plants. (United States)

    Brar, Hargeet K; Bhattacharyya, Madan K


    Plants do not produce antibodies. However, plants can correctly assemble functional antibody molecules encoded by mammalian antibody genes. Many plant diseases are caused by pathogen toxins. One such disease is the soybean sudden death syndrome (SDS). SDS is a serious disease caused by the fungal pathogen Fusarium virguliforme. The pathogen, however, has never been isolated from diseased foliar tissues. Thus, one or more toxins produced by the pathogen have been considered to cause foliar SDS. One of these possible toxins, FvTox1, was recently identified. We investigated whether expression of anti-FvTox1 single-chain variable-fragment (scFv) antibody in transgenic soybean can confer resistance to foliar SDS. We have created two scFv antibody genes, Anti-FvTox1-1 and Anti-FvTox1-2, encoding anti-FvTox1 scFv antibodies from RNAs of a hybridoma cell line that expresses mouse monoclonal anti-FvTox1 7E8 antibody. Both anti-FvTox1 scFv antibodies interacted with an antigenic site of FvTox1 that binds to mouse monoclonal anti-FvTox1 7E8 antibody. Binding of FvTox1 by the anti-FvTox1 scFv antibodies, expressed in either Escherichia coli or transgenic soybean roots, was initially verified on nitrocellulose membranes. Expression of anti-FvTox1-1 in stable transgenic soybean plants resulted in enhanced foliar SDS resistance compared with that in nontransgenic control plants. Our results suggest that i) FvTox1 is an important pathogenicity factor for foliar SDS development and ii) expression of scFv antibodies against pathogen toxins could be a suitable biotechnology approach for protecting crop plants from toxin-induced diseases.

  12. Metacompreensão e inteligência: um estudo correlacional com estudantes do ensino fundamental Metacomprehension and intelligence: a correlate study with elementary students

    Directory of Open Access Journals (Sweden)

    Maria Cristina Rodrigues Azevedo Joly


    Full Text Available Este estudo objetivou buscar evidências de validade para a Escala de Metacompreensão (META-FII por meio da correlação com o WISC-III. Participaram 61 estudantes, 59,0% do gênero feminino, regularmente matriculadas em uma escola da rede pública de ensino de São Paulo. As idades dos participantes variaram de 10 a 13 anos (M = 11,79; DP = 1,13. A META-FII foi aplicada coletivamente em uma primeira sessão e o WISC-III em duas sessões posteriores, individualmente. Os resultados evidenciaram diferença significativa quanto ao gênero apenas para o QI compreensão verbal. Foram encontradas correlações positivas entre os QIs, verbal, compreensão verbal e total com a META-FII o que atribui a evidência de validade para a escala de metacompreensão.This study aimed to get validity evidences of Metacomprehension Scale (META-FII by WISC-III correlation test. The participants were 61 students, 59,0% were female that were at public elementary school in São Paulo state. The age variation was from 10 through 13 years old (M = 11,79; SD = 1,13. The META-FII scale was collectively administered in the first session and the WISC-III in two post sessions, individually. The results showed significant difference to genre only at verbal comprehension QI. Verbal, verbal comprehension and total QI were positive correlated with META-FII scale which give to the scale validity evidences.

  13. Mutações predisponentes à trombofilia em indivíduos de Minas Gerais - Brasil com suspeita clínica de trombose Predisposing thrombophilic mutations in individuals with clinical suspicion of thrombosis from Minas Gerais, Brazil

    Directory of Open Access Journals (Sweden)

    Sabrina P. Guimarães


    Full Text Available A trombose é reconhecidamente uma doença de caráter multifatorial. Sua ocorrência está intimamente relacionada à presença de fatores genéticos e adquiridos que concorrem isoladamente ou em associação para o seu desencadeamento. No entanto, a frequência dos fatores genéticos pode variar de acordo com a origem étnica e com outros aspectos epidemiológicos dos grupos de indivíduos e populações estudadas. No Brasil, dados referentes a indivíduos brasileiros e em especial do estado de Minas Gerais são escassos. O objetivo do presente estudo foi investigar a frequência das mutações fator V Leiden e G20210A no gene protrombina em 1.103 indivíduos com suspeita clínica de trombofilia, empregando a técnica da reação em cadeia da polimerase seguida de restrição enzimática (PCR-RFLP. Os dados foram analisados usando-se o programa Epi Info versão 6.04. A amostra consistiu de 76,16% mulheres e 23,84% homens, com média de idade de 43,06± 14,65. A mutação fator V Leiden foi observada em heterozigose em 7,52% dos indivíduos e em 0,36% em homozigose. A mutação G20210A no gene da protrombina apresentou-se em heterozigose em 5,90% dos indivíduos e em homozigose em 0,18%. O presente trabalho mostra a importância dos testes genéticos conforme o perfil da população analisada, ressaltando informações epidemiológicas da população brasileira e benefícios clínicos.Thrombosis is known to be a multifactorial disease. Its incidence is directly related to the presence of genetic and acquired factors that concur separately or in association to its appearance. However, the frequency of genetic factors can vary according to ethnic background and with other epidemiological aspects of populations. Data from Brazilian individuals and especially those from the State of Minas Gerais are scarce. The present study aims at investigating the frequencies of the factor v Leiden and the G20210G prothrombin gene mutations of 1103 individuals

  14. Prediction of ischemic stroke in young Indians: is thrombophilia profiling a way out? (United States)

    Chatterjee, Tathagata; Gupta, Neha; Choudhry, Ved Prakash; Behari, Madhuri; Saxena, Renu; Ashraf, Mohammad Z


    Stroke in the young is attributed to the prevalence of thrombophilia, however, reports explaining the cause mechanisms from Indian populations are largely not known. The information about the association of inherited thrombophilia and occurrence of stroke is still missing. Therefore, we describe here 52 cases of young ischemic stroke of which 22 cases were of recurrent stroke and 30 cases of first episode stroke along with an equal number of healthy controls. Imaging techniques (CT/MRI/Doppler studies) were used to identify the type and location of infarcts among various regions of the brain. All the patients and controls were screened for hypercoagulable state by employing Pro C global test. Those tested positive for the latter were evaluated for conventional thrombophilic factors, activity levels of protein C and protein S, antithrombin III levels, plasma homocysteine levels and presence of activated protein C resistance, lupus anticoagulant, methylenetetrahydrofolate reductase (MTHFR C677T) and prothrombin G20210A polymorphisms. Out of 52 cases there were 22 cases of recurrent stroke and 30 cases of first ischemic stroke. Infarcts were single in 39 out of 52 cases and multiple in 13 cases. Among the different regions of brain internal capsule infarcts were seen in 13 of 52 (25%) cases, and cerebellum, basal ganglion and midbrain infarcts were seen in five cases (9.6%) each and remaining infarcts were in other anatomical regions of the brain. Left middle cerebral artery territory was involved in 17 of 52 (32.7%) cases. The prevalence of individual thrombophilia among cases ranged from 28.8% (15/52) for protein S and 11.5% (6/52) for protein C deficiencies respectively. All cases of protein C were protein S deficient. Five cases of protein C deficiency patients were of 25 years and younger as compared with one case in the at least 25 years age group. Plasma homocysteine levels were elevated in three cases (5.7%) as compared with normal levels in controls

  15. A patient with C protein deficiency and multiple thromboses. case report Paciente con deficiencia de proteína C y múltiples trombosis: reporte de caso

    Directory of Open Access Journals (Sweden)

    Alejandro Román González


    Full Text Available Inherited thrombophilias are an important group of diseases that should be taken into account in the study of patients with thromboembolic disease, particularly in those whose clinical presentation includes frequent and recurrent thrombotic episodes at young age, in unusual sites, and a familial history of thrombosis. We report the case of a patient with C protein deficiency which developed deep venous thromboses of both legs when he was 36 and 37 years old. At 51 years of age he suffered from mesenteric thrombosis requiring surgical treatment and small intestine transplantation. His father had deep venous thrombosis. This is the first report of C protein deficiency in the Colombian literature. Other inherited thrombophilias such as the G20210A mutation in the prothrombin gene and actor V Leiden were absent. Se debe considerar un estado de hipercoagulabilidad primaria o trombofilia heredada en los pacientes con enfermedad tromboembólica venosa. La sospecha clínica se debe dirigir a los pacientes con presentación temprana, recurrente, familiar o en sitios anatómicos poco usuales. En este reporte se describe el caso de un paciente con déficit de proteína C de la coagulación, quien desarrolló trombosis venosa profunda del miembro inferior derecho a los 36 años y un año después, trombosis venosa profunda del miembro inferior izquierdo. A la edad de 51 años presentó trombosis de vasos mesentéricos que condujo a una resección intestinal extensa lo que obligó a un trasplante de intestino delgado. Su padre había presentado trombosis venosa de los miembros inferiores. Se descartó la presencia asociada de la mutación G20210A de la protrombina y del Factor V Leiden. Hasta donde sabemos, es el primer caso de deficiencia de proteína C de la coagulación informado en la literatura colombiana.

  16. inherited thrombophilia and recurrent pregnancy loss Trombofilias heredadas y pérdida gestacional recurrente

    Directory of Open Access Journals (Sweden)

    Angela Patricia Cadavid Jaramillo


    Full Text Available One of the causes of recurrent pregnancy loss is thrombophilia, defined as a tendency to thrombosis or hypercoagulability, with various clinical manifestations dependent on the vascular region affected by the absence of blood flow. Thrombophilias can be classified as inherited or acquired, according to the nature of their cause. The former are factor V Leiden, the prothrombin G20210A, the methilenetetrahydrofolate reductase C677T, deficiencies of the natural anticoagulants antithrombin III, protein C and protein S, dysfibrinogens and homocystinuria. The group of acquired thrombophilias includes antiphospholipid syndrome, activated protein C resistance without alterations in the gene of factor V and mild or moderate hyperhomocysteinemia. This article reviews several recent studies looking for association between different thrombophilias and recurrent pregnancy loss. Also diagnostic, prophylactic and therapeutic recommendations are included, for women with thrombophilia and pregnancy loss Una de las causas de pérdida gestacional recurrente es la trombofilia, que se define como una tendencia a la trombosis o hipercoagulabilidad, con variabilidad en las manifestaciones clínicas dependiente de la región vascular afectada por la ausencia de flujo sanguíneo. Las trombofilias se pueden clasificar como heredadas y adquiridas de acuerdo con la naturaleza de su causa. Entre las trombofilias heredadas están el factor V Leiden, la protrombina G20210A, la metilentetrahidrofolato reductasa C677T, las deficiencias de los anticoagulantes naturales antitrombina III, proteína C y proteína S, las disfibrinogenemias y la homocistinuria. En el grupo de las trombofilias adquiridas se encuentran el síndrome antifosfolípido, la resistencia a la proteína C activada sin alteraciones en el gen del factor V y la hiperhomocisteinemia leve o moderada. Este artículo es una revisión de la literatura de estudios recientes que han buscado la asociación entre las

  17. Fruit and vegetable intake in the Mexican population: results from the Mexican National Health and Nutrition Survey 2006 Consumo de frutas y verduras en la población mexicana


    Ivonne Ramírez-Silva; Rivera, Juan A.; Xochitl Ponce; Mauricio Hernández-Ávila


    OBJECTIVE: To quantify fruit and vegetable (FV) dietary intake in the Mexican population and compliance with international recommendations. MATERIAL AND METHODS: FV dietary intake (FV-DI) and compliance with international recommendations were obtained in a representative sample of a Mexican population ages 1-59 years old using dietary data from the Mexican National Health and Nutrition Survey 2006 (ENSANUT 2006). RESULTS: Average FV-DI for different age groups range from 61 to 72 g for fruits...

  18. Targeting Cell Surface Proteins in Molecular Photoacoustic Imaging to Detect Ovarian Cancer Early (United States)


    Skin; Trachea, Breast, Salivary Brain, Spinal Cord Colon, Intestine , Salivary, Trachea, Uterus Number of normal tissue types demonstrating...1. Express antigen in mammalian system 2. Iteratively screen yeast scFv libraries and identify high affinity scFv 3. Confirm specificity of scFv by

  19. Using a correction factor to correct for overreporting in a food-frequency questionnaire does not improve biomarker-assessed validity of estimates for fruit and vegetable consumption

    NARCIS (Netherlands)

    Bogers, R.P.; Dagnelie, P.C.; Westerterp, K.R.; Kester, A.D.M.; Klaveren, van J.D.; Bast, A.; Brandt, van den P.A.


    To correct for overreporting of fruit and vegetable (FV) consumption in a food-frequency questionnaire, summary questions about consumption of main FV groups are often used to calculate correction factors. This study compared the ability to rank people according to their FV intake of those summary q

  20. Administrative and School Nutrition Perspectives of Salad Bar Operations in Public Schools (United States)

    Andersen, Lori; Myers, Leann; O'Malley, Keelia; Rose, Donald; Johnson, Carolyn C.


    Purposes/Objectives: Fruit and vegetable (F/V) consumption may aid in childhood obesity prevention. F/V consumption in youth is low. School-based salad bars (SBs) may improve F/V access in youth. The purpose of this study was to explore administrative and school nutrition personnel perspectives related to adoption and continued implementation of…

  1. The impact of fruit and vegetable intake on weight management (United States)

    Fruit and vegetables (FV) are important sources of phytochemicals, dietary fiber, and low energy density, and their consumption may be protective against obesity. Despite these potential benefits of FV consumption on human health, rates of FV intake remain low throughout the world. This chapter revi...

  2. Do descriptive norms related to parents and friends predict fruit and vegetable intake similarly among 11-year-old girls and boys? (United States)

    Lehto, Elviira; Ray, Carola; Haukkala, Ari; Yngve, Agneta; Thorsdottir, Inga; Roos, Eva


    We examined whether there are sex differences in children's fruit and vegetable (FV) intake and in descriptive norms (i.e. perceived FV intake) related to parents and friends. We also studied whether friends' impact is as important as that of parents on children's FV intake. Data from the PRO GREENS project in Finland were obtained from 424 children at the age 11 years at baseline. At baseline, 2009 children filled in a questionnaire about descriptive norms conceptualised as perceived FV intake of their parents and friends. They also filled in a validated FFQ that assessed their FV intake both at baseline and in the follow-up in 2010. The associations were examined with multi-level regression analyses with multi-group comparisons. Girls reported higher perceived FV intake of friends and higher own fruit intake at baseline, compared with boys, and higher vegetable intake both at baseline and in the follow-up. Perceived FV intake of parents and friends was positively associated with both girls' and boys' FV intake in both study years. The impact of perceived fruit intake of the mother was stronger among boys. The change in children's FV intake was affected only by perceived FV intake of father and friends. No large sex differences in descriptive norms were found, but the impact of friends on children's FV intake can generally be considered as important as that of parents. Future interventions could benefit from taking into account friends' impact as role models on children's FV intake.

  3. Estimation of vegetation fraction in arid areas using ALOS imagery

    NARCIS (Netherlands)

    Matkan, A.A.; Darvishzadeh, R.; Hosseiniasl, A.; Ebrahimi, M.


    Fraction of vegetation (Fv) plays an important role in ecosystems. Estimation of Fv is essential for drought monitoring, natural resources studies, estimation of soil erosion volume etc. The aim of this study is to estimate Fv in an arid area in Iran using ALOS Imagery (June 2008). In order to find

  4. Creation and Evaluation of a Single-chain Antibody Tetramer that Targets Brain Endothelial Cells. (United States)

    Zhang, Xiaobin; Wang, Xin Xiang; Shusta, Eric V


    Antibodies that target and internalize into blood-brain barrier (BBB) endothelial cells offer promise as drug delivery agents. Previously, we identified a single-chain antibody (scFvA) capable of binding to the BBB. In an attempt to improve the binding and internalization properties of the single chain antibody (scFvA), a biotinylation tag (Avitag) was fused to scFvA and the protein secreted by yeast. The scFvA-Avitag could be biotinylated by yeast-displayed BirA enzyme and biotinylated scFvA-Avitag could be used to create scFv tetramers. Tetramerization of scFvA improved the internalization of scFvA into BBB endothelial cells, and biotinylated scFvA-Avitag could also be used to target streptavidin-coated quantum dots for BBB endothelial cell internalization. Perfusing the rat brain with scFvA-tetramer confirmed that the antigen targeted by scFvA is distributed on blood side of the BBB, suggesting the potential for downstream application of scFvA in brain-targeted drug delivery.

  5. When Fruits and Vegetables Are Optional, Elementary School Children Choose Processed over Whole Offerings (United States)

    Amin, Sarah A.; Yon, Bethany A.; Taylor, Jennifer C.; Johnson, Rachel K.


    Purpose/Objectives: Increasing children's fruit and vegetable (FV) consumption is an important goal for the National School Lunch Program (NSLP). In 2012 the NSLP began requiring students to select a FV. The objective of this study was to compare children's FV choices in two school cafeteria environments a year before these new USDA regulations…

  6. Antitumor activity of Pleurotus ostreatus polysaccharide fractions on Ehrlich tumor and Sarcoma 180. (United States)

    Facchini, Jean Mary; Alves, Endi Pricila; Aguilera, Charlise; Gern, Regina Maria Miranda; Silveira, Marcia Luciane Lange; Wisbeck, Elisabeth; Furlan, Sandra Aparecida


    The medicinal properties of fungi of the genus Pleurotus have attracted great interest due to their therapeutic properties. Polysaccharides synthesized by Pleurotus, including the β-glucans are considered the main responsible for its therapeutic properties. This study aimed to evaluate the efficacy of polysaccharidic fractions extracted from mycelial biomass of Pleurotus ostreatus DSM 1833 in inhibiting the development of Ehrlich Tumor (ET) and Sarcoma 180 (S-180). FC, FI and FII fractions provided 60.6, 76.5 and 73.6% of ET inhibition, respectively (mean value of about 70%) while FS, FIII-1 and FIII-2 showed no inhibition against ET. FII and FIII-2 resulted in 85.6 and 93.6% (mean value of about 90%) while FIII-1, FC and FS resulted in 54.1 and 0%, respectively, of S-180 inhibition. The yields of the fractions FS, FI, FII, FIII-1 and FIII-2 obtained from P. ostreatus mycelial biomass were 11.6, 1.3, 0.4, 0.65 and 0.35%, respectively. FII fraction (30mg/kg) apparently had no toxic effect on healthy animals, since no difference between the body weights of animals in substance control (SC) and negative control (NC) groups was observed.

  7. 26 CFR 301.9100-7T - Time and manner of making certain elections under the Tax Reform Act of 1986. (United States)


    ... taxable year for which the election is made. 201(a), 1802(a) 168(h)(6)(F)(ii), 168(j) (as in effect before... development of a mine or other natural deposit (other than an oil, gas, or geothermal well) located outside... governed by the trust laws of the State of Minnesota, receives royalties from iron ore leases, and income...

  8. serrulatus Venom on Murine Macrophage Functions in Vitro

    Directory of Open Access Journals (Sweden)

    Vera L. Petricevich


    highest levels of IL-1α, IL-β, and TNF were observed after 12 hours and that (b the maximum levels of IFN-γ and NO were observed after 72 hours. Taken together, these data indicate that fractions have a differential immunomodulating effect on macrophage secretion, and that FII is a potent activator of TNF production of macrophages.

  9. DMPD: RIG-I: tri-ing to discriminate between self and non-self RNA. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17307033 RIG-I: tri-ing to discriminate between self and non-self RNA. Bowie AG, Fi...-I: tri-ing to discriminate between self and non-self RNA. PubmedID 17307033 Title RIG-I: tri-ing to discriminate between self... and non-self RNA. Authors Bowie AG, Fitzgerald KA. Publication

  10. 78 FR 47459 - Self-Regulatory Organizations; The NASDAQ Stock Market LLC; Notice of Filing and Immediate... (United States)


    ... whose provision this proposal is modeled.\\3\\ \\3\\ See PHLX Rule 1092(f)(ii). Securities Exchange Act... Customers, for purposes of Chapter V, Section 6. See Chapter I, Section 1(a)(48). These changes should... 10 at $2.50 vs. S10 at $3.00). If not given a choice, Public Customer A would be naked short 10 calls...

  11. 78 FR 47471 - Self-Regulatory Organizations; NASDAQ OMX BX, Inc.; Notice of Filing and Immediate Effectiveness... (United States)


    ... (``PHLX''), on whose provision this proposal is modeled.\\3\\ \\3\\ See PHLX Rule 1092(f)(ii). Securities... Customers, for purposes of Chapter V, Section 6. See Chapter I, Section 1(a)(49). These changes should... would be naked short 10 calls at $3.00 that are now offered at $6.00 (a $3,000 loss). These examples...

  12. Fosfomycin Resistance in Escherichia coli, Pennsylvania, USA. (United States)

    Alrowais, Hind; McElheny, Christi L; Spychala, Caressa N; Sastry, Sangeeta; Guo, Qinglan; Butt, Adeel A; Doi, Yohei


    Fosfomycin resistance in Escherichia coli is rare in the United States. An extended-spectrum β-lactamase-producing E. coli clinical strain identified in Pennsylvania, USA, showed high-level fosfomycin resistance caused by the fosA3 gene. The IncFII plasmid carrying this gene had a structure similar to those found in China, where fosfomycin resistance is commonly described.

  13. The second zinc-finger domain of poly(ADP-ribose) polymerase determines specificity for single-stranded breaks in DNA.

    NARCIS (Netherlands)

    G. Gradwohl; J.M. de Murcia; M. Molinete; F. Simonin; M.H.M. Koken (Marcel); J.H.J. Hoeijmakers (Jan); G. de Murcia


    textabstractPoly(ADP-ribose) polymerase (EC is a zinc-binding protein that specifically binds to a DNA strand break in a zinc-dependent manner. We describe here the cloning and expression in Escherichia coli of a cDNA fragment encoding the two putative zinc fingers (FI and FII) domain of t

  14. Purification and characterization of novel fibrinolytic proteases as potential antithrombotic agents from earthworm Perionyx excavatus

    NARCIS (Netherlands)

    phan, T.T.B.; Ta, T.D.; Nguyen, D.T.X.; Broek, van den L.A.M.


    Six protease fractions, namely FI, FII, FIII-1, FIII-2, FIII-3 and FIV, were isolated from Perionyx excavatus earthworm biomass by acetone precipitation, followed by serial chromatography using anion exchange, hydrophobic interaction and size exclusion chromatography. All fractions exhibited strong

  15. 12 CFR 308.203 - Order to dismiss a director or senior executive officer. (United States)


    ... executive officer under § 38(f)(2)(F)(ii) of the FDI Act, the FDIC shall also serve a copy of the directive... the bank based on supervisory criteria other than capital, pursuant to section 38(g) of the FDI...

  16. Frame approximation of pseudo-inverse operators

    DEFF Research Database (Denmark)

    Christensen, Ole


    Let T denote an operator on a Hilbert space (H, [.,.]), and let {f(i)}(i=1)(infinity) be a frame for the orthogonal complement of the kernel NT. We construct a sequence of operators {Phi (n)} of the form Phi (n) (.) = Sigma (n)(i=1) [., g(t)(n)]f(i) which converges to the psuedo-inverse T+ of T i...

  17. Disparities in the frequency of fruit and vegetable consumption by socio-demographic and lifestyle characteristics in Canada

    Directory of Open Access Journals (Sweden)

    Sharaf Mesbah F


    Full Text Available Abstract Background The health benefits of adequate fruit and vegetable (F&V consumption are significant and widely documented. However, many individuals self-report low F&V consumption frequency per day. This paper examines the disparities in the frequency of F&V consumption by socio-demographic and lifestyle characteristics. Method This study uses a representative sample of 93,719 individuals from the Canadian Community Health Survey (2007. A quantile regression model is estimated in order to capture the differential effects of F&V determinants across the conditional distribution of F&V consumption. Results The conditional and unconditional analyses reveal the existence of a socioeconomic gradient in F&V consumption frequency, in which the low income-education groups consume F&V less frequently than the high income-education groups. We also find significant disparities in F&V consumption frequency by demographic and lifestyle characteristics. The frequency of F&V consumption is relatively lower among: males, those in middle age, singles, smokers, individuals with weak social interaction and households with no children. The quantile regression results show that the association between F&V consumption frequency, and socio-demographic and lifestyle factors varies significantly along the conditional F&V consumption distribution. In particular, individual educational attainment is positively and significantly associated with F&V consumption frequency across different parts of the F&V distribution, while the income level matters only over the lower half of the distribution. F&V consumption follows a U-shaped pattern across the age categories. Those aged 30-39, 40-49 and 50-59 years consume F&V less frequently than those aged 18-29 years. The smallest F&V consumption is among the middle aged adults (40-49. Conclusions Understanding the socio-demographic and lifestyle characteristics of individuals with low F&V consumption frequency could increase the

  18. Pulmonary Embolism Mimicking Community Acquired Pneumonia: A Case Report

    Directory of Open Access Journals (Sweden)

    Ibrahim Koc


    Full Text Available Acute pulmonary trombo embolism is a form of venous thromboembolism that is common and sometimes even may be fatal. Patients might present with variable clinical presentation and often have non-specific complaints which make the diagnosis challenging. Here we aimed to report a thirty years old male who was diagnosed with community acquired pneumonia but further investigations revealed pulmonary embolism. A thirty years old male presented to our clinic with right sided chest pain and shortness of breath. Chest radiograph revealed right sided consolidations and pleural effusion. His physical examination revealed high body temperature (38 C° and oxygen saturation on room air was 85 %. The patient did not respond to the antibiotherapy and oxygen supply. Computed tomography angiography of the chest revealed right sided pulmonary embolism with pneumonia. Blood and sputum cultures revealed no bacteria. Cardiovascular disease panel revealed heterozygous mutation in prothrombine G20210A and metilentetrahidrofolate reductase (MTHFR C677T. In conclusion pulmonary embolism may mimic community acquired pneumonia thus clinicians must be carefull during the diagnostic process.

  19. Unexplained infertility: association with inherited thrombophilia. (United States)

    Fatini, Cinzia; Conti, Lucia; Turillazzi, Valentina; Sticchi, Elena; Romagnuolo, Ilaria; Milanini, Maria Novella; Cozzi, Cinzia; Abbate, Rosanna; Noci, Ivo


    Unexplained infertility represents one of the most common diagnoses in fertility care. Attention is being paid to the association between inherited thrombophilia and infertility causes. In this study we investigated the prevalence of inherited thrombophilia according to infertility causes. We studied Prothrombin gene G20210A mutation, Factor V Leiden, deficiencies in protein S and C and antithrombin in 930 Caucasian infertile women referred to Fertility Center of the Department of Sciences for Woman and Child's Health, University of Florence, of whom 230 with unexplained, 195 female and 283 male infertility, and in 240 women who have conceived naturally without hormonal stimulation therapy. A significant relationship between inherited thrombophilia [OR 95%CI 1.97 (1.05-3.68), p = 0.03] and unexplained infertility was observed, whereas no association between thrombophilia and female and male infertility was found. Significantly higher prevalence of prothrombin gene mutation in unexplained infertile women in comparison to that observed in fertile women was observed (5.7% vs 2.1% p = 0.04); the prevalence of the other thrombophilia determinants was higher, even if not significantly, in the unexplained infertile group. This study demonstrates the relationship between inherited thrombophilia and unexplained infertility, thus suggesting the contribution of genetic components in modulating unexplained infertility, behind anovulation, male and tubal factor. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. Factor V G1691A (Leiden is a major etiological factor in Egyptian Budd-Chiari syndrome patients

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    Tawhida Y. Abdel Ghaffar


    Full Text Available Objective: Budd-Chiari syndrome is a multifactorial disease in which several prothrombotic disorders may predispose patients to the development of thrombosis at this uncommon location (hepatic veins. The aim of this study was to determine the prevalence and characteristics of inherited thrombophilia in Egyptian Budd-Chiari syndrome patients.Materials and Methods: The study included 47 Budd-Chiari syndrome patients (20 children and 27 adults. Genotyping of Factor V G1691A (Leiden, prothrombin G20210A (PT, and methylenetetrahydrofolate reductase C677T were performed using real-time PCR and fluorescence melting curve detection analysis.Results: Factor V Leiden was observed in 29 patients (61.7%. It is the only factor that caused Budd-Chiari syndrome in 18 of the patients and in 5 of the patients with inferior vena cava involvement. Myeloproliferative disease was noted in 12 (25.5% patients, antiphospholipid syndrome in 5 (10.6%, and Behcet’s disease in 3 (6.4%. Interestingly, 3 of the children with Budd-Chiari syndrome had lipid storage disease.Conclusion: Factor V Leiden was a major etiological factor in Egyptian Budd-Chiari syndrome patients, which may have been related to the high frequency of this mutation in the study region. Factor V Leiden was also a strong thrombophilic factor and the leading cause of inferior vena cava thrombosis in these patients. Lipid storage disease should be included as a risk factor for Budd-Chiari syndrome.

  1. Primary thrombophilia in Mexico: a single tertiary referral hospital experience. (United States)

    Lacayo-Leñero, Dennis; Hernández-Hernández, Darinel; Valencia-Martínez, Andrés; Barrales-Benítez, Olga; Vargas-Ruiz, Angel G


    Thrombophilia is a complex hypercoagulable state that increases the risk of thrombosis. Most reports in medical literature of the Mexican population with this disease lack statistical validity. Therefore, the aim of this study is to describe the prevalence of primary thrombophilia in a tertiary referral hospital in Mexico. This is a study of patients referred to our hospital because of a hypercoagulable state and who later on were diagnosed with primary thrombophilia. The thrombophilia workup included methylenetetrahydrofolate reductase (MTHFR) C677T, antiphospholipid antibodies, protein C, protein S, antithrombin, factor VIII, factor V Leiden, prothrombin mutation G20210A, activated protein C resistance, JAK2 V617F and homocysteine. Ninety-five individuals were tested. The MTHFR C677T polymorphism was the most frequent anomaly in 84.1% of the tested individuals. There was a relatively low prevalence of factor V Leiden (5.2%) and anticoagulant protein deficiency (8.3%). The MTHFR C677T polymorphism has a very high prevalence compared with the low prevalence of anticoagulant protein deficiency and factor V Leiden mutation in Mexicans.

  2. Association between Thrombophilia and Repeated Assisted Reproductive Technology Failures

    Directory of Open Access Journals (Sweden)

    Kobra Hamdi


    Full Text Available Purpose: This study was performed to investigate the incidence of thrombophilic gene mutations in repeated assisted reproductive technology (ART failures. Methods: The prevalence of mutated genes in the patients with a history of three or more previous ART failures was compared with the patients with a history of successful pregnancy following ARTs. The study group included 70 patients, 34 with three or more previously failed ARTs (A and control group consisted of 36 patients with successful pregnancy following ARTs (B. All patients were tested for the presence of mutated thrombophilic genes including factor V Leiden (FVL, Methylenetetrahydrofolate reductase (MTHFR and Prothrombin (G20210A using real-time polymerase chain reaction (RT- PCR. Results: Mutation of FVL gene was detected in 5.9% women of group A (2 of 34 compared with 2.8% women (1 of 36 of control group (P = 0.6. Mutation of MTHFR gene was found in 35.3% (12 cases as compared with 50% (18 cases of control (35.3% versus 50%; P = 0.23. Regarding Prothrombin, only control group had 5.6% mutation (P = 0.49. No significant differences were detected in the incidences of FVL, Prothrombin and MTHFR in the study group A compared with the control group B. Conclusion: The obtained results suggest that thrombophilia does not have a significant effect in ART failures.

  3. Genetic aspects of the antiphospholipid syndrome: An update. (United States)

    Sebastiani, Gian Domenico; Iuliano, Annamaria; Cantarini, Luca; Galeazzi, Mauro


    Studies on the immunogenetic predisposition to antiphospholipid syndrome (APS) and on other non-genetic and epigenetic factors are summarised and discussed. Family studies suggest a genetic predisposition to APS. It appears that this genetic predisposition is in part accounted for by the HLA system, the most consistent associations being those with DR4 and DRw53. Furthermore, it appears that lupus anticoagulant (LA) and anticardiolipin (aCL) antibodies are both associated with the same HLA antigens. Population studies suggest that HLA genes have a role in conferring susceptibility to develop primary APS, with some differences in different ethnic groups. Other genes, outside the MHC, give their contribution to the development of this autoimmune syndrome, such as IRF5, STAT4 and those related to inherited thrombophilia--factor V Leiden and G20210A prothrombin polymorphisms. Finally, post-transcriptional modifications of anti-beta2GPI antibodies could be implicated too. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Diagnosis and treatment strategies of thrombophilic risk factors

    Directory of Open Access Journals (Sweden)

    Murat Albayrak


    Full Text Available Thrombophilia is defined as the general name for a group of genetic and acquired situations, arising from defects of hemostasis mechanism and generating tendency to thrombosis. Examples of the acquired risk factors that increase the tendency to thrombosis are venous catheters, sepsis, surgery, hyperlipidemia, congestive heart disease, increased lipoprotein a, old age, antiphospholipid syndrome, nephrotic syndrome, hyperviscosity, chronic myeloproliferative neoplasms, paroxysmal nocturnal hemoglobinuria, heparin induced thrombocytopenia, vasculitis, immobility, obesity, major surgery, trauma, burns, malignancy, pregnancy and oral contraceptive usage. Genetic tendency to venous thromboembolism in early ages, even without any known cause, is named as thrombophilia. Factor V Leiden, prothrombin G20210A, methylenetetrahydrofolate reductase gene mutations, factor VIII elevation, protein C, protein S and antithrombin deficiency parameters are suggested for evaluation, in patient groups suspected of inherited thrombophilia. Detection of inherited thrombophilic factors in selected patient groups is a guide in developing treatment strategies and in establishing prognosis. Due to the genetic heterogeneities, each society should determine their thrombophilic risk pool. Thus, with the determination of the risk factors, unnecessary assessments will be prevented and a cost-effective approach can be developed.

  5. Methylene tetrahydrofolate reductase gene mutation together with anticardiolipin antibody during pregnancy: a case report

    Directory of Open Access Journals (Sweden)

    Egle Couto

    Full Text Available CONTEXT: High plasmatic homocysteine levels have been associated with arterial and venous thrombosis. The C677T methylene tetrahydrofolate reductase (MTHFR gene mutation is one of the known causes for high homocysteine levels in plasma. Anticardiolipin antibody (ACA is also associated with thrombosis and, along with other clinical complications such as recurrent abortion and stillbirth, is part of the antiphospholipid syndrome. DESIGN: Case report. CASE REPORT: A 19-year-old woman with two gestations and one parity (G2P1 had exhibited deep venous thrombosis in her previous puerperal period. Investigation of thrombophilic factors revealed ACA-IgM and heterozygous C677T mutation in the MTHFR gene. Lupus anticoagulant, protein C, protein S and antithrombin III deficiencies, and Leiden factor V and the G20210A mutation in the prothrombin gene, were not detected. The patient received 55,000 IU of subcutaneous heparin daily, from the 15th to the 36th week of pregnancy, when vaginal delivery took place. There were no clinical complications during the puerperal period and she was discharged three days after delivery, while still using oral anticoagulants.

  6. Thrombophilia

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    Maurizio Zangari


    Full Text Available Thrombophilia or hypercoagulable state is a clinical condition characterized by a tendency to develop venous (less frequently arterial thrombosis. The development of a venous thromboembolic episode (VTE is the result of environmental risk factors such as age, male sex, obesity, the exposure to “risk periods” of immobilization, trauma, cancer, pregnancy or the use of exogenous hormones or antineoplastic medications often on a background of a congenital procoagulant state. Table 1 summarizes the most frequently inherited and acquired thrombophilic conditions in a population of patients with a first episode of VTE.In patients with venous thrombosis before the early nineties a biologic cause of thrombophilia was detectable in only 5% to 15% of cases and was confined to deficiencies of antithrombin, protein C, and protein S. The discovery of two prothrombotic mutations prevalent in white populations, the factor V-Arg506Gln mutation (factor V Leiden and the prothrombin G20210A mutation has significantly increased the number of patients with recognizable hereditary risk factor. The antiphospholipid antibody syndrome and elevated plasma homocysteine levels are also frequently identifiable risk factors in patients presenting with venous as well as arterial thrombosis.

  7. Differential risks in men and women for first and recurrent venous thrombosis: the role of genes and environment. (United States)

    Roach, R E J; Cannegieter, S C; Lijfering, W M


    Men have a higher risk of first and recurrent venous thrombosis than do women. However, the pathophysiology underlying this phenomenon is as yet unknown. In this review article, we assessed the prevalence and strength of genetic and acquired risk factors for venous thrombosis for men and women separately, because it is likely that either a difference in effect or distribution of a risk factor explains the risk difference between the sexes. We also summarized the sex-specific results of previous studies on the risk of first and recurrent venous thrombosis. Few explanations for the sex difference were found. The major factor, explaining about 20% difference in population-attributable fraction, was body height. No difference in prevalence or strength for other venous thrombosis risk factors was observed, such as plaster cast immobilization, hospitalization, surgery, trauma, malignancy, hyperhomocysteinemia, factor V Leiden, prothrombin G20210A, or blood group non-O. Alternative explanations for the sex difference are hypothesized in this review, including X- or Y-linked mutations or a mutation on a gene with a sex-specific effect. Future studies should focus on the sex-specific risk of venous thrombosis to unravel the pathophysiology and thereby improve sex-specific treatment and prevention strategies. Even so, male sex can be used as a tool through which individuals at increased risk of first or recurrent venous thrombosis may be identified.

  8. Lack of Association between Recurrent Pregnancy Loss and Inherited Thrombophilia in a Group of Colombian Patients (United States)

    Cardona, Henry; Castañeda, Serguei A.; Cardona Maya, Wálter; Alvarez, Leonor; Gómez, Joaquín; Gómez, Jorge; Torres, José; Tobón, Luis; Bedoya, Gabriel; Cadavid, Ángela P.


    Studies have shown an association between recurrent pregnancy loss and inherited thrombophilia in Caucasian populations, but there is insufficient knowledge concerning triethnic populations such as the Colombian. The aim of this study was to evaluate whether inherited thrombophilia is associated with recurrent pregnancy loss. Methods. We conducted a case-control study of 93 patients with recurrent pregnancy loss (cases) and 206 healthy multiparous women (controls) in a Colombian subpopulation. Three single nucleotide polymorphisms (SNPs) markers of the inherited thrombophilias factor V Leiden, prothrombin G20210A, and methylenetetrahydrofolate reductase C677T were genotyped by PCR-RFLP. Activated protein C resistance and plasma levels of antithrombin, protein C, and protein S were also measured. Results. The frequency of thrombophilia-associated SNPs, activated protein C resistance, and anticoagulant protein deficiencies, was low overall, except for the methylenetetrahydrofolate reductase C677T SNP. The differences between patients and controls had no statistical significance. Conclusion. Our study confirms the low prevalence of inherited thrombophilias in non-Caucasian populations and it is unlikely that the tested thrombophilias play a role in the pathogenesis of recurrent pregnancy loss in this Colombian population. PMID:22577540

  9. Prothrombotic Gene Polymorphisms in Young Patients with Cerebrovascular Accident

    Directory of Open Access Journals (Sweden)

    Kuyaþ Hekimler Öztürk


    Full Text Available Aim: Cerebrovascular diseases are complex multifactorial disorders showing an increased incidence with increasing age and affected by genetic and environmental factors. Although risk factors for cerebrovascular diseases include age, sex, lineage, hypertension, diabetes mellitus, hypercholesterolemia; in young cerebrovascular patients below age 45, genetic factors may also contribute to the etiology. In this retrospective study, prothrombotic gene polymorphisms which are thought to be related with formation of disease in young adults with cerebrovascular accident (CVA were investigated. Material and Method: In the current study, Methylenetetrahydropholate Reductase (MTHFR C677T and A129C; Prothrombin (Factor II G20210A; Factor V Leiden G1691A prothrombotic gene polymorphisms were evaluated for 43 young patients under the age of 45 with cerebrovascular accident history. Result: For 43 young patients with cerebrovascular incident history, the frequency of following polymorphisms were determined as follows; MTHFR C677T polymorphism heterozygous frequency is 46.1%, homozygous frequency is 9.3%; MTHFR A1298C polymorphism heterozygous frequency is 39.47%, homozygous frequency is 26.31%; Prothrombin polymorphism heterozygous and homozygous frequency is 2.3%; FactorV Leiden polymorphism heterozygous frequency is 9.3%. Discussion: After evaluation the experimental results, we believe that MTHFR gene C677T and A1298C polymorphisms might be risk factors in CVAs. It was observed that cigarette usage, hypertension and existence of family story in addition to these polymorphisms increase the available risk.

  10. Possible incorrect genotyping of heterozygous factor V Leiden and Prothrombin 20210 gene mutations by the GeneXpert assay. (United States)

    Marturano, Alessandro; Bury, Loredana; Gresele, Paolo


    The GeneXpert analyzer is a hands-off system for the detection of Factor V Leiden and of Prothrombin G20210A (GPRO) gene thrombophilic mutations. Although the system is efficient and easy to use, we report the rare possibility of incorrect genotyping. 1648 samples were evaluated using the GeneXpert HemosIL Factor II and Factor V assay: 1319 were freshly analyzed while 329 were frozen, thawed and diluted with saline prior to analysis to avoid clogging of the instrument syringe. Two samples, both heterozygous, one for the factor V Leiden and the other for the GPRO gene, were incorrectly genotyped as homozygous for the relative mutation. Inspection of the Ct values and amplification curves and genotyping with PCR revealed the correct genotype as heterozygous for factor V Leiden and GPRO mutation. The GeneXpert HemosIL Factor II and Factor V assay is an automated, fast genotyping assay requiring almost no sample manipulation, advantageous characteristics if compared with other PCR-based methods. However, an inattentive use of it can generate incorrect diagnosis. A careful handling of the sample, in particular correct dilution of frozen/thawed samples before analysis, and the inspection of the amplification curves and Ct values are required to avoid artifacts. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Inherited Genetic Markers for Thrombophilia in Northeastern Iran (a Clinical-Based Report

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    Fatemeh Keify


    Full Text Available Background: Thrombophilia is a main predisposition to thrombosis due to a procoagulant state. Several point mutations play key roles in blood-clotting disorders, which are grouped under the term thrombophilia. These thrombophilic mutations are methylenetetrahydrofolate reductase (MTHFR, C677T, and A1298C, factor V Leiden (G1691A, prothrombin gene mutation (factor II, G20210A, and plasminogen activator inhibitor (PAI. In the present study, we assessed the prevalence of the above thrombophilia markers in patients with recurrent pregnancy loss or first and second trimester abortions, infertility, and failed in vitro fertilization (IVF. Methods: This study was conducted among 457 cases those were referred to detect the inherited genetic markers for thrombophilia. Markers for MTHFR, Factor II, and Factor V were assessed by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP, and PAI was assessed by Amplification Refractory Mutation System (ARMS-PCR. Results: Two hundred sixty cases (56.89% were diagnosed as having at least one thrombophilia marker, whereas 197 cases (43.11% had no thrombophilia markers and were normal. Conclusion: According to the current study, the pattern of abnormal genetic markers for thrombophilia in northeastern Iran demonstrates the importance of genetic evaluations in patients who show clinical abnormalities with recurrent spontaneous abortion (RSA or other serious obstetric complications.

  12. The 2007 John Charnley Award. Factors leading to low prevalence of DVT and pulmonary embolism after THA: analysis of genetic and prothrombotic factors. (United States)

    Kim, Young-Hoo; Kim, Jun-Shik


    We evaluated 136 hips (104 patients) to determine the prevalence of and contributing factors in deep vein thrombosis (DVT) and pulmonary embolism (PE) in those who were not given thromboprophylaxis when undergoing primary cementless total hip arthroplasty (THA). We performed coagulation assays, a full blood count, blood typing, and serum chemical profile tests for all patients on three separate occasions. Molecular genetic testing was performed preoperatively to detect the genetic traits involving DVT. DVT was diagnosed by roentgenographic venography, and PE was diagnosed by perfusion lung scanning. Our patients had a low prevalence of DVT and no patient had PE. Patients with bilateral THA had similar rates (p = 0.158; CI, -0.134-0.125) of venographic DVT as patients with unilateral THA (16 of 65 or 25% versus 12 of 72 or 17% respectively). We observed a relationship between DVT and factor V Leiden mutation, antithrombin-III level, and prothrombin promoter G20210A mutation. We saw no relationship between DVT and coagulation or thrombophilic data. We conclude combinations of absent thrombophilic polymorphisms with low clinical prothrombotic risk factors led to low prevalence of DVT and virtually absent PE after THA in the current series of patients, who had not received any form of prophylactic or therapeutic treatment for DVT.

  13. [Hypercoagulable workup in ophthalmology. When and what]. (United States)

    Muñoz-Negrete, F J; Casas-Lleras, P; Pérez-López, M; Rebolleda, G


    Most ophthalmologic disorders secondary to hypercoagulabe state are due to the confluence of congenital and adquired factors. A systematic workup is mandatory. Most of congenital coagulation disorders cause venous trombosis and are inherited autosomal dominantly. In order of frequency these are factor V Leiden mutation (activated protein C resistance), G20210A mutation of the prothrombin gen and protein C, protein S, and antithrombin III deficiencies. Sickle cell anemia can determine arerial and venous thrombosis. In relation with arterial occlusion, the markers most frequently involved are homcysteine fasting levels and the markers of antiphospholipid antibody syndrome. Both of them can also determine venous thrombosis. Several acquired factors can lead to hypoercoagulable state, especially hyperhomocysteinemia, antiphospholipid antibody syndrome, hepatic disease, alcohol and tobacco intake, oral contraceptives, immobilization, surgeries and malignancies. In central venous occlusion is only necessary to rule out hyperhomocysteinemia and antiphospholipid antibody syndrome in young patients without known risk factors. In central artery occlusion, hypercoagulable workup is only recommended for patients less than 50 years-old with unknown emboli source. In this cases protein C, protein S, and antithrombin III deficiencies, homocystein, sickle cell disease and antiphospholipid antibody syndrome will ruled out. In non arteritic ischemic optic neuropathy hypercoagulable work up is not necessary. In amaurosis fugax without known emboli source, it is recommended to rule out etiologies of arterial occlusion, especially antithrombin III deficiencies, homocystein, sickle cell disease and antiphospholipid antibody syndrome.

  14. Frequency of Thrombophilic Gene Mutations in Patients with Deep Vein Thrombosis and in Women with Recurrent Pregnancy Loss

    Directory of Open Access Journals (Sweden)

    Elgari Mahmoud Mohamed


    Full Text Available Thrombophilia may be anticipated by single or combined hereditary defects in encoding genes factor V, Prothrombin, and MTHFR. The aim of this study was to determine the prevalence and associated risks of V Leiden (G1691A, Prothrombin (G20210A, and MTHFR (C677T mutations in Saudi women with Deep Vein Thrombosis (DVT and women with recurrent pregnancy loss (RPL. Protein C and protein S activity were measured to determine combined effects, if any. We examined 60 women with a history of DVT and 60 with RPL, extracted DNA from EDTA blood and determined three mutations by using multiplex PCR reactions followed by Strip Assay KIT. Pro C Global assay was used to determine the cutoff value [PCATNR = 0.80]. Protein C/S chromogenic assay was used to estimate protein C and S percentages. Frequency of Factor V Leiden G/A genotype in patients with DVT 7 (11.6% had a significant association for DVT χ2 (OR = 5.1, P = 0.03. In women with RPL the three mutations did not show any significant association, levels of Protein C, protein S and PCAT-NR in patient groups not different from controls (P > 0.05. In conclusion, we recommend expanding on these data to provide larger-scale studies.

  15. Lack of Association between Recurrent Pregnancy Loss and Inherited Thrombophilia in a Group of Colombian Patients

    Directory of Open Access Journals (Sweden)

    Henry Cardona


    Full Text Available Studies have shown an association between recurrent pregnancy loss and inherited thrombophilia in Caucasian populations, but there is insufficient knowledge concerning triethnic populations such as the Colombian. The aim of this study was to evaluate whether inherited thrombophilia is associated with recurrent pregnancy loss. Methods. We conducted a case-control study of 93 patients with recurrent pregnancy loss (cases and 206 healthy multiparous women (controls in a Colombian subpopulation. Three single nucleotide polymorphisms (SNPs markers of the inherited thrombophilias factor V Leiden, prothrombin G20210A, and methylenetetrahydrofolate reductase C677T were genotyped by PCR-RFLP. Activated protein C resistance and plasma levels of antithrombin, protein C, and protein S were also measured. Results. The frequency of thrombophilia-associated SNPs, activated protein C resistance, and anticoagulant protein deficiencies, was low overall, except for the methylenetetrahydrofolate reductase C677T SNP. The differences between patients and controls had no statistical significance. Conclusion. Our study confirms the low prevalence of inherited thrombophilias in non-Caucasian populations and it is unlikely that the tested thrombophilias play a role in the pathogenesis of recurrent pregnancy loss in this Colombian population.

  16. Association between isotretinoin use and central retinal vein occlusion in an adolescent with minor predisposition for thrombotic incidents: a case report

    Directory of Open Access Journals (Sweden)

    Labiris Georgios


    Full Text Available Abstract Introduction We report an adolescent boy with minimal pre-existing risk for thromboses who suffered central retinal vein occlusion associated with isotretinoin use for acne. To the best of our knowledge, this is the first well documented case of this association. Case presentation An otherwise healthy 17-year-old white man who was treated with systemic isotretinoin for recalcitrant acne was referred with central retinal vein occlusion in one eye. Although a detailed investigation was negative, DNA testing revealed that the patient was a heterozygous carrier of the G20210A mutation of the prothrombin gene. Despite the fact that this particular mutation is thought to represent only a minor risk factor for thromboses, it is probable that isotretinoin treatment greatly increased the risk of a vaso-occlusive incident in this patient. Conclusion Isotretinoin use may be associated with sight- and life-threatening thrombotic adverse effects even in young patients with otherwise minimal thrombophilic risk. Physicians should be aware of such potential dangers.

  17. Social, attitudinal and behavioural correlates of fruit and vegetable consumption among Cypriot adolescents. (United States)

    Loucaides, Constantinos A; Jago, Russell; Theophanous, Maria


    To examine the prevalence and correlates of fruit and vegetable (FV) consumption in Cypriot adolescents. A cross-sectional study. The Republic of Cyprus. A total of 1966 adolescents with a mean age of 14·7 (SD 2·2) years from nine elementary (n 448), six middle (n 657), five high (n 475) and five technical/vocational schools (n 386) in Cyprus. Participants completed a questionnaire assessing FV consumption using a two-item screening measure and a number of social, attitudinal and behavioural correlates of FV consumption. Overall, 19·3% of adolescents reported consuming five or more portions of FV daily, with elementary and middle school students more likely to meet recommendations (23·8% and 24·4%, respectively) compared with high and technical/vocational school students (14·0% and 12·5%, respectively). Consuming five or more portions of FV was associated with preference for FV (OR = 2·2), family eating patterns (OR = 1·5), friends' FV consumption (OR = 1·2) and school support for FV consumption (OR = 0·8). Consuming at least one portion of fruit daily was significantly associated with preference for FV (OR = 2·0) and family eating patterns (OR = 1·7). Consuming at least one portion of vegetables daily was associated with preference for FV (OR = 4·2) and eating while watching television (OR = 0·8). Targeting individual and family-based components may enhance the effectiveness of intervention programmes to promote FV consumption.

  18. Single Chain Fragment Variable Recombinant Antibody Functionalized Gold Nanoparticles for a Highly Sensitive Colorimetric Immunoassay (United States)

    Liu, Yang; Liu, Yi; Raymond, Raymond L.; Zeng, Xiangqun


    In this report, the peptide linker connecting scFv VH and VL domains were genetically modified to contain different amino acids (i.e. cysteine (scFv-cys) or histidines ( scFv-his)) to enable the scFv to adsorb or self-assemble onto the gold nanoparticles (NPs). The scFv-cys stabilized gold NPs were used to develop a highly sensitive colorimetric immunosensor. The scFv-cys stabilized gold NPs were characterized by UV-vis spectra, transmission electron microscope (TEM) and FT-IR. After adding the antigen rabbit IgG, the solution of scFv-cys stabilized gold NPs shows obvious visible color change from deep red to light purple due to the aggregation of the gold nanoparticles. Based on the colorimetric aggregation of scFv-cys stabilized gold NPs, the immunosensor exhibits high sensitivity with detection limit of 1.7 nM and good specificity. The good properties of the colorimetric aggregation immunosensor would be attributed to the small size of scFv and the covalent link between the scFv and gold NPs that improve the better orientation and enhance the probe density. With the advantages of speed, simplicity and specificity, the colorimetric immunoassay based on the functionalized scFv stabilized gold NPs represents a promising approach for protein analysis and clinical diagnostics. PMID:19327975

  19. Comparison of Chlorophyll Fluorescence Parameters in Four Cultivars of Phyllostachys nigra%4个紫竹栽培类型叶绿素荧光特性的比较

    Institute of Scientific and Technical Information of China (English)

    时燕; 黄耀华; 郭小勤; 林新春; 方伟


    紫竹(Phyllostachys nigra)为多用途经济竹种,研究利用叶绿素荧光测定技术测定大径紫竹、1a紫、2a紫、3a紫4个紫竹栽培类型的叶绿素荧光参数并进行比较.结果表明:4个紫竹栽培类型间叶绿素荧光参数除qP外均达到显著或极显著水平,光系统Ⅱ功能存在差异.3a紫的Fv、Fv/Fm、Fv/Fo、Yield、ETR、qP、NPQ值最高,其次为1 a紫、2 a紫,大径紫竹最低,说明3 a紫具有较好的光合生理功能,1 a紫、2 a紫次之,大径紫竹最差:相关分析表明:Fo与Fv/Fm、Fv/Fo,Fm与qP、NPQ显著负相关或极显著负相关;Fm与Fv、Fv/Fm、Fv/Fo,Fv与Fv/Fm、Fv/Fo,Fv/Fm与Fv/Fo,Fv/Fo与ETR,F与Fm',Yield与ETR、qP,ETR与qP,qP与NPQ显著正相关或极显著正相关.

  20. The association between motivation and fruit and vegetable intake: The moderating role of social support. (United States)

    McSpadden, Kate E; Patrick, Heather; Oh, April Y; Yaroch, Amy L; Dwyer, Laura A; Nebeling, Linda C


    Despite knowing that fruit and vegetable (FV) intake promotes health and well-being, few U.S. adults meet current guidelines. Thus, understanding people's motivation for FV intake is important for predicting dietary behavior. Applying self-determination theory, the goal of this study was to examine the role of social support as a potential moderator of the link between autonomous and controlled motivations and FV intake. Cross-sectional data from 2959 adults in the United States were analyzed. Autonomous motivation and perceived social support were positively associated with FV intake, while controlled motivation was negatively associated with FV intake. Additionally, there was evidence that the negative association between controlled motivation and FV intake was attenuated by higher levels of perceived social support. Findings suggest the need for a more comprehensive approach to understanding the role of motivation in health behaviors like FV intake and the potential roles played by friends and family in these motivational processes.

  1. Accelerating of Image Retrieval in CBIR System with Relevance Feedback

    Directory of Open Access Journals (Sweden)

    Radosavljević Vladan


    Full Text Available Content-based image retrieval (CBIR system with relevance feedback, which uses the algorithm for feature-vector (FV dimension reduction, is described. Feature-vector reduction (FVR exploits the clustering of FV components for a given query. Clustering is based on the comparison of magnitudes of FV components of a query. Instead of all FV components describing color, line directions, and texture, only their representative members describing FV clusters are used for retrieval. In this way, the "curse of dimensionality" is bypassed since redundant components of a query FV are rejected. It was shown that about one tenth of total FV components (i.e., the reduction of 90% is sufficient for retrieval, without significant degradation of accuracy. Consequently, the retrieving process is accelerated. Moreover, even better balancing between color and line/texture features is obtained. The efficiency of FVR CBIR system was tested over TRECVid 2006 and Corel 60 K datasets.

  2. Accelerating of Image Retrieval in CBIR System with Relevance Feedback

    Directory of Open Access Journals (Sweden)

    Branimir Reljin


    Full Text Available Content-based image retrieval (CBIR system with relevance feedback, which uses the algorithm for feature-vector (FV dimension reduction, is described. Feature-vector reduction (FVR exploits the clustering of FV components for a given query. Clustering is based on the comparison of magnitudes of FV components of a query. Instead of all FV components describing color, line directions, and texture, only their representative members describing FV clusters are used for retrieval. In this way, the “curse of dimensionality” is bypassed since redundant components of a query FV are rejected. It was shown that about one tenth of total FV components (i.e., the reduction of 90% is sufficient for retrieval, without significant degradation of accuracy. Consequently, the retrieving process is accelerated. Moreover, even better balancing between color and line/texture features is obtained. The efficiency of FVR CBIR system was tested over TRECVid 2006 and Corel 60 K datasets.

  3. Thrombomodulin-dependent effect of factor V Leiden mutation on the cross-linking of α2-plasmin inhibitor to fibrin and its consequences on fibrinolysis. (United States)

    Koncz, Zsuzsa; Bagoly, Zsuzsa; Haramura, Gizella; Mezei, Zoltán A; Muszbek, László


    It has been shown that thrombomodulin (TM) considerably delays factor XIII (FXIII) activation and this effect is abrogated by Factor V Leiden (FV(Leiden)) mutation. The aim of the study was to explore the effect of TM on the cross-linking of α(2)-plasmin inhibitor (α(2)-PI) to fibrin in plasma samples of different FV genotypes and how this effect is related to the impaired fibrinolysis of FV(Leiden) carriers. In the plasma samples of fifteen individuals with different FV genotypes and in FV deficient plasma supplemented with wild type FV or FV(Leiden) coagulation was initiated by recombinant human tissue factor and phospholipids with or without recombinant human TM (rhTM). In the recovered clots the extent of α(2)-PI-fibrin cross-linking was evaluated by Western blotting and quantitative densitometry. The effect of rhTM on tissue plasminogen activator (tPA) induced clot lysis was measured by turbidimetric method. rhTM significantly delayed the formation of α(2)-PI-fibrin α-chain heterodimers/oligomers in plasma samples containing wild type FV. This effect of rhTM was impaired in the presence of FV(Leiden). rhTM delayed tPA-induced clot lysis and this effect of rhTM was more pronounced in plasma containing FV(Leiden). When TAFIa was inhibited by potato carboxypeptidase inhibitor, rhTM accelerated clot lysis in the presence of wild type FV, which is explained by the delayed α(2)-PI-fibrin cross-linking. This effect of rhTM did not prevail in the presence of FV(Leiden). FV(Leiden) abrogates the delaying effect of rhTM on α(2)-PI-fibrin cross-linking, which contributes to the impaired fibrinolysis observed in FV(Leiden) carriers. Copyright © 2012 Elsevier Ltd. All rights reserved.

  4. Expression of Anti-ASGPR ScFv Targeting Melittin and Its Anti-tumor Efficacy in Vitro%去唾液酸糖蛋白受体单链抗体靶向蜂毒肽的体外抑瘤效应研究

    Institute of Scientific and Technical Information of China (English)

    曹利民; 沈关心; 赵晓蓉; 叶庆; 雷萍; 吴砂; 代维; 朱慧芬; 朱荫昌; 司进


    目的探讨抗去唾液酸糖蛋白受体(ASGPR)单链抗体C1与蜂毒肽(Melittin)重组蛋白C1M靶向抑制肝癌细胞的效果.方法将含C1M/ pGC的XL1-Blue转化菌通过IPTG诱导表达.表达产物用Ni2+螯合柱亲和纯化,免疫组织化学技术分析重组蛋白C1M的抗原结合能力,四氮唑盐(MTT)法检测C1M对肝癌细胞株HepG2的增殖抑制作用.结果原核表达质粒C1M/ pGC在XL1-Blue中获得有效表达;表达产物以可溶性形式存在;纯化的C1M相对分子量为29.4×103;免疫组化结果表明C1M能有效识别去唾液酸糖蛋白受体,与HepG2(C1M)共培养3 d,细胞生长抑制率达92.0%.结论在大肠埃希菌中成功表达C1M,位于C端的Melittin蜂毒肽能有效抑制肿瘤细胞的生长,为体内研究Melittin的靶向抗肝癌效果奠定了基础.

  5. 去唾液酸糖蛋白受体H1亚单位的表达、纯化及其单链抗体的筛选与鉴定%Expression and purification of the human asialoglycoprotein receptor H1 subunit and selection of anti-CRDH1 scFv

    Institute of Scientific and Technical Information of China (English)

    曹利民; 司进; 朱荫昌; 王炜煜; 赵晓蓉; 叶庆; 李文涵; 朱慧芬; 沈关心


    目的去唾液酸糖蛋白受体(ASGPR)H1亚单位的重组、表达、纯化及其单链抗体的筛选与鉴定.方法将ASGPR H1亚单位糖识别区基因(CRDH1)定向克隆至原核表达载体pET-32c经IPTG诱导表达,表达产物用Ni2+螯合柱亲和纯化,免疫印迹技术(WB)检测;用纯化的CRDH1对人源噬菌体抗体库进行4轮固相筛选,阳性克隆用表达标记(Trx-His-s tag)进行差异筛选.取阳性噬菌体C3感染宿主菌HB2151,37℃振荡培养过夜,终浓度为1 mmol/L的IPTG诱导表达,12%SDS-PAGE与Western blot鉴定以及免疫组化鉴定纯化的单链抗体C3与肝细胞结合的特性.结果CRDH1/pET-32c在原核系统经诱导表达出相对分子质量(Mr)约35×103大小的融合蛋白,以包涵体的形式存在;通过Ni2+亲和柱纯化获得CRDH1融合蛋白;经4轮筛选和差异筛选后,有2株可分泌性表达与CRDH1特异性结合的单链抗体,DNA序列分析表明单链抗体重链基因和轻链基因分别属于人免疫球蛋白VH3家族和VKI家族.噬菌体C3经诱导表达出Mr约28×103大小的融合蛋白,可溶性分泌表达于培养基中;通过Ni2+亲和柱纯化获得单链抗体,ELISA、Western blot表明单链抗体C3能较好结合rCRDH1,免疫组化显示该单链抗体可与肝癌细胞、肝细胞特异结合.结论成功表达ASGPR并筛选出其人源单链抗体.该单链抗体可与表达的rCRDH1以及肝细胞和肝癌细胞特异性结合,提示对肝脏疾病的靶向治疗有潜在的应用价值.

  6. Erratum to: Ungersma SE, Pacheco G, Ho C, Yee SF, Ross J, van Bruggen N, Peale FV Jr, Ross S, Carano RA. Vessel imaging with viable tumor analysis for quantification of tumor angiogenesis. Magn Reson Med 2010;63:1637–1647. (United States)

    Ungersma, Sharon E; Pacheco, Glenn; Ho, Calvin; Yee, Sharon Fong; Ross, Jed; van Bruggen, Nicholas; Peale, Franklin V; Ross, Sarajane; Carano, Richard A D


    Imaging of tumor microvasculature has become an important tool for studying angiogenesis and monitoring antiangiogenic therapies. Ultrasmall paramagnetic iron oxide contrast agents for indirect imaging of vasculature offer a method for quantitative measurements of vascular biomarkers such as vessel size index, blood volume, and vessel density (Q). Here, this technique is validated with direct comparisons to ex vivo micro-computed tomography angiography and histologic vessel measurements, showing significant correlations between in vivo vascular MRI measurements and ex vivo structural vessel measurements. The sensitivity of the MRI vascular parameters is also demonstrated, in combination with a multispectral analysis technique for segmenting tumor tissue to restrict the analysis to viable tumor tissue and exclude regions of necrosis. It is shown that this viable tumor segmentation increases sensitivity for detection of significant effects on blood volume and Q by two antiangiogenic therapeutics [anti-vascular endothelial growth factor (anti-VEGF) and anti-neuropilin-1] on an HM7 colorectal tumor model. Anti-vascular endothelial growth factor reduced blood volume by 36±3% (p<0.0001) and Q by 52±3% (p<0.0001) at 48 h post-treatment; the effects of anti-neuropilin-1 were roughly half as strong with a reduction in blood volume of 18±6% (p<0.05) and a reduction in Q of 33±5% (p<0.05) at 48 h post-treatment.

  7. 公允价值呈报方式对上市物流企业会计信息价值的影响%Study on Influence of FV-based Presenting Method on Accounting Information Value of Listed Logistics Enterprises

    Institute of Scientific and Technical Information of China (English)



    针对更改公允价值变动呈报方式会对上市物流企业财务报告可读性和会计信息透明度产生影响的问题,综合分析了金融资产公允价值变动呈报方式变化的影响,构建了一组价值相关模型和收益相关模型,检验金融资产公允价值变动呈报方式对会计信息价值的影响。研究结果证明,在利润表中增加金融资产公允价值变动数据能够增强会计信息价值,能够显著提高财务报告的可读性和显著降低投资者估值成本,为完善会计准则提供参考。%In this paper, we comprehensively analyzed the influence of the changes of the presenting methods of the changes in fair value of financial assets on the readability and transparency of the financial statements of the listed logistics enterprises, built the relevant value and income models to verify the claims made, and found that, by adding in the income statement the data reflecting the changes in the fair value of the financial assets, the value of the accounting information of the enterprises was enhanced.

  8. Simultaneous inhibition of epidermal growth factor receptor (EGFR) signaling and enhanced activation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor-mediated apoptosis induction by an scFv : sTRAIL fusion protein with specificity for human EGFR

    NARCIS (Netherlands)

    Bremer, E; Samplonius, DF; van Genne, L; Dijkstra, MH; Kroesen, BJ; de Leij, LFMH; Helfrich, W


    Epidermal growth factor receptor (EGFR) signaling inhibition by monoclonal antibodies and EGFR-specific tyrosine kinase inhibitors has shown clinical efficacy in cancer by restoring susceptibility of tumor cells to therapeutic apoptosis induction. Tumor necrosis factor-related apoptosis-inducing lig

  9. Fruits and vegetables as a healthier snack throughout the day among families with older children: findings from a survey of parent-child dyads. (United States)

    Smith, Teresa M; Pinard, Courtney A; Byker Shanks, Carmen; Wethington, Holly; Blanck, Heidi M; Yaroch, Amy L


    Most U.S. youth fail to eat the recommended amount of fruits and vegetables (FV) however many consume too many calories as added sugars and solid fats, often as snacks. The aim of this study was to assess factors associated with serving FV as snacks and with meals using parent-child dyads. A cross-sectional sample of U.S. children aged 9 to 18, and their caregiver/parent (n=1522) were part of a Consumer Panel of households for the 2008 YouthStyles mail survey. Chi-square test of independence and multivariable logistic regression were used to assess associations between serving patterns of FV as snacks with variations in serving patterns, and covariates including dietary habits. Most parents (72%) reported serving FV at meals and as snacks. Fruit was most frequently served as a snack during the day (52%) and vegetables were most frequently served as a snack during the day (22%) but rarely in the morning. Significant differences in child FV intake existed among FV as a snack serving patterns by parents. Compared to children whose parents served FV only at meals, children whose parents reported serving FV as snacks in addition to meals were significantly more likely to have consumed FV the day before (using a previous day screener), Psnacks, may increase FV intake among older children and adolescents.

  10. The Fragaria vesca homolog of suppressor of overexpression of constans1 represses flowering and promotes vegetative growth. (United States)

    Mouhu, Katriina; Kurokura, Takeshi; Koskela, Elli A; Albert, Victor A; Elomaa, Paula; Hytönen, Timo


    In the annual long-day plant Arabidopsis thaliana, suppressor of overexpression of constans1 (SOC1) integrates endogenous and environmental signals to promote flowering. We analyzed the function and regulation of the SOC1 homolog (Fragaria vesca [Fv] SOC1) in the perennial short-day plant woodland strawberry (Fragaria vesca). We found that Fv SOC1 overexpression represses flower initiation under inductive short days, whereas its silencing causes continuous flowering in both short days and noninductive long days, similar to mutants in the floral repressor Fv terminal flower1 (Fv TFL1). Molecular analysis of these transgenic lines revealed that Fv SOC1 activates Fv TFL1 in the shoot apex, leading to the repression of flowering in strawberry. In parallel, Fv SOC1 regulates the differentiation of axillary buds to runners or axillary leaf rosettes, probably through the activation of gibberellin biosynthetic genes. We also demonstrated that Fv SOC1 is regulated by photoperiod and Fv flowering locus T1, suggesting that it plays a central role in the photoperiodic control of both generative and vegetative growth in strawberry. In conclusion, we propose that Fv SOC1 is a signaling hub that regulates yearly cycles of vegetative and generative development through separate genetic pathways.

  11. Cell cycle requirements for transduction by foamy virus vectors compared to those of oncovirus and lentivirus vectors. (United States)

    Trobridge, Grant; Russell, David W


    Retroviral vectors based on foamy viruses (FV) are efficient gene delivery vehicles for therapeutic and research applications. While previous studies have shown that FV vectors transduce quiescent cell cultures more efficiently than oncoviral vectors, their specific cell cycle requirements have not been determined. Here we compare the transduction frequencies of FV vectors with those of onco- and lentiviral vectors in nondividing and dividing normal human fibroblasts by several methods. FV vectors transduced serum-deprived fibroblast cultures more efficiently than oncoretroviral vectors and at rates comparable to those of lentiviral vectors. However, in these cultures FV vectors only transduced a subpopulation of proliferating cells, as determined by bromodeoxyuridine staining for DNA synthesis. In contrast to lentiviral vectors, FV vectors were unable to transduce human fibroblasts arrested by aphidicolin (G(1)/S phase) or gamma-irradiation (G(2) phase), and a partial cell cycle that included mitosis but not DNA synthesis was required. We could not determine if mitosis facilitated nuclear entry of FV vectors, since cell-free vector preparations contained long terminal repeat circles, precluding their use as nuclear markers. In contrast to oncoviral vectors, both FV and lentiviral vectors efficiently transduced G(0) fibroblasts that were later stimulated to divide. In the case of FV vectors, this was due to the persistence of a stable transduction intermediate in quiescent cells. Our findings support the use of FV vectors as a safe and effective alternative to lentiviral vectors for ex vivo transduction of stem cells that are quiescent during culture but divide following transplantation.

  12. In vitro caries lesion rehardening and enamel fluoride uptake from fluoride varnishes as a function of application mode. (United States)

    Lippert, Frank; Hara, Anderson T; Martinez-Mier, Esperanza Angeles; Zero, Domenick T


    To study the laboratory predicted anticaries efficacy of five commercially available fluoride varnishes (FV) by determining their ability to reharden and to deliver fluoride to an early caries lesion when applied directly or in close vicinity to the lesion (halo effect). Early caries lesions were created in 80 polished bovine enamel specimens. Specimens were allocated to five FV groups (n = 16) based on Knoop surface microhardness (KHN) after lesion creation. All tested FV claimed to contain 5% sodium fluoride and were: CavityShield, Enamel Pro, MI Varnish, Prevident and Vanish. FV were applied (10 +/- 2 mg per lesion) to eight specimens per FV group (direct application); the remaining eight specimens received no FV but were later exposed to fluoride released from specimens which received a FV treatment (indirect application). Specimens were paired again and placed into containers (one per FV). Artificial saliva was added and containers placed into an incubator (27 hours at 37 degrees C). Subsequently, FV was carefully removed using chloroform. Specimens were exposed to fresh artificial saliva again (67 hours at 37 degrees C). KHN was measured and differences to baseline values calculated. Enamel fluoride uptake (EFU) was determined using the acid etch technique. Data were analyzed using two-way ANOVA. The two-way ANOVA highlighted significant interactions between FV vs. application mode, for both deltaKHN and EFU (P Vanish did not differ. Prevident exhibited a higher EFU when applied directly, but little difference in rehardening.


    Institute of Scientific and Technical Information of China (English)

    Wang Ying; Chen Wei; Li Xu


    Objective To obtain the gene of murine Single chain Fv fragment (ScFv) against human cervical cancer and to express it in E. coli. Methods The variable region gene fragments of the heavy and light chains, which were amplified respectively using recombinant DNA techniques from CsA125 hybridoma cells, were spliced together through a flexible linker to ScFv against human cervical cancer. The ScFv genes were then cloned into expression vector pCANTAB 5E and expressed in E. coli HB2151 and TG1 respectively. The soluble ScFv were characterized by SDS PAGE and Western blot. The antigen-binding activities of the soluble and phage displayed ScFv were assayed by ELISA and cell immunohistochemical analysis. Results The expressed ScFv antibodies were soluble and phage displayed. The soluble ScFv secreted and expressed in E. coli HB2151 induced by IPTG were confirmed with SDS-PAGE, Western blot and ELISA. The specific binding capacity of the soluble and phage displayed ScFv to the surface associated antigen of human cervical cancer cell line was further confirmed with immunohistochemical studies. Conclusion The soluble and phage displayed ScFv expressed in E. coli against human cervical cancer showed high, specific affinity for the cervical cancer cell line surface associated antigen.

  14. A conjugate of an anti-midkine single-chain variable fragment to doxorubicin inhibits tumor growth

    Directory of Open Access Journals (Sweden)

    Shuli Zhao


    Full Text Available Doxorubicin (DOX was conjugated to a single-chain variable fragment (scFv against human midkine (MK, and the conjugate (scFv-DOX was used to target the chemotherapeutic agent to a mouse solid tumor model in which the tumor cells expressed high levels of human MK. The His-tagged recombinant scFv was expressed in bacteria, purified by metal affinity chromatography, and then conjugated to DOX using oxidative dextran (Dex as a linker. The molecular formula of this immunoconjugate was scFv(Dex1.3(DOX20. In vitro apoptosis assays showed that the scFv-DOX conjugate was more cytotoxic against MK-transfected human adenocarcinoma cells (BGC823-MK than untransfected cells (55.3 ± 2.4 vs 22.4 ± 3.8% for three independent experiments. Nude mice bearing BGC823-MK solid tumors received scFv-DOX or equivalent doses of scFv + DOX for 2 weeks and tumor growth was more effectively inhibited by the scFv-DOX conjugate than by scFv + DOX (51.83% inhibition vs 40.81%. Histological analysis of the tumor tissues revealed that the highest levels of DOX accumulated in tumors from mice treated with scFv-DOX and this resulted in more extensive tumor cell death than in animals treated with the equivalent dose of scFv + DOX. These results show that the scFv-DOX conjugate effectively inhibited tumor growth in vivo and suggest that antigen-specific scFv may be competent drug-carriers.

  15. Implementation of strategies to increase adolescents' access to fruit and vegetables at school

    DEFF Research Database (Denmark)

    Aarestrup, Anne Kristine; Jørgensen, Thea Suldrup; Jørgensen, Sanne Ellegaard;


    BACKGROUND: Access to fruit and vegetables (FV) is associated with adolescents' FV consumption. However, little is known about implementation of strategies to increase access to FV at schools. We examined the implementation of two environmental components designed to increase access to FV at Danish...... schools. METHODS: We used data from 20 intervention schools involved in the school-based multicomponent Boost trial targeting 13-year-olds' FV consumption. The environmental components at school included daily provision of free FV and promotion of a pleasant eating environment. Questionnaire data...... was collected by the end of the nine-month intervention period among 1,121 pupils (95%), from all school principals (n = 20) and half way through the intervention period and by the end of the intervention among 114 teachers (44%). The implementation of the components was examined descriptively using...

  16. Synthesis and thermolysis of Cp*(C5Me4CH2)TiR complexes

    NARCIS (Netherlands)

    Luinstra, GA; Brinkmann, PHP; Teuben, JH; Luinstra, Gerrit A.


    Substitution of the chloride in Cp*FvTiCl with MR (Fv = C5Me4CH2; R = Me, CH2SiMe3, CH2CMe3, CH = CH2, M = Li; R = CH2Ph, M = K; R = C3H5, M = MgCl; R = Ph, M = Na . NaCl) gives Cp*FvTiR. NMR spectroscopic evidence points towards a series of structurally related compounds with a bent-sandwich

  17. Efficient intracellular retrotransposition of an exogenous primate retrovirus genome


    Heinkelein, Martin; Pietschmann, Thomas; Jármy, Gergely; Dressler, Marco; Imrich, Horst; Thurow, Jana; Lindemann, Dirk,; Bock, Michael; Moebes, Astrid; Roy, Jacqueline; Herchenröder, Ottmar; Rethwilm, Axel


    The foamy virus (FV) subgroup of Retroviridae reverse transcribe their RNA (pre-)genome late in the replication cycle before leaving an infected cell. We studied whether a marker gene-transducing FV vector is able to shuttle to the nucleus and integrate into host cell genomic DNA. While a potential intracellular retrotransposition of vectors derived from other retroviruses was below the detection limit of our assay, we found that up to 5% of cells transfected with the FV vector were stably tr...

  18. Production of a human single-chain variable fragment antibody against esophageal carcinoma

    Institute of Scientific and Technical Information of China (English)

    Ming-Yan Xu; Xiao-Hu Xu; Geng-Zhen Chen; Xiao-Ling Deng; Jonathan Li; Xiao-Jun Yu; Mei-Zhen Chen


    AIM: To construct a phage display library of human singlechain variable fragment (scFv) antibodies associated with esophageal cancer and to preliminarily screen a scFv antibody against esophageal cancer.METHODS: Total RNA extracted from metastatic lymph nodes of esophageal cancer patients was used to construct a scFv gene library. Rescued by M13K07 helper phage, the scFv phage display library was constructed. esophageal cancer cell line Eca 109 and normal human esophageal epithelial cell line (NHEEC) were used for panning and subtractive panning of the scFv phage display library to obtain positive phage clones. Soluble scFv was expressed in E.coli HB2151 which was transfected with the positive phage clone, then purified by affinity chromatography.Relative molecular mass of soluble scFv was estimated by Western blotting, its bioactivity was detected by cell ELISA assay. Sequence of scFv was determined using the method of dideoxynucleotide sequencing.RESULTS: The size of scFv gene library was approximately 9×106 clones. After four rounds of panning with Eca109 and three rounds of subtractive panning with NHEEC cells, 25 positive phage clones were obtained. Soluble scFv was found to have a molecular mass of 31 ku and was able to bind to Eca109 cells, but not to HeLa and NHEEC cells. Variable heavy (VH) gene from one of the positive clones was shown to be derived from the γ chain subgroup Ⅳ of immunoglobulin, and variable light (VL) gene from the κchain subgroup I of immunoglobulin.CONCLUSION: A human scFv phage display library can be constructed from the metastatic lymph nodes of esophageal cancer patients. A whole human scFv against esophageal cancer shows some bioactivity.

  19. Effect of Salinity on Chlorophyll Fluorescence and Chlorophyll Content of the Desert Shrub Calotropis procera


    , A. Akhkha; , T. Boutraa


    The effect of salinity stress on the efficiency of the photosynthetic apparatus in the desert shrub Calotropis procera Aiton (family: Asclepiadaceae) was investigated using chlorophyll fluorescence technique. This technique allowed the determination of a number of chlorophyll fluorescence parameters such as initial fluorescence Fo, maximum fluorescence Fm and variable fluorescence Fv. Furthermore, the effect of salinity stress on other chlorophyll fluorescence parameters (Fv / Fo and Fv / Fm ...

  20. Complete Sequence of a F33:A-:B- Conjugative Plasmid Carrying the oqxAB, fosA3 and blaCTX-M-55 Elements from a Foodborne Escherichia coli Strain

    Directory of Open Access Journals (Sweden)

    Marcus Ho-yin Wong


    Full Text Available This study reports the complete sequence of pE80, a conjugative IncFII plasmid recovered from an E. coli strain isolated from chicken meat. This plasmid harbors multiple resistance determinants including oqxAB, fosA3, blaCTX-M-55 and blaTEM-1, and is a close variant of the recently reported p42-2 element, which was recovered from E. coli of veterinary source. Recovery of pE80 constitutes evidence that evolution or genetic re-arrangement of IncFII type plasmids residing in animal-borne organisms is an active event, which involves acquisition and integration of foreign resistance elements into the plasmid backbone. Dissemination of these plasmids may further compromise the effectiveness of current antimicrobial strategies.

  1. Acquired antiprothrombin antibodies: an unusual cause of bleeding. (United States)

    Carvalho, Cristiana; Viveiro, Carolina; Maia, Paulo; Rezende, Teresa


    Acquired inhibitors of coagulation causing bleeding manifestations are rare in children. They emerge, normally in the context of autoimmune diseases or drug ingestion, but transient and self-limiting cases can occur after viral infection. We describe, an otherwise healthy, 7-year-old girl who had gingival bleeding after a tooth extraction. The prothrombin time (PT) and the activated partial thromboplastin time (APTT) were both prolonged with evidence of an immediate acting inhibitor (lupic anticoagulant). Further coagulation studies demonstrated prothrombin (FII) deficiency and prothrombin directed (FII) antibodies. The serological tests to detect an underlying autoimmune disease were all negative. The coagulation studies normalised alongside the disappearance of the antibody. This article presents lupus anticoagulant hypoprothrombinaemia syndrome (LAHS) as a rare case of acquired bleeding diathesis in childhood.

  2. Dietary changes in fasting levels of factor VII coagulant activity (FVII:C) are accompanied by changes in factor VII protein and other vitamin K-dependent proteins

    DEFF Research Database (Denmark)

    Bladbjerg, Else-Marie; Tholstrup, T; Marckmann, P


    each. Fasting blood samples were collected before and after the dietary regimen and analysed for triglycerides, FVII:C, and protein concentrations of FVII, FII, FX, protein C, CRP, albumin, fibrinogen, and F1 + 2. FVII:C was significantly reduced on diet S compared with diet ML. This was accompanied...... by a decrease in FVII protein, F1 + 2 and the vitamin K-dependent proteins FII, FX, and protein C. In contrast, no changes were observed in triglycerides, FVII:C/FVII:Ag, albumin and CRP. Fibrinogen was increased on diet S compared with diet ML. Our findings suggest that the change in fasting FVII:C was part...... of a general change in concentrations of vitamin K-dependent proteins. Udgivelsesdato: 1995-Feb...

  3. Production and characterization of a single-chain antibody of anti-CD3%抗CD3单链抗体基因克隆表达及生物活性鉴定

    Institute of Scientific and Technical Information of China (English)

    陈秀芹; 阎锡蕴


    The genes encoding antibody heavy and light chain variable regions(VH and VL)were cloned by RT-PCR from OKT3 hybridoma cells,which produced anti-CD3 moleclonal antibody.The VH and VL genes were fused and become a single chain Fv(scFv).The scFv gene was cloned into pCANTAB5E vector and expressed on bacterial phage surface.By three panning rounds,we have obtained two single-chain antibodys that specific for CD3.The antiCD3 scFv wil be a reagent fox diagnosis and therapy of immuno-disorder.

  4. A VL-linker-VH Orientation Dependent Single Chain Variable Antibody Fragment Against Rabies Virus G Protein with Enhanced Neutralizing Potency in vivo. (United States)

    Cheng, Yue; Li, Zhuang; Xi, Hualong; Gu, Tiejun; Yuan, Ruosen; Chen, Xiaoxu; Jiang, Chunlai; Kong, Wei; Wu, Yongge


    Lethal rabies can be prevented effectively by post-exposure prophylactic (PEP) with rabies immunoglobulin (RIG). Single-chain variable fragment (scFv), which is composed of a variable heavy chain (VH) and variable light chain (VL) connected by a peptide linker, may be developed as alternative to RIG for neutralizing rabies virus (RV). However, our previously constructed scFv (FV57S) with the (NH2) VH-linker-VL (COOH) orientation showed a lower neutralizing potency than its parent RIG. This orientation may inhibit FV57S from refolding into an intact and correct conformation. Therefore, the RFV57S protein with a VL-linker-VH orientation was constructed based on FV57S. A HIS tag was incorporated to aid in purification and detection of RFV57S and FV57S. However, abilities of RFV57S and FV57S to bind with the anti-HIS tag mAb were different. Therefore, a novel direct ELISA was established by utilizing a biotin-labeled truncated glycoprotein of RV. Although with similar stability and in vitro neutralizing potency as FV57S, RFV57S showed enhanced binding ability, affinity and in vivo protective efficacy against lethal dose of RV. Our studies support the feasibility of developing a scFv with reversed orientation and provide a novel method for evaluating the binding ability, stability and affinity of engineered antibodies recognizing linear epitope.

  5. Perceived social-ecological factors associated with fruit and vegetable purchasing, preparation, and consumption among young adults. (United States)

    Graham, Dan J; Pelletier, Jennifer E; Neumark-Sztainer, Dianne; Lust, Katherine; Laska, Melissa N


    Most young adults do not consume recommended levels of fruits and vegetables (F/V), and interventions to increase F/V-related behaviors among this understudied population are needed. Therefore, it is important to identify correlates of F/V intake among young adults to guide intervention development. This cross-sectional study used data from an online survey to identify factors related to young adults' F/V purchasing, preparation, and consumption, and to explore between-factor relationships using mediation analysis. In 2010, 1,201 college students in Minnesota completed questionnaires assessing F/V behaviors as well as perceptions of F/V-related individual, social, and environmental factors. Factor analysis identified questionnaire items assessing similar constructs. Seven factors were identified (personal barriers, F/V knowledge, family, friends, neighborhood, access barriers, and campus) and evaluated for relationships with F/V purchasing, preparation, and consumption using linear regression. Results revealed that perceived personal barriers (eg, lacking cooking skills) were inversely related to all F/V outcomes. Perception that family and friends eat healthfully and neighborhood access to F/V were positively related to all outcomes. Individual-, social-, and environment-level perceptions were related to purchasing, preparation, and consumption, and the effects of these factors were similar when accounting for mediated effects. Factors at all three levels and the ways in which these various factors operate together may be important to consider in future efforts to improve F/V behaviors among young adults.

  6. Novel Selenium-containing Human Single-chain Variable Fragment with Glutathione Peroxidase Activity from Computer-aided Molecular Design

    Institute of Scientific and Technical Information of China (English)

    WANG Cheng; MU Ying; L(U) Shao-wu; LUO Gui-min; WAN Pei; GONG Ping-sheng; L(U) Li-min; XU Ya-wei; ZHAO Yang; HE Bo; ZHAO Gang; YAN Gang-lin


    In order to enhance the glutathione peroxidase(GPX) catalytic activity of the selenium-containing single-chain variable fragments(Se-scFv),a novel human scFv was designed on the basis of the structure of human antibody and optimized via bioinformatics methods such as homologous sequence analysis,three-dimensional(3D)model building,binding-site analysis and docking.The DNA sequence of the new human scFv was synthesized and cloned into the expression vector pET22b(+),then the scFv protein was expressed in soluble form in Escherichia coli BL21(DE3) and purified by Ni2+-immobilized metal affinity chromatography(IMAC).The serine residue of scFv in the active site was converted into selenocysteine(Sec) with the chemical modification method,thus,the human Se-scFv with GPX activity was obtained.The GPX activity of the Se-scFv protein was characterized.Compared with other Se-scFv,the new human Se-scFv showed similar efficiency for catalyzing the reduction of hydrogen peroxide by glutathione.It exhibited pH and temperature dependent catalytic activity and a typical ping-pong kinetic mechanism.

  7. Characterization of antibodies in single-chain format against the E7 oncoprotein of the Human papillomavirus type 16 and their improvement by mutagenesis

    Directory of Open Access Journals (Sweden)

    Accardi Luisa


    Full Text Available Abstract Background Human papillomaviruses (HPV are the etiological agents of cervical cancer. The viral E7 protein plays a crucial role in viral oncogenesis. Many strategies have been explored to block the E7 oncoprotein activity. The single-chain variable antibody fragments (scFvs are valuable tools in cancer immunotherapy and can be used as "intracellular antibodies" to knock out specific protein functions. For both in vivo and in vitro employment, the scFv intrinsic solubility and stability are important to achieve long-lasting effects. Here we report the characterization in terms of reactivity, solubility and thermal stability of three anti-HPV16 E7 scFvs. We have also analysed the scFv43 sequence with the aim of improving stability and then activity of the antibody, previously shown to have antiproliferative activity when expressed in HPV16-positive cells. Methods The three anti-HPV16 E7 scFv 32, 43 51 were selected from the ETH-2 "phage-display" library. Thermal stability was evaluated with ELISA by determining the residual activity of each purified scFv against the recombinant HPV16 E7, after incubation in the presence of human seroalbumine for different time-intervals at different temperatures. Sequence analysis of the scFvs was performed with BLAST and CLUSTALL programs. The scFv43 aminoacid changes were reverted back to the consensus sequence from the immunoglobuline database by site-directed mutagenesis. ScFv solubility was evaluated with Western blotting by determining their relative amounts in the soluble and insoluble fractions of both prokaryotic and eukaryotic systems. Results ScFv51 was the most thermally stable scFv considered. Sequence analysis of the most reactive scFv43 has evidenced 2 amino acid changes possibly involved in molecule stability, in the VH and VL CDR3 regions respectively. By mutagenesis, two novel scFv43-derived scFvs were obtained, scFv43 M1 and M2. ScFv43 M2 showed to have improved thermal stability and

  8. Adolescent Overweight and Obesity: Links to Socioeconomic Status and Fruit and Vegetable Intakes

    Directory of Open Access Journals (Sweden)

    Jihyun You


    Full Text Available Whether adolescent overweight/obesity is linked to socioeconomic status (SES and fruit and vegetable (F/V intakes has not been confirmed. We aimed to determine whether there is an association between SES and adolescent overweight/obesity and to test the mediating effect of F/V intakes. This cross-sectional study included the data of 63,111 adolescents extracted from the 2013 Korea Youth Risk Behavior Web-based Survey. Overweight/obesity was defined as a body mass index ≥ 85th percentile, while F/V intakes were categorized as high (recommended levels: ≥1 fruit serving and ≥3 vegetable servings per day versus low. Among girls, low SES (beta = 0.50, p < 0.001 and F/V intakes (beta = −0.17, p = 0.038 were both significantly associated with overweight/obesity; the former association was significantly mediated by F/V intakes (Sobel test: z = 2.00, p = 0.046. Among boys, neither SES nor F/V intakes was significantly associated with overweight/obesity. Adolescent overweight/obesity was significantly linked to low SES and F/V intakes among girls only; low SES indirectly increased the risk of overweight/obesity via low F/V intakes. Therefore, promoting F/V intakes for socially disadvantaged girls should be prioritized as a population-based strategy for preventing adolescent overweight/obesity in South Korea.

  9. Characterization and functional analysis of Calmodulin and Calmodulin-like genes in Fragaria vesca

    Directory of Open Access Journals (Sweden)

    Kai Zhang


    Full Text Available Calcium is a universal messenger that is involved in the modulation of diverse developmental and adaptive processes in response to various stimuli. Calmodulin (CaM and calmodulin-like (CML proteins are major calcium sensors in all eukaryotes, and they have been extensively investigated for many years in plants and animals. However, little is known about CaMs and CMLs in woodland strawberry (Fragaria vesca. In this study, we performed a genome-wide analysis of the strawberry genome and identified 4 CaM and 36 CML genes. Bioinformatics analyses, including gene structure, phylogenetic tree, synteny and three-dimensional model assessments, revealed the conservation and divergence of FvCaMs and FvCMLs, thus providing insight regarding their functions. In addition, the transcript abundance of four FvCaM genes and the four most related FvCML genes were examined in different tissues and in response to multiple stress and hormone treatments. Moreover, we investigated the subcellular localization of several FvCaMs and FvCMLs, revealing their potential interactions based on the localizations and potential functions. Furthermore, overexpression of five FvCaM and FvCML genes could not induce a hypersensitive response, but four of the five genes could increase resistance to Agrobacterium tumefaciens in Nicotiana benthamiana leaves. This study provides evidence for the biological roles of FvCaM and CML genes, and the results lay the foundation for future functional studies of these genes.

  10. Large Animal Models for Foamy Virus Vector Gene Therapy

    Directory of Open Access Journals (Sweden)

    Peter A. Horn


    Full Text Available Foamy virus (FV vectors have shown great promise for hematopoietic stem cell (HSC gene therapy. Their ability to efficiently deliver transgenes to multi-lineage long-term repopulating cells in large animal models suggests they will be effective for several human hematopoietic diseases. Here, we review FV vector studies in large animal models, including the use of FV vectors with the mutant O6-methylguanine-DNA methyltransferase, MGMTP140K to increase the number of genetically modified cells after transplantation. In these studies, FV vectors have mediated efficient gene transfer to polyclonal repopulating cells using short ex vivo transduction protocols designed to minimize the negative effects of ex vivo culture on stem cell engraftment. In this regard, FV vectors appear superior to gammaretroviral vectors, which require longer ex vivo culture to effect efficient transduction. FV vectors have also compared favorably with lentiviral vectors when directly compared in the dog model. FV vectors have corrected leukocyte adhesion deficiency and pyruvate kinase deficiency in the dog large animal model. FV vectors also appear safer than gammaretroviral vectors based on a reduced frequency of integrants near promoters and also near proto-oncogenes in canine repopulating cells. Together, these studies suggest that FV vectors should be highly effective for several human hematopoietic diseases, including those that will require relatively high percentages of gene-modified cells to achieve clinical benefit.

  11. Gamification of Dietary Decision-Making in an Elementary-School Cafeteria: e93872

    National Research Council Canada - National Science Library

    Brooke A Jones; Gregory J Madden; Heidi J Wengreen; Sheryl S Aguilar; E Anne Desjardins


    .... The present demonstration project used a behaviorally based gamification approach to develop an intervention designed to increase FV consumption while minimizing material and labor costs to the school...

  12. Gamification of dietary decision-making in an elementary-school cafeteria

    National Research Council Canada - National Science Library

    Jones, Brooke A; Madden, Gregory J; Wengreen, Heidi J; Aguilar, Sheryl S; Desjardins, E Anne


    .... The present demonstration project used a behaviorally based gamification approach to develop an intervention designed to increase FV consumption while minimizing material and labor costs to the school...

  13. Operation CROSSROADS-1946 (United States)


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  14. Integrated Medical Information Technology System (IMITS): Information and Clinical Technologies for the Advancement of Healthcare (United States)


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  15. Property Accountability Task Force (United States)



  16. A Functional Inspection Model for the Immeasurable Potential Failure State

    Institute of Scientific and Technical Information of China (English)

    ZHU Wen-ge; LI Shi-qi; ZHAO Di


    Functional inspection is a type of preventive maintenance of Reliability Centered Maintenance (RCM). We, in this paper, establish a functional inspection model(FIM)--the cost model and the availability model for the immeasurable potential failure state based on the delay time concept. This model can be used t