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Sample records for coli inclusion bodies

  1. Quality control of inclusion bodies in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Schweder Thomas

    2010-05-01

    Full Text Available Abstract Background Bacterial inclusion bodies (IBs are key intermediates for protein production. Their quality affects the refolding yield and further purification. Recent functional and structural studies have revealed that IBs are not dead-end aggregates but undergo dynamic changes, including aggregation, refunctionalization of the protein and proteolysis. Both, aggregation of the folding intermediates and turnover of IBs are influenced by the cellular situation and a number of well-studied chaperones and proteases are included. IBs mostly contain only minor impurities and are relatively homogenous. Results IBs of α-glucosidase of Saccharomyces cerevisiae after overproduction in Escherichia coli contain a large amount of (at least 12 different major product fragments, as revealed by two-dimensional polyacrylamide gel electrophoresis (2D PAGE. Matrix-Assisted-Laser-Desorption/Ionization-Time-Of-Flight Mass-Spectrometry (MALDI-ToF MS identification showed that these fragments contain either the N- or the C-terminus of the protein, therefore indicate that these IBs are at least partially created by proteolytic action. Expression of α-glucosidase in single knockout mutants for the major proteases ClpP, Lon, OmpT and FtsH which are known to be involved in the heat shock like response to production of recombinant proteins or to the degradation of IB proteins, clpP, lon, ompT, and ftsH did not influence the fragment pattern or the composition of the IBs. The quality of the IBs was also not influenced by the sampling time, cultivation medium (complex and mineral salt medium, production strategy (shake flask, fed-batch fermentation process, production strength (T5-lac or T7 promoter, strain background (K-12 or BL21, or addition of different protease inhibitors during IB preparation. Conclusions α-glucosidase is fragmented before aggregation, but neither by proteolytic action on the IBs by the common major proteases, nor during downstream IB

  2. Dean vortex membrane microfiltration and diafiltration of rBDNF E. coli inclusion bodies

    NARCIS (Netherlands)

    Schutyser, M.A.I.; Rupp, R.; Wideman, J.; Belfort, G.

    2002-01-01

    Cross-flow microfiltration (CMF) and diafiltration were used to concentrate and purify recombinant Brain-Derived Neutrophic Factor (rBDNF) inclusion bodies from an E. coli cell suspension and a homogenized E. coli cell suspension (homogenate/lysate). Although these processes have been tested

  3. Expression of nattokinase in Escherichia coli and renaturation of its inclusion body.

    Science.gov (United States)

    Ni, He; Guo, Peng-Cheng; Jiang, Wei-Ling; Fan, Xiao-Min; Luo, Xiang-Yu; Li, Hai-Hang

    2016-08-10

    Nattokinase is an important fibrinolytic enzyme with therapeutic applications for cardiovascular diseases. The full-length and mature nattokinase genes were cloned from Bacillus subtilis var. natto and expressed in pQE30 vector in Escherichia coli. The full-length gene expressed low nattokinase activity in the intracellular soluble and the medium fractions. The mature gene expressed low soluble nattokinase activity and large amount insoluble protein in inclusion bodies without enzyme activity. Large amount of refolding solutions (RSs) at different pH values were screening and RS-10 and RS-11 at pH 9 were selected to refold nattokinase inclusion bodies. The recombinant cells were lysed with 0.1mg/mL lysozyme and ultrasonic treatment. After centrifugation, the pellete was washed twice with 20mM Tris-HCl buffer (pH 7.5) containing 1% Triton X-100 to purify the inclusion bodies. The inclusion bodies were dissolved in water at pH 12.0 and refolded with RS-10. The refolded proteins showed 42.8IU/mg and 79.3IU/mg fibrinolytic activity by the traditional dilution method (20-fold dilution into RS-10) and the directly mixing the protein solution with equal volume RS-10, respectively, compared to the 52.0IU/mg of total water-soluble proteins from B. subtilis var. natto. This work demonstrated that the inclusion body of recombinant nattokinase expressed in E. coli could be simply refolded to the natural enzyme activity level by directly mixing the protein solution with equal volume refolding solution. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Application of an E. coli signal sequence as a versatile inclusion body tag.

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    Jong, Wouter S P; Vikström, David; Houben, Diane; van den Berg van Saparoea, H Bart; de Gier, Jan-Willem; Luirink, Joen

    2017-03-21

    Heterologous protein production in Escherichia coli often suffers from bottlenecks such as proteolytic degradation, complex purification procedures and toxicity towards the expression host. Production of proteins in an insoluble form in inclusion bodies (IBs) can alleviate these problems. Unfortunately, the propensity of heterologous proteins to form IBs is variable and difficult to predict. Hence, fusing the target protein to an aggregation prone polypeptide or IB-tag is a useful strategy to produce difficult-to-express proteins in an insoluble form. When screening for signal sequences that mediate optimal targeting of heterologous proteins to the periplasmic space of E. coli, we observed that fusion to the 39 amino acid signal sequence of E. coli TorA (ssTorA) did not promote targeting but rather directed high-level expression of the human proteins hEGF, Pla2 and IL-3 in IBs. Further analysis revealed that ssTorA even mediated IB formation of the highly soluble endogenous E. coli proteins TrxA and MBP. The ssTorA also induced aggregation when fused to the C-terminus of target proteins and appeared functional as IB-tag in E. coli K-12 as well as B strains. An additive effect on IB-formation was observed upon fusion of multiple ssTorA sequences in tandem, provoking almost complete aggregation of TrxA and MBP. The ssTorA-moiety was successfully used to produce the intrinsically unstable hEGF and the toxic fusion partner SymE, demonstrating its applicability as an IB-tag for difficult-to-express and toxic proteins. We present proof-of-concept for the use of ssTorA as a small, versatile tag for robust E. coli-based expression of heterologous proteins in IBs.

  5. Kinetics of inclusion body formation and its correlation with the characteristics of protein aggregates in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Arun K Upadhyay

    Full Text Available The objective of the research was to understand the structural determinants governing protein aggregation into inclusion bodies during expression of recombinant proteins in Escherichia coli. Recombinant human growth hormone (hGH and asparaginase were expressed as inclusion bodies in E.coli and the kinetics of aggregate formation was analyzed in details. Asparaginase inclusion bodies were of smaller size (200 nm and the size of the aggregates did not increase with induction time. In contrast, the seeding and growth behavior of hGH inclusion bodies were found to be sequential, kinetically stable and the aggregate size increased from 200 to 800 nm with induction time. Human growth hormone inclusion bodies showed higher resistance to denaturants and proteinase K degradation in comparison to those of asparaginase inclusion bodies. Asparaginase inclusion bodies were completely solubilized at 2-3 M urea concentration and could be refolded into active protein, whereas 7 M urea was required for complete solubilization of hGH inclusion bodies. Both hGH and asparaginase inclusion bodies showed binding with amyloid specific dyes. In spite of its low β-sheet content, binding with dyes was more prominent in case of hGH inclusion bodies than that of asparaginase. Arrangements of protein molecules present in the surface as well as in the core of inclusion bodies were similar. Hydrophobic interactions between partially folded amphiphillic and hydrophobic alpha-helices were found to be one of the main determinants of hGH inclusion body formation. Aggregation behavior of the protein molecules decides the nature and properties of inclusion bodies.

  6. Kinetics of Inclusion Body Formation and Its Correlation with the Characteristics of Protein Aggregates in Escherichia coli

    Science.gov (United States)

    Upadhyay, Arun K.; Murmu, Aruna; Singh, Anupam; Panda, Amulya K.

    2012-01-01

    The objective of the research was to understand the structural determinants governing protein aggregation into inclusion bodies during expression of recombinant proteins in Escherichia coli. Recombinant human growth hormone (hGH) and asparaginase were expressed as inclusion bodies in E.coli and the kinetics of aggregate formation was analyzed in details. Asparaginase inclusion bodies were of smaller size (200 nm) and the size of the aggregates did not increase with induction time. In contrast, the seeding and growth behavior of hGH inclusion bodies were found to be sequential, kinetically stable and the aggregate size increased from 200 to 800 nm with induction time. Human growth hormone inclusion bodies showed higher resistance to denaturants and proteinase K degradation in comparison to those of asparaginase inclusion bodies. Asparaginase inclusion bodies were completely solubilized at 2–3 M urea concentration and could be refolded into active protein, whereas 7 M urea was required for complete solubilization of hGH inclusion bodies. Both hGH and asparaginase inclusion bodies showed binding with amyloid specific dyes. In spite of its low β-sheet content, binding with dyes was more prominent in case of hGH inclusion bodies than that of asparaginase. Arrangements of protein molecules present in the surface as well as in the core of inclusion bodies were similar. Hydrophobic interactions between partially folded amphiphillic and hydrophobic alpha-helices were found to be one of the main determinants of hGH inclusion body formation. Aggregation behavior of the protein molecules decides the nature and properties of inclusion bodies. PMID:22479486

  7. Refolding in high hydrostatic pressure of recombinant proteins from inclusion bodies in Escherichia Coli

    International Nuclear Information System (INIS)

    Balduino, Keli Nunes

    2009-01-01

    The expression of proteins as inclusion bodies in bacteria is a widely used alternative for production of recombinant protein. However, the aggregation is a problem often encountered during refolding of these proteins. High hydrostatic pressure are able to solubilise the inclusion bodies in the presence of low concentrations of denaturant reagents, encouraging refolding protein with high efficiency and reduce costs. This work aims to refolding of recombinant proteins expressed in Escherichia coli from inclusion bodies using high hydrostatic pressure. Three toxins, all featuring five or more disulfide bonds were studied: NXH8, Natterin 2 and Bothropstoxin 1. Suspensions of inclusion bodies of the three proteins were pressurized to 2000 bars for 16 hours. The buffers were optimized for refolding of the three proteins. The buffer used in the refolding of NXH8 was 50 mM Tris HCl, pH 9.0 with proportion of 1GSH: 4GSSG at a concentration of 6 mM and 2 M GdnHCl. Inclusion bodies were used in O.D. (A600nm) of 0.5. After refolding process, dialysis was performed at pH 7.0. The final yield of obtaining soluble NXH8 was 40% (28,6 mg of soluble NXH8/L of culture medium). The refolding of Bothropstoxin 1 was obtained in refolding buffer of Tris HCl 50 mM, pH 7,5 with proportion of 2 GSH: GSSG 3 and concentration of 3 mM and 1 M GdnHCl. Use with a suspension of O.D. (A600nm) of 0.5. The final yield of recovery of Bothropstoxin 1 refolded was 32% (9,2 mg of refolded Bothropstoxin 1/L of culture medium). The refolding of Natterin 2 was performed in the refolding buffer: 20 mM Tris HCl pH 9.0 at a ratio of 2 GSH: 3GSSG and concentration of 10 mM and 1 M GdnHCl and inclusion bodies O.D. (A600nm) of 6.0. The yield of Natterin 2 refolded was 20% (3,7 mg/L of culture medium). Physico-chemical and biological analysis were performed by SDS-PAGE, western blot, scanning electron microscopy, biological tests in vivo and in vitro and structural. The analysis conducted in NXH8 did not show

  8. Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process.

    Science.gov (United States)

    Singh, Anupam; Upadhyay, Vaibhav; Upadhyay, Arun Kumar; Singh, Surinder Mohan; Panda, Amulya Kumar

    2015-03-25

    Formation of inclusion bodies in bacterial hosts poses a major challenge for large scale recovery of bioactive proteins. The process of obtaining bioactive protein from inclusion bodies is labor intensive and the yields of recombinant protein are often low. Here we review the developments in the field that are targeted at improving the yield, as well as quality of the recombinant protein by optimizing the individual steps of the process, especially solubilization of the inclusion bodies and refolding of the solubilized protein. Mild solubilization methods have been discussed which are based on the understanding of the fact that protein molecules in inclusion body aggregates have native-like structure. These methods solubilize the inclusion body aggregates while preserving the native-like protein structure. Subsequent protein refolding and purification results in high recovery of bioactive protein. Other parameters which influence the overall recovery of bioactive protein from inclusion bodies have also been discussed. A schematic model describing the utility of mild solubilization methods for high throughput recovery of bioactive protein has also been presented.

  9. Purification of inclusion bodies using PEG precipitation under denaturing conditions to produce recombinant therapeutic proteins from Escherichia coli.

    Science.gov (United States)

    Chen, Huanhuan; Li, Ninghuan; Xie, Yueqing; Jiang, Hua; Yang, Xiaoyi; Cagliero, Cedric; Shi, Siwei; Zhu, Chencen; Luo, Han; Chen, Junsheng; Zhang, Lei; Zhao, Menglin; Feng, Lei; Lu, Huili; Zhu, Jianwei

    2017-07-01

    It has been documented that the purification of inclusion bodies from Escherichia coli by size exclusion chromatography (SEC) may benefit subsequent refolding and recovery of recombinant proteins. However, loading volume and the high cost of the column limits its application in large-scale manufacturing of biopharmaceutical proteins. We report a novel process using polyethylene glycol (PEG) precipitation under denaturing conditions to replace SEC for rapid purification of inclusion bodies containing recombinant therapeutic proteins. Using recombinant human interleukin 15 (rhIL-15) as an example, inclusion bodies of rhIL-15 were solubilized in 7 M guanidine hydrochloride, and rhIL-15 was precipitated by the addition of PEG 6000. A final concentration of 5% (w/v) PEG 6000 was found to be optimal to precipitate target proteins and enhance recovery and purity. Compared to the previously reported S-200 size exclusion purification method, PEG precipitation was easier to scale up and achieved the same protein yields and quality of the product. PEG precipitation also reduced manufacturing time by about 50 and 95% of material costs. After refolding and further purification, the rhIL-15 product was highly pure and demonstrated a comparable bioactivity with a rhIL-15 reference standard. Our studies demonstrated that PEG precipitation of inclusion bodies under denaturing conditions holds significant potential as a manufacturing process for biopharmaceuticals from E. coli protein expression systems.

  10. Online analysis of protein inclusion bodies produced in E. coli by monitoring alterations in scattered and reflected light.

    Science.gov (United States)

    Ude, Christian; Ben-Dov, Nadav; Jochums, André; Li, Zhaopeng; Segal, Ester; Scheper, Thomas; Beutel, Sascha

    2016-05-01

    The online monitoring of recombinant protein aggregate inclusion bodies during microbial cultivation is an immense challenge. Measurement of scattered and reflected light offers a versatile and non-invasive measurement technique. Therefore, we investigated two methods to detect the formation of inclusion bodies and monitor their production: (1) online 180° scattered light measurement (λ = 625 nm) using a sensor platform during cultivation in shake flask and (2) online measurement of the light reflective interference using a porous Si-based optical biosensor (SiPA). It could be shown that 180° scattered light measurement allows monitoring of alterations in the optical properties of Escherichia coli BL21 cells, associated with the formation of inclusion bodies during cultivation. A reproducible linear correlation between the inclusion body concentration of the non-fluorescent protein human leukemia inhibitory factor (hLIF) carrying a thioredoxin tag and the shift ("Δamp") in scattered light signal intensity was observed. This was also observed for the glutathione-S-transferase-tagged green fluorescent protein (GFP-GST). Continuous online monitoring of reflective interference spectra reveals a significant increase in the bacterium refractive index during hLIF production in comparison to a non-induced reference that coincide with the formation of inclusion bodies. These online monitoring techniques could be applied for fast and cost-effective screening of different protein expression systems.

  11. Expression of Mycobacterium tuberculosis Protein Tyrosine Phosphatase B in Escherichia coli and Its Recovery from Inclusion Body

    Directory of Open Access Journals (Sweden)

    Lalu Rudyat Telly Savalas

    2017-12-01

    Full Text Available The present study aims at expressing and partially purifying PtpB in active form. To achieve this, Mtb PtpB gene has been cloned into pET30a vector and overexpressed in Escherichia coli BL 21(DE3 under IPTG induction in the form of an inclusion body. Following resolubilization by urea and dialysis, the resulted PtpB has been shown to be active against para-Nitrophenyl phosphate.  It is concluded that the resulted PtpB has had been recovered from inclusion body to give the active form of the enzyme, and thus the success in overexpressing PtpB provides the required material to investigate the biochemical properties of the pathogen virulence factor further. 

  12. Anti-aggregatory effect of cyclodextrins in the refolding process of recombinant growth hormones from Escherichia coli inclusion bodies

    DEFF Research Database (Denmark)

    Bajorunaite, Egle; Cirkovas, Andrejus; Radzevicius, Kostas

    2009-01-01

    Cyclodextrins with different ring size and ring substituents were tested for recombinant mink and porcine growth hormones aggregation suppression in the refolding process from Escherichia coli inclusion bodies. Methyl-β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin show a positive effect...... on the aggregation suppression of both proteins. The influence of different methyl-β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin concentrations on the renaturation yield of both growth hormones was investigated. Moreover, methyl-β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin suppress not only folding...

  13. Access to gram scale amounts of functional globular adiponectin from E. coli inclusion bodies by alkaline-shock solubilization.

    Science.gov (United States)

    Heiker, John T; Klöting, Nora; Blüher, Matthias; Beck-Sickinger, Annette G

    2010-07-16

    The adipose tissue derived protein adiponectin exerts anti-diabetic, anti-inflammatory and anti-atherosclerotic effects. Adiponectin serum concentrations are in the microgram per milliliter range in healthy humans and inversely correlate with obesity and metabolic disorders. Accordingly, raising circulating adiponectin levels by direct administration may be an intriguing strategy in the treatment of obesity-related metabolic disorders. However production of large amounts of recombinant adiponectin protein is a primary obstacle so far. Here, we report a novel method for large amount production of globular adiponectin from E. coli inclusion bodies utilizing an alkaline-shock solubilization method without chaotropic agents followed by precipitation of the readily renaturing protein. Precipitation of the mildly solubilized protein capitalizes on advantages of inclusion body formation. This approach of inclusion body protein recovery provides access to gram scale amounts of globular adiponectin with standard laboratory equipment avoiding vast dilution or dialysis steps to neutralize the pH and renature the protein, thus saving chemicals and time. The precipitated protein is readily renaturing in buffer, is of adequate purity without a chromatography step and shows biological activity in cultured MCF7 cells and significantly lowered blood glucose levels in mice with streptozotocin induced type 1 diabetes. Copyright 2010 Elsevier Inc. All rights reserved.

  14. A camelid nanobody against EGFR was easily obtained through refolding of inclusion body expressed in Escherichia coli.

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    Xu, Li; Song, Xiaoyu; Jia, Lingyun

    2017-11-01

    Using anti-EGFR (epidermal growth factor receptor) nanobody is a good choice for diagnoses and therapeutics for high EGFR expression diseases. In the present study, the percentage composition of anti-EGFR nanobody attained 25% of the total cell protein expressed in Escherichia coli BL21 (DE3). However, almost all nanobodies were expressed as inclusion bodies. To acquire active nanobodies, a series of dilution refolding procedures were optimized after inclusion bodies were dissolved into 6 M urea and purified with immobilized metal affinity chromatography. The results showed the refolding rate of the anti-EGFR nanobodies attained to 73%, and about 100 mg nanobodies were refolded from 1 L cells under the conditions that the initial nanobody concentration was 0.3 mg/mL, the dilution speed was 2.5 mL/Min, the dilution buffer was Tris-HCl at pH 8.0, the additives were 0.2 M Arg, 5 mM reduced glutathione (GSH), and 1 mM oxidized glutathione (GSSG). Then the activity of the refolded nanobodies was confirmed. The results showed that the refolded anti-EGFR nanobodies, in a dose-dependent manner, bounded to the tumor cell surface of A431 and MCF-7 and significantly inhibited the proliferation of A431 caused by the epidermal growth factor. Our study provides a facile method to rapidly, efficiently, and massively prepare anti-EGFR antibodies and promotes anti-EGFR-based recognition in cancer diagnoses and therapeutics. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

  15. MB109 as bioactive human bone morphogenetic protein-9 refolded and purified from E. coli inclusion bodies

    Science.gov (United States)

    2014-01-01

    Background The development of chemical refolding of transforming growth factor-beta (TGF-β) superfamily ligands has been instrumental to produce the recombinant proteins for biochemical studies and exploring the potential of protein therapeutics. The osteogenic human bone morphogenetic protein-2 (hBMP-2) and its Drosophila DPP homolog were the early successful cases of refolding into functional form. Despite the similarity in their three dimensional structure and amino acid sequences, several other TGF-β superfamily ligands could not be refolded readily by the same methods. Results Here, we report a comprehensive study on the variables of a rapid-dilution refolding method, including the concentrations of protein, salt, detergent and redox agents, pH, refolding duration and the presence of aggregation suppressors and host-cell contaminants, in order to identify the optimal condition to refold human BMP-9 (hBMP-9). To produce a recombinant form of hBMP-9 in E. coli cells, a synthetic codon-optimized gene was designed to encode the mature domain of hBMP-9 (Ser320 – Arg429) directly behind the first methionine, which we herein referred to as MB109. An effective purification scheme was also developed to purify the refolded MB109 to homogeneity with a final yield of 7.8 mg from 100 mg of chromatography-purified inclusion bodies as a starting material. The chemically refolded MB109 binds to ALK1, ActRIIb and BMPRII receptors with relatively high affinity as compared to other Type I and Type II receptors based on surface plasmon resonance analysis. Smad1-dependent luciferase assay in C2C12 cells shows that the MB109 has an EC50 of 0.61 ng/mL (25 pM), which is nearly the same as hBMP-9. Conclusion MB109 is prone to be refolded as non-functional dimer and higher order multimers in most of the conditions tested, but bioactive MB109 dimer can be refolded with high efficiency in a narrow window, which is strongly dependent on the pH, refolding duration, the presence of

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

    Science.gov (United States)

    Zhao, Mingzhi; Wu, Feilin; Xu, Ping

    2015-12-01

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

  17. Inclusion body myositis.

    OpenAIRE

    Garlepp, M J; Mastaglia, F L

    1996-01-01

    The idiopathic inflammatory myopathies (IIM) are a heterogenous group of rare disorders that share many similarities. In addition to sporadic inclusion body myositis (IBM), these include dematomyositis (DM), polymyositis (PM), and autoimmune necrotizing myopathy (NM). For discussion of later three disorders, the reader is referred to the IIM review in this issue. IBM is the most common IIM after age 50. It typically presents with chronic insidious proximal leg and/or distal arm asymmetric mus...

  18. Inclusion body myositis.

    Science.gov (United States)

    Dimachkie, Mazen M; Barohn, Richard J

    2014-08-01

    The idiopathic inflammatory myopathies (IIMs) are a heterogeneous group of rare disorders that share many similarities. In addition to sporadic inclusion body myositis (IBM), these include dermatomyositis, polymyositis, and autoimmune necrotizing myopathy. IBM is the most common IIM after age 50 years. Muscle histopathology shows endomysial inflammatory exudates surrounding and invading nonnecrotic muscle fibers often accompanied by rimmed vacuoles and protein deposits. It is likely that IBM is has a prominent degenerative component. This article reviews the evolution of knowledge in IBM, with emphasis on recent developments in the field, and discusses ongoing clinical trials. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Inclusion-Body Myositis: Diagnosis

    Science.gov (United States)

    ... for MDA Blog Donate Search MDA.org Close Inclusion-Body Myositis (IBM) Diagnosis As with other muscle diseases, a doctor diagnoses inclusion-body myositis (IBM) by considering the individual’s personal ...

  20. Purification and refolding of anti-T-antigen single chain antibodies (scFvs) expressed in Escherichia coli as inclusion bodies.

    Science.gov (United States)

    Yuasa, Noriyuki; Koyama, Tsubasa; Fujita-Yamaguchi, Yoko

    2014-02-01

    T-antigen (Galβ1-3GalNAcα-1-Ser/Thr) is an oncofetal antigen that is commonly expressed as a carbohydrate determinant in many adenocarcinomas. Since it is associated with tumor progression and metastasis, production of recombinant antibodies specific for T-antigen could lead to the development of cancer diagnostics and therapeutics. Previously, we isolated and characterized 11 anti-T-antigen phage clones from a phage library displaying human single-chain antibodies (scFvs) and purified one scFv protein, 1G11. More recently, we purified and characterized 1E8 scFv protein using a Drosophila S2 expression system. In the current study, four anti-T-antigen scFv genes belonging to Groups 1-4 were purified from inclusion bodies expressed in Escherichia coli cells. Inclusion bodies isolated from E. coli cells were denatured in 3.5 M Gdn-HCl. Solubilized His-tagged scFv proteins were purified using Ni(2+)-Sepharose column chromatography in the presence of 3.5 M Gdn-HCl. Purified scFv proteins were refolded according to a previously published method of step-wise dialysis. Two anti-T-antigen scFv proteins, 1E6 and 1E8 that belong to Groups 1 and 2, respectively, were produced in sufficient amounts, thus allowing further characterization of their binding activity with T-antigen. Specificity and affinity constants determined using enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR), respectively, provided evidence that both 1E8 and 1E6 scFv proteins are T-antigen specific and suggested that 1E8 scFv protein has a higher affinity for T-antigen than 1E6 scFv protein.

  1. INCLUSION BODY MYOSITIS

    Directory of Open Access Journals (Sweden)

    Luh Yeni Laksmini

    2014-09-01

    Full Text Available Inclusion body myositis (IBM merupakan penyakit inflamasi pada otot yang bersifat progresif dengan penyebab yang tidak diketahui dan tidak menunjukkan respon yang baik terhadap berbagai terapi. Gambaran histopatologi IBM ditandai dengan infiltrat sel-sel limfosit diantara ruangan endomisial, di dalam otot dan di sekitar otot dengan fokus-fokus inklusi di dalam miosit (rimmed vacuole serta beberapa serat otot terlihat atrofi dan nekrosis. Dilaporkan wanita, usia 46 tahun dengan IBM. Keluhan utama pasien berupa kelemahan pada kedua tangan, kaki kanan terasa berat jika diangkat sehingga susah berjalan. Pemeriksaan saraf sensorik ekstremitas dekstra dan sinistra dalam batas normal. Pemeriksaan enzim cretinine kinase meningkat secara dramatik. Pemeriksaan histopatologi dari biospi otot gastrocnemius menunjukkan gambaran yang sesuai untuk IBM dan telah dilakukan penanganan dengan pemberian oral methilprednisolon 3x32 mg dan mecobalmin 1x500ìg intravena, namun tidak menunjukkan respon yang baik terhadap terapi dan akhirnya pasien meninggal. [MEDICINA 2013;44:118-123].

  2. The disulfide-rich Metridia luciferase refolded from E. coli inclusion bodies reveals the properties of a native folded enzyme produced in insect cells.

    Science.gov (United States)

    Markova, Svetlana V; Larionova, Marina D; Gorbunova, Darya A; Vysotski, Eugene S

    2017-10-01

    The bioluminescence of a marine copepod Metridia longa is determined by a small secreted coelenterazine-dependent luciferase that uses coelenterazine as a substrate of enzymatic reaction to generate light (λ max =480nm). To date, four different isoforms of the luciferase differing in size, sequences, and properties have been cloned by functional screening. All of them contain ten conserved Cys residues that suggests up to five SS intramolecular bonds per luciferase molecule. Whereas the use of copepod luciferases as bioluminescent reporters in biomedical research in vivo is growing from year to year, their application for in vitro assays is still limited by the difficulty in obtaining significant amounts of luciferase. The most cost-effective host for producing recombinant proteins is Escherichia coli. However, prokaryotic and eukaryotic cells maintain the reductive environment in cytoplasm that hinders the disulfide bond formation and consequently the proper folding of luciferase. Here we report the expression of the MLuc7 isoform of M. longa luciferase in E. coli cells and the efficient procedure for refolding from inclusion bodies yielding a high-active monomeric protein. Furthermore, in a set of identical experiments we demonstrate that bioluminescent and structural features of MLuc7 produced in bacterial cells are identical to those of MLuc7 isoform produced from culture medium of insect cells. Although the yield of high-purity protein is only 6mg/L, the application of E. coli cells to produce the luciferase is simpler and more cost-effective than the use of insect cells. We expect that the suggested technology of Metridia luciferase production allows obtaining of sufficient amounts of protein both for the development of novel in vitro analytical assays with the use of MLuc7 as a label and for structural studies. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Refolding in high hydrostatic pressure of recombinant proteins from inclusion bodies in Escherichia Coli; Renaturacao em altas pressoes hidrostaticas de proteinas recombinantes agregadas em corpos de inclusao produzidos em Escherichia Coli

    Energy Technology Data Exchange (ETDEWEB)

    Balduino, Keli Nunes

    2009-07-01

    The expression of proteins as inclusion bodies in bacteria is a widely used alternative for production of recombinant protein. However, the aggregation is a problem often encountered during refolding of these proteins. High hydrostatic pressure are able to solubilise the inclusion bodies in the presence of low concentrations of denaturant reagents, encouraging refolding protein with high efficiency and reduce costs. This work aims to refolding of recombinant proteins expressed in Escherichia coli from inclusion bodies using high hydrostatic pressure. Three toxins, all featuring five or more disulfide bonds were studied: NXH8, Natterin 2 and Bothropstoxin 1. Suspensions of inclusion bodies of the three proteins were pressurized to 2000 bars for 16 hours. The buffers were optimized for refolding of the three proteins. The buffer used in the refolding of NXH8 was 50 mM Tris HCl, pH 9.0 with proportion of 1GSH: 4GSSG at a concentration of 6 mM and 2 M GdnHCl. Inclusion bodies were used in O.D. (A600nm) of 0.5. After refolding process, dialysis was performed at pH 7.0. The final yield of obtaining soluble NXH8 was 40% (28,6 mg of soluble NXH8/L of culture medium). The refolding of Bothropstoxin 1 was obtained in refolding buffer of Tris HCl 50 mM, pH 7,5 with proportion of 2 GSH: GSSG 3 and concentration of 3 mM and 1 M GdnHCl. Use with a suspension of O.D. (A600nm) of 0.5. The final yield of recovery of Bothropstoxin 1 refolded was 32% (9,2 mg of refolded Bothropstoxin 1/L of culture medium). The refolding of Natterin 2 was performed in the refolding buffer: 20 mM Tris HCl pH 9.0 at a ratio of 2 GSH: 3GSSG and concentration of 10 mM and 1 M GdnHCl and inclusion bodies O.D. (A600nm) of 6.0. The yield of Natterin 2 refolded was 20% (3,7 mg/L of culture medium). Physico-chemical and biological analysis were performed by SDS-PAGE, western blot, scanning electron microscopy, biological tests in vivo and in vitro and structural. The analysis conducted in NXH8 did not show

  4. Expression and purification of a novel therapeutic single-chain variable fragment antibody against BNP from inclusion bodies of Escherichia coli.

    Science.gov (United States)

    Bu, Dawei; Zhou, Yuwei; Tang, Jian; Jing, Fang; Zhang, Wei

    2013-12-01

    Abnormal brain natriuretic peptide (BNP) secretion is regarded as the dominating mechanism of cerebral salt wasting syndrome (CSW), which results from a renal loss of sodium and water during intracranial disease leading to hyponatremia. Scale preparation of therapeutic single-chain variable fragment (scFv) that can neutralize elevated circulating BNP may have potential value for clinical use. In this report, we used a recently isolated humanized anti-BNP scFv fragment (3C1) as model antibody (Ab) to evaluate the potential of scale production of this therapeutic protein. The truncated gene encoding for scFv fragment cloned in pET22b (+) was mainly overexpressed as inclusion bodies in Escherichia coli (E. coli) Rosetta (DE3) pLysS cells. The insoluble fragment was solubilized and purified by Ni-NTA agarose resin under denaturation conditions, and recovered via an effective refolding buffer containing 50 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1 mM EDTA, 0.5 M arginine, 2 mM GSH, 1 mM GSSG, and 5% glycerol. The refolded scFv fragment was concentrated by PEG20000, and dialyzed in PBS (containing 5% glycerol, pH 7.4). The final yield was approximately 10.2 mg active scFv fragment per liter of culture (3.4 g wet weight cells). The scFv fragment was more than 95% pure assessed by SDS-PAGE assay. Recombinant scFv fragment with His tag displayed its immunoreactivity with anti-His tag Ab by western blotting. ELISA showed the scFv fragment specifically bound to BNP, and it displayed similar activity as the traditional anti-BNP monoclonal Ab (mAb). Thus, the current strategy allows convenient small-scale production of this therapeutic protein. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Inclusion body myositis. Clinical aspects

    NARCIS (Netherlands)

    Cox, Fieke Maria Elisabeth

    2014-01-01

    Sporadische inclusion body myositis (IBM) is een van de meest voor voorkomende verworven spierziekte die ontstaat na het 50e levensjaar. In dit proefschrift worden de klinische aspecten van sporadische IBM beschreven. Uit de studie met betrekking tot het natuurlijk beloop blijkt dat de ziekte niet

  6. [Solubilization Specificities Interferon beta-1b from Inclusion Bodies].

    Science.gov (United States)

    Zhuravko, A S; Kononova, N V; Bobruskin, A I

    2015-01-01

    A new solubilization method of recombinant interferon beta-1b (IFNβ-1b) from the inclusion bodies was developed. This method allows to extract the target protein selectively in the solutions of different alcohols, such as ethanol, propanol and isopropanol. It was shown that the more effective IFNβ-1b solubilization was achieved in the 55% propanol solution. This method allowed to extract the target protein from inclusion bodies around 85-90%, and significantly reduced Escherichia coli content in the solubilizate, in comparison with standard methods.

  7. Isolation of cell-free bacterial inclusion bodies.

    Science.gov (United States)

    Rodríguez-Carmona, Escarlata; Cano-Garrido, Olivia; Seras-Franzoso, Joaquin; Villaverde, Antonio; García-Fruitós, Elena

    2010-09-17

    Bacterial inclusion bodies are submicron protein clusters usually found in recombinant bacteria that have been traditionally considered as undesirable products from protein production processes. However, being fully biocompatible, they have been recently characterized as nanoparticulate inert materials useful as scaffolds for tissue engineering, with potentially wider applicability in biomedicine and material sciences. Current protocols for inclusion body isolation from Escherichia coli usually offer between 95 to 99% of protein recovery, what in practical terms, might imply extensive bacterial cell contamination, not compatible with the use of inclusion bodies in biological interfaces. Using an appropriate combination of chemical and mechanical cell disruption methods we have established a convenient procedure for the recovery of bacterial inclusion bodies with undetectable levels of viable cell contamination, below 10⁻¹ cfu/ml, keeping the particulate organization of these aggregates regarding size and protein folding features. The application of the developed protocol allows obtaining bacterial free inclusion bodies suitable for use in mammalian cell cultures and other biological interfaces.

  8. Impact of Glycerol as Carbon Source onto Specific Sugar and Inducer Uptake Rates and Inclusion Body Productivity in E. coli BL21(DE3

    Directory of Open Access Journals (Sweden)

    Julian Kopp

    2017-12-01

    Full Text Available The Gram-negative bacterium E. coli is the host of choice for a multitude of used recombinant proteins. Generally, cultivation is easy, media are cheap, and a high product titer can be obtained. However, harsh induction procedures using isopropyl β-d-1 thiogalactopyranoside as inducer are often referred to cause stress reactions, leading to a phenomenon known as “metabolic” or “product burden”. These high expressions of recombinant proteins mainly result in decreased growth rates and cell lysis at elevated induction times. Therefore, approaches tend to use “soft” or “tunable” induction with lactose and reduce the stress level of the production host. The usage of glucose as energy source in combination with lactose as induction reagent causes catabolite repression effects on lactose uptake kinetics and as a consequence reduced product titer. Glycerol—as an alternative carbon source—is already known to have positive impact on product formation when coupled with glucose and lactose in auto-induction systems, and has been referred to show no signs of repression when cultivated with lactose concomitantly. In recent research activities, the impact of different products on the lactose uptake using glucose as carbon source was highlighted, and a mechanistic model for glucose-lactose induction systems showed correlations between specific substrate uptake rate for glucose or glycerol (qs,C and the maximum specific lactose uptake rate (qs,lac,max. In this study, we investigated the mechanistic of glycerol uptake when using the inducer lactose. We were able to show that a product-producing strain has significantly higher inducer uptake rates when being compared to a non-producer strain. Additionally, it was shown that glycerol has beneficial effects on viability of cells and on productivity of the recombinant protein compared to glucose.

  9. Soni-removal of nucleic acids from inclusion bodies.

    Science.gov (United States)

    Neerathilingam, Muniasamy; Mysore, Sumukh; Gandham, Sai Hari A

    2014-05-23

    Inclusion bodies (IBs) are commonly formed in Escherichia coli due to over expression of recombinant proteins in non-native state. Isolation, denaturation and refolding of these IBs is generally performed to obtain functional protein. However, during this process IBs tend to form non-specific interactions with sheared nucleic acids from the genome, thus getting carried over into downstream processes. This may hinder the refolding of IBs into their native state. To circumvent this, we demonstrate a methodology termed soni-removal which involves disruption of nucleic acid-inclusion body interaction using sonication; followed by solvent based separation. As opposed to conventional techniques that use enzymes and column-based separations, soni-removal is a cost effective alternative for complete elimination of buried and/or strongly bound short nucleic acid contaminants from IBs. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  10. Chemical Assistance in Refolding of Bacterial Inclusion Bodies

    Directory of Open Access Journals (Sweden)

    Mona Alibolandi

    2011-01-01

    Full Text Available Escherichia coli is one of the most widely used hosts for the production of recombinant proteins but insoluble expression of heterologous proteins is a major bottleneck in production of recombinant proteins in E. coli. In vitro refolding of inclusion body into proteins with native conformations is a solution for this problem but there is a need for optimization of condition for each protein specifically. Several approaches have been described for in vitro refolding; most of them involve the use of additives for assisting correct folding. Cosolutes play a major role in refolding process and can be classified according to their function as aggregation suppressors and folding enhancers. This paper presents a review of additives that are used in refolding process of insoluble recombinant proteins in small scale and industrial processes.

  11. In vitro folding of inclusion body proteins.

    Science.gov (United States)

    Rudolph, R; Lilie, H

    1996-01-01

    Insoluble, inactive inclusion bodies are frequently formed upon recombinant protein production in transformed microorganisms. These inclusion bodies, which contain the recombinant protein in an highly enriched form, can be isolated by solid/liquid separation. After solubilization, native proteins can be generated from the inactive material by using in vitro folding techniques. New folding procedures have been developed for efficient in vitro reconstitution of complex hydrophobic, multidomain, oligomeric, or highly disulfide-bonded proteins. These protocols take into account process parameters such as protein concentration, catalysis of disulfide bond formation, temperature, pH, and ionic strength, as well as specific solvent ingredients that reduce unproductive side reactions. Modification of the protein sequence has been exploited to improve in vitro folding.

  12. Bacterial Inclusion Bodies: Discovering Their Better Half.

    Science.gov (United States)

    Rinas, Ursula; Garcia-Fruitós, Elena; Corchero, José Luis; Vázquez, Esther; Seras-Franzoso, Joaquin; Villaverde, Antonio

    2017-09-01

    Bacterial inclusion bodies (IBs) are functional, non-toxic amyloids occurring in recombinant bacteria showing analogies with secretory granules of the mammalian endocrine system. The scientific interest in these mesoscale protein aggregates has been historically masked by their status as a hurdle in recombinant protein production. However, progressive understanding of how the cell handles the quality of recombinant polypeptides and the main features of their intriguing molecular organization has stimulated the interest in inclusion bodies and spurred their use in diverse technological fields. The engineering and tailoring of IBs as functional protein particles for materials science and biomedicine is a good example of how formerly undesired bacterial byproducts can be rediscovered as promising functional materials for a broad spectrum of applications. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Genetics Home Reference: familial encephalopathy with neuroserpin inclusion bodies

    Science.gov (United States)

    ... Home Health Conditions FENIB Familial encephalopathy with neuroserpin inclusion bodies Printable PDF Open All Close All Enable ... expand/collapse boxes. Description Familial encephalopathy with neuroserpin inclusion bodies ( FENIB ) is a disorder that causes progressive ...

  14. Dynamic recruitment of active proteasomes into polyglutamine initiated inclusion bodies

    NARCIS (Netherlands)

    Schipper-Krom, Sabine; Juenemann, Katrin; Jansen, Anne H.; Wiemhoefer, Anne; van den Nieuwendijk, Rianne; Smith, Donna L.; Hink, Mark A.; Bates, Gillian P.; Overkleeft, Hermen; Ovaa, Huib; Reits, Eric

    2014-01-01

    Neurodegenerative disorders such as Huntington's disease are hallmarked by neuronal intracellular inclusion body formation. Whether proteasomes are irreversibly recruited into inclusion bodies in these protein misfolding disorders is a controversial subject. In addition, it has been proposed that

  15. Refolding techniques for recovering biologically active recombinant proteins from inclusion bodies.

    Science.gov (United States)

    Yamaguchi, Hiroshi; Miyazaki, Masaya

    2014-02-20

    Biologically active proteins are useful for studying the biological functions of genes and for the development of therapeutic drugs and biomaterials in a biotechnology industry. Overexpression of recombinant proteins in bacteria, such as Escherichia coli, often results in the formation of inclusion bodies, which are protein aggregates with non-native conformations. As inclusion bodies contain relatively pure and intact proteins, protein refolding is an important process to obtain active recombinant proteins from inclusion bodies. However, conventional refolding methods, such as dialysis and dilution, are time consuming and, often, recovered yields of active proteins are low, and a trial-and-error process is required to achieve success. Recently, several approaches have been reported to refold these aggregated proteins into an active form. The strategies largely aim at reducing protein aggregation during the refolding procedure. This review focuses on protein refolding techniques using chemical additives and laminar flow in microfluidic chips for the efficient recovery of active proteins from inclusion bodies.

  16. Application of preparative disk gel electrophoresis for antigen purification from inclusion bodies.

    Science.gov (United States)

    Okegawa, Yuki; Koshino, Masanori; Okushima, Teruya; Motohashi, Ken

    2016-02-01

    Specific antibodies are a reliable tool to examine protein expression patterns and to determine the protein localizations within cells. Generally, recombinant proteins are used as antigens for specific antibody production. However, recombinant proteins from mammals and plants are often overexpressed as insoluble inclusion bodies in Escherichia coli. Solubilization of these inclusion bodies is desirable because soluble antigens are more suitable for injection into animals to be immunized. Furthermore, highly purified proteins are also required for specific antibody production. Plastidic acetyl-CoA carboxylase (ACCase: EC 6.4.1.2) from Arabidopsis thaliana, which catalyzes the formation of malonyl-CoA from acetyl-CoA in chloroplasts, formed inclusion bodies when the recombinant protein was overexpressed in E. coli. To obtain the purified protein to use as an antigen, we applied preparative disk gel electrophoresis for protein purification from inclusion bodies. This method is suitable for antigen preparation from inclusion bodies because the purified protein is recovered as a soluble fraction in electrode running buffer containing 0.1% sodium dodecyl sulfate that can be directly injected into immune animals, and it can be used for large-scale antigen preparation (several tens of milligrams). Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Renal pathophysiologic role of cortical tubular inclusion bodies.

    Science.gov (United States)

    Radi, Zaher A; Stewart, Zachary S; Grzemski, Felicity A; Bobrowski, Walter F

    2013-01-01

    Renal tubular inclusion bodies are rarely associated with drug administration. The authors describe the finding of renal cortical tubular intranuclear and intracytoplasmic inclusion bodies associated with the oral administration of a norepinephrine/serotonin reuptake inhibitor (NSRI) test article in Sprague-Dawley (SD) rats. Rats were given an NSRI daily for 4 weeks, and kidney histopathologic, ultrastructural pathology, and immunohistochemical examinations were performed. Round eosinophilic intranuclear inclusion bodies were observed histologically in the tubular epithelial cells of the renal cortex in male and female SD rats given the NSRI compound. No evidence of degeneration or necrosis was noted in the inclusion-containing renal cells. By ultrastructural pathology, inclusion bodies consisted of finely granular, amorphous, and uniformly stained nonmembrane-bound material. By immunohistochemistry, inclusion bodies stained positive for d-amino acid oxidase (DAO) protein. In addition, similar inclusion bodies were noted in the cytoplasmic tubular epithelial compartment by ultrastructural and immunohistochemical examination.  This is the first description of these renal inclusion bodies after an NSRI test article administration in SD rats. Such drug-induced renal inclusion bodies are rat-specific, do not represent an expression of nephrotoxicity, represent altered metabolism of d-amino acids, and are not relevant to human safety risk assessment.

  18. Phenotypic variability within the inclusion body spectrum of basophilic inclusion body disease and neuronal intermediate filament inclusion disease in frontotemporal lobar degenerations with FUS-positive inclusions.

    Science.gov (United States)

    Gelpi, Ellen; Lladó, Albert; Clarimón, Jordi; Rey, Maria Jesús; Rivera, Rosa Maria; Ezquerra, Mario; Antonell, Anna; Navarro-Otano, Judith; Ribalta, Teresa; Piñol-Ripoll, Gerard; Pérez, Anna; Valldeoriola, Francesc; Ferrer, Isidre

    2012-09-01

    Basophilic inclusion body disease and neuronal intermediate filament inclusion disease (NIFID) are rare diseases included among frontotemporal lobar degenerations with FUS-positive inclusions (FTLD-FUS). We report clinical and pathologic features of 2 new patients and reevaluate neuropathologic characteristics of 2 previously described cases, including an early-onset case of basophilic inclusion body disease (aged 38 years) with a 5-year disease course and abundant FUS-positive inclusion bodies and 3 NIFID cases. One NIFID case (aged 37 years) presented with early-onset psychiatric disturbances and rapidly progressive cognitive decline. Two NIFID cases had later onset (aged 64 years and 70 years) and complex neurologic deficits. Postmortem neuropathologic studies in late-onset NIFID cases disclosed α-internexin-positive "hyaline conglomerate"-type inclusions that were positive with 1 commercial anti-FUS antibody directed to residues 200 and 250, but these were negative to amino acids 90 and 220 of human FUS. Early-onset NIFID had similar inclusions that were positive with both commercial anti-FUS antibodies. Genetic testing performed on all cases revealed no FUS gene mutations. These findings indicate that phenotypic variability in NIFID, including clinical manifestations and particular neuropathologic findings, may be related to the age at onset and individual differences in the evolution of lesions.

  19. Inclusion bodies are a site of ebolavirus replication.

    Science.gov (United States)

    Hoenen, Thomas; Shabman, Reed S; Groseth, Allison; Herwig, Astrid; Weber, Michaela; Schudt, Gordian; Dolnik, Olga; Basler, Christopher F; Becker, Stephan; Feldmann, Heinz

    2012-11-01

    Inclusion bodies are a characteristic feature of ebolavirus infections in cells. They contain large numbers of preformed nucleocapsids, but their biological significance has been debated, and they have been suggested to be aggregates of viral proteins without any further biological function. However, recent data for other viruses that produce similar structures have suggested that inclusion bodies might be involved in genome replication and transcription. In order to study filovirus inclusion bodies, we fused mCherry to the ebolavirus polymerase L, which is found in inclusion bodies. The resulting L-mCherry fusion protein was functional in minigenome assays and incorporated into virus-like particles. Importantly, L-mCherry fluorescence in transfected cells was readily detectable and distributed in a punctate pattern characteristic for inclusion bodies. A recombinant ebolavirus encoding L-mCherry instead of L was rescued and showed virtually identical growth kinetics and endpoint titers to those for wild-type virus. Using this virus, we showed that the onset of inclusion body formation corresponds to the onset of viral genome replication, but that viral transcription occurs prior to inclusion body formation. Live-cell imaging further showed that inclusion bodies are highly dynamic structures and that they can undergo dramatic reorganization during cell division. Finally, by labeling nascent RNAs using click technology we showed that inclusion bodies are indeed the site of viral RNA synthesis. Based on these data we conclude that, rather than being inert aggregates of nucleocapsids, ebolavirus inclusion bodies are in fact complex and dynamic structures and an important site at which viral RNA replication takes place.

  20. Yeast prions form infectious amyloid inclusion bodies in bacteria

    Directory of Open Access Journals (Sweden)

    Espargaró Alba

    2012-06-01

    Full Text Available Abstract Background Prions were first identified as infectious proteins associated with fatal brain diseases in mammals. However, fungal prions behave as epigenetic regulators that can alter a range of cellular processes. These proteins propagate as self-perpetuating amyloid aggregates being an example of structural inheritance. The best-characterized examples are the Sup35 and Ure2 yeast proteins, corresponding to [PSI+] and [URE3] phenotypes, respectively. Results Here we show that both the prion domain of Sup35 (Sup35-NM and the Ure2 protein (Ure2p form inclusion bodies (IBs displaying amyloid-like properties when expressed in bacteria. These intracellular aggregates template the conformational change and promote the aggregation of homologous, but not heterologous, soluble prionogenic molecules. Moreover, in the case of Sup35-NM, purified IBs are able to induce different [PSI+] phenotypes in yeast, indicating that at least a fraction of the protein embedded in these deposits adopts an infectious prion fold. Conclusions An important feature of prion inheritance is the existence of strains, which are phenotypic variants encoded by different conformations of the same polypeptide. We show here that the proportion of infected yeast cells displaying strong and weak [PSI+] phenotypes depends on the conditions under which the prionogenic aggregates are formed in E. coli, suggesting that bacterial systems might become useful tools to generate prion strain diversity.

  1. Tunable geometry of bacterial inclusion bodies as substrate materials for tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    GarcIa-Fruitos, Elena; Seras-Franzoso, JoaquIn; Vazquez, Esther; Villaverde, Antonio [CIBER en BioingenierIa, Biomateriales y Nanomedicina, Bellaterra, 08193 Barcelona (Spain); Institut de Biotecnologia i de Biomedicina and Departament de Genetica i de Microbiologia, Universitat Autonoma de Barcelona, 08193 Bellaterra (Cerdanyola del Valles), Barcelona (Spain)

    2010-05-21

    A spectrum of materials for biomedical applications is produced in bacteria, and some of them, such as metals or polyhydroxyalkanoates, are straightforwardly obtained as particulate entities. We have explored the biofabrication process of bacterial inclusion bodies, particulate proteinaceous materials (ranging from 50 to 500 nm in diameter) recently recognized as suitable for surface topographical modification and tissue engineering. Inclusion bodies have been widely described as spherical or pseudo-spherical particles with only minor morphological variability, mostly restricted to their size. Here we have identified a cellular gene in Escherichia coli (clpP) that controls the in vivo fabrication process of inclusion bodies. In the absence of the encoded protease, the dynamics of protein deposition is perturbed, resulting in unusual tear-shaped particles with enhanced surface-volume ratios. This fact modifies the ability of inclusion bodies to promote mammalian cell attachment and differentiation upon surface decoration. The implications of the genetic control of inclusion body geometry are discussed in the context of their biological fabrication and regarding the biomedical potential of these protein clusters in regenerative medicine.

  2. Tunable geometry of bacterial inclusion bodies as substrate materials for tissue engineering

    International Nuclear Information System (INIS)

    GarcIa-Fruitos, Elena; Seras-Franzoso, JoaquIn; Vazquez, Esther; Villaverde, Antonio

    2010-01-01

    A spectrum of materials for biomedical applications is produced in bacteria, and some of them, such as metals or polyhydroxyalkanoates, are straightforwardly obtained as particulate entities. We have explored the biofabrication process of bacterial inclusion bodies, particulate proteinaceous materials (ranging from 50 to 500 nm in diameter) recently recognized as suitable for surface topographical modification and tissue engineering. Inclusion bodies have been widely described as spherical or pseudo-spherical particles with only minor morphological variability, mostly restricted to their size. Here we have identified a cellular gene in Escherichia coli (clpP) that controls the in vivo fabrication process of inclusion bodies. In the absence of the encoded protease, the dynamics of protein deposition is perturbed, resulting in unusual tear-shaped particles with enhanced surface-volume ratios. This fact modifies the ability of inclusion bodies to promote mammalian cell attachment and differentiation upon surface decoration. The implications of the genetic control of inclusion body geometry are discussed in the context of their biological fabrication and regarding the biomedical potential of these protein clusters in regenerative medicine.

  3. Towards revealing the structure of bacterial inclusion bodies.

    Science.gov (United States)

    Wang, Lei

    2009-01-01

    Protein aggregation is a widely observed phenomenon in human diseases, biopharmaceutical production, and biological research. Protein aggregates are generally classified as highly ordered, such as amyloid fibrils, or amorphous, such as bacterial inclusion bodies. Amyloid fibrils are elongated filaments with diameters of 6-12 nm, they are comprised of residue-specific cross-beta structure, and display characteristic properties, such as binding with amyloid-specific dyes. Amyloid fibrils are associated with dozens of human pathological conditions, including Alzheimer disease and prion diseases. Distinguished from amyloid fibrils, bacterial inclusion bodies display apparent amorphous morphology. Inclusion bodies are formed during high-level recombinant protein production, and formation of inclusion bodies is a major concern in biotechnology. Despite of the distinctive morphological difference, bacterial inclusion bodies have been found to have some amyloid-like properties, suggesting that they might contain structures similar to amyloid-like fibrils. Recent structural data further support this hypothesis, and this review summarizes the latest progress towards revealing the structural details of bacterial inclusion bodies.

  4. Inclusion bodies of aggregated hemosiderins in liver macrophages.

    Science.gov (United States)

    Hayashi, Hisao; Tatsumi, Yasuaki; Wakusawa, Shinya; Shigemasa, Ryota; Koide, Ryoji; Tsuchida, Ken-Ichi; Morotomi, Natsuko; Yamashita, Tetsuji; Kumagai, Kotaro; Ono, Yukiya; Hayashi, Kazuhiko; Ishigami, Masatoshi; Goto, Hidemi; Kato, Ayako; Kato, Koichi

    2017-12-01

    Hemosiderin formation is a structural indication of iron overload. We investigated further adaptations of the liver to excess iron. Five patients with livers showing iron-rich inclusions larger than 2 µm were selected from our database. The clinical features of patients and structures of the inclusions were compared with those of 2 controls with mild iron overload. All patients had severe iron overload with more than 5000 ng/mL of serum ferritin. Etiologies were variable, from hemochromatosis to iatrogenic iron overload. Their histological stages were either portal fibrosis or cirrhosis. Inclusion bodies were ultra-structurally visualized as aggregated hemosiderins in the periportal macrophages. X-ray analysis always identified, in addition to a large amount of iron complexes including oxygen and phosphorus, a small amount of copper and sulfur in the mosaic matrixes of inclusions. There were no inclusions in the control livers. Inclusion bodies, when the liver is loaded with excess iron, may appear in the macrophages as isolated organella of aggregated hemosiderins. Trace amounts of copper-sulfur complexes were always identified in the mosaic matrices of the inclusions, suggesting cuproprotein induction against excess iron. In conclusion, inclusion formation in macrophages may be an adaptation of the liver loaded with excess iron.

  5. Role of the disaggregase ClpB in processing of proteins aggregated as inclusion bodies.

    Science.gov (United States)

    Zblewska, Kamila; Krajewska, Joanna; Zolkiewski, Michal; Kędzierska-Mieszkowska, Sabina

    2014-08-01

    Overproduction of heterologous proteins in bacterial systems often results in the formation of insoluble inclusion bodies (IBs), which is a major impediment in biochemical research and biotechnology. In principle, the activity of molecular chaperones could be employed to gain control over the IB formation and to improve the recombinant protein yields, but the potential of each of the major bacterial chaperones (DnaK/J, GroEL/ES, and ClpB) to process IBs has not been fully established yet. We investigated the formation of inclusion bodies (IBs) of two aggregation-prone proteins, VP1LAC and VP1GFP, overproduced in Escherichiacoli in the presence and absence of the chaperone ClpB. We found that both ClpB isoforms, ClpB95 and ClpB80 accumulated in E. coli cells during the production of IBs. The amount of IB proteins increased in the absence of ClpB. ClpB supported the resolubilization and reactivation of the aggregated VP1LAC and VP1GFP in E. coli cells. The IB disaggregation was optimal in the presence of both ClpB95 and ClpB80. Our results indicate an essential role of ClpB in controlling protein aggregation and inclusion body formation in bacteria. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Refolding Techniques for Recovering Biologically Active Recombinant Proteins from Inclusion Bodies

    Directory of Open Access Journals (Sweden)

    Hiroshi Yamaguchi

    2014-02-01

    Full Text Available Biologically active proteins are useful for studying the biological functions of genes and for the development of therapeutic drugs and biomaterials in a biotechnology industry. Overexpression of recombinant proteins in bacteria, such as Escherichia coli, often results in the formation of inclusion bodies, which are protein aggregates with non-native conformations. As inclusion bodies contain relatively pure and intact proteins, protein refolding is an important process to obtain active recombinant proteins from inclusion bodies. However, conventional refolding methods, such as dialysis and dilution, are time consuming and, often, recovered yields of active proteins are low, and a trial-and-error process is required to achieve success. Recently, several approaches have been reported to refold these aggregated proteins into an active form. The strategies largely aim at reducing protein aggregation during the refolding procedure. This review focuses on protein refolding techniques using chemical additives and laminar flow in microfluidic chips for the efficient recovery of active proteins from inclusion bodies.

  7. Emerging therapeutic options for sporadic inclusion body myositis

    Directory of Open Access Journals (Sweden)

    Alfano LN

    2015-09-01

    Full Text Available Lindsay N Alfano, Linda P Lowes Nationwide Children’s Hospital, Center for Gene Therapy, Columbus, OH, USA Abstract: Sporadic inclusion body myositis is the most common inflammatory muscle disorder preferentially affecting males over the age of 40 years. Progressive muscle weakness of the finger flexors and quadriceps muscles results in loss of independence with activities of daily living and eventual wheelchair dependence. Initial signs of disease are often overlooked and can lead to mis- or delayed diagnosis. The underlying cause of disease is unknown, and disease progression appears refractory to available treatment options. This review discusses the clinical presentation of inclusion body myositis and the current efforts in diagnosis, and focuses on the current state of research for both nonpharmacological and pharmacological treatment options for this patient group. Keywords: myositis, inclusion body myositis, inflammatory myopathy, treatment, function, outcomes

  8. Dynamic recruitment of active proteasomes into polyglutamine initiated inclusion bodies.

    Science.gov (United States)

    Schipper-Krom, Sabine; Juenemann, Katrin; Jansen, Anne H; Wiemhoefer, Anne; van den Nieuwendijk, Rianne; Smith, Donna L; Hink, Mark A; Bates, Gillian P; Overkleeft, Hermen; Ovaa, Huib; Reits, Eric

    2014-01-03

    Neurodegenerative disorders such as Huntington's disease are hallmarked by neuronal intracellular inclusion body formation. Whether proteasomes are irreversibly recruited into inclusion bodies in these protein misfolding disorders is a controversial subject. In addition, it has been proposed that the proteasomes may become clogged by the aggregated protein fragments, leading to impairment of the ubiquitin-proteasome system. Here, we show by fluorescence pulse-chase experiments in living cells that proteasomes are dynamically and reversibly recruited into inclusion bodies. As these recruited proteasomes remain catalytically active and accessible to substrates, our results challenge the concept of proteasome sequestration and impairment in Huntington's disease, and support the reported absence of proteasome impairment in mouse models of Huntington's disease. Copyright © 2013 Federation of European Biochemical Societies. All rights reserved.

  9. Inclusion bodies in loggerhead erythrocytes are associated with unstable hemoglobin and resemble human Heinz bodies.

    Science.gov (United States)

    Basile, Filomena; Di Santi, Annalisa; Caldora, Mercedes; Ferretti, Luigi; Bentivegna, Flegra; Pica, Alessandra

    2011-08-01

    The aim of this study was to clarify the role of the erythrocyte inclusions found during the hematological screening of loggerhead population of the Mediterranean Sea. We studied the erythrocyte inclusions in blood specimens collected from six juvenile and nine adult specimens of the loggerhead turtle, Caretta caretta, from the Adriatic and Tyrrhenian Seas. Our study indicates that the percentage of mature erythrocytes containing inclusions ranged from 3 to 82%. Each erythrocyte contained only one round inclusion body. Inclusion bodies stained with May Grünwald-Giemsa show that their cytochemical and ultrastructure characteristics are identical to those of human Heinz bodies. Because Heinz bodies originate from the precipitation of unstable hemoglobin (Hb) and cause globular osmotic resistance to increase, we analyzed loggerhead Hb using electrophoresis and high-performance liquid chromatography to detect and quantitate Hb fractions. We also tested the resistance of Hb to alkaline pH, heat, isopropanol denaturation, and globular osmosis. Our hemogram results excluded the occurrence of any infection, which could be associated with an inclusion body, in all the specimens. Negative Feulgen staining indicated that the inclusion bodies are not derived from DNA fragmentation. We hypothesize that amino acid substitutions could explain why loggerhead Hb precipitates under normal physiologic conditions, forming Heinz bodies. The identification of inclusion bodies in loggerhead erythrocytes allow us to better understand the haematological characteristics and the physiology of these ancient reptiles, thus aiding efforts to conserve such an endangered species. Copyright © 2011 Wiley-Liss, Inc., A Wiley Company.

  10. Preparative Protein Production from Inclusion Bodies and Crystallization: A Seven-Week Biochemistry Sequence

    Science.gov (United States)

    Peterson, Megan J.; Snyder, W. Kalani; Westerman, Shelley; McFarland, Benjamin J.

    2011-01-01

    We describe how to produce and purify proteins from E. coli inclusion bodies by adapting versatile, preparative-scale techniques to the undergraduate laboratory schedule. This seven-week sequence of experiments fits into an annual cycle of research activity in biochemistry courses. Recombinant proteins are expressed as inclusion bodies, which are collected, washed, then solubilized in urea. Stepwise dialysis to dilute urea over the course of a week produces refolded protein. Column chromatography is used to purify protein into fractions, which are then analyzed with gel electrophoresis and concentration assays. Students culminate the project by designing crystallization trials in sitting-drop trays. Student evaluation of the experience has been positive, listing 5–12 new techniques learned, which are transferrable to graduate research in academia and industry. PMID:21691428

  11. Lewy and his inclusion bodies: Discovery and rejection

    Directory of Open Access Journals (Sweden)

    Eliasz Engelhardt

    Full Text Available ABSTRACT Fritz Jacob Heinrich Lewy described the pathology of Paralysis agitans [Parkinson disease] and was the first to identify eosinophilic inclusion bodies in neurons of certain brain nuclei, later known as Lewy bodies, the pathological signature of the Lewy body diseases. In 1912, he published his seminal study, followed soon after by an update paper, and 10 years later, in 1923, by his voluminous book, where he exhaustively described the subject. The publication provided extensive information on the pathology of Paralysis agitans, and the entirely novel finding of eosinophilic inclusion bodies, which would become widely recognized and debated in the future. His discovery was acknowledged by important researchers who even named the structure after him. However, after his last publication on the issue, inexplicably, he never mentioned his histopathological discovery again. Despite several hypotheses, the reasons that led him to neglect (reject the structure which he so preeminently described have remained elusive.

  12. Physical function and muscle strength in sporadic inclusion body myositis

    DEFF Research Database (Denmark)

    Jørgensen, Anders N; Aagaard, Per; Nielsen, Jakob L

    2017-01-01

    INTRODUCTION: In this study, self-reported physical function, functional capacity, and isolated muscle function were investigated in sporadic inclusion body myositis (sIBM) patients. METHODS: The 36-item Short Form (SF-36) Health Survey and 2-min walk test (2MWT), timed up & go test (TUG), and 30-s...

  13. [The practice guideline 'Dermatomyositis, polymyositis and sporadic inclusion body myositis'

    NARCIS (Netherlands)

    Hoogendijk, J.E.; Bijlsma, J.W.J.; Engelen, B.G.M. van; Lindeman, E.J.M.; Royen-Kerkhof, A. van; Rie, M.A. de; Visser, M. de; Jennekens, F.G.I.

    2005-01-01

    This guideline presents recommendations for the diagnosis and treatment of dermatomyositis, polymyositis and sporadic inclusion body myositis (sIBM) according to the best available evidence. Characteristic skin abnormalities can be sufficient for the diagnosis of dermatomyositis. In case of doubt, a

  14. Richtlijn 'dermatomyositis, polymyositis en sporadische "inclusion body"-myositis'

    NARCIS (Netherlands)

    Hoogendijk, J. E.; Bijlsma, J. W. J.; van Engelen, B. G. M.; Lindeman, E.; van Royen-Kerkhof, A.; de Rie, M. A.; de Visser, M.; Jennekens, F. G. I.

    2005-01-01

    This guideline presents recommendations for the diagnosis and treatment of dermatomyositis, polymyositis and sporadic inclusion body myositis (sIBM) according to the best available evidence. Characteristic skin abnormalities can be sufficient for the diagnosis of dermatomyositis. In case of doubt, a

  15. [Inclusion Bodies are Formed in SFTSV-infected Human Macrophages].

    Science.gov (United States)

    Jin, Cong; Song, Jingdong; Han, Ying; Li, Chuan; Qiu, Peihong; Liang, Mifang

    2016-01-01

    The severe fever with thrombocytopenia syndrome virus (SFTSV) is a new member in the genus Phlebovirus of the family Bunyaviridae identified in China. The SFTSV is also the causative pathogen of an emerging infectious disease: severe fever with thrombocytopenia syndrome. Using immunofluorescent staining and confocal microscopy, the intracellular distribution of nucleocapsid protein (NP) in SFTSV-infected THP-1 cells was investigated with serial doses of SFTSV at different times after infection. Transmission electron microscopy was used to observe the ultrafine intracellular structure of SFTSV-infected THP-1 cells at different times after infection. SFTSV NP could form intracellular inclusion bodies in infected THP-1 cells. The association between NP-formed inclusion bodies and virus production was analyzed: the size of the inclusion body formed 3 days after infection was correlated with the viral load in supernatants collected 7 days after infection. These findings suggest that the inclusion bodies formed in SFTSV-infected THP-1 cells could be where the SFTSV uses host-cell proteins and intracellular organelles to produce new viral particles.

  16. Enhanced expression and purification of camelid single domain VHH antibodies from classical inclusion bodies.

    Science.gov (United States)

    Maggi, Maristella; Scotti, Claudia

    2017-08-01

    Single domain antibodies (sdAbs) are small antigen-binding domains derived from naturally occurring, heavy chain-only immunoglobulins isolated from camelid and sharks. They maintain the same binding capability of full-length IgGs but with improved thermal stability and permeability, which justifies their scientific, medical and industrial interest. Several described recombinant forms of sdAbs have been produced in different hosts and with different strategies. Here we present an optimized method for a time-saving, high yield production and extraction of a poly-histidine-tagged sdAb from Escherichia coli classical inclusion bodies. Protein expression and extraction were attempted using 4 different methods (e.g. autoinducing or IPTG-induced soluble expression, non-classical and classical inclusion bodies). The best method resulted to be expression in classical inclusion bodies and urea-mediated protein extraction which yielded 60-70 mg/l bacterial culture. The method we here describe can be of general interest for an enhanced and efficient heterologous expression of sdAbs for research and industrial purposes. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. [In vitro renaturation of proteins from inclusion bodies].

    Science.gov (United States)

    Porowińska, Dorota; Marszałek, Ewelina; Wardęcka, Paulina; Komoszyński, Michał

    2012-06-11

    Recombinant proteins and enzymes are commonly used in many areas of our life, such as diagnostics, industry and medicine, due to heterologous synthesis in prokaryotic expression systems. However, a high expression level of foreign protein in bacteria cells results in formation of inactive and insoluble aggregates--inclusion bodies. Reactivation of aggregated proteins is a complex and time-consuming process. Every protein requires experimental optimization of the process conditions. The choice of the refolding method depends on the type of recombinant protein and its physical, chemical and biological properties. Recovery of the activity of proteins accumulated in inclusion bodies can be divided into 4 steps: 1) inclusion bodies isolation, 2) solubilization of aggregates, 3) renaturation, 4) purification of catalytically active molecules. Efficiency of the refolding process depends on many physical factors and chemical and biological agents. The above parameters determine the time of the folding and prevent protein aggregation. They also assist the folding and have an influence on the solubility and stability of native molecules. To date, dilution, dialysis and chromatography are the most often used methods for protein refolding.

  18. Membrane and inclusion body targeting of lyssavirus matrix proteins.

    Science.gov (United States)

    Pollin, Reiko; Granzow, Harald; Köllner, Bernd; Conzelmann, Karl-Klaus; Finke, Stefan

    2013-02-01

    Lyssavirus matrix proteins (M) support virus budding and have accessory functions that may contribute to host cell manipulation and adaptation to specific hosts. Here, we show that rabies virus (RABV) and European Bat Lyssavirus Type 1 (EBLV-1) M proteins differ in targeting and accumulation at cellular membranes. In contrast to RABV M, EBLV-1 M expressed from authentic EBLV-1 or chimeric RABV accumulated at the Golgi apparatus. Chimeric M proteins revealed that Golgi association depends on the integrity of the entire EBLV-1 M protein. Since RABV and EBLV-1 M differ in the use of cellular membranes for particle formation, differential membrane targeting and transport of M might determine the site of virus production. Moreover, both RABV and EBLV-1 M were for the first time detected within the nucleus and in Negri body-like inclusions bodies. Whereas nuclear M may imply hitherto unknown functions of lyssavirus M in host cell manipulation, the presence of M in inclusion bodies may correlate with regulatory functions of M in virus RNA synthesis. The data strongly support a model in which targeting of lyssavirus M proteins to distinctintracellular sites is a key determinant of diverse features in lyssavirus replication, host adaptation and pathogenesis. © 2012 Blackwell Publishing Ltd.

  19. Employment of colorimetric enzyme assay for monitoring expression and solubility of GST fusion proteins targeted to inclusion bodies.

    Science.gov (United States)

    Mačinković, Igor S; Abughren, Mohamed; Mrkic, Ivan; Grozdanović, Milica M; Prodanović, Radivoje; Gavrović-Jankulović, Marija

    2013-12-01

    High levels of recombinant protein expression can lead to the formation of insoluble inclusion bodies. These complex aggregates are commonly solubilized in strong denaturants, such as 6-8M urea, although, if possible, solubilization under milder conditions could facilitate subsequent refolding and purification of bioactive proteins. Commercially available GST-tag assays are designed for quantitative measurement of GST activity under native conditions. GST fusion proteins accumulated in inclusion bodies are considered to be undetectable by such assays. In this work, solubilization of recombinantly produced proteins was performed in 4M urea. The activity of rGST was assayed in 2M urea and it was shown that rGST preserves 85% of its activity under such denaturing conditions. A colorimetric GST activity assay with 1-chloro-2, 4-dinitrobenzene (CDNB) was examined for use in rapid detection of expression targeted to inclusion bodies and for the identification of inclusion body proteins which can be solubilized in low concentrations of chaotropic agents. Applicability of the assay was evaluated by tracking protein expression of two GST-fused allergens of biopharmaceutical value in E. coli, GST-Der p 2 and GST-Mus a 5, both targeted to inclusion bodies. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Positive in vitro wound healing effects of functional inclusion bodies of a lipoxygenase from the Mexican axolotl.

    Science.gov (United States)

    Stamm, Anne; Strauß, Sarah; Vogt, Peter; Scheper, Thomas; Pepelanova, Iliyana

    2018-04-07

    AmbLOXe is a lipoxygenase, which is up-regulated during limb-redevelopment in the Mexican axolotl, Ambystoma mexicanum, an animal with remarkable regeneration capacity. Previous studies have shown that mammalian cells transformed with the gene of this epidermal lipoxygenase display faster migration and wound closure rate during in vitro wound healing experiments. In this study, the gene of AmbLOXe was codon-optimized for expression in Escherichia coli and was produced in the insoluble fraction as protein aggregates. These inclusion bodies or nanopills were shown to be reservoirs containing functional protein during in vitro wound healing assays. For this purpose, functional inclusion bodies were used to coat cell culture surfaces prior cell seeding or were added directly to the medium after cells reached confluence. In both scenarios, AmbLOXe inclusion bodies led to faster migration rate and wound closure, in comparison to controls containing either no AmbLOXe or GFP inclusion bodies. Our results demonstrate that AmbLOXe inclusion bodies are functional and may serve as stable reservoirs of this enzyme. Nevertheless, further studies with soluble enzyme are also necessary in order to start elucidating the exact molecular substrates of AmbLOXe and the biochemical pathways involved in the wound healing effect.

  1. Genetics Home Reference: inclusion body myopathy with early-onset Paget disease and frontotemporal dementia

    Science.gov (United States)

    ... Share: Email Facebook Twitter Home Health Conditions IBMPFD Inclusion body myopathy with early-onset Paget disease and ... Javascript to view the expand/collapse boxes. Description Inclusion body myopathy with early-onset Paget disease and ...

  2. Amyloid-linked cellular toxicity triggered by bacterial inclusion bodies

    International Nuclear Information System (INIS)

    Gonzalez-Montalban, Nuria; Villaverde, Antonio; Aris, Anna

    2007-01-01

    The aggregation of proteins in the form of amyloid fibrils and plaques is the characteristic feature of some pathological conditions ranging from neurodegenerative disorders to systemic amyloidoses. The mechanisms by which the aggregation processes result in cell damage are under intense investigation but recent data indicate that prefibrillar aggregates are the most proximate mediators of toxicity rather than mature fibrils. Since it has been shown that prefibrillar forms of the nondisease-related misfolded proteins are highly toxic to cultured mammalian cells we have studied the cytoxicity associated to bacterial inclusion bodies that have been recently described as protein deposits presenting amyloid-like structures. We have proved that bacterial inclusion bodies composed by a misfolding-prone β-galactosidase fusion protein are clearly toxic for mammalian cells but the β-galactosidase wild type enzyme forming more structured thermal aggregates does not impair cell viability, despite it also binds and enter into the cells. These results are in the line that the most cytotoxic aggregates are early prefibrilar assemblies but discard the hypothesis that the membrane destabilization is Key event to subsequent disruption of cellular processes, such as ion balance, oxidative state and the eventually cell death

  3. Catalytically-active inclusion bodies-Carrier-free protein immobilizates for application in biotechnology and biomedicine.

    Science.gov (United States)

    Krauss, Ulrich; Jäger, Vera D; Diener, Martin; Pohl, Martina; Jaeger, Karl-Erich

    2017-09-20

    Bacterial inclusion bodies (IBs) consist of unfolded protein aggregates and represent inactive waste products often accumulating during heterologous overexpression of recombinant genes in Escherichia coli. This general misconception has been challenged in recent years by the discovery that IBs, apart from misfolded polypeptides, can also contain substantial amounts of active and thus correctly or native-like folded protein. The corresponding catalytically-active inclusion bodies (CatIBs) can be regarded as a biologically-active sub-micrometer sized biomaterial or naturally-produced carrier-free protein immobilizate. Fusion of polypeptide (protein) tags can induce CatIB formation paving the way towards the wider application of CatIBs in synthetic chemistry, biocatalysis and biomedicine. In the present review we summarize the history of CatIBs, present the molecular-biological tools that are available to induce CatIB formation, and highlight potential lines of application. In the second part findings regarding the formation, architecture, and structure of (Cat)IBs are summarized. Finally, an overview is presented about the available bioinformatic tools that potentially allow for the prediction of aggregation and thus (Cat)IB formation. This review aims at demonstrating the potential of CatIBs for biotechnology and hopefully contributes to a wider acceptance of this promising, yet not widely utilized, protein preparation. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Formation of distinct inclusion bodies by inhibition of ubiquitin-proteasome and autophagy-lysosome pathways

    International Nuclear Information System (INIS)

    Lee, Junho; Yang, Kyu-Hwan; Joe, Cheol O.; Kang, Seok-Seong

    2011-01-01

    Research highlights: → Distinct inclusion bodies are developed by inhibition of UPP and ALP. → The inclusion bodies differ in morphology, localization and formation process. → The inclusion bodies are distinguishable by the localization of TSC2. → Inhibition of both UPP and ALP simultaneously induces those inclusion bodies. -- Abstract: Accumulation of misfolded proteins is caused by the impairment of protein quality control systems, such as ubiquitin-proteasome pathway (UPP) and autophagy-lysosome pathway (ALP). In this study, the formation of inclusion bodies was examined after the blockade of UPP and/or ALP in A549 cells. UPP inhibition induced a single and large inclusion body localized in microtubule-organizing center. Interestingly, however, ALP inhibition generated dispersed small inclusion bodies in the cytoplasm. Tuberous sclerosis complex 2 was selectively accumulated in the inclusion bodies of UPP-inhibited cells, but not those of ALP-inhibited cells. Blockade of transcription and translation entirely inhibited the formation of inclusion body induced by UPP inhibition, but partially by ALP inhibition. Moreover, the simultaneous inhibition of two protein catabolic pathways independently developed two distinct inclusion bodies within a single cell. These findings clearly demonstrated that dysfunction of each catabolic pathway induced formation and accumulation of unique inclusion bodies on the basis of morphology, localization and formation process in A549 cells.

  5. Sporadic inclusion body myositis: the genetic contributions to the pathogenesis

    Science.gov (United States)

    2014-01-01

    Sporadic inclusion body myositis (sIBM) is the commonest idiopathic inflammatory muscle disease in people over 50 years old. It is characterized by slowly progressive muscle weakness and atrophy, with typical pathological changes of inflammation, degeneration and mitochondrial abnormality in affected muscle fibres. The cause(s) of sIBM are still unknown, but are considered complex, with the contribution of multiple factors such as environmental triggers, ageing and genetic susceptibility. This review summarizes the current understanding of the genetic contributions to sIBM and provides some insights for future research in this mysterious disease with the advantage of the rapid development of advanced genetic technology. An international sIBM genetic study is ongoing and whole-exome sequencing will be applied in a large cohort of sIBM patients with the aim of unravelling important genetic risk factors for sIBM. PMID:24948216

  6. Functional impairment in patients with sporadic Inclusion Body Myositis.

    Science.gov (United States)

    Dunlap, Heather V; Macneil, Lauren G; Tarnopolsky, Mark A

    2014-03-01

    We conducted a retrospective chart review of 53 patients diagnosed with sporadic Inclusion Body Myositis (sIBM) who have been followed at the McMaster Neuromuscular Clinic since 1996. We reviewed patient medical histories in order to compare our findings with similar cohorts, and analyzed quantitative strength data to determine functionality in guiding decisions related to gait assistive devices. Patient information was acquired through retrospective clinic chart review. Our study found knee extension strength decreased significantly as patients transitioned to using more supportive gait assistive devices (P cane)(P Falls and fear of falling poses a significant threat to patient physical well-being. The prevalence of dysphagia increased as patients required more supportive gait devices, and finally a significant negative correlation was found between time after onset and creatine kinase (CK) levels (P falling would be beneficial in preventing future falls and improving long-term patient outcomes.

  7. Seborrheic inclusion cyst of the skin positive for cytoplasmic inclusion bodies and HPV antigen.

    Science.gov (United States)

    Terada, Tadashi

    2012-01-01

    Seborrheic inclusion cyst (SIC) is a very rare variant of epidermal cyst of the skin. SIC shows seborrheic keratosis (SK)-like lesion in epidermal cyst. SIC is extremely rare; only 6 case reports have been published in the English literature. However, no immunohistochemical study of SIC has been reported. A 41-year-old Japanese man noticed a subcutaneous tumor in the neck. Physical examination showed slightly mobile tumor in the subcutaneous tissue, and total excision was performed. Grossly, the tumor (1 x 1 x 0.8 cm) was cyst containing atheromatous keratin. Microscopically, the lesion is a cyst containing keratins. About one half of the cyst showed features of epidermal cyst consisting of mature squamous epithelium with granular layers. The other one half showed SK-like epidermal proliferation. The SK-like area showed basaloid cell proliferation with pseudohorn cysts. No significant atypia was noted. Many eosinophilic cytoplasmic inclusion bodies were noted in the SK-like area. Immunohistochemically, the SK-like area was positive for pancytokeratin AE1/3, pancytokeratin CAM5.2, p63, and Ki-67 (labeling=8%) and HPV, but negative for p53. The pathological diagnosis was SIC.

  8. Muscle sonography in six patients with hereditary inclusion body myopathy

    International Nuclear Information System (INIS)

    Adler, Ronald S.; Garolfalo, Giovanna; Paget, Stephen; Kagen, Lawrence

    2008-01-01

    To evaluate the morphological changes of muscle with sonography in six patients affected by hereditary inclusion body myopathy (HIBM). We studied a group of six Persian Jews diagnosed with HIBM. All were homozygous for the GNE mutation M712T. Ultrasonographic examinations of the quadriceps femoris and hamstring muscle groups were performed. A follow-up ultrasound examination was performed, after an interval of 3 years, in four of these patients. Muscles were assessed subjectively as to echogenicity, determined by gray-scale assessment, and loss of normal muscle morphology. Power Doppler sonography (PDS) was used to assess vascularity. A sonographic finding of central atrophy and peripheral sparing resulting in a target-like appearance was noted in the hamstring compartment of all six patients. The quadriceps compartment also showed involvement of the rectus femoris of all patients, which, in some cases, was the only muscle involved in the quadriceps. Vascularity was markedly reduced in the affected areas, with blood flow demonstrated in the peripherally spared areas. The severity of atrophy increased with disease duration. In this case series, we describe a new sonographic finding as well as document progression of HIBM disease, which has generally been described as quadriceps sparing. The myopathic target lesion, as well as isolated rectus femoris atrophy, may provide a useful adjunct to disease diagnosis. (orig.)

  9. Botulinum toxin alleviates dysphagia of patients with inclusion body myositis.

    Science.gov (United States)

    Schrey, Aleksi; Airas, Laura; Jokela, Manu; Pulkkinen, Jaakko

    2017-09-15

    Oropharyngeal dysphagia is a disabling and undertreated symptom that often occurs in patients with sporadic inclusion body myositis (s-IBM). In this study, we examined the effect of botulinum neurotoxin A (BoNT-A) injections to the cricopharyngeus muscle (CPM) of patients with s-IBM and dysphagia. A single-center retrospective study involving 40 biopsy-proven s-IBM-patients treated in the District of Southwest Finland from 2000 to 2013. The incidence of dysphagia, rate of aspirations, rate of aspiration pneumonias and treatment results of dysphagia were analyzed. Patients treated for dysphagia were evaluated before and after surgery by video-fluoroscopy and/or using a questionnaire. Twenty-five of the 40 s-IBM patients (62.5%) experienced dysphagia. BoNT-A was injected a median of 2 times (range 1-7) in 12 patients with dysphagia. Before the injections 7 patients reported aspiration, none afterwards. The corresponding figures for aspiration pneumonia were 3 and 0. All of these patients had normal swallowing function 12months (median, range 2-60) after the last injection. BoNT-A injections to the CPM alleviate the dysphagia of s-IBM patients reversibly and appear to reduce the rate of aspiration effectively. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Evaluation and construction of diagnostic criteria for inclusion body myositis

    Science.gov (United States)

    Mammen, Andrew L.; Amato, Anthony A.; Weiss, Michael D.; Needham, Merrilee

    2014-01-01

    Objective: To use patient data to evaluate and construct diagnostic criteria for inclusion body myositis (IBM), a progressive disease of skeletal muscle. Methods: The literature was reviewed to identify all previously proposed IBM diagnostic criteria. These criteria were applied through medical records review to 200 patients diagnosed as having IBM and 171 patients diagnosed as having a muscle disease other than IBM by neuromuscular specialists at 2 institutions, and to a validating set of 66 additional patients with IBM from 2 other institutions. Machine learning techniques were used for unbiased construction of diagnostic criteria. Results: Twenty-four previously proposed IBM diagnostic categories were identified. Twelve categories all performed with high (≥97%) specificity but varied substantially in their sensitivities (11%–84%). The best performing category was European Neuromuscular Centre 2013 probable (sensitivity of 84%). Specialized pathologic features and newly introduced strength criteria (comparative knee extension/hip flexion strength) performed poorly. Unbiased data-directed analysis of 20 features in 371 patients resulted in construction of higher-performing data-derived diagnostic criteria (90% sensitivity and 96% specificity). Conclusions: Published expert consensus–derived IBM diagnostic categories have uniformly high specificity but wide-ranging sensitivities. High-performing IBM diagnostic category criteria can be developed directly from principled unbiased analysis of patient data. Classification of evidence: This study provides Class II evidence that published expert consensus–derived IBM diagnostic categories accurately distinguish IBM from other muscle disease with high specificity but wide-ranging sensitivities. PMID:24975859

  11. Protein misfolding specifies recruitment to cytoplasmic inclusion bodies.

    Science.gov (United States)

    Bersuker, Kirill; Brandeis, Michael; Kopito, Ron R

    2016-04-25

    Inclusion bodies (IBs) containing aggregated disease-associated proteins and polyubiquitin (poly-Ub) conjugates are universal histopathological features of neurodegenerative diseases. Ub has been proposed to target proteins to IBs for degradation via autophagy, but the mechanisms that govern recruitment of ubiquitylated proteins to IBs are not well understood. In this paper, we use conditionally destabilized reporters that undergo misfolding and ubiquitylation upon removal of a stabilizing ligand to examine the role of Ub conjugation in targeting proteins to IBs that are composed of an N-terminal fragment of mutant huntingtin, the causative protein of Huntington's disease. We show that reporters are excluded from IBs in the presence of the stabilizing ligand but are recruited to IBs after ligand washout. However, we find that Ub conjugation is not necessary to target reporters to IBs. We also report that forced Ub conjugation by the Ub fusion degradation pathway is not sufficient for recruitment to IBs. Finally, we find that reporters and Ub conjugates are stable at IBs. These data indicate that compromised folding states, rather than conjugation to Ub, can specify recruitment to IBs. © 2016 Bersuker et al.

  12. Refolding of proteins from inclusion bodies: rational design and recipes.

    Science.gov (United States)

    Basu, Anindya; Li, Xiang; Leong, Susanna Su Jan

    2011-10-01

    The need to develop protein biomanufacturing platforms that can deliver proteins quickly and cost-effectively is ever more pressing. The rapid rate at which genomes can now be sequenced demands efficient protein production platforms for gene function identification. There is a continued need for the biotech industry to deliver new and more effective protein-based drugs to address new diseases. Bacterial production platforms have the advantage of high expression yields, but insoluble expression of many proteins necessitates the development of diverse and optimised refolding-based processes. Strategies employed to eliminate insoluble expression are reviewed, where it is concluded that inclusion bodies are difficult to eliminate for various reasons. Rational design of refolding systems and recipes are therefore needed to expedite production of recombinant proteins. This review article discusses efforts towards rational design of refolding systems and recipes, which can be guided by the development of refolding screening platforms that yield both qualitative and quantitative information on the progression of a given refolding process. The new opportunities presented by light scattering technologies for developing rational protein refolding buffer systems which in turn can be used to develop new process designs armed with better monitoring and controlling functionalities are discussed. The coupling of dynamic and static light scattering methodologies for incorporation into future bioprocess designs to ensure delivery of high-quality refolded proteins at faster rates is also discussed.

  13. Packaging protein drugs as bacterial inclusion bodies for therapeutic applications

    Directory of Open Access Journals (Sweden)

    Villaverde Antonio

    2012-06-01

    Full Text Available Abstract A growing number of insights on the biology of bacterial inclusion bodies (IBs have revealed intriguing utilities of these protein particles. Since they combine mechanical stability and protein functionality, IBs have been already exploited in biocatalysis and explored for bottom-up topographical modification in tissue engineering. Being fully biocompatible and with tuneable bio-physical properties, IBs are currently emerging as agents for protein delivery into mammalian cells in protein-replacement cell therapies. So far, IBs formed by chaperones (heat shock protein 70, Hsp70, enzymes (catalase and dihydrofolate reductase, grow factors (leukemia inhibitory factor, LIF and structural proteins (the cytoskeleton keratin 14 have been shown to rescue exposed cells from a spectrum of stresses and restore cell functions in absence of cytotoxicity. The natural penetrability of IBs into mammalian cells (reaching both cytoplasm and nucleus empowers them as an unexpected platform for the controlled delivery of essentially any therapeutic polypeptide. Production of protein drugs by biopharma has been traditionally challenged by IB formation. However, a time might have arrived in which recombinant bacteria are to be engineered for the controlled packaging of therapeutic proteins as nanoparticulate materials (nanopills, for their extra- or intra-cellular release in medicine and cosmetics.

  14. Integrated continuous processing of proteins expressed as inclusion bodies: GCSF as a case study.

    Science.gov (United States)

    Kateja, Nikhil; Agarwal, Harshit; Hebbi, Vishwanath; Rathore, Anurag S

    2017-07-01

    Affordability of biopharmaceuticals continues to be a challenge, particularly in developing economies. This has fuelled advancements in manufacturing that can offer higher productivity and better economics without sacrificing product quality in the form of an integrated continuous manufacturing platform. While platform processes for monoclonal antibodies have existed for more than a decade, development of an integrated continuous manufacturing process for bacterial proteins has received relatively scant attention. In this study, we propose an end-to-end integrated continuous downstream process (from inclusion bodies to unformulated drug substance) for a therapeutic protein expressed in Escherichia coli as inclusion body. The final process consisted of a continuous refolding in a coiled flow inverter reactor directly coupled to a three-column periodic counter-current chromatography for capture of the product followed by a three-column con-current chromatography for polishing. The continuous bioprocessing train was run uninterrupted for 26 h to demonstrate its capability and the resulting output was analyzed for the various critical quality attributes, namely product purity (>99%), high molecular weight impurities (<0.5%), host cell proteins (<100 ppm), and host cell DNA (<10 ppb). All attributes were found to be consistent over the period of operation. The developed assembly offers smaller facility footprint, higher productivity, fewer hold steps, and significantly higher equipment and resin utilization. The complexities of process integration in the context of continuous processing have been highlighted. We hope that the study presented here will promote development of highly efficient, universal, end-to-end, fully continuous platforms for manufacturing of biotherapeutics. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:998-1009, 2017. © 2016 American Institute of Chemical Engineers.

  15. Luminescent conjugated oligothiophenes for sensitive fluorescent assignment of protein inclusion bodies.

    Science.gov (United States)

    Klingstedt, Therése; Blechschmidt, Cristiane; Nogalska, Anna; Prokop, Stefan; Häggqvist, Bo; Danielsson, Olof; Engel, W King; Askanas, Valerie; Heppner, Frank L; Nilsson, K Peter R

    2013-03-18

    Small hydrophobic ligands identifying intracellular protein deposits are of great interest, as protein inclusion bodies are the pathological hallmark of several degenerative diseases. Here we report that fluorescent amyloid ligands, termed luminescent conjugated oligothiophenes (LCOs), rapidly and with high sensitivity detect protein inclusion bodies in skeletal muscle tissue from patients with sporadic inclusion body myositis (s-IBM). LCOs having a conjugated backbone of at least five thiophene units emitted strong fluorescence upon binding, and showed co-localization with proteins reported to accumulate in s-IBM protein inclusion bodies. Compared with conventional amyloid ligands, LCOs identified a larger fraction of immunopositive inclusion bodies. When the conjugated thiophene backbone was extended with terminal carboxyl groups, the LCO revealed striking spectral differences between distinct protein inclusion bodies. We conclude that 1) LCOs are sensitive, rapid and powerful tools for identifying protein inclusion bodies and 2) LCOs identify a wider range of protein inclusion bodies than conventional amyloid ligands. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Ultrastructure of inclusion bodies in annulus cells in the degenerating human intervertebral disc.

    Science.gov (United States)

    Gruber, H E; Hanley, E N

    2009-06-01

    The rough endoplasmic reticulum (rER) of the cell has an architectural editing function that checks whether protein structure and three-dimensional assembly have occurred properly prior to export of newly synthesized material out of the cell. If these have been faulty, the material is retained within the rER as an inclusion body. Inclusion bodies have been identified previously in chondrocytes and osteoblasts in chondrodysplasias and osteogenesis imperfecta. Inclusion bodies in intervertebral disc cells, however, have only recently been recognized. Our objectives were to use transmission electron microscopy to analyze more fully inclusion bodies in the annulus pulposus and to study the extracellular matrix (ECM) surrounding cells containing inclusion bodies. ECM frequently encapsulated cells with inclusion bodies, and commonly contained prominent banded aggregates of Type VI collagen. Inclusion body material had several morphologies, including relatively smooth, homogeneous material, or a rougher, less homogeneous feature. Such findings expand our knowledge of the fine structure of the human disc cell and ECM during disc degeneration, and indicate the potential utility of ultrastructural identification of discs with intracellular inclusion bodies as a screening method for molecular studies directed toward identification of defective gene products in degenerating discs.

  17. Bacterial inclusion bodies as potential synthetic devices for pathogen recognition and a therapeutic substance release.

    Science.gov (United States)

    Talafová, Klaudia; Hrabárová, Eva; Chorvát, Dušan; Nahálka, Jozef

    2013-02-07

    Adhesins of pathogens recognise the glycans on the host cell and mediate adherence. They are also crucial for determining the tissue preferences of pathogens. Currently, glyco-nanomaterials provide potential tool for antimicrobial therapy. We demonstrate that properly glyco-tailored inclusion bodies can specifically bind pathogen adhesins and release therapeutic substances. In this paper, we describe the preparation of tailored inclusion bodies via the conjugation of indicator protein aggregated to form inclusion bodies with soluble proteins. Whereas the indicator protein represents a remedy, the soluble proteins play a role in pathogen recognition. For conjugation, glutaraldehyde was used as linker. The treatment of conjugates with polar lysine, which was used to inactivate the residual glutaraldehyde, inhibited unwanted hydrophobic interactions between inclusion bodies. The tailored inclusion bodies specifically interacted with the SabA adhesin from Helicobacter pylori aggregated to form inclusion bodies that were bound to the sialic acids decorating the surface of human erythrocytes. We also tested the release of indicator proteins from the inclusion bodies using sortase A and Ssp DNAB intein self-cleaving modules, respectively. Sortase A released proteins in a relatively short period of time, whereas the intein cleavage took several weeks. The tailored inclusion bodies are promising "nanopills" for biomedical applications. They are able to specifically target the pathogen, while a self-cleaving module releases a soluble remedy. Various self-cleaving modules can be enabled to achieve the diverse pace of remedy release.

  18. Production and purification of avian antibodies (IgYs from inclusion bodies of a recombinant protein central in NAD+ metabolism

    Directory of Open Access Journals (Sweden)

    Paula A. Moreno-González

    2013-08-01

    Full Text Available The use of hens for the production of polyclonal antibodies reduces animal intervention and moreover yields a higher quantity of antibodies than other animal models.  The phylogenetic distance between bird and mammal antigens, often leads to more specific avian antibodies than their mammalian counterparts.Since a large amount of antigen is required for avian antibody production, the use of recombinant proteins for this procedure has been growing faster over the last years. Nevertheless, recombinant protein production through heterologous systems frequently prompts the protein to precipitate, forming insoluble aggregates of limited utility (inclusion bodies. A methodology for the production of avian polyclonal antibodies, using recombinant protein from inclusion bodies is presented in this article.In order to produce the antigen, a recombinant Nicotinamide mononucleotide adenylyltransferase from Giardia intestinalis (His-GiNMNAT was expressed in Escherichia coli.  The protein was purified through solubilization from inclusion bodies prior to its renaturalization.  Antibodies were purified from egg yolk of immunized hens by water dilution, followed by ammonium sulfate precipitation and thiophilic affinity chromatography.The purified antibodies were tested against His-GiNMNAT protein in Western blot essays. From one egg yolk, 14.4 mg of highly pure IgY were obtained; this antibody was able to detect 15ng of His-GiNMNAT.  IgY specificity was improved by means of antigen affinity purification, allowing its use for parasite protein recognition.

  19. Should body image programs be inclusive? A focus group study of college students.

    Science.gov (United States)

    Ciao, Anna C; Ohls, Olivia C; Pringle, Kevin D

    2018-01-01

    Most evidence-based body image programs for college students (e.g., the Body Project) are designed for female-only audiences, although body dissatisfaction is not limited to female-identified individuals. Furthermore, programs do not explicitly discuss diversity, although individuals with marginalized gender, racial, and sexual identities may be particularly vulnerable to body image disturbances. Making programs more inclusive may increase their disseminability. This qualitative study examined the feasibility of adapting the Body Project for universal and inclusive use with college students. Participants (N = 36; M age = 21.66 years; 73% female-identified; 20% sexual minority; 23% racial minority) attended one of five semi-structured focus groups to explore the inclusivity of appearance-based cultural norms using adapted Body Project activities and discuss the feasibility of universal and inclusive interventions. Inductive qualitative content analysis with three-rater consensus identified focus group themes. There was consensus that inclusive interventions could have a positive impact (broadening perspectives, normalizing body image concerns, increasing awareness) despite potential barriers (poor diversity representation, vulnerability). There was strong consensus regarding advice for facilitating inclusive interventions (e.g., skilled facilitation, education, increasing diversity). Results suggest that inclusive body image programs are desirable and provide a framework for creating the EVERYbody Project, a program for more universal audiences. © 2017 Wiley Periodicals, Inc.

  20. Inclusion bodies as potential vehicles for recombinant protein delivery into epithelial cells

    Science.gov (United States)

    2012-01-01

    Background We present the potential of inclusion bodies (IBs) as a protein delivery method for polymeric filamentous proteins. We used as cell factory a strain of E. coli, a conventional host organism, and keratin 14 (K14) as an example of a complex protein. Keratins build the intermediate filament cytoskeleton of all epithelial cells. In order to build filaments, monomeric K14 needs first to dimerize with its binding partner (keratin 5, K5), which is then followed by heterodimer assembly into filaments. Results K14 IBs were electroporated into SW13 cells grown in culture together with a “reporter” plasmid containing EYFP labeled keratin 5 (K5) cDNA. As SW13 cells do not normally express keratins, and keratin filaments are built exclusively of keratin heterodimers (i.e. K5/K14), the short filamentous structures we obtained in this study can only be the result of: a) if both IBs and plasmid DNA are transfected simultaneously into the cell(s); b) once inside the cells, K14 protein is being released from IBs; c) released K14 is functional, able to form heterodimers with EYFP-K5. Conclusions Soluble IBs may be also developed for complex cytoskeletal proteins and used as nanoparticles for their delivery into epithelial cells. PMID:22624805

  1. Formation of distinct inclusion bodies by inhibition of ubiquitin-proteasome and autophagy-lysosome pathways.

    Science.gov (United States)

    Lee, Junho; Yang, Kyu-Hwan; Joe, Cheol O; Kang, Seok-Seong

    2011-01-14

    Accumulation of misfolded proteins is caused by the impairment of protein quality control systems, such as ubiquitin-proteasome pathway (UPP) and autophagy-lysosome pathway (ALP). In this study, the formation of inclusion bodies was examined after the blockade of UPP and/or ALP in A549 cells. UPP inhibition induced a single and large inclusion body localized in microtubule-organizing center. Interestingly, however, ALP inhibition generated dispersed small inclusion bodies in the cytoplasm. Tuberous sclerosis complex 2 was selectively accumulated in the inclusion bodies of UPP-inhibited cells, but not those of ALP-inhibited cells. Blockade of transcription and translation entirely inhibited the formation of inclusion body induced by UPP inhibition, but partially by ALP inhibition. Moreover, the simultaneous inhibition of two protein catabolic pathways independently developed two distinct inclusion bodies within a single cell. These findings clearly demonstrated that dysfunction of each catabolic pathway induced formation and accumulation of unique inclusion bodies on the basis of morphology, localization and formation process in A549 cells. Copyright © 2010 Elsevier Inc. All rights reserved.

  2. Creating Inclusive Physical Activity Spaces: The Case of Body-Positive Yoga.

    Science.gov (United States)

    Pickett, Andrew C; Cunningham, George B

    2017-09-01

    Within the modern cultural climate, those in larger bodies face high levels of weight stigma, particularly in sport and physical activity spaces, which serves as a strong barrier to their participation. However, given the strong link between physical activity and general health and well-being for participants, it is important to explore strategies that encourage participation of these individuals. Thus, the current research examined strategies that physical activity instructors use to develop inclusive exercise spaces for all body sizes. This study employed a series of semistructured qualitative interviews (n = 9) with instructors of body-inclusive yoga classes to explore the ways in which they encourage participation for those in larger bodies. Emergent themes from the current study suggested support for 6 factors for creating body-inclusive physical activity spaces: authentic leadership, a culture of inclusion, a focus on health, inclusive language, leader social activism, and a sense of community. This study revealed that leaders must intentionally cultivate inclusion in their spaces to encourage those in nonconforming bodies to participate. These findings have important health and management implications for the sport and physical activity context and provide a basic outline of practical strategies that practitioners can use to foster inclusion in their spaces.

  3. TDP-43 inclusion bodies formed in bacteria are structurally amorphous, non-amyloid and inherently toxic to neuroblastoma cells.

    Directory of Open Access Journals (Sweden)

    Claudia Capitini

    Full Text Available Accumulation of ubiquitin-positive, tau- and α-synuclein-negative intracellular inclusions of TDP-43 in the central nervous system represents the major hallmark correlated to amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions. Such inclusions have variably been described as amorphous aggregates or more structured deposits having an amyloid structure. Following the observations that bacterial inclusion bodies generally consist of amyloid aggregates, we have overexpressed full-length TDP-43 and C-terminal TDP-43 in E. coli, purified the resulting full-length and C-terminal TDP-43 containing inclusion bodies (FL and Ct TDP-43 IBs and subjected them to biophysical analyses to assess their structure/morphology. We show that both FL and Ct TDP-43 aggregates contained in the bacterial IBs do not bind amyloid dyes such as thioflavin T and Congo red, possess a disordered secondary structure, as inferred using circular dichroism and infrared spectroscopies, and are susceptible to proteinase K digestion, thus possessing none of the hallmarks for amyloid. Moreover, atomic force microscopy revealed an irregular structure for both types of TDP-43 IBs and confirmed the absence of amyloid-like species after proteinase K treatment. Cell biology experiments showed that FL TDP-43 IBs were able to impair the viability of cultured neuroblastoma cells when added to their extracellular medium and, more markedly, when transfected into their cytosol, where they are at least in part ubiquitinated and phosphorylated. These data reveal an inherently high propensity of TDP-43 to form amorphous aggregates, which possess, however, an inherently high ability to cause cell dysfunction. This indicates that a gain of toxic function caused by TDP-43 deposits is effective in TDP-43 pathologies, in addition to possible loss of function mechanisms originating from the cellular mistrafficking of the protein.

  4. 40 CFR 180.1149 - Inclusion bodies of the multi-nuclear polyhedrosis virus of Anagrapha falcifera; exemption from...

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Inclusion bodies of the multi-nuclear... Inclusion bodies of the multi-nuclear polyhedrosis virus of Anagrapha falcifera; exemption from the requirement of a tolerance. The microbial pest control agent inclusion bodies of the multi-nuclear...

  5. Formation of active inclusion bodies induced by hydrophobic self-assembling peptide GFIL8.

    Science.gov (United States)

    Wang, Xu; Zhou, Bihong; Hu, Weike; Zhao, Qing; Lin, Zhanglin

    2015-06-16

    In the last few decades, several groups have observed that proteins expressed as inclusion bodies (IBs) in bacteria could still be biologically active when terminally fused to an appropriate aggregation-prone partner such as pyruvate oxidase from Paenibacillus polymyxa (PoxB). More recently, we have demonstrated that three amphipathic self-assembling peptides, an alpha helical peptide 18A, a beta-strand peptide ELK16, and a surfactant-like peptide L6KD, have properties that induce target proteins into active IBs. We have developed an efficient protein expression and purification approach for these active IBs by introducing a self-cleavable intein molecule. In this study, the self-assembling peptide GFIL8 (GFILGFIL) with only hydrophobic residues was analyzed, and this peptide effectively induced the formation of cytoplasmic IBs in Escherichia coli when terminally attached to lipase A and amadoriase II. The protein aggregates in cells were confirmed by transmission electron microscopy analysis and retained ~50% of their specific activities relative to the native counterparts. We constructed an expression and separation coupled tag (ESCT) by incorporating an intein molecule, the Mxe GyrA intein. Soluble target proteins were successfully released from active IBs upon cleavage of the intein between the GFIL8 tag and the target protein, which was mediated by dithiothreitol. A variant of GFIL8, GFIL16 (GFILGFILGFILGFIL), improved the ESCT scheme by efficiently eliminating interference from the soluble intein-GFIL8 molecule. The yields of target proteins at the laboratory scale were 3.0-7.5 μg/mg wet cell pellet, which is comparable to the yields from similar ESCT constructs using 18A, ELK16, or the elastin-like peptide tag scheme. The all-hydrophobic self-assembling peptide GFIL8 induced the formation of active IBs in E. coli when terminally attached to target proteins. GFIL8 and its variant GFIL16 can act as a "pull-down" tag to produce purified soluble proteins with

  6. Characterization of the 4,6-α-glucanotransferase GTFB enzyme of Lactobacillus reuteri 121 isolated from inclusion bodies.

    Science.gov (United States)

    Bai, Yuxiang; van der Kaaij, Rachel Maria; Woortman, Albert Jan Jacob; Jin, Zhengyu; Dijkhuizen, Lubbert

    2015-06-09

    The GTFB enzyme of the probiotic bacterium Lactobacillus reuteri 121 is a 4,6-α-glucanotransferase of glycoside hydrolase family 70 (GH70; http://www.cazy.org ). Contrary to the glucansucrases in GH70, GTFB is unable to use sucrose as substrate, but instead converts malto-oligosaccharides and starch into isomalto-/malto- polymers that may find application as prebiotics and dietary fibers. The GTFB enzyme expresses well in Escherichia coli BL21 Star (DE3), but mostly accumulates in inclusion bodies (IBs) which generally contain wrongly folded protein and inactive enzyme. Denaturation followed by refolding, as well as ncIB preparation were used for isolation of active GTFB protein from inclusion bodies. Soluble, refolded and ncIB GTFB were compared using activity assays, secondary structure analysis by FT-IR, and product analyses by NMR, HPAEC and SEC. Expression of GTFB in E. coli yielded > 100 mg/l relatively pure and active but mostly insoluble GTFB protein in IBs, regardless of the expression conditions used. Following denaturing, refolding of GTFB protein was most efficient in double distilled H2O. Also, GTFB ncIBs were active, with approx. 10 % of hydrolysis activity compared to the soluble protein. When expressed as units of activity obtained per liter E. coli culture, the total amount of ncIB GTFB expressed possessed around 180 % hydrolysis activity and 100 % transferase activity compared to the amount of soluble GTFB enzyme obtained from one liter culture. The product profiles obtained for the three GTFB enzyme preparations were similar when analyzed by HPAEC and NMR. SEC investigation also showed that these 3 enzyme preparations yielded products with similar size distributions. FT-IR analysis revealed extended β-sheet formation in ncIB GTFB providing an explanation at the molecular level for reduced GTFB activity in ncIBs. The thermostability of ncIB GTFB was relatively high compared to the soluble and refolded GTFB. In view of their relatively high yield

  7. A bibliography of high energy two-body and inclusive scattering data

    International Nuclear Information System (INIS)

    Gault, F.D.; Read, B.J.; Roberts, R.G.

    1977-09-01

    A bibliography is presented of the data on high energy two-body and quasi-two-body final state scattering processes. This updated edition also covers one and two-particle inclusive production. It contains references to those published papers whose main purpose is to provide data on high energy two-body and inclusive hadronic scattering cross-sections rather than just properties of the produced particles. It covers the leading high energy physics journals and the period up to June 1977. The entries are grouped by process in the order indicated in the Table of Contents, and an author index is also provided. (author)

  8. Reassessment of inclusion body-based production as a versatile opportunity for difficult-to-express recombinant proteins.

    Science.gov (United States)

    Hoffmann, Daniel; Ebrahimi, Mehrdad; Gerlach, Doreen; Salzig, Denise; Czermak, Peter

    2017-11-10

    The production of recombinant proteins in the microbial host Escherichia coli often results in the formation of cytoplasmic protein inclusion bodies (IBs). Proteins forming IBs are often branded as difficult-to-express, neglecting that IBs can be an opportunity for their production. IBs are resistant to proteolytic degradation and contain up to 90% pure recombinant protein, which does not interfere with the host metabolism. This is especially advantageous for host-toxic proteins like antimicrobial peptides (AMPs). IBs can be easily isolated by cell disruption followed by filtration and/or centrifugation, but conventional techniques for the recovery of soluble proteins from IBs are laborious. New approaches therefore simplify protein recovery by optimizing the production process conditions, and often include mild resolubilization methods that either increase the yield after refolding or avoid the necessity of refolding all together. For the AMP production, the IB-based approach is ideal, because these peptides often have simple structures and are easy to refold. The intentional IB production of almost every protein can be achieved by fusing recombinant proteins to pull-down tags. This review discusses the techniques available for IB-based protein production before considering technical approaches for the isolation of IBs from E. coli lysates followed by efficient protein resolubilization which ideally omits further refolding. The techniques are evaluated in terms of their suitability for the process-scale production and downstream processing of recombinant proteins and are discussed for AMP production as an example.

  9. Association of inclusion body myositis with T cell large granular lymphocytic leukaemia

    DEFF Research Database (Denmark)

    Greenberg, Steven A; Pinkus, Jack L; Amato, Anthony A

    2016-01-01

    SEE HOHLFELD AND SCHULZE-KOOPS DOI101093/BRAIN/AWW053 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: Inclusion body myositis and T cell large granular lymphocytic leukaemia are rare diseases involving pathogenic cytotoxic CD8+ T cells. After encountering four patients with both disorders, we...... prospectively screened 38 patients with inclusion body myositis for the presence of expanded large granular lymphocyte populations by standard clinical laboratory methods (flow cytometry, examination of blood smears, and T cell receptor gene rearrangements), and performed muscle immunohistochemistry for CD8, CD......57, and TIA1. Most (22/38; 58%) patients with inclusion body myositis had aberrant populations of large granular lymphocytes in their blood meeting standard diagnostic criteria for T cell large granular lymphocytic leukaemia. These T cell populations were clonal in 20/20 patients and stably present...

  10. Microfluidic chips with multi-junctions: an advanced tool in recovering proteins from inclusion bodies.

    Science.gov (United States)

    Yamaguchi, Hiroshi; Miyazaki, Masaya

    2015-01-01

    Active recombinant proteins are used for studying the biological functions of genes and for the development of therapeutic drugs. Overexpression of recombinant proteins in bacteria often results in the formation of inclusion bodies, which are protein aggregates with non-native conformations. Protein refolding is an important process for obtaining active recombinant proteins from inclusion bodies. However, the conventional refolding method of dialysis or dilution is time-consuming and recovered active protein yields are often low, and a cumbersome trial-and-error process is required to achieve success. To circumvent these difficulties, we used controllable diffusion through laminar flow in microchannels to regulate the denaturant concentration. This method largely aims at reducing protein aggregation during the refolding procedure. This Commentary introduces the principles of the protein refolding method using microfluidic chips and the advantage of our results as a tool for rapid and efficient recovery of active recombinant proteins from inclusion bodies.

  11. Cytosolic 5′-nucleotidase 1A autoantibody profile and clinical characteristics in inclusion body myositis

    Science.gov (United States)

    Rietveld, A; Pye, S R; Mariampillai, K; Benveniste, O; Peeters, M T J; Miller, J A L; Hanna, M G; Machado, P M; Parton, M J; Gheorghe, K R; Badrising, U A; Lundberg, I E; Sacconi, S; Herbert, M K; McHugh, N J; Lecky, B R F; Brierley, C; Hilton-Jones, D; Lamb, J A; Roberts, M E; Cooper, R G; Saris, C G J; Pruijn, G J M; Chinoy, H; van Engelen, B G M

    2017-01-01

    Objectives Autoantibodies directed against cytosolic 5′-nucleotidase 1A have been identified in many patients with inclusion body myositis. This retrospective study investigated the association between anticytosolic 5′-nucleotidase 1A antibody status and clinical, serological and histopathological features to explore the utility of this antibody to identify inclusion body myositis subgroups and to predict prognosis. Materials and methods Data from various European inclusion body myositis registries were pooled. Anticytosolic 5′-nucleotidase 1A status was determined by an established ELISA technique. Cases were stratified according to antibody status and comparisons made. Survival and mobility aid requirement analyses were performed using Kaplan-Meier curves and Cox proportional hazards regression. Results Data from 311 patients were available for analysis; 102 (33%) had anticytosolic 5′-nucleotidase 1A antibodies. Antibody-positive patients had a higher adjusted mortality risk (HR 1.89, 95% CI 1.11 to 3.21, p=0.019), lower frequency of proximal upper limb weakness at disease onset (8% vs 23%, adjusted OR 0.29, 95% CI 0.12 to 0.68, p=0.005) and an increased prevalence of excess of cytochrome oxidase deficient fibres on muscle biopsy analysis (87% vs 72%, adjusted OR 2.80, 95% CI 1.17 to 6.66, p=0.020), compared with antibody-negative patients. Interpretation Differences were observed in clinical and histopathological features between anticytosolic 5′-nucleotidase 1A antibody positive and negative patients with inclusion body myositis, and antibody-positive patients had a higher adjusted mortality risk. Stratification of inclusion body myositis by anticytosolic 5′-nucleotidase 1A antibody status may be useful, potentially highlighting a distinct inclusion body myositis subtype with a more severe phenotype. PMID:28122761

  12. Cytosolic 5'-nucleotidase 1A autoantibody profile and clinical characteristics in inclusion body myositis.

    Science.gov (United States)

    Lilleker, J B; Rietveld, A; Pye, S R; Mariampillai, K; Benveniste, O; Peeters, M T J; Miller, J A L; Hanna, M G; Machado, P M; Parton, M J; Gheorghe, K R; Badrising, U A; Lundberg, I E; Sacconi, S; Herbert, M K; McHugh, N J; Lecky, B R F; Brierley, C; Hilton-Jones, D; Lamb, J A; Roberts, M E; Cooper, R G; Saris, C G J; Pruijn, G J M; Chinoy, H; van Engelen, B G M

    2017-05-01

    Autoantibodies directed against cytosolic 5'-nucleotidase 1A have been identified in many patients with inclusion body myositis. This retrospective study investigated the association between anticytosolic 5'-nucleotidase 1A antibody status and clinical, serological and histopathological features to explore the utility of this antibody to identify inclusion body myositis subgroups and to predict prognosis. Data from various European inclusion body myositis registries were pooled. Anticytosolic 5'-nucleotidase 1A status was determined by an established ELISA technique. Cases were stratified according to antibody status and comparisons made. Survival and mobility aid requirement analyses were performed using Kaplan-Meier curves and Cox proportional hazards regression. Data from 311 patients were available for analysis; 102 (33%) had anticytosolic 5'-nucleotidase 1A antibodies. Antibody-positive patients had a higher adjusted mortality risk (HR 1.89, 95% CI 1.11 to 3.21, p=0.019), lower frequency of proximal upper limb weakness at disease onset (8% vs 23%, adjusted OR 0.29, 95% CI 0.12 to 0.68, p=0.005) and an increased prevalence of excess of cytochrome oxidase deficient fibres on muscle biopsy analysis (87% vs 72%, adjusted OR 2.80, 95% CI 1.17 to 6.66, p=0.020), compared with antibody-negative patients. Differences were observed in clinical and histopathological features between anticytosolic 5'-nucleotidase 1A antibody positive and negative patients with inclusion body myositis, and antibody-positive patients had a higher adjusted mortality risk. Stratification of inclusion body myositis by anticytosolic 5'-nucleotidase 1A antibody status may be useful, potentially highlighting a distinct inclusion body myositis subtype with a more severe phenotype. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  13. Blood-flow restricted resistance training in patients with sporadic inclusion body myositis

    DEFF Research Database (Denmark)

    Jørgensen, A.; Aagaard, P.; Frandsen, U.

    2018-01-01

    Objectives: To investigate the effect of 12 weeks of low-load blood-flow restricted resistance (BFR) training on self-reported and objective physical function, and maximal muscle strength in patients with sporadic inclusion body myositis (sIBM). Method: Twenty-two patients with sIBM were randomized......), which was used to measure self-reported physical function. All patients performed physical function tests (2-Minute Walk Test, Timed Up and Go, and 30-Second Chair Stand), completed the Inclusion Body Myositis Functional Rating Scale (IBMFRS), and were tested for isolated knee extensor muscle strength...

  14. Human Metapneumovirus Induces Formation of Inclusion Bodies for Efficient Genome Replication and Transcription.

    Science.gov (United States)

    Cifuentes-Muñoz, Nicolás; Branttie, Jean; Slaughter, Kerri Beth; Dutch, Rebecca Ellis

    2017-12-15

    Human metapneumovirus (HMPV) causes significant upper and lower respiratory disease in all age groups worldwide. The virus possesses a negative-sense single-stranded RNA genome of approximately 13.3 kb encapsidated by multiple copies of the nucleoprotein (N), giving rise to helical nucleocapsids. In addition, copies of the phosphoprotein (P) and the large RNA polymerase (L) decorate the viral nucleocapsids. After viral attachment, endocytosis, and fusion mediated by the viral glycoproteins, HMPV nucleocapsids are released into the cell cytoplasm. To visualize the subsequent steps of genome transcription and replication, a fluorescence in situ hybridization (FISH) protocol was established to detect different viral RNA subpopulations in infected cells. The FISH probes were specific for detection of HMPV positive-sense RNA (+RNA) and viral genomic RNA (vRNA). Time course analysis of human bronchial epithelial BEAS-2B cells infected with HMPV revealed the formation of inclusion bodies (IBs) from early times postinfection. HMPV IBs were shown to be cytoplasmic sites of active transcription and replication, with the translation of viral proteins being closely associated. Inclusion body formation was consistent with an actin-dependent coalescence of multiple early replicative sites. Time course quantitative reverse transcription-PCR analysis suggested that the coalescence of inclusion bodies is a strategy to efficiently replicate and transcribe the viral genome. These results provide a better understanding of the steps following HMPV entry and have important clinical implications. IMPORTANCE Human metapneumovirus (HMPV) is a recently discovered pathogen that affects human populations of all ages worldwide. Reinfections are common throughout life, but no vaccines or antiviral treatments are currently available. In this work, a spatiotemporal analysis of HMPV replication and transcription in bronchial epithelial cell-derived immortal cells was performed. HMPV was shown to

  15. Analysis of magnetite crystals and inclusion bodies inside magnetotactic bacteria from different environmental locations

    Science.gov (United States)

    Oestreicher, Z.; Lower, B.; Lower, S.; Bazylinski, D. A.

    2011-12-01

    Biomineralization occurs throughout the living world; a few common examples include iron oxide in chiton teeth, calcium carbonate in mollusk shells, calcium phosphate in animal bones and teeth, silica in diatom shells, and magnetite crystals inside the cells of magnetotactic bacteria. Biologically controlled mineralization is characterized by biominerals that have species-specific properties such as: preferential crystallographic orientation, consistent particle size, highly ordered spatial locations, and well-defined composition and structure. It is well known that magnetotactic bacteria synthesize crystals of magnetite inside of their cells, but how they mineralize the magnetite is poorly understood. Magnetosomes have a species-specific morphology that is due to specific proteins involved in the mineralization process. In addition to magnetite crystals, magnetotactic bacteria also produce inclusion bodies or granules that contain different elements, such as phosphorus, calcium, and sulfur. In this study we used the transmission electron microscope to analyze the structure of magnetite crystals and inclusion bodies from different species of magnetotactic bacteria in order to determine the composition of the inclusion bodies and to ascertain whether or not the magnetite crystals contain elements other than iron and oxygen. Using energy dispersive spectroscopy we found that different bacteria from different environments possess inclusion bodies that contain different elements such as phosphorus, calcium, barium, magnesium, and sulfur. These differences may reflect the conditions of the environment in which the bacteria inhabit.

  16. Comparison of weakness progression in inclusion body myositis during treatment with methotrexate or placebo

    NARCIS (Netherlands)

    Badrising, UA; Maat-Schieman, MLC; Ferrari, MD; Zwinderman, AH; Wessels, JAM; Breedveld, FC; van Doorn, PA; van Engelen, BGM; Hoogendijk, JE; Howeler, CJ; de Jager, AE; Jennekens, FGI; Koehler, PJ; de Visser, M; Viddeleer, A; Verschuuren, JJ; Wintzen, AR

    We investigated whether 5 to 20mg per week oral methotrexate could slow down disease progression in 44 patients with inclusion body myositis in a randomized double-blind placebo-controlled study over 48 weeks. Mean change of quantitative muscle strength testing sum scores was the primary study

  17. Epidemiology of inclusion body myositis in the Netherlands : A nationwide study

    NARCIS (Netherlands)

    Badrising, UA; Maat-Schieman, M; van Duinen, SG; Breedveld, F; van Doorn, P; van Engelen, B; van den Hoogen, F; Hoogendijk, J; Howeler, C; de Jager, A; Jennekens, F; Koehler, P; van der Leeuw, H; de Visser, M; Verschuuren, JJ; Wintzen, AR

    2000-01-01

    Epidemiologic data on inclusion body myositis (IBM) are scarce, and possibly biased, because they are derived from larger neuromuscular centers. The present nationwide collaborative cross-sectional study, which culminated on July 1, 1999, resulted in identification of 76 patients with IBM and the

  18. Myositis with endomysial cell invasion indicates inclusion body myositis even if other criteria are not fulfilled

    NARCIS (Netherlands)

    Van de Vlekkert, J.; Hoogendijk, J. E.; de Visser, M.

    2015-01-01

    The objective of this study was to investigate if patients with endomysial mononuclear cell infiltrates invading non-necrotic fibers have a disease course consistent with inclusion body myositis (IBM), irrespective of other histopathological and clinical characteristics. All patients with a muscle

  19. A large planetary body inferred from diamond inclusions in a ureilite meteorite.

    Science.gov (United States)

    Nabiei, Farhang; Badro, James; Dennenwaldt, Teresa; Oveisi, Emad; Cantoni, Marco; Hébert, Cécile; El Goresy, Ahmed; Barrat, Jean-Alix; Gillet, Philippe

    2018-04-17

    Planetary formation models show that terrestrial planets are formed by the accretion of tens of Moon- to Mars-sized planetary embryos through energetic giant impacts. However, relics of these large proto-planets are yet to be found. Ureilites are one of the main families of achondritic meteorites and their parent body is believed to have been catastrophically disrupted by an impact during the first 10 million years of the solar system. Here we studied a section of the Almahata Sitta ureilite using transmission electron microscopy, where large diamonds were formed at high pressure inside the parent body. We discovered chromite, phosphate, and (Fe,Ni)-sulfide inclusions embedded in diamond. The composition and morphology of the inclusions can only be explained if the formation pressure was higher than 20 GPa. Such pressures suggest that the ureilite parent body was a Mercury- to Mars-sized planetary embryo.

  20. Purification and characterization of naturally occurring HIV-1 (South African subtype C) protease mutants from inclusion bodies.

    Science.gov (United States)

    Maseko, Sibusiso B; Natarajan, Satheesh; Sharma, Vikas; Bhattacharyya, Neelakshi; Govender, Thavendran; Sayed, Yasien; Maguire, Glenn E M; Lin, Johnson; Kruger, Hendrik G

    2016-06-01

    Human immunodeficiency virus (HIV) infections in sub-Saharan Africa represent about 56% of global infections. Many studies have targeted HIV-1 protease for the development of drugs against AIDS. Recombinant HIV-1 protease is used to screen new drugs from synthetic compounds or natural substances. Along with the wild type (C-SA) we also over-expressed and characterized two mutant forms from patients that had shown resistance to protease inhibitors. Using recombinant DNA technology, we constructed three recombinant plasmids in pGEX-6P-1 and expressed them containing a sequence encoding wild type HIV protease and two mutants (I36T↑T contains 100 amino acids and L38L↑N↑L contains 101 amino acids). These recombinant proteins were isolated from inclusion bodies by using QFF anion exchange and GST trap columns. In SDS-PAGE, we obtained these HIV proteases as single bands of approximately 11.5, 11.6 and 11.7 kDa for the wild type, I36T↑Tand L38L↑N↑L mutants, respectively. The enzyme was recovered efficiently (0.25 mg protein/L of Escherichia coli culture) and had high specific activity of 2.02, 2.20 and 1.33 μmol min(-1) mg(-1) at an optimal pH of 5 and temperature of 37 °C for the wild type, I36T↑T and L38L↑N↑L, respectively. The method employed here provides an easy and rapid purification of the HIV-1(C-SA) protease from the inclusion bodies, with high yield and high specific activities. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. The late endocytic Rab39a GTPase regulates the interaction between multivesicular bodies and chlamydial inclusions.

    Science.gov (United States)

    Gambarte Tudela, Julian; Capmany, Anahi; Romao, Maryse; Quintero, Cristian; Miserey-Lenkei, Stephanie; Raposo, Graca; Goud, Bruno; Damiani, Maria Teresa

    2015-08-15

    Given their obligate intracellular lifestyle, Chlamydia trachomatis ensure that they have access to multiple host sources of essential lipids by interfering with vesicular transport. These bacteria hijack Rab6-, Rab11- and Rab14-controlled trafficking pathways to acquire sphingomyelin from the Golgi complex. Another important source of sphingolipids, phospholipids and cholesterol are multivesicular bodies (MVBs). Despite their participation in chlamydial inclusion development and bacterial replication, the molecular mechanisms mediating the interaction between MVBs and chlamydial inclusions remain unknown. In the present study, we demonstrate that Rab39a labels a subset of late endocytic vesicles - mainly MVBs - that move along microtubules. Moreover, Rab39a is actively recruited to chlamydial inclusions throughout the pathogen life cycle by a bacterial-driven process that depends on the Rab39a GTP- or GDP-binding state. Interestingly, Rab39a participates in the delivery of MVBs and host sphingolipids to maturing chlamydial inclusions, thereby promoting inclusion growth and bacterial development. Taken together, our findings indicate that Rab39a favours chlamydial replication and infectivity. This is the first report showing that a late endocytic Rab GTPase is involved in chlamydial infection development. © 2015. Published by The Company of Biologists Ltd.

  2. Integrated process development-a robust, rapid method for inclusion body harvesting and processing at the microscale level.

    Science.gov (United States)

    Walther, Cornelia; Kellner, Martin; Berkemeyer, Matthias; Brocard, Cécile; Dürauer, Astrid

    2017-10-21

    Escherichia coli stores large amounts of highly pure product within inclusion bodies (IBs). To take advantage of this beneficial feature, after cell disintegration, the first step to optimal product recovery is efficient IB preparation. This step is also important in evaluating upstream optimization and process development, due to the potential impact of bioprocessing conditions on product quality and on the nanoscale properties of IBs. Proper IB preparation is often neglected, due to laboratory-scale methods requiring large amounts of materials and labor. Miniaturization and parallelization can accelerate analyses of individual processing steps and provide a deeper understanding of up- and downstream processing interdependencies. Consequently, reproducible, predictive microscale methods are in demand. In the present study, we complemented a recently established high-throughput cell disruption method with a microscale method for preparing purified IBs. This preparation provided results comparable to laboratory-scale IB processing, regarding impurity depletion, and product loss. Furthermore, with this method, we performed a "design of experiments" study to demonstrate the influence of fermentation conditions on the performance of subsequent downstream steps and product quality. We showed that this approach provided a 300-fold reduction in material consumption for each fermentation condition and a 24-fold reduction in processing time for 24 samples.

  3. Investigation of the phase morphology of bacterial PHA inclusion bodies by contrast variation SANS

    International Nuclear Information System (INIS)

    Russell, R.A.; Holden, P.J.; Garvey, C.J.; Wilde, K.L.; Hammerton, K.M.; Foster, L.J.

    2006-01-01

    Under growth-limiting conditions, many bacteria are able to metabolise excess organic acids into polyhydroxyalkanoates (PHA) and store these polymers as intracellular inclusions until the return of favourable conditions. Various models have been proposed for the macromolecular organisation of the boundary layer surrounding the polymer, and contrast-variation small-angle neutron scattering (SANS) was used to study its organisation. Inclusions formed by Pseudomonas oleovorans under hydrogenating conditions showed lowest scattering intensity at ca. 20% D 2 O. The inclusions consist of protein and membrane lipids in the boundary layer and polyhydroxyoctanoate (lipid) in the inclusion body. At 20% D 2 O the contributions of lipids were contrast matched with the solvent, indicating that lipids contributed the bulk of the scattering intensity observed at other D 2 O/H 2 O ratios. These results are inconsistent with a model of the boundary layer which proposed outer and inner layers of crystalline protein lattice sandwiching a membrane lipid membrane layer [E.S. Stuart, R.W. Lenz, R.C. Fuller, Can J Microbiol 41(Suppl 1) (1995) 84-93], and is more consistent with a model consisting of a lipid monolayer containing embedded proteins [U. Pieper-furst, M.H. Madkour, F. Mayer, A. Steinbuchel, J. Bacteriol. 176 (1994) 4328-4337.] By altering the H/D content of the precursors, we were able to collect SANS data from preparations of both deuterated and H/D copolymer inclusions, where initial PHA produced was hydrogenated followed by deuteration. Deuterated inclusions showed minimum intensity above 90% D 2 O/H 2 O whereas the sequentially produced copolymer (assumed to be in a core/shell arrangement) displayed minimum scattering some 20% lower, which is consistent with the increased hydrogenation of the boundary layer expected from its synthesis during supply of hydrogenated followed by deuterated precursors

  4. Mineralization of alpha-1-antitrypsin inclusion bodies in Mmalton alpha-1-antitrypsin deficiency.

    Science.gov (United States)

    Callea, Francesco; Giovannoni, Isabella; Francalanci, Paola; Boldrini, Renata; Faa, Gavino; Medicina, Daniela; Nobili, Valerio; Desmet, Valeer J; Ishak, Kamal; Seyama, Kuniaki; Bellacchio, Emanuele

    2018-05-16

    Alpha-1-antitrypsin (AAT) deficiency (AATD) of Z, Mmalton, Siiyama type is associated with liver storage of the mutant proteins and liver disease. The Z variant can be diagnosed on isoelectric focusing (IEF) while Mmalton and Siiyama may be missed or misdiagnosed with this technique. Therefore, molecular analysis is mandatory for their characterization. In particular, that holds true for the Mmalton variant as on IEF profile it resembles the wild M2 subtype. This is a retrospective analysis involving review of medical records and of liver biopsy specimens from a series of Mmalton, Z and Siiyama Alpha-1-antitrypsin deficiency patients. The review has been implemented by additional histological stains, electron microscopic observations and 3-D modeling studies of the sites of the mutations. Z, Mmalton and Siiyama liver specimen contained characteristic intrahepatocytic PAS-D globules. The globules differed in the three variants as only Mmalton cases showed dark basophilic precipitates within the AAT inclusions. The precipitates were visualized in haematoxylin-eosin (H.E.) stained preparations and corresponded to calcium precipitates as demonstrated by von Kossa staining. On immunohistochemistry, ZAAT inclusions were stained by polyclonal as well as monoclonal noncommercial anti-AAT antibody (AZT11), whilst Mmalton and Siiyama inclusion bodies remained negative with the monoclonal anti-Z antibody. 3-D protein analysis allowed to predict more severe misfolding of the Mmalton molecule as compared to Z and Siiyama that could trigger anomalous interaction with endoplasmic reticulum chaperon proteins, namely calcium binding proteins. Mmalton AAT inclusion bodies contain calcium precipitates inside them that allow the differential diagnosis with Siiyama and ZAAT inclusions in routine histological sections. The study has confirmed the specificity of the monoclonal AZT11 for the Z mutant. Thus, the combination of these two features is crucial for the distinction between the

  5. Columbid herpesvirus-1 in two Cooper's hawks (Accipiter cooperii) with fatal inclusion body disease.

    Science.gov (United States)

    Pinkerton, Marie E; Wellehan, James F X; Johnson, April J; Childress, April L; Fitzgerald, Scott D; Kinsel, Michael J

    2008-07-01

    We report two separate naturally occurring cases of fatal herpesviral disease in Cooper's Hawks (Accipiter cooperii). Gross lesions included splenomegaly and hepatomegaly, with diffuse pale mottling or scattered small white foci. Histologic lesions included splenic and hepatic necrosis associated with eosinophilic intranuclear inclusion bodies characteristic of herpesvirus. In one case, necrosis and inclusions were also noted in bone marrow, thymus, bursa of Fabricius, thyroid gland, parathyroid gland, ceca, and the enteric system. Transmission electron microscopy demonstrated viral particles typical of herpesvirus within hepatocyte nuclei and budding from the nuclear membrane. Herpesviral DNA was amplified via polymerase chain reaction (PCR) of paraffin-embedded liver and spleen, and sequence data were consistent with columbid herpesvirus-1, an alphaherpesvirus of Rock Pigeons (Columba livia). PCR results provide evidence that this disease is transmitted to raptors via Rock Pigeons, most likely through ingestion of Rock Pigeons as prey.

  6. Inclusion bodies induced by bean rugose mosaic virus seen under light microscopy

    Directory of Open Access Journals (Sweden)

    Carmen Rivera

    2000-12-01

    Full Text Available Two types of inclusion bodies were consistently observed under light microscopy in bean (Phaseolus vulgaris leaf tissue infected with bean rugose mosaic virus (BRMV, a species of the genus Comovirus, family Comoviridae. One type consisted of vacuolated inclusions found mainly in the cytoplasm of epidermal cells. The other type consisted of abundant crystalloid inclusions of different sizes and shapes found consistently in glandular hairs, guard cells, phloem tissue, xylem elements and occasionally in epidermal and mesophyll tissues. The two types of inclusion bodies stained with Azure A and Luxol Brilliant Green Bl-Calcomine Orange 2RS (O-G, and were similar to those seen to be caused by other species of comoviruses.Se observaron dos tipos de inclusiones virales, mediante microscopia de luz, en hojas de plantas de frijol (Phaseolus vulgaris previamente infectadas con el virus del mosaico rugoso del frijol ("bean rugose mosaic comovirus", BRMV, especie del género Comovirus, familia Comoviridae. Se hallaron inclusiones vesiculadas, principalmente en el citoplasma de células de la epidermis, y abundantes inclusiones cristalinas de diferentes formas y tamaños siempre en células guarda, tricomas glandulares, floema, elementos del xilema y ocasionalmente en células epidérmicas y del mesófilo. Ambos tipos de inclusiones tiñeron con Azure A y con la tinción, verde naranja (Luxol Brilliant Green BL-Calcomine Orange 2 RS conocida como OG, y son similares a las inclusiones inducidas por otras especies del género Comovirus.

  7. Infantile digital fibromatosis (inclusion body fibromatosis observed in a baby without finger involvement

    Directory of Open Access Journals (Sweden)

    Avni Kaya

    2013-01-01

    Full Text Available A 9-day-old male baby was hospitalized after his birth due to some swells under the skin. The hard consistency nodules observed under the skin all over the body of the patient were of different size, and presented lesions, among which the biggest was 1 × 1 cm. No lesions were observed on the fingers. By superficial ultrasonography, multiple isoechoic hypoechoic lesions were observed among the muscle plan. In thoracolumbar magnetic resonance imaging, multiple massif lesions retaining peripheral contrast (the biggest was 1.7 × 1.4 cm large had been observed under the skin muscle plans, between the muscles of the extremities. The biopsy was positive for smooth muscle actin, but negative for desmin, S100, and CD34. These findings were diagnosed as infantile digital fibromatosis (IDF (inclusion body fibromatosis. The case was presented with an objective to illustrate and remind that IDF can be observed in babies without finger involvement.

  8. Functional organization of cytoplasmic inclusion bodies in cells infected by respiratory syncytial virus.

    Science.gov (United States)

    Rincheval, Vincent; Lelek, Mickael; Gault, Elyanne; Bouillier, Camille; Sitterlin, Delphine; Blouquit-Laye, Sabine; Galloux, Marie; Zimmer, Christophe; Eleouet, Jean-François; Rameix-Welti, Marie-Anne

    2017-09-15

    Infection of cells by respiratory syncytial virus induces the formation of cytoplasmic inclusion bodies (IBs) where all the components of the viral RNA polymerase complex are concentrated. However, the exact organization and function of these IBs remain unclear. In this study, we use conventional and super-resolution imaging to dissect the internal structure of IBs. We observe that newly synthetized viral mRNA and the viral transcription anti-terminator M2-1 concentrate in IB sub-compartments, which we term "IB-associated granules" (IBAGs). In contrast, viral genomic RNA, the nucleoprotein, the L polymerase and its cofactor P are excluded from IBAGs. Live imaging reveals that IBAGs are highly dynamic structures. Our data show that IBs are the main site of viral RNA synthesis. They further suggest that shortly after synthesis in IBs, viral mRNAs and M2-1 transiently concentrate in IBAGs before reaching the cytosol and suggest a novel post-transcriptional function for M2-1.Respiratory syncytial virus (RSV) induces formation of inclusion bodies (IBs) sheltering viral RNA synthesis. Here, Rincheval et al. identify highly dynamic IB-associated granules (IBAGs) that accumulate newly synthetized viral mRNA and the viral M2-1 protein but exclude viral genomic RNA and RNA polymerase complexes.

  9. Expression, characterization of a novel nitrilase PpL19 from Pseudomonas psychrotolerans with S-selectivity toward mandelonitrile present in active inclusion bodies.

    Science.gov (United States)

    Sun, Huihui; Gao, Wenyuan; Wang, Hualei; Wei, Dongzhi

    2016-03-01

    To identify a novel nitrilase with S-selectivity toward mandelonitrile that can produce (S)-mandelic acid in one step. A novel nitrilase PpL19 from Pseudomonas psychrotolerans L19 was discovered by genome mining. It showed S-selectivity with an enantiomeric excess of 52.7 % when used to hydrolyse (R, S)-mandelonitrile. No byproduct was observed. PpL19 was overexpressed in Escherichia coli BL21 (DE3) and formed inclusion bodies that were active toward mandelonitrile and stable across a broad range of temperature and pH. In addition, PpL19 hydrolysed nitriles with diverse structures; arylacetonitriles were the optimal substrates. Homology modelling and docking studies of both enantiomers of mandelonitrile in the active site of nitrilase PpL19 shed light on the enantioselectivity. A novel nitrilase PpL19 from P. psychrotolerans L19 was mined and distinguished from other nitrilases as it was expressed as an active inclusion body and showed S-selectivity toward mandelonitrile.

  10. Hepato nephropathology associated with inclusion body hepatitis complicated with citrinin mycotoxicosis in a broiler farm

    Directory of Open Access Journals (Sweden)

    Asok Kumar Mariappan

    2018-02-01

    Full Text Available Aim: Mortality in a broiler chicken farm was investigated for identifying the cause of mortality. Materials and Methods: A broiler farm with a population of 16000 succumbed to a disease outbreak. Clinical signs, vaccination history and mortality, were recorded. Necropsy examination and microscopic examination were carried out along with toxicological and molecular studies. Results: The clinical signs in the affected broiler birds were of non-specific nature with a total mortality of 26.39%. Postmortem examination and microscopical findings revealed hepatitis with basophilic intranuclear inclusion, splenitis, myocarditis, and nephritis. Glomerulonephritis was the prominent renal pathology recorded in this study. Polymerase chain reaction test confirmed the presence of fowl adenovirus (FAdV genome in the target organs, and toxicological examination by thin-layer chromatography revealed the presence of a toxic level of citrinin in the feed samples. Conclusion: Based on various diagnostic investigations, the mortality in the flock was attributed to inclusion body hepatitis (IBH complicated with citrinin mycotoxicosis. Thus, apart from liver pathology which occurs in a classical IBH cases, glomerulonephritis too occurs which are also a prominent finding which pathologists often miss. Thus, kidneys should also be examined histologically to assess the microscopic tissue alterations in poultry suspected for IBH along with a mycotoxicological analysis of feed. This will definitely throw light on the synergistic pathology elicited and exhibited by FAdV and mycotoxins in the poultry.

  11. Asymptomatic hyper-creatine-kinase-emia as sole manifestation of inclusion body myositis

    Directory of Open Access Journals (Sweden)

    Josef Finsterer

    2013-06-01

    Full Text Available Sporadic inclusion body myositis (sIBM usually manifests with painless weakness of the hand, finger and hip flexors. Absence of symptoms or signs, but mild hyper-CK-emia as the sole manifestation of IBM, has not been reported. We report the case of a 73-year-old male who presented with asymptomatic recurrent hyper-CK-emia ranging from 200 to 1324 U/L (n<171 U/L, since 10 years. Clinical neurologic investigation, nerve conduction studies and EMG were non-informative. Muscle biopsy surprisingly revealed sIBM. sIBM may be asymptomatic and may manifest with hyper-CK-emia exclusively. So, it has to be included in the differential diagnoses of asymptomatic hyper-CK-emia.

  12. Detection of novel divergent arenaviruses in boid snakes with inclusion body disease in The Netherlands.

    Science.gov (United States)

    Bodewes, R; Kik, M J L; Raj, V Stalin; Schapendonk, C M E; Haagmans, B L; Smits, S L; Osterhaus, A D M E

    2013-06-01

    Arenaviruses are bi-segmented negative-stranded RNA viruses, which were until recently only detected in rodents and humans. Now highly divergent arenaviruses have been identified in boid snakes with inclusion body disease (IBD). Here, we describe the identification of a new species and variants of the highly divergent arenaviruses, which were detected in tissues of captive boid snakes with IBD in The Netherlands by next-generation sequencing. Phylogenetic analysis of the complete sequence of the open reading frames of the four predicted proteins of one of the detected viruses revealed that this virus was most closely related to the recently identified Golden Gate virus, while considerable sequence differences were observed between the highly divergent arenaviruses detected in this study. These findings add to the recent identification of the highly divergent arenaviruses in boid snakes with IBD in the United States and indicate that these viruses also circulate among boid snakes in Europe.

  13. Efficient inclusion body processing using chemical extraction and high gradient magnetic fishing

    DEFF Research Database (Denmark)

    Heebøll-Nielsen, Anders; Choe, W.S.; Middelberg, A.P.J.

    2003-01-01

    of the product-loaded supports with the aid of high gradient magnetic fields. The manufacture and testing of two types of micron-sized nonporous superparamagnetic metal chelator particles derivatized with iminodiacetic acid is described. In small-scale adsorption studies conducted with a hexahistidine tagged...... at a 60-fold increased scale using the high gradient magnetic fishing (HGMF) system to collect loaded Cu2+-chelator particles following batch adsorption of L1. Over 70% of the initial Ll present was recovered within the HGMF rig in a highly clarified form in two batch elution cycles with an overall......In this study we introduce a radical new approach for the recovery of proteins expressed in the form of inclusion bodies, involving W chemical extraction from the host cells, (ii) adsorptive capture of the target protein onto small magnetic adsorbents, and (iii) subsequent rapid collection...

  14. Dynamic phosphorylation of Ebola virus VP30 in NP-induced inclusion bodies.

    Science.gov (United States)

    Lier, Clemens; Becker, Stephan; Biedenkopf, Nadine

    2017-12-01

    Zaire Ebolavirus (EBOV) causes a severe feverish disease with high case fatality rates. Transcription of EBOV is dependent on the activity of the nucleocapsid protein VP30 which represents an essential viral transcription factor. Activity of VP30 is regulated via phosphorylation at six N-terminal serine residues. Recent data demonstrated that dynamic phosphorylation and dephosphorylation of serine residue 29 is essential for transcriptional support activity of VP30. To analyze the spatio/temporal dynamics of VP30 phosphorylation, we generated a peptide antibody recognizing specifically VP30 phosphorylated at serine 29. Using this antibody we could demonstrate that (i) the majority of VP30 molecules in EBOV-infected cells is dephosphorylated at the crucial position serine 29, (ii) both, VP30 phosphorylation and dephosphorylation take place in viral inclusion bodies that are induced by the nucleoprotein NP and (iii) NP influences the phosphorylation state of VP30. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Inclusion bodies of recombinant Epstein-Barr virus capsid antigen p18 as potential immobilized antigens in enzyme immunoassays for detection of nasopharyngeal carcinoma.

    Science.gov (United States)

    Lim, Chun Shen; Goh, Siang Ling; Kariapper, Leena; Krishnan, Gopala; Lim, Yat-Yuen; Ng, Ching Ching

    2015-08-25

    Development of indirect enzyme-linked immunosorbent assays (ELISAs) often utilizes synthetic peptides or recombinant proteins from Escherichia coli as immobilized antigens. Because inclusion bodies (IBs) formed during recombinant protein expression in E. coli are commonly thought as misfolded aggregates, only refolded proteins from IBs are used to develop new or in-house diagnostic assays. However, the promising utilities of IBs as nanomaterials and immobilized enzymes as shown in recent studies have led us to explore the potential use of IBs of recombinant Epstein-Barr virus viral capsid antigen p18 (VCA p18) as immobilized antigens in ELISAs for serologic detection of nasopharyngeal carcinoma (NPC). Thioredoxin fusion VCA p18 (VCA-Trx) and IBs of VCA p18 without fusion tags (VCA-IBs) were purified from E. coli. The diagnostic performances of IgG/VCA-IBs, IgG/VCA-Denat-IBs (using VCA-IBs coated in 8mol/l urea), IgG/VCA-Trx, and IgG/VCA-Peptide assays were compared by screening 100 NPC case-control pairs. The IgG/VCA-Denat-IBs assay showed the best area under the receiver operating characteristic curve (AUC: 0.802; p<0.05), while the AUCs for the IgG/VCA-IBs, IgG/VCA-Trx, and IgG/VCA-Peptide assays were comparable (AUC: 0.740, 0.727, and 0.741, respectively). We improved the diagnostic performance of the ELISA significantly using IBs of recombinant VCA p18. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Cooperation between two ClpB isoforms enhances the recovery of the recombinant {beta}-galactosidase from inclusion bodies

    Energy Technology Data Exchange (ETDEWEB)

    Guenther, Izabela [Department of Biochemistry, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk (Poland); Zolkiewski, Michal [Department of Biochemistry, Kansas State University, Manhattan, KS 66506 (United States); Kedzierska-Mieszkowska, Sabina, E-mail: kedzie@biotech.ug.gda.pl [Department of Biochemistry, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk (Poland)

    2012-10-05

    Highlights: Black-Right-Pointing-Pointer An important role of synergistic cooperation between the two ClpB isoforms. Black-Right-Pointing-Pointer Both ClpB isoforms are associated with IBs of {beta}-galactosidase. Black-Right-Pointing-Pointer ClpB is a key chaperone in IB protein release. -- Abstract: Bacterial ClpB is a molecular chaperone that solubilizes and reactivates aggregated proteins in cooperation with the DnaK chaperone system. The mechanism of protein disaggregation mediated by ClpB is linked to translocation of substrates through the central channel within the ring-hexameric structure of ClpB. Two isoforms of ClpB are produced in vivo: the full-length ClpB95 and the truncated ClpB80 (ClpB{Delta}N), which does not contain the N-terminal domain. The functional specificity of the two ClpB isoforms and the biological role of the N-terminal domain are still not fully understood. Recently, it has been demonstrated that ClpB may achieve its full potential as an aggregate-reactivating chaperone through the functional interaction and synergistic cooperation of its two isoforms. It has been found that the most efficient resolubilization and reactivation of stress-aggregated proteins occurred in the presence of both ClpB95 and ClpB80. In this work, we asked if the two ClpB isoforms functionally cooperate in the solubilization and reactivation of proteins from insoluble inclusion bodies (IBs) in Escherichia coli cells. Using the model {beta}-galactosidase fusion protein (VP1LAC), we found that solubilization and reactivation of enzymes entrapped in IBs occurred more efficiently in the presence of ClpB95 with ClpB80 than with either ClpB95 or ClpB80 alone. The two isoforms of ClpB chaperone acting together enhanced the solubility and enzymatic activity of {beta}-galactosidase sequestered into IBs. Both ClpB isoforms were associated with IBs of {beta}-galactosidase, what demonstrates their affinity to this type of aggregates. These results demonstrate a synergistic

  17. A monoclonal antibody to inclusion body disease of cranes virus enabling specific immunohistochemistry and competitive ELISA

    Science.gov (United States)

    Letchworth, G.J.; Fishel, J.R.; Hansen, W.R.

    1997-01-01

    Inclusion body disease of cranes (IBDC) herpesvirus kills some infected cranes and persists in convalescent animals. To enable further study and rapid identification of carrier animals, we developed a monoclonal antibody (MAb) to IBDC virus and used it in immunohistochemistry and a competitive enzyme-linked immunosorbent assay (ELISA). We used conventional techniques to make murine MAbs directed against IBDC virus purified from infected duck embryo cells. Hybridomas reacting in an ELISA with IBDC virus but not uninfected duck embryo cells were characterized by radioimmunoprecipitation, in situ immunohistochemistry, and competitive ELISA with neutralizing and nonneutralizing crane sera. MAb 2C11 immunoprecipitated 59-, 61-, and 110-kD proteins from IBDC virus-infected but not uninfected cells and stained glutaraldehyde-fixed IBDC virus plaques but not surrounding uninfected duck embryo cells in vitro. Antibody 2C11 did not react with duck embryo cells infected with falcon herpesvirus, psittacine herpesvirus, infectious laryngotracheitis, pigeon herpesvirus, or duck plague virus. A competitive ELISA using antibody 2C11 identified most sera that were positive in the neutralization test. This antibody will be useful in further characterizing IBDC virus, its pathogenesis, and its natural history.

  18. Capillary gel electrophoresis for the quantification and purity determination of recombinant proteins in inclusion bodies.

    Science.gov (United States)

    Espinosa-de la Garza, Carlos E; Perdomo-Abúndez, Francisco C; Campos-García, Víctor R; Pérez, Néstor O; Flores-Ortiz, Luis F; Medina-Rivero, Emilio

    2013-09-01

    In this work, a high-resolution CGE method for quantification and purity determination of recombinant proteins was developed, involving a single-component inclusion bodies (IBs) solubilization solution. Different recombinant proteins expressed as IBs were used to show method capabilities, using recombinant interferon-β 1b as the model protein for method validation. Method linearity was verified in the range from 0.05 to 0.40 mg/mL and a determination coefficient (r(2) ) of 0.99 was obtained. The LOQs and LODs were 0.018 and 0.006 mg/mL, respectively. RSD for protein content repeatability test was 2.29%. In addition, RSD for protein purity repeatability test was 4.24%. Method accuracy was higher than 90%. Specificity was confirmed, as the method was able to separate recombinant interferon-β 1b monomer from other aggregates and impurities. Sample content and purity was demonstrated to be stable for up to 48 h. Overall, this method is suitable for the analysis of recombinant proteins in IBs according to the attributes established on the International Conference for Harmonization guidelines. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. An efficient procedure for the expression and purification of HIV-1 protease from inclusion bodies.

    Science.gov (United States)

    Nguyen, Hong-Loan Thi; Nguyen, Thuy Thi; Vu, Quy Thi; Le, Hang Thi; Pham, Yen; Trinh, Phuong Le; Bui, Thuan Phuong; Phan, Tuan-Nghia

    2015-12-01

    Several studies have focused on HIV-1 protease for developing drugs for treating AIDS. Recombinant HIV-1 protease is used to screen new drugs from synthetic compounds or natural substances. However, large-scale expression and purification of this enzyme is difficult mainly because of its low expression and solubility. In this study, we constructed 9 recombinant plasmids containing a sequence encoding HIV-1 protease along with different fusion tags and examined the expression of the enzyme from these plasmids. Of the 9 plasmids, pET32a(+) plasmid containing the HIV-1 protease-encoding sequence along with sequences encoding an autocleavage site GTVSFNF at the N-terminus and TEV plus 6× His tag at the C-terminus showed the highest expression of the enzyme and was selected for further analysis. The recombinant protein was isolated from inclusion bodies by using 2 tandem Q- and Ni-Sepharose columns. SDS-PAGE of the obtained HIV-1 protease produced a single band of approximately 13 kDa. The enzyme was recovered efficiently (4 mg protein/L of cell culture) and had high specific activity of 1190 nmol min(-1) mg(-1) at an optimal pH of 4.7 and optimal temperature of 37 °C. This procedure for expressing and purifying HIV-1 protease is now being scaled up to produce the enzyme on a large scale for its application. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Chaperone-mediated autophagy components are upregulated in sporadic inclusion-body myositis muscle fibres.

    Science.gov (United States)

    Cacciottolo, M; Nogalska, A; D'Agostino, C; Engel, W K; Askanas, V

    2013-12-01

    Sporadic inclusion-body myositis (s-IBM) is an age-associated degenerative muscle disease. Characteristic features are muscle-fibre vacuolization and intramuscle-fibre accumulations of multiprotein aggregates, which may result from the demonstrated impairments of the 26S proteasome and autophagy. Chaperone-mediated autophagy (CMA) is a selective form of lysosomal degradation targeting proteins carrying the KFERQ motif. Lysosome-associated membrane protein type 2A (LAMP2A) and the heat-shock cognate protein 70 (Hsc70) constitute specific CMA components. Neither CMA components nor CMA activity has been studied in normal or disease human muscle, to our knowledge. We studied CMA components by immunocytochemistry, immunoblots, real-time PCR and immunoprecipitation in: (a) 16 s-IBM, nine aged-matched normal and nine disease control muscle biopsies; and (b) cultured human muscle fibres (CHMFs) with experimentally inhibited activities of either the 26S proteasome or autophagy. Compared with age-matched controls, in s-IBM muscle, LAMP2A and Hsc70 were on a given transverse section accumulated as aggregates in approximately 5% of muscle fibres, where they (a) colocalized with each other and α-synuclein (α-syn), a CMA-targeted protein; and (b) were bound to each other and to α-syn by immunoprecipitation. By immunoblots, LAMP2A was increased sevenfold P pathogenic aspect in s-IBM. © 2013 British Neuropathological Society.

  1. An Amino Acid of Human Parainfluenza Virus Type 3 Nucleoprotein Is Critical for Template Function and Cytoplasmic Inclusion Body Formation

    Science.gov (United States)

    Zhang, Shengwei; Chen, Longyun; Zhang, Guangyuan; Yan, Qin; Yang, Xiaodan; Ding, Binbin; Tang, Qiaopeng; Sun, Shengjun; Hu, Zhulong

    2013-01-01

    The nucleoprotein (N) and phosphoprotein (P) interaction of nonsegmented negative-strand RNA viruses is essential for viral replication; this includes N0-P (N0, free of RNA) interaction and the interaction of N-RNA with P. The precise site(s) within N that mediates the N-P interaction and the detailed regulating mechanism, however, are less clear. Using a human parainfluenza virus type 3 (HPIV3) minigenome assay, we found that an N mutant (NL478A) did not support reporter gene expression. Using in vivo and in vitro coimmunoprecipitation, we found that NL478A maintains the ability to form NL478A0-P, to self-assemble, and to form NL478A-RNA but that NL478A-RNA does not interact with P. Using an immunofluorescence assay, we found that N-P interaction provides the minimal requirement for the formation of cytoplasmic inclusion bodies, which contain viral RNA, N, P, and polymerase in HPIV3-infected cells. NL478A was unable to form inclusion bodies when coexpressed with P, but the presence of N rescued the ability of NL478A to form inclusion bodies and the transcriptional function of NL478A, thereby suggesting that hetero-oligomers formed by N and NL478A are functional and competent to form inclusion bodies. Furthermore, we found that NL478A is also defective in virus growth. To our knowledge, we are the first to use a paramyxovirus to identify a precise amino acid within N that is critical for N-RNA and P interaction but not for N0-P interaction for the formation of inclusion bodies, which appear to be bona fide sites of RNA synthesis. PMID:24027324

  2. Dynamic JUNQ inclusion bodies are asymmetrically inherited in mammalian cell lines through the asymmetric partitioning of vimentin.

    Science.gov (United States)

    Ogrodnik, Mikołaj; Salmonowicz, Hanna; Brown, Rachel; Turkowska, Joanna; Średniawa, Władysław; Pattabiraman, Sundararaghavan; Amen, Triana; Abraham, Ayelet-chen; Eichler, Noam; Lyakhovetsky, Roman; Kaganovich, Daniel

    2014-06-03

    Aging is associated with the accumulation of several types of damage: in particular, damage to the proteome. Recent work points to a conserved replicative rejuvenation mechanism that works by preventing the inheritance of damaged and misfolded proteins by specific cells during division. Asymmetric inheritance of misfolded and aggregated proteins has been shown in bacteria and yeast, but relatively little evidence exists for a similar mechanism in mammalian cells. Here, we demonstrate, using long-term 4D imaging, that the vimentin intermediate filament establishes mitotic polarity in mammalian cell lines and mediates the asymmetric partitioning of damaged proteins. We show that mammalian JUNQ inclusion bodies containing soluble misfolded proteins are inherited asymmetrically, similarly to JUNQ quality-control inclusions observed in yeast. Mammalian IPOD-like inclusion bodies, meanwhile, are not always inherited by the same cell as the JUNQ. Our study suggests that the mammalian cytoskeleton and intermediate filaments provide the physical scaffold for asymmetric inheritance of dynamic quality-control JUNQ inclusions. Mammalian IPOD inclusions containing amyloidogenic proteins are not partitioned as effectively during mitosis as their counterparts in yeast. These findings provide a valuable mechanistic basis for studying the process of asymmetric inheritance in mammalian cells, including cells potentially undergoing polar divisions, such as differentiating stem cells and cancer cells.

  3. Calcium dysregulation, functional calpainopathy, and endoplasmic reticulum stress in sporadic inclusion body myositis.

    Science.gov (United States)

    Amici, David R; Pinal-Fernandez, Iago; Mázala, Davi A G; Lloyd, Thomas E; Corse, Andrea M; Christopher-Stine, Lisa; Mammen, Andrew L; Chin, Eva R

    2017-03-22

    Sporadic inclusion body myositis (IBM) is the most common primary myopathy in the elderly, but its pathoetiology is still unclear. Perturbed myocellular calcium (Ca 2+ ) homeostasis can exacerbate many of the factors proposed to mediate muscle degeneration in IBM, such as mitochondrial dysfunction, protein aggregation, and endoplasmic reticulum stress. Ca 2+ dysregulation may plausibly be initiated in IBM by immune-mediated membrane damage and/or abnormally accumulating proteins, but no studies to date have investigated Ca 2+ regulation in IBM patients. We first investigated protein expression via immunoblot in muscle biopsies from IBM, dermatomyositis, and non-myositis control patients, identifying several differentially expressed Ca 2+ -regulatory proteins in IBM. Next, we investigated the Ca 2+ -signaling transcriptome by RNA-seq, finding 54 of 183 (29.5%) genes from an unbiased list differentially expressed in IBM vs. controls. Using an established statistical approach to relate genes with causal transcription networks, Ca 2+ abundance was considered a significant upstream regulator of observed whole-transcriptome changes. Post-hoc analyses of Ca 2+ -regulatory mRNA and protein data indicated a lower protein to transcript ratio in IBM vs. controls, which we hypothesized may relate to increased Ca 2+ -dependent proteolysis and decreased protein translation. Supporting this hypothesis, we observed robust (4-fold) elevation in the autolytic activation of a Ca 2+ -activated protease, calpain-1, as well as increased signaling for translational attenuation (eIF2a phosphorylation) downstream of the unfolded protein response. Finally, in IBM samples we observed mRNA and protein under-expression of calpain-3, the skeletal muscle-specific calpain, which broadly supports proper Ca 2+ homeostasis. Together, these data provide novel insight into mechanisms by which intracellular Ca 2+ regulation is perturbed in IBM and offer evidence of pathological downstream effects.

  4. Overlapping features of polymyositis and inclusion body myositis in HIV-infected patients

    Science.gov (United States)

    Lloyd, Thomas E.; Pinal-Fernandez, Iago; Michelle, E. Harlan; Christopher-Stine, Lisa; Pak, Katherine; Sacktor, Ned

    2017-01-01

    Objective: To characterize patients with myositis with HIV infection. Methods: All HIV-positive patients with myositis seen at the Johns Hopkins Myositis Center from 2003 to 2013 were included in this case series. Muscle biopsy features, weakness pattern, serum creatine kinase (CK) level, and anti–nucleotidase 1A (NT5C1A) status of HIV-positive patients with myositis were assessed. Results: Eleven of 1,562 (0.7%) patients with myositis were HIV-positive. Myositis was the presenting feature of HIV infection in 3 patients. Eight of 11 patients had weakness onset at age 45 years or less. The mean time from the onset of weakness to the diagnosis of myositis was 3.6 years (SD 3.2 years). The mean of the highest measured CK levels was 2,796 IU/L (SD 1,592 IU/L). On muscle biopsy, 9 of 10 (90%) had endomysial inflammation, 7 of 10 (70%) had rimmed vacuoles, and none had perifascicular atrophy. Seven of 11 (64%) patients were anti-NT5C1A-positive. Upon presentation, all had proximal and distal weakness. Five of 6 (83%) patients followed 1 year or longer on immunosuppressive therapy had improved proximal muscle strength. However, each eventually developed weakness primarily affecting wrist flexors, finger flexors, knee extensors, or ankle dorsiflexors. Conclusions: HIV-positive patients with myositis may present with some characteristic polymyositis features including young age at onset, very high CK levels, or proximal weakness that improves with treatment. However, all HIV-positive patients with myositis eventually develop features most consistent with inclusion body myositis, including finger and wrist flexor weakness, rimmed vacuoles on biopsy, or anti-NT5C1A autoantibodies. PMID:28283597

  5. Intracellular organisation of polyhydroxyalkanoate inclusion bodies: a role for small angle neutron scattering?

    International Nuclear Information System (INIS)

    Foster, L.J.R.; Holden, P.J.; Garvey, C.J.; Russell, R.A.; Stone, D.J.M.

    2003-01-01

    Full text: Polyhydroxyalkanoates (PHAs) are a diverse family of bacterially produced biopolyesters. Their biodegradability, and in some cases biocompatibility, suggest applications ranging from bioplastics to biomedical implantation devices. Despite extensive interest in their production and potential applications, little is known about their intracellular organisation. Microbial PHAs are synthesised by microorganisms under conditions of nutrient stress and can comprise up to 90% of the dry cell mass. The formation and organisation of these PHA inclusion bodies requires clarification. Such investigations have important implications for the biotechnological production of PHAs in microbes and other organisms, for downstream processing and in vitro precision polymerisation. Morphological and biochemical evidence supports two different models for the intracellular organisation of PHAs. Steinbuchel and coworkers propose a simple model of amorphous PHA enclosed by a single protein membrane consisting of structural proteins (PHAsins) and enzymes responsible for synthesis and degradation. In contrast, Fuller and coworkers have theorised a more complex system of PHA encompassed by a PHAsin bilayer separated by phospholipid. The polymerase and depolymerase enzymes are proposed to be associated with an incomplete inner PHAsin layer. It may be that such models are genera or species specific, since both proposals were derived from research on different species producing different types of PHA. Our initial investigations have focussed on in vivo deuteration of polyhydroxyoctanoate, produced by Pseudomonas oleovorans, both in fermentation on natural and deuterated substrates and during Small Angle Neutron Scattering by whole cells using AUSANS. The nature of the structural questions and our preliminary findings including contrast variation data will be discussed

  6. Intravenous immune globulin in hereditary inclusion body myopathy: a pilot study

    Directory of Open Access Journals (Sweden)

    Dorward Heidi

    2007-01-01

    Full Text Available Abstract Background Hereditary Inclusion Body Myopathy (HIBM is an autosomal recessive, adult onset, non-inflammatory neuromuscular disorder with no effective treatment. The causative gene, GNE, codes for UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, which catalyzes the first two reactions in the synthesis of sialic acid. Reduced sialylation of muscle glycoproteins, such as α-dystroglycan and neural cell adhesion molecule (NCAM, has been reported in HIBM. Methods We treated 4 HIBM patients with intravenous immune globulin (IVIG, in order to provide sialic acid, because IgG contains 8 μmol of sialic acid/g. IVIG was infused as a loading dose of 1 g/kg on two consecutive days followed by 3 doses of 400 mg/kg at weekly intervals. Results For all four patients, mean quadriceps strength improved from 19.0 kg at baseline to 23.2 kg (+22% directly after IVIG loading to 25.6 kg (+35% at the end of the study. Mean shoulder strength improved from 4.1 kg at baseline to 5.9 kg (+44% directly after IVIG loading to 6.0 kg (+46% at the end of the study. The composite improvement for 8 other muscle groups was 5% after the initial loading and 19% by the end of the study. Esophageal motility and lingual strength improved in the patients with abnormal barium swallows. Objective measures of functional improvement gave variable results, but the patients experienced improvements in daily activities that they considered clinically significant. Immunohistochemical staining and immunoblotting of muscle biopsies for α-dystroglycan and NCAM did not provide consistent evidence for increased sialylation after IVIG treatment. Side effects were limited to transient headaches and vomiting. Conclusion The mild benefits in muscle strength experienced by HIBM patients after IVIG treatment may be related to the provision of sialic acid supplied by IVIG. Other sources of sialic acid are being explored as treatment options for HIBM.

  7. Avian Adenoviruses Infections with Special Attention to Inclusion Body Hepatitis/ Hydropericardium Syndrome and Egg Drop Syndrome

    Directory of Open Access Journals (Sweden)

    Hafez Mohamed Hafez*

    2011-04-01

    Full Text Available The first avian adenovirus (AAV associated with clinical disease was isolated from an outbreak of respiratory disease in quail in 1950 (Olson, 1950. Since that time, AAVs have been found in all types and breeds of chickens and from a variety of other avian species. The infections may be asymptomatic or associated with several clinical and pathological conditions. Vertical transmission via the egg is the most common way of transmission. Also horizontal transmission through faeces, contaminated egg trays, crates and trucks play a role in the infection route. Studies have demonstrated the presence of antibodies in healthy poultry, and viruses have been isolated from normal birds. Avian adenoviruses in chickens are the etiological agents of 2 diseases known as inclusion body hepatitis (IBH and hydropericardium syndrome (HP. In some cases each condition is observed separately, however, recently the 2 conditions have frequently been observed as a single entity; therefore, the name hepatitis hydropericardium has been widely used to describe the pathologic condition. The syndrome is an acute disease of young chickens associated with anemia, haemorrhagic disorders, hydropericardium and high mortality. Egg-Drop-Syndrome (EDS is caused also by an adenovirus. The disease is characterised by a severe drop in egg production as well as the production of shell-less, thin-shelled, discoloured or misshapen eggs in apparently healthy birds. Ducks and geese are the natural host of the EDS virus. It was first described in chickens in the 1970s and spread to several countries world wide. The birds usually do not show any other signs of disease, and mortality is not expected. There is no specific treatment of the AAV infections. Active immunization by vaccination using an inactivated is wide spread.

  8. Sleep disordered breathing in a cohort of patients with sporadic inclusion body myositis.

    Science.gov (United States)

    Della Marca, Giacomo; Sancricca, Cristina; Losurdo, Anna; Di Blasi, Chiara; De Fino, Chiara; Morosetti, Roberta; Broccolini, Aldobrando; Testani, Elisa; Scarano, Emanuele; Servidei, Serenella; Mirabella, Massimiliano

    2013-08-01

    The aims of the study were: (1) to evaluate subjective sleep quality and daytime sleepiness in patients affected by sporadic inclusion-body myositis (IBM); (2) to define the sleep and sleep-related respiratory pattern in IBM patients. Thirteen consecutive adult patients affected by definite IBM were enrolled, six women and seven men, mean age 66.2 ± 11.1 years (range: 50-80). Diagnosis was based on clinical and muscle biopsy studies. All patients underwent subjective sleep evaluation (Pittsburgh Sleep Quality Index, PSQI and Epworth Sleepiness Scale, ESS), oro-pharingo-esophageal scintigraphy, pulmonary function tests, psychometric measures, anatomic evaluation of upper airways, and laboratory-based polysomnography. Findings in IBM patients were compared to those obtained from a control group of 25 healthy subjects (13 men and 12 women, mean age 61.9 ± 8.6 years). Disease duration was >10 years in all. Mean IBM severity score was 28.8 ± 5.4 (range 18-36). Dysphagia was present in 10 patients. Nine patients had PSQI scores ≥ 5; patients had higher mean PSQI score (IBM: 7.2 ± 4.7, CONTROLS: 2.76 ± 1.45, p=0.005); one patient (and no controls) had EES>9. Polysomnography showed that IBM patients, compared to controls, had lower sleep efficiency (IBM: 78.8 ± 12.0%, 94.0 ± 4.5%, p<0.001), more awakenings (IBM: 11.9 ± 11.0, CONTROLS: 5.2 ± 7.5, p=0.009) and increased nocturnal time awake (IBM: 121.2 ± 82.0 min., 46.12 ± 28.8 min., p=0.001). Seven Patients (and no controls) had polysomnographic findings consistent with sleep disordered breathing (SDB). Data suggest that sleep disruption, and in particular SDB, might be highly prevalent in IBM. Data indicate that IBM patients have poor sleep and high prevalence of SDB. Copyright © 2013 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.

  9. Alternative preparation of inclusion bodies excludes interfering non-protein contaminants and improves the yield of recombinant proinsulin.

    Science.gov (United States)

    Mackin, Robert B

    2014-01-01

    The goal of simple, high-yield expression and purification of recombinant human proinsulin has proven to be a considerable challenge. First, proinsulin forms inclusion bodies during bacterial expression. While this phenomenon can be exploited as a capture step, conventionally prepared inclusion bodies contain significant amounts of non-protein contaminants that interfere with subsequent chromatographic purification. Second, the proinsulin molecules within the inclusion bodies are incorrectly folded, and likely cross-linked to one another, making it difficult to quantify the amount of expressed proinsulin. Third, proinsulin is an intermediate between the initial product of ribosomal translation (preproinsulin) and the final product secreted by pancreatic beta cells (insulin). Therefore, to be efficiently produced in bacteria, it must be produced as an N-terminally extended fusion protein, which has to be converted to authentic proinsulin during the purification scheme. To address all three of these problems, while simultaneously streamlining the procedure and increasing the yield of recombinant proinsulin, we have made three substantive modifications to our previous method for producing proinsulin:.•Conditions for the preparation of inclusion bodies have been altered so contaminants that interfere with semi-preparative reversed-phase chromatography are excluded while the proinsulin fusion protein is retained at high yield.•Aliquots are taken following important steps in the procedure and the quantity of proinsulin-related polypeptide in the sample is compared to the amount present prior to that step.•Final purification is performed using a silica-based reversed-phase matrix in place of a polystyrene-divinylbenzene-based matrix.

  10. OUTPATIENT PHYSICAL THERAPY EVALUATION AND TREATMENT OF A PATIENT DIAGNOSED WITH SPORADIC INCLUSION BODY MYOSITIS: A CASE STUDY

    Directory of Open Access Journals (Sweden)

    Tyler Harrigfeld

    2017-08-01

    Full Text Available Background: Sporadic inclusion body myositis is an autoimmune and degenerative disorder of skeletal muscle that affects people at random. It most commonly begins as progressive weakness and atrophy of lower extremity musculature, beginning with the proximal leg. These impairments in body structure adversely affect the performance of functional activities and mobility, resulting in a progressive decrease in independence and participation both at home and in the community. Physical therapy attempts to minimize these effects through educational and procedural interventions focused on treating impairments and limitations. The purpose of this case study was to provide a description of the physical therapy management of a patient diagnosed with sporadic inclusion body myositis. Case Summary: The patient was a 66-year-old male who was diagnosed with sporadic inclusion body myositis with a chief complaint of weakness and fall risk. He presented with generalized lower extremity weakness and atrophy of bilateral quadriceps, as well as impaired balance and increasing fatigue with activity. Therapeutic exercise, home exercise program, balance, gait, and stair training were delivered to address these impairments. Patient outcomes showed improvement in balance and safety with functional activities. Discussion: The patient was seen for seven visits that were 45 – 60 minutes in length, over a five-week period. The patient made subjective reports of improvement in functional activities and balance; however many objective outcome measures could not be reassessed. There is a need for further research on this population to determine the effectiveness and parameters of physical therapy interventions. Conclusion: Physical therapy may have helped improve balance as well as subjective reports from the patient of increased feeling of confidence while navigating stairs.

  11. Efficient system of artificial oil bodies for functional expression and purification of recombinant nattokinase in Escherichia coli.

    Science.gov (United States)

    Chiang, Chung-Jen; Chen, Hong-Chen; Chao, Yun-Peng; Tzen, Jason T C

    2005-06-15

    Nattokinase, a serine protease, and pronattokinase, when expressed in Escherichia coli, formed insoluble aggregates without enzymatic activity. For functional expression and purification, nattokinase or pronattokinase was first overexpressed in E. coli as an insoluble recombinant protein linked to the C terminus of oleosin, a structural protein of seed oil bodies, by an intein fragment. Artificial oil bodies were reconstituted with triacylglycerol, phospholipid, and the insoluble recombinant protein thus formed. Soluble nattokinase was subsequently released through self-splicing of intein induced by temperature alteration, with the remaining oleosin-intein residing in oil bodies and the leading propeptide of pronattokinase, when present, spontaneously cleaved in the process. Active nattokinase with fibrinolytic activity was harvested by concentrating the supernatant. Nattokinase released from oleosin-intein-pronattokinase exhibited 5 times higher activity than that released from oleosin-intein-nattokinase, although the production yields were similar in both cases. Furthermore, active nattokinase could be harvested in the same system by fusing pronattokinase to the N terminus of oleosin via a different intein linker, with self-splicing induced by 1,4-dithiothreitol. These results have shown a great potential of this system for bacterial expression and purification of functional recombinant proteins.

  12. On the reconstruction of inclusions in a heat conductive body from dynamical boundary data over a finite time interval

    International Nuclear Information System (INIS)

    Ikehata, Masaru; Kawashita, Mishio

    2010-01-01

    The enclosure method was originally introduced for inverse problems concerning non-destructive evaluation governed by elliptic equations. It was developed as one of the useful approaches in inverse problems and applied for various equations. In this paper, an application of the enclosure method to an inverse initial boundary value problem for a parabolic equation with a discontinuous coefficient is given. A simple method to extract the depth of unknown inclusions in a heat conductive body from a single set of the temperature and heat flux on the boundary observed over a finite time interval is introduced. Other related results with infinitely many data are also reported. One of them gives the minimum radius of the open ball centred at a given point that contains the inclusions. The formula for the minimum radius is newly discovered

  13. Effects of Inclusion Levels of Wheat Bran and Body Weight on Ileal and Fecal Digestibility in Growing Pigs

    Science.gov (United States)

    Huang, Q.; Su, Y. B.; Li, D. F.; Liu, L.; Huang, C. F.; Zhu, Z. P.; Lai, C. H.

    2015-01-01

    The objective of this study was to determine the effects of graded inclusions of wheat bran (0%, 9.65%, 48.25% wheat bran) and two growth stages (from 32.5 to 47.2 kg and 59.4 to 78.7 kg, respectively) on the apparent ileal digestibility (AID), apparent total tract digestibility (ATTD) and hindgut fermentation of nutrients and energy in growing pigs. Six light pigs (initial body weight [BW] 32.5±2.1 kg) and six heavy pigs (initial BW 59.4±3.2 kg) were surgically prepared with a T-cannula in the distal ileum. A difference method was used to calculate the nutrient and energy digestibility of wheat bran by means of comparison with a basal diet consisting of corn-soybean meal (0% wheat bran). Two additional diets were formulated by replacing 9.65% and 48.25% wheat bran by the basal diet, respectively. Each group of pigs was allotted to a 6×3 Youden square design, and pigs were fed to three experimental diets during three 11-d periods. Hindgut fermentation values were calculated as the differences between ATTD and AID values. For the wheat bran diets, the AID and ATTD of dry matter (DM), ash, organic matter (OM), carbohydrates (CHO), gross energy (GE), and digestible energy (DE) decreased with increasing inclusion levels of wheat bran (pdigestibility of wheat bran in 9.65% inclusion level due to the coefficient of variation (CV) of the nutrient and energy digestibility being higher at 9.65% compared to 48.25% inclusion level of wheat bran. Digestible energy content of wheat bran at 48.25% inclusion level (4.8 and 6.7 MJ/kg of DM, respectively) fermented by hindgut was significantly higher (pdigestibility of some nutrients in pigs, while it positively affects the DE fermentation in the hindgut. PMID:25925062

  14. Detection and prevalence of boid inclusion body disease in collections of boas and pythons using immunological assays.

    Science.gov (United States)

    Chang, L; Fu, D; Stenglein, M D; Hernandez, J A; DeRisi, J L; Jacobson, E R

    2016-12-01

    Inclusion body disease (IBD) of boas and pythons is characterized by the intracytoplasmic accumulation of an antigenic 68 kDa viral protein IBDP, more recently known as the nucleoprotein (NP) of the reptarenaviruses. Blood samples of 131 captive boas and pythons (53 boa constrictors, Boa constrictor; 35 rainbow boas, Epicrates cenchria; 22 ball pythons, Python regius; 5 carpet pythons, Morelia spilota; 6 Burmese pythons, Python bivittatus; 4 Jamaican boas, Epicrates subflavus; 5 anacondas, Eunectes spp.; and 1 green tree python, Morelia viridis) were obtained from 28 collections in the USA. Diagnosis of IBD was initially made by the identification of eosinophilic intracytoplasmic inclusion bodies in hematoxylin and eosin (HE) stained blood films and isolated peripheral white blood cells (PWBC). The overall prevalence of IBD in study snakes was 25/131 or 19% (95% CI = 12.4%, 25.8%) with boa constrictors being more commonly infected (22/53 or 41.5%; 95% CI = 28.2%, 54.8%) than other species in this study. Of the 22 IBD positive boa constrictors, 87% were clinically healthy, 13% had various signs of chronic illness, and none showed signs of central nervous system disease. Using a validated monoclonal anti-NP antibody, NP was confirmed within the isolated PWBC by immunohistochemical staining and Western blots. The presence of reptarenaviruses within blood samples of 27 boa constrictors and three rainbow boas was also assessed by PCR. Among boa constrictors, very good agreements were shown between the observation of inclusion bodies (by HE stain) and the presence of NP (by immunohistochemistry, kappa = 0.92; and Western blots, kappa = 0.89), or the presence of reptarenaviruses (by PCR; kappa = 0.92). Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Injection of Botulinum Toxin a to Upper Esophageal Sphincter for Oropharyngeal Dysphagia in Two Patients with Inclusion Body Myositis

    Directory of Open Access Journals (Sweden)

    Louis WC Liu

    2004-01-01

    Full Text Available Inclusion body myositis (IBM is a progressive degenerative skeletal muscle disease leading to weakening and atrophy of both proximal and distal muscles. Dysphagia is reported in up to 86% of IBM patients. Surgical cricopharyngeal myotomy may be effective for cricopharyngeal dysphagia and there is one published report that botulinum toxin A, injected into the cricopharyngeus muscle using a hypopharyngoscope under general anesthesia, relieved IBM-associated dysphagia. This report presents the first documentation of botulinum toxin A injection into the upper esophageal sphincter using a flexible esophagogastroduodenoscope under conscious sedation, to reduce upper esophageal sphincter pressure and successfully alleviate oropharyngeal dysphagia in two IBM patients.

  16. L-arginine mediated renaturation enhances yield of human, α6 Type IV collagen non-collagenous domain from bacterial inclusion bodies.

    Science.gov (United States)

    Gunda, Venugopal; Boosani, Chandra Shekhar; Verma, Raj Kumar; Guda, Chittibabu; Sudhakar, Yakkanti Akul

    2012-10-01

    The anti-angiogenic, carboxy terminal non-collagenous domain (NC1) derived from human Collagen type IV alpha 6 chain, [α6(IV)NC1] or hexastatin, was earlier obtained using different recombinant methods of expression in bacterial systems. However, the effect of L-arginine mediated renaturation in enhancing the relative yields of this protein from bacterial inclusion bodies has not been evaluated. In the present study, direct stirring and on-column renaturation methods using L-arginine and different size exclusion chromatography matrices were applied for enhancing the solubility in purifying the recombinant α6(IV)NC1 from bacterial inclusion bodies. This methodology enabled purification of higher quantities of soluble protein from inclusion bodies, which inhibited endothelial cell proliferation, migration and tube formation. Thus, the scope for L-arginine mediated renaturation in obtaining higher yields of soluble, biologically active NC1 domain from bacterial inclusion bodies was evaluated.

  17. Neutral weak-current two-body contributions in inclusive scattering from {sup 12}C

    Energy Technology Data Exchange (ETDEWEB)

    Lovato, Alessandro [ANL; Gandolfi, Stefano [LANL; Carlson, Joseph [LANL; Pieper, S. C. [ANL; Schiavilla, Rocco [JLAB, ODU

    2014-05-01

    An {\\it ab initio} calculation of the sum rules of the neutral weak response functions in $^{12}$C is reported, based on a realistic Hamiltonian, including two- and three-nucleon potentials, and on realistic currents, consisting of one- and two-body terms. We find that the sum rules of the response functions associated with the longitudinal and transverse components of the (space-like) neutral current are largest and that a significant portion ($\\simeq 30$\\%) of the calculated strength is due to two-body terms. This fact may have implications for the MiniBooNE and other neutrino quasi-elastic scattering data on nuclei.

  18. Effects of Inclusion Levels of Wheat Bran and Body Weight on Ileal and Fecal Digestibility in Growing Pigs

    Directory of Open Access Journals (Sweden)

    Q. Huang

    2015-06-01

    Full Text Available The objective of this study was to determine the effects of graded inclusions of wheat bran (0%, 9.65%, 48.25% wheat bran and two growth stages (from 32.5 to 47.2 kg and 59.4 to 78.7 kg, respectively on the apparent ileal digestibility (AID, apparent total tract digestibility (ATTD and hindgut fermentation of nutrients and energy in growing pigs. Six light pigs (initial body weight [BW] 32.5±2.1 kg and six heavy pigs (initial BW 59.4±3.2 kg were surgically prepared with a T-cannula in the distal ileum. A difference method was used to calculate the nutrient and energy digestibility of wheat bran by means of comparison with a basal diet consisting of corn-soybean meal (0% wheat bran. Two additional diets were formulated by replacing 9.65% and 48.25% wheat bran by the basal diet, respectively. Each group of pigs was allotted to a 6×3 Youden square design, and pigs were fed to three experimental diets during three 11-d periods. Hindgut fermentation values were calculated as the differences between ATTD and AID values. For the wheat bran diets, the AID and ATTD of dry matter (DM, ash, organic matter (OM, carbohydrates (CHO, gross energy (GE, and digestible energy (DE decreased with increasing inclusion levels of wheat bran (p<0.05. While only AID of CHO and ATTD of DM, ash, OM, CHO, GE, and DE content differed (p<0.05 when considering the BW effect. For the wheat bran ingredient, there was a wider variation effect (p<0.01 on the nutrient and energy digestibility of wheat bran in 9.65% inclusion level due to the coefficient of variation (CV of the nutrient and energy digestibility being higher at 9.65% compared to 48.25% inclusion level of wheat bran. Digestible energy content of wheat bran at 48.25% inclusion level (4.8 and 6.7 MJ/kg of DM, respectively fermented by hindgut was significantly higher (p<0.05 than that in 9.65% wheat bran inclusion level (2.56 and 2.12 MJ/kg of DM, respectively, which was also affected (p<0.05 by two growth stages

  19. Comparative study to develop a single method for retrieving wide class of recombinant proteins from classical inclusion bodies.

    Science.gov (United States)

    Padhiar, Arshad Ahmed; Chanda, Warren; Joseph, Thomson Patrick; Guo, Xuefang; Liu, Min; Sha, Li; Batool, Samana; Gao, Yifan; Zhang, Wei; Huang, Min; Zhong, Mintao

    2018-03-01

    The formation of inclusion bodies (IBs) is considered as an Achilles heel of heterologous protein expression in bacterial hosts. Wide array of techniques has been developed to recover biochemically challenging proteins from IBs. However, acquiring the active state even from the same protein family was found to be an independent of single established method. Here, we present a new strategy for the recovery of wide sub-classes of recombinant protein from harsh IBs. We found that numerous methods and their combinations for reducing IB formation and producing soluble proteins were not effective, if the inclusion bodies were harsh in nature. On the other hand, different practices with mild solubilization buffers were able to solubilize IBs completely, yet the recovery of active protein requires large screening of refolding buffers. With the integration of previously reported mild solubilization techniques, we proposed an improved method, which comprised low sarkosyl concentration, ranging from 0.05 to 0.1% coupled with slow freezing (- 1 °C/min) and fast thaw (room temperature), resulting in greater solubility and the integrity of solubilized protein. Dilution method was employed with single buffer to restore activity for every sub-class of recombinant protein. Results showed that the recovered protein's activity was significantly higher compared with traditional solubilization/refolding approach. Solubilization of IBs by the described method was proved milder in nature, which restored native-like conformation of proteins within IBs.

  20. Clustering of water bodies in unpolluted and polluted environments based on Escherichia coli phylogroup abundance using a simple interaction database

    Directory of Open Access Journals (Sweden)

    Nancy de Castro Stoppe

    2014-12-01

    Full Text Available Different types of water bodies, including lakes, streams, and coastal marine waters, are often susceptible to fecal contamination from a range of point and nonpoint sources, and have been evaluated using fecal indicator microorganisms. The most commonly used fecal indicator is Escherichia coli, but traditional cultivation methods do not allow discrimination of the source of pollution. The use of triplex PCR offers an approach that is fast and inexpensive, and here enabled the identification of phylogroups. The phylogenetic distribution of E. coli subgroups isolated from water samples revealed higher frequencies of subgroups A1 and B2(3 in rivers impacted by human pollution sources, while subgroups D1 and D2 were associated with pristine sites, and subgroup B1 with domesticated animal sources, suggesting their use as a first screening for pollution source identification. A simple classification is also proposed based on phylogenetic subgroup distribution using the w-clique metric, enabling differentiation of polluted and unpolluted sites.

  1. Structure and Stability of Molecular Crystals with Many-Body Dispersion-Inclusive Density Functional Tight Binding.

    Science.gov (United States)

    Mortazavi, Majid; Brandenburg, Jan Gerit; Maurer, Reinhard J; Tkatchenko, Alexandre

    2018-01-18

    Accurate prediction of structure and stability of molecular crystals is crucial in materials science and requires reliable modeling of long-range dispersion interactions. Semiempirical electronic structure methods are computationally more efficient than their ab initio counterparts, allowing structure sampling with significant speedups. We combine the Tkatchenko-Scheffler van der Waals method (TS) and the many-body dispersion method (MBD) with third-order density functional tight-binding (DFTB3) via a charge population-based method. We find an overall good performance for the X23 benchmark database of molecular crystals, despite an underestimation of crystal volume that can be traced to the DFTB parametrization. We achieve accurate lattice energy predictions with DFT+MBD energetics on top of vdW-inclusive DFTB3 structures, resulting in a speedup of up to 3000 times compared with a full DFT treatment. This suggests that vdW-inclusive DFTB3 can serve as a viable structural prescreening tool in crystal structure prediction.

  2. A simple strategy for the purification of native recombinant full-length human RPL10 protein from inclusion bodies.

    Science.gov (United States)

    Pereira, Larissa M; Silva, Luana R; Alves, Joseane F; Marin, Nélida; Silva, Flavio Sousa; Morganti, Ligia; Silva, Ismael D C G; Affonso, Regina

    2014-09-01

    The L10 ribosomal protein (RPL10) plays a role in the binding of the 60 S and 40 S ribosomal subunits and in mRNA translation. The evidence indicates that RPL10 also has multiple extra-ribosomal functions, including tumor suppression. Recently, the presence of RPL10 in prostate and ovarian cancers was evaluated, and it was demonstrated to be associated with autistic disorders and premature ovarian failure. In the present work, we successfully cloned and expressed full-length human RPL10 (hRPL10) protein and isolated inclusion bodies containing this protein that had formed under mild growth conditions. The culture produced 376mg of hRPL10 protein per liter of induced bacterial culture, of which 102.4mg was present in the soluble fraction, and 25.6mg was recovered at approximately 94% purity. These results were obtained using a two-step process of non-denaturing protein extraction from pelleted inclusion bodies. We studied the characteristics of this protein using circular dichroism spectroscopy and by monitoring the changes induced by the presence or absence of zinc ions using fluorescence spectrometry. The results demonstrated that the protein obtained using these non-conventional methods retained its secondary and tertiary structure. The conformational changes induced by the incorporation of zinc suggested that this protein could interact with Jun or the SH3 domain of c-yes. The results suggested that the strategy used to obtain hRPL10 is simple and could be applied to obtaining other proteins that are susceptible to degradation. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Immunohistochemical Detection of a Unique Protein within Cells of Snakes Having Inclusion Body Disease, a World-Wide Disease Seen in Members of the Families Boidae and Pythonidae

    Science.gov (United States)

    Chang, Li-Wen; Fu, Ann; Wozniak, Edward; Chow, Marjorie; Duke, Diane G.; Green, Linda; Kelley, Karen; Hernandez, Jorge A.; Jacobson, Elliott R.

    2013-01-01

    Inclusion body disease (IBD) is a worldwide disease in captive boa constrictors (boa constrictor) and occasionally in other snakes of the families Boidae and Pythonidae. The exact causative agent(s) and pathogenesis are not yet fully understood. Currently, diagnosis of IBD is based on the light microscopic identification of eosinophilic intracytoplasmic inclusion bodies in hematoxylin and eosin stained tissues or blood smears. An antigenically unique 68 KDa protein was identified within the IBD inclusion bodies, called IBD protein. A validated immuno-based ante-mortem diagnostic test is needed for screening snakes that are at risk of having IBD. In this study, despite difficulties in solubilizing semi-purified inclusion bodies, utilizing hybridoma technology a mouse anti-IBD protein monoclonal antibody (MAB) was produced. The antigenic specificity of the antibody was confirmed and validated by western blots, enzyme-linked immunosorbent assay, immuno-transmission electron microscopy, and immunohistochemical staining. Paraffin embedded tissues of IBD positive and negative boa constrictors (n=94) collected from 1990 to 2011 were tested with immunohistochemical staining. In boa constrictors, the anti-IBDP MAB had a sensitivity of 83% and specificity of 100% in detecting IBD. The antibody also cross-reacted with IBD inclusion bodies in carpet pythons (Morelia spilota) and a ball python (python regius). This validated antibody can serve as a tool for the development of ante-mortem immunodiagnostic tests for IBD. PMID:24340066

  4. Immunohistochemical detection of a unique protein within cells of snakes having inclusion body disease, a world-wide disease seen in members of the families Boidae and Pythonidae.

    Directory of Open Access Journals (Sweden)

    Li-Wen Chang

    Full Text Available Inclusion body disease (IBD is a worldwide disease in captive boa constrictors (boa constrictor and occasionally in other snakes of the families Boidae and Pythonidae. The exact causative agent(s and pathogenesis are not yet fully understood. Currently, diagnosis of IBD is based on the light microscopic identification of eosinophilic intracytoplasmic inclusion bodies in hematoxylin and eosin stained tissues or blood smears. An antigenically unique 68 KDa protein was identified within the IBD inclusion bodies, called IBD protein. A validated immuno-based ante-mortem diagnostic test is needed for screening snakes that are at risk of having IBD. In this study, despite difficulties in solubilizing semi-purified inclusion bodies, utilizing hybridoma technology a mouse anti-IBD protein monoclonal antibody (MAB was produced. The antigenic specificity of the antibody was confirmed and validated by western blots, enzyme-linked immunosorbent assay, immuno-transmission electron microscopy, and immunohistochemical staining. Paraffin embedded tissues of IBD positive and negative boa constrictors (n=94 collected from 1990 to 2011 were tested with immunohistochemical staining. In boa constrictors, the anti-IBDP MAB had a sensitivity of 83% and specificity of 100% in detecting IBD. The antibody also cross-reacted with IBD inclusion bodies in carpet pythons (Morelia spilota and a ball python (python regius. This validated antibody can serve as a tool for the development of ante-mortem immunodiagnostic tests for IBD.

  5. Estudo comparativo das inclusões do alastrim e da variola vera A comparison of the inclusion bodies of alastrim and variola vera

    Directory of Open Access Journals (Sweden)

    C. Magarinos Torres

    1935-09-01

    Full Text Available Resulta das descripções e documentos examinados que as inclusões cytoplasmaticas da variola vera, no material humano examinado, apresentam caracteres geraes que poderão ser assim resumidos: 1. intensa coloração pela safranina nos preparados pelo methodo de Unna modificado (Fig. 42, mostrando a inclusão matiz semelhante ao dos nucleolos da mesma cellula. 2. reacção predominantemente acidophila nos preparados pela hematoxylina-eosina, traduzida pela tonalidade rosea ou vermelha de coloração (Figs. 27, 29, 30, 31 e 31. 3. frequente multiplicidade de inclusões de fórma e dimensões variadas na mesma cellula (Figs. 29 e 30, as maiores inclusões (Figs. 29 e 32, sendo menores que as grandes inclusões cytoplasmaticas solitarias do alastrim (Figs. 15, 16 e 18. É a regra, ainda, observar-se desapparecimento de qualquer estructura no cytoplasma das cellulas com inclusões em zona muito extensa, tornando-se difficil explicar tal aspecto unicamente pela retracção das inclusões no acto da fixação (Figs. 31 e 32. Quanto ás inclusões intranucleares, ellas se apresentam sob tres aspectos, dois dos quaes bem reconhecidos por Luger e Lauda (1926. Em um primeiro aspecto, o nucleo conserva, em parte, o reticulo de linina e encerra uma massa irregular (Fig. 35 ou, então, pequenas massas e granulos de dimensões variaveis (Figs. 33 e 34 constituidos por material acidophilo que ali não existe em condições normaes. A membrana nuclear tem espessura visinha do normal. Em um segundo aspecto, a inclusão occupa a totalidade do nucleoplasma (Fig. 3, apenas separado da membrana nuclear, em alguns casos (Fig. 38, por um estreito espaço claro (zona de retracção. Por vezes apresenta um aspecto homogeneo (Fig. 37; outras vezes a inclusão mostra pequenas areas chromophobas ( Fig. 38. A hyperchromatose da membrana nuclear é accentuada, tanto neste como no aspecto seguinte. No terceiro aspecto (aspecto corpuscular (Figs. 39, 40, 48 e 50, a inclus

  6. Upon Infection the Cellular WD Repeat-containing Protein 5 (WDR5) Localizes to Cytoplasmic Inclusion Bodies and Enhances Measles Virus Replication.

    Science.gov (United States)

    Ma, Dzwokai; George, Cyril X; Nomburg, Jason; Pfaller, Christian K; Cattaneo, Roberto; Samuel, Charles E

    2017-12-13

    Replication of negative-strand RNA viruses occurs in association with discrete cytoplasmic foci called inclusion bodies. Whereas inclusion bodies represent a prominent subcellular structure induced by viral infection, our knowledge of the cellular protein components involved in inclusion body formation and function is limited. Using measles virus-infected HeLa cells, we found that the WD repeat-containing protein 5 (WDR5), a subunit of histone H3 lysine 4 methyltransferases, was selectively recruited to virus-induced inclusion bodies. Furthermore, WDR5 was found in complexes containing viral proteins associated with RNA replication. WDR5 was not detected with mitochondria, stress granules, or other known secretory or endocytic compartments of infected cells. WDR5 deficiency decreased both viral protein production and infectious virus yields. Interferon production was modestly increased in WDR5 deficient cells. Thus, our study identifies WDR5 as a novel viral inclusion body-associated cellular protein and suggests a role for WDR5 in promoting viral replication. IMPORTANCE Measles virus is a human pathogen that remains a global concern with more than 100,000 measles-related deaths annually despite the availability of an effective vaccine. As measles continues to cause significant morbidity and mortality, understanding the virus-host interactions at the molecular level that affect virus replication efficiency is important for development and optimization of treatment procedures. Measles virus is an RNA virus that encodes six genes and replicates in the cytoplasm of infected cells in discrete cytoplasmic replication bodies, though little is known of the biochemical nature of these structures. Here we show that the cellular protein WDR5 is enriched in the cytoplasmic viral replication factories and enhances virus growth. WDR5-containing protein complex includes viral proteins responsible for viral RNA replication. Thus, we have identified WDR5 as a host factor that

  7. Inclusion Body Myositis: A Case Presenting with Respiratory Failure and Autopsy Findings Leading to the Hypothesis of a Paraneoplastic Cause.

    Science.gov (United States)

    Dardis, Christopher; Antezana, Ariel; Tanji, Kurenai; Maccabee, Paul J

    2017-06-23

    BACKGROUND Sporadic inclusion body myositis (IBM) is the most common acquired myopathy seen in adults aged over 50 years, with a prevalence estimated at between 1 and 70 per million. Weakness of the diaphragm with loss of vital capacity is almost universal in IBM. This is almost always asymptomatic. When respiratory complications occur, they are most often due to aspiration. Respiratory failure due to diaphragmatic weakness is exceptionally rare, particularly as the presenting symptom of the disease. It is not currently considered to be a paraneoplastic syndrome. CASE REPORT Our patient presented with hypercarbic respiratory failure. This is the first such reported case without signs of weakness elsewhere of which we are aware. We suspected IBM based on her history of progressive weakness and findings on electromyography. There was a delay of 5 years in obtaining biopsy for confirmation, during which she presented with recurrent episodes of respiratory failure despite using non-invasive ventilation. An autopsy revealed the presence of papillary thyroid carcinoma with spread to local lymph nodes. On the basis that these co-morbidities are unlikely to have occurred by chance (we estimate 1×10-17), we hypothesize that IBM may be a paraneoplastic condition. We acknowledge that proof would require demonstrating a pathogenic antibody. CONCLUSIONS IBM should be considered in older patients (age >45) presenting with otherwise unexplained respiratory failure. A workup for possible malignancy in this setting appears reasonable.

  8. Mutant LRRK2 Toxicity in Neurons Depends on LRRK2 Levels and Synuclein But Not Kinase Activity or Inclusion Bodies

    Science.gov (United States)

    Skibinski, Gaia; Nakamura, Ken; Cookson, Mark R.

    2014-01-01

    By combining experimental neuron models and mathematical tools, we developed a “systems” approach to deconvolve cellular mechanisms of neurodegeneration underlying the most common known cause of Parkinson's disease (PD), mutations in leucine-rich repeat kinase 2 (LRRK2). Neurons ectopically expressing mutant LRRK2 formed inclusion bodies (IBs), retracted neurites, accumulated synuclein, and died prematurely, recapitulating key features of PD. Degeneration was predicted from the levels of diffuse mutant LRRK2 that each neuron contained, but IB formation was neither necessary nor sufficient for death. Genetic or pharmacological blockade of its kinase activity destabilized LRRK2 and lowered its levels enough to account for the moderate reduction in LRRK2 toxicity that ensued. By contrast, targeting synuclein, including neurons made from PD patient-derived induced pluripotent cells, dramatically reduced LRRK2-dependent neurodegeneration and LRRK2 levels. These findings suggest that LRRK2 levels are more important than kinase activity per se in predicting toxicity and implicate synuclein as a major mediator of LRRK2-induced neurodegeneration. PMID:24403142

  9. Mortality and Causes of Death in Patients with Sporadic Inclusion Body Myositis: Survey Study Based on the Clinical Experience of Specialists in Australia, Europe and the USA

    NARCIS (Netherlands)

    Price, Mark A.; Barghout, Victoria; Benveniste, Olivier; Christopher-Stine, Lisa; Corbett, Alastair; de Visser, Marianne; Hilton-Jones, David; Kissel, John T.; Lloyd, Thomas E.; Lundberg, Ingrid E.; Mastaglia, Francis; Mozaffar, Tahseen; Needham, Merrilee; Schmidt, Jens; Sivakumar, Kumaraswamy; DeMuro, Carla; Tseng, Brian S.

    2016-01-01

    There is a paucity of data on mortality and causes of death (CoDs) in patients with sporadic inclusion body myositis (sIBM), a rare, progressive, degenerative, inflammatory myopathy that typically affects those aged over 50 years. Based on patient records and expertise of clinical specialists, this

  10. Effects of blood-flow-restricted resistance training on muscle function in a 74-year-old male with sporadic inclusion body myositis

    DEFF Research Database (Denmark)

    Jørgensen, Anders Nørkær; Aagaard, P; Nielsen, J L

    2016-01-01

    Sporadic inclusion body myositis (sIBM) is a systemic disease that is characterized by substantial skeletal muscle weakness and muscle inflammation, leading to impaired physical function. The objective was to investigate the effect of low-load resistance exercise with concurrent partial blood flow...

  11. High pH solubilization and chromatography-based renaturation and purification of recombinant human granulocyte colony-stimulating factor from inclusion bodies.

    Science.gov (United States)

    Li, Ming; Fan, Hua; Liu, Jiahua; Wang, Minhong; Wang, Lili; Wang, Chaozhan

    2012-03-01

    Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is a very efficient therapeutic protein drug which has been widely used in human clinics to treat cancer patients suffering from chemotherapy-induced neutropenia. In this study, rhG-CSF was solubilized from inclusion bodies by using a high-pH solution containing low concentration of urea. It was found that solubilization of the rhG-CSF inclusion bodies greatly depended on the buffer pH employed; alkalic pH significantly favored the solubilization. In addition, when small amount of urea was added to the solution at high pH, the solubilization was further enhanced. After solubilization, the rhG-CSF was renatured with simultaneous purification by using weak anion exchange, strong anion exchange, and hydrophobic interaction chromatography, separately. The results indicated that the rhG-CSF solubilized by the high-pH solution containing low concentration of urea had much higher mass recovery than the one solubilized by 8 M urea when using anyone of the three refolding methods employed in this work. In the case of weak anion exchange chromatography, the high pH solubilized rhG-CSF could get a mass recovery of 73%. The strategy of combining solubilization of inclusion bodies at high pH with refolding of protein using liquid chromatography may become a routine method for protein production from inclusion bodies.

  12. Estudo comparativo das inclusões do alastrim e da vacina no macaco (Macacus rhesus A comparison of the inclusion bodies of alastrim and vaccinia in the monkey (Macacus rhesus

    Directory of Open Access Journals (Sweden)

    C. Magarinos Torres

    1934-02-01

    Full Text Available Vesiculas e pustulas contendo numerosas inclusões citoplasmicas nas celulas epidermicas, foram regularmente produzidas no macaco (Macacus rhesus, quer com o virus do alastrim, quer com o da vacina, após inoculação endovenosa e sem previa escarificação. O virus do alastrim parece menos virulento para essa especie de macaco que o da vacina. Ao passo que 12 macacos rhesus injetados por via endovenosa com sete amostras diferentes de virus do alastrim, após apresentarem com regularidade um infecção experimental, sobreviveram e se conservaram em boa saúde, a injecção endovenosa do virus da vacina recentemente preparado (polpa bruta produziu a morte em 2, dentre 4 animais experimentados. 2. - Foram notadas diferenças pequenas, mas nitidas, na morfologia das inclusões do alastrim e da vacina, em material fixado no liquido de Helly, incluido em parafina e corado pela hematoxilina-eosina. Dizem elas respeito ao numero de inclusões encontradas em cada celula epidermica e às suas reações de coloração. 3. - As inclusões do alastrim, quando apresentam grandes dimensões, conservam-se unicas ou solitarias no citoplasma das celulas epidermicas do macaco rhesus, e coram-se em tonalidade que varia do azul escuro ao cinzento-azulado. Comtudo, em celulas que sofreram necrose, ou naquelas contendo 2 a 4 inclusões de pequenas dimensões, por vezes elas se mostram coradas em roseo. 4. - As inclusões da vacina, quando em faze adeantada de desenvolvimento, são multiplas nas celulas epidermicas do macaco rhesus e mostram, regularmente, uma policromatofilia caracteristica.1. - Vesicles and pustules containing numerous cytoplasmic inclusion bodies within the epidermal cells were regularly produced in monkeys (Macacus rhesus by intravenous inoculation either of alastrim virus or of recently prepared vaccine emulsion, no previous scarifications being required. Alastrim virus seems less virulent for this species of monkey than the virus of vaccinia is

  13. Kinetics of viral load and erythrocytic inclusion body formation in pacific herring artificially infected with erythrocytic necrosis virus

    Science.gov (United States)

    Glenn, Jolene A.; Emmenegger, Eveline J.; Grady, Courtney A.; Roon, Sean R.; Gregg, Jacob L.; Conway, Carla M.; Winton, James R.; Hershberger, Paul K.

    2012-01-01

    Viral erythrocytic necrosis (VEN) is a condition that affects marine and anadromous fish species, including herrings and salmonids, in the Atlantic and Pacific oceans. Infection is frequently associated with severe anemia and causes episodic mortality among wild and hatchery fish when accompanied by additional stressors; VEN can be presumptively diagnosed by (1) light microscopic identification of a single characteristic—a round, magenta-colored, 0.8-μm-diameter inclusion body (IB) within the cytoplasm of erythrocytes and their precursors on Giemsa-stained blood films; or (2) observation (via transmission electron microscopy [TEM]) of the causative iridovirus, erythrocytic necrosis virus (ENV), within erythrocytes or their precursors. To better understand the kinetics of VEN, specific-pathogen-free Pacific herring Clupea pallasii were infected with ENV by intraperitoneal injection. At 1, 4, 7, 10, 14, 21, and 28 d postexposure, samples of blood, spleen, and kidney were collected and assessed (1) via light microscopy for the number of intracytoplasmic IBs in blood smears and (2) via TEM for the number of virions within erythrocytes. The mean prevalence of intracytoplasmic IBs in the blood cells increased from 0% at 0–4 d postexposure to 94% at 28 d postexposure. Viral load within circulating red blood cells peaked at 7 d postexposure, fell slightly, and then reached a plateau. However, blood cells observed within the kidney and spleen tissues demonstrated high levels of ENV between 14 and 28 d postexposure. The results indicate that the viral load within erythrocytes does not correlate well with IB prevalence and that the virus can persist in infected fish for more than 28 d.

  14. Corpos de inclusão citoplasmática: estudo em diversas doenças e revisão da literatura Inclusion cytoplasmic bodies: a study in several diseases and a literature review

    Directory of Open Access Journals (Sweden)

    Rosana Herminia Scola

    1996-06-01

    Full Text Available Estudamos 16 casos entre 1400 biópsias musculares que apresentavam vacúolos marginados, cujo aspecto histológico sugeria corpos de inclusão citoplasmáticos. Procuramos correlacionar os dados clínicos, laboratoriais e histopatológicos, a fim de determinar a especificidade dos corpos de inclusão citoplasmáticos para determinadas doenças. A creatinaquinase mostrou-se elevada em 10 casos. A eletromiografia foi anormal em todos os casos. A histoquímica muscular em 5 casos revelou uma miopatia, em 7 padrão misto, em dois desinervação e em 2 casos miopatia inflamatória. A microscopia eletrônica demonstrou a presença de filamentos em 8 casos (nucleares, dispersos no citoplasma ou na região subsarcolemal. Os pacientes foram classificados conforme a história clínica, hereditariedade, dados laboratoriais, eletrofisiológicos, histoquímicos e microscopia eletrônica Encontramos miosite com corpos de inclusão citoplasmática (4 casos, atrofia muscular espinhal juvenil (6 casos, miopatias distais (3 casos, distrofia de cinturas pélvica e escapular (2 casos e polineuropatia periférica (1 caso. Apresentamos revisão sobre a patogenia, formação e possível etiologia dos vacúolos marginados e sua relação com as diversas entidades em que foram detectados, sugerindo que não são específicos para uma única doença.Among 1400 muscle biopsies, we studied 16 cases with rimmed vacuoles, whose histology suggests cytoplasm inclusion bodies. We tried to correlate the clinical, laboratory and histopatological data in order to verify the specificity of cytoplasm inclusion bodies to certain diseases. The creatinekinase was increased in 10 cases. In all cases electromyography was abnormal. Muscle histochemistry revealed myopathy in 5 cases, mixed pattern in 7, denervation in 2 and in 2 cases, inflammatory myopathy. Electron microscopy showed the presence of filaments in 8 cases (nuclear, disseminated in cytoplasm or in the subsarcolemmal region

  15. Purification and Characterization of Recombinant Vaccinia L1R Protein from Escherichia coli

    Science.gov (United States)

    2016-08-01

    RECOMBINANT VACCINIA L1R PROTEIN FROM ESCHERICHIA COLI 1. INTRODUCTION 1.1 Background Vaccinia virus (VACV) is the active component of the...the preparation of the recombinant VACV L1R protein fragment by denaturing , refolding, and purifying material expressed into inclusion bodies in...PURIFICATION AND CHARACTERIZATION OF RECOMBINANT VACCINIA L1R PROTEIN FROM ESCHERICHIA COLI ECBC-TR-1370

  16. Children with moderate-high infection with Entamoeba coli have higher percentage of body and abdominal fat than non-infected children.

    Science.gov (United States)

    Zavala, G A; García, O P; Campos-Ponce, M; Ronquillo, D; Caamaño, M C; Doak, C M; Rosado, J L

    2016-12-01

    Intestinal parasites, virus and bacterial infections are positively associated with obesity and adiposity in vitro and in animal models, but conclusive evidence of this relationship in humans is lacking. The aim of this cross-sectional study was to determine differences in adiposity between infected and non-infected children, with a high prevalence of intestinal parasitic infection and obesity. A total of 296 school-aged children (8.0 ± 1.5 years) from a rural area in Querétaro, Mexico, participated in this study. Anthropometry (weight, height and waist circumference) and body fat (DXA) were measured in all children. A fresh stool sample was collected from each child and analysed for parasites. Questionnaires related to socioeconomic status and clinical history were completed by caretakers. Approximately 11% of the children were obese, and 19% were overweight. The overall prevalence of infection was 61%. Ascaris lumbricoides was the most prevalent soil transmitted helminth (16%) followed by hookworm. Entamoeba coli was the predominant protozoa (20%) followed by Endolimax nana, Balantidium coli, Entamoeba histolytica/dispar, Iodamoeba bütschlii and Giardia lamblia. Children with moderate-heavy infection of E. coli had significantly higher waist circumference, waist-to-height ratio, body and abdominal fat than children not infected or with light-intensity infection (p fat deposition and thereby have long-term consequences on human health. Further studies are needed to better understand if E. coli contributes directly to fat deposition and possible mechanisms. © 2015 World Obesity Federation.

  17. The P2 of Wheat yellow mosaic virus rearranges the endoplasmic reticulum and recruits other viral proteins into replication-associated inclusion bodies.

    Science.gov (United States)

    Sun, Liying; Andika, Ida Bagus; Shen, Jiangfeng; Yang, Di; Chen, Jianping

    2014-06-01

    Viruses commonly modify host endomembranes to facilitate biological processes in the viral life cycle. Infection by viruses belonging to the genus Bymovirus (family Potyviridae) has long been known to induce the formation of large membranous inclusion bodies in host cells, but their assembly and biological roles are still unclear. Immunoelectron microscopy of cells infected with the bymovirus Wheat yellow mosaic virus (WYMV) showed that P1, P2 and P3 are the major viral protein constituents of the membranous inclusions, whereas NIa-Pro (nuclear inclusion-a protease) and VPg (viral protein genome-linked) are probable minor components. P1, P2 and P3 associated with the endoplasmic reticulum (ER), but only P2 was able to rearrange ER and form large aggregate structures. Bioinformatic analyses and chemical experiments showed that P2 is an integral membrane protein and depends on the active secretory pathway to form aggregates of ER membranes. In planta and in vitro assays demonstrated that P2 interacts with P1, P3, NIa-Pro or VPg and recruits these proteins into the aggregates. In vivo RNA labelling using WYMV-infected wheat protoplasts showed that the synthesis of viral RNAs occurs in the P2-associated inclusions. Our results suggest that P2 plays a major role in the formation of membranous compartments that house the genomic replication of WYMV. © 2013 BSPP AND JOHN WILEY & SONS LTD.

  18. Constraining the Thermochronological History of the IAB Parent Body: High Resolution Ar-40-Ar-39 Ages on Plagioclase Separates from Silicate Inclusions of IAB Meteorites

    Science.gov (United States)

    Vogel, N.; Renne, P. R.

    2004-12-01

    The processes that led to the assembly of primitive inclusions in a once molten metal matrix as represented by IAB meteorites have not yet been fully understood [1]. Ar-Ar dating of the inclusions provides important information about the thermal history of the IAB parent body [e.g., 2, 3], but the analysis of bulk inclusions, the standard procedure in the past, is often impaired by excess 40Ar and redistribution or loss of K and/or Ar during the history of the meteoriod and in the reactor. To minimize these problems, we prepared from silicate inclusions of four IABs pure plagioclase separates of different grain sizes and quality grades. On these we performed high resolution stepwise Ar-40-Ar-39 dating. Preliminary ages for the different separates of the inclusions are, in Ma, 4540(11) to 4459(12) for Caddo County, 4500(20) to 4380(30) for Landes, 4440(50) to 4340(30) for Ocotillo, and 4480(40) to 4200(30) and 4430(30) to 4300(30) for CDC2 and CDC1, respectively. The age ranges might reflect the residence time of each inclusion in the K-Ar blocking temperature range (ca. 600 K), and is narrowest for Caddo County, being also the oldest inclusion studied by us. Assuming that IABs resulted from a collision of a molten metal body with a chondritic planetesimal [4], Caddo County could represent a surface sample explaining the early and fast cooling, whereas the other samples might have been buried deeper within the IAB body, subject to prolonged residence at elevated temperatures. If IABs formed in impact metal melt pools peppered with chondritic host material [5] the different cooling ages, and age ranges recorded in each inclusion could reflect residence times in a certain metal melt pool, which indirectly would translate into pool sizes and the energies released by the previous impacts. Also, there may have been more than one IAB parent body. Whatever process led to the formation of IAB meteorites was active already very early in the history of the solar system, in

  19. Inclusion in political and public life: the experiences of people with intellectual disability on government disability advisory bodies in Australia.

    Science.gov (United States)

    Frawley, Patsie; Bigby, Christine

    2011-03-01

    Civil and political participation lies at the core of citizenship. Increasingly, people with intellectual disability are members of disability advisory bodies. This study investigated the political orientations of advisory body members with intellectual disability, their participatory experiences, and the types of support they received. The 9 people with intellectual disability who in 2005 were members of disability advisory bodies at a state, national, and Victorian local government level were interviewed, together with 12 other members or secretariat staff of these bodies. Observations were also conducted of advisory body meetings. The political perspective of members with intellectual disability varied, but all had a background in self-advocacy. They found the work hard but rewarding and encountered both practical and intangible obstacles to participation. Members received varying types of practical support, but a supportive collegial milieu was characteristic among members who felt most confident about their participation. The milieu, structures, and processes of advisory bodies must all be adjusted to accommodate people with intellectual disability if they are to participate meaningfully.

  20. Sodium phenylbutyrate reverses lysosomal dysfunction and decreases amyloid-β42 in an in vitro-model of inclusion-body myositis.

    Science.gov (United States)

    Nogalska, Anna; D'Agostino, Carla; Engel, W King; Askanas, Valerie

    2014-05-01

    Sporadic inclusion-body myositis (s-IBM) is a severe, progressive muscle disease for which there is no enduring treatment. Pathologically characteristic are vacuolated muscle fibers having: accumulations of multi-protein aggregates, including amyloid-β(Aβ) 42 and its toxic oligomers; increased γ-secretase activity; and impaired autophagy. Cultured human muscle fibers with experimentally-impaired autophagy recapitulate some of the s-IBM muscle abnormalities, including vacuolization and decreased activity of lysosomal enzymes, accompanied by increased Aβ42, Aβ42 oligomers, and increased γ-secretase activity. Sodium phenylbutyrate (NaPB) is an orally bioavailable small molecule approved by the FDA for treatment of urea-cycle disorders. Here we describe that NaPB treatment reverses lysosomal dysfunction in an in vitro model of inclusion-body myositis, involving cultured human muscle fibers. NaPB treatment improved lysosomal activity, decreased Aβ42 and its oligomers, decreased γ-secretase activity, and virtually prevented muscle-fiber vacuolization. Accordingly, NaPB might be considered a potential treatment of s-IBM patients. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Establishment of mouse model of MYH9 disorders: heterozygous R702C mutation provokes macrothrombocytopenia with leukocyte inclusion bodies, renal glomerulosclerosis and hearing disability.

    Science.gov (United States)

    Suzuki, Nobuaki; Kunishima, Shinji; Ikejiri, Makoto; Maruyama, Shoichi; Sone, Michihiko; Takagi, Akira; Ikawa, Masahito; Okabe, Masaru; Kojima, Tetsuhito; Saito, Hidehiko; Naoe, Tomoki; Matsushita, Tadashi

    2013-01-01

    Nonmuscle myosin heavy chain IIA (NMMHCIIA) encoded by MYH9 is associated with autosomal dominantly inherited diseases called MYH9 disorders. MYH9 disorders are characterized by macrothrombocytopenia and very characteristic inclusion bodies in granulocytes. MYH9 disorders frequently cause nephritis, sensorineural hearing disability and cataracts. One of the most common and deleterious mutations causing these disorders is the R702C missense mutation. We generated knock-in mice expressing the Myh9 R702C mutation. R702C knock-in hetero mice (R702C+/- mice) showed macrothrombocytopenia. We studied megakaryopoiesis of cultured fetal liver cells of R702C+/- mice and found that proplatelet formation was impaired: the number of proplatelet tips was decreased, proplatelet size was increased, and proplatelet shafts were short and enlarged. Although granulocyte inclusion bodies were not visible by May-Grünwald Giemsa staining, immunofluorescence analysis indicated that NMMHCIIA proteins aggregated and accumulated in the granulocyte cytoplasm. In other organs, R702C+/- mice displayed albuminuria which increased with age. Renal pathology examination revealed glomerulosclerosis. Sensory hearing loss was indicated by lowered auditory brainstem response. These findings indicate that Myh9 R702C knock-in mice mirror features of human MYH9 disorders arising from the R702C mutation.

  2. Further research on the biological function of inclusion bodies of Anomala cuprea entomopoxvirus, with special reference to the effect on the insecticidal activity of a Bacillus thuringiensis formulation.

    Science.gov (United States)

    Mitsuhashi, Wataru; Asano, Shoji; Miyamoto, Kazuhisa; Wada, Sanae

    2014-01-01

    Entomopoxviruses (EVs) form two types of inclusion body: spheroids, which contain virions, and spindles, which do not. The authors tested whether the spindles from a coleopteran EV, Anomala cuprea EV (ACEV), enhanced the insecticidal activity of a commercial Bacillus thuringiensis (Bt) formulation and the susceptibility of scarabaeid pest species in Japan to the virus's spheroids, to assess whether ACEV inclusion bodies are potential biological control agents for pest insects. Peroral inoculation with both ACEV spindles and the Bt toxin only or the complete Bt formulation shortened the survival and increased the mortality of treated insects compared with those of insects inoculated with Bt without the spindles (8-38 h of decrease in LT50 values among assays). ACEV showed high infectivity to a major scarabaeid pest species in Japanese sugar cane fields. The results suggest that spindles or the constituent protein fusolin can be used as a coagent with Bt formulations, and that fusolin coexpression with a Bt toxin in crops might improve the insecticidal efficacy. In addition, the spheroids are potential biocontrol agents for some scarabaeid pests that are not easy to control because of their underground habitation. © 2013 Society of Chemical Industry.

  3. Continuous processing of recombinant proteins: Integration of inclusion body solubilization and refolding using simulated moving bed size exclusion chromatography with buffer recycling.

    Science.gov (United States)

    Wellhoefer, Martin; Sprinzl, Wolfgang; Hahn, Rainer; Jungbauer, Alois

    2013-12-06

    An integrated process which combines continuous inclusion body dissolution with NaOH and continuous matrix-assisted refolding based on closed-loop simulated moving bed size exclusion chromatography was designed and experimentally evaluated at laboratory scale. Inclusion bodies from N(pro) fusion pep6His and N(pro) fusion MCP1 from high cell density fermentation were continuously dissolved with NaOH, filtered and mixed with concentrated refolding buffer prior to refolding by size exclusion chromatography (SEC). This process enabled an isocratic operation of the simulated moving bed (SMB) system with a closed-loop set-up with refolding buffer as the desorbent buffer and buffer recycling by concentrating the raffinate using tangential flow filtration. With this continuous refolding process, we increased the refolding and cleavage yield of both model proteins by 10% compared to batch dilution refolding. Furthermore, more than 99% of the refolding buffer of the raffinate could be recycled which reduced the buffer consumption significantly. Based on the actual refolding data, we compared throughput, productivity, and buffer consumption between two batch dilution refolding processes - one using urea for IB dissolution, the other one using NaOH for IB dissolution - and our continuous refolding process. The higher complexity of the continuous refolding process was rewarded with higher throughput and productivity as well as significantly lower buffer consumption compared to the batch dilution refolding processes. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. Inclusion of Body Bias Effect in SPICE Modeling of 4H-SiC Integrated Circuit Resistors

    Science.gov (United States)

    Neudeck, Philip G.

    2017-01-01

    The DC electrical behavior of n-type 4H-SiC resistors used for realizing 500 degrees Celsius durable integrated circuits (ICs) is studied as a function of substrate bias and temperature. Improved fidelity electrical simulation is described using SPICE NMOS model to simulate resistor substrate body bias effect that is absent from the SPICE semiconductor resistor model.

  5. Inclusion of Body-Bias Effect in SPICE Modeling of 4H-SiC Integrated Circuit Resistors

    Science.gov (United States)

    Neudeck, Philip G.

    2017-01-01

    The DC electrical behavior of n-type 4H-SiC resistors used for realizing 500 C durable integrated circuits (ICs) is studied as a function of substrate bias and temperature. Improved fidelity electrical simulation is described using SPICE NMOS model to simulate resistor substrate body bias effect that is absent from the SPICE semiconductor resistor model.

  6. Effects of inclusion levels of Indigofera sp. on feed intake, digestibility and body weight gain in kids fed Brachiaria ruziziensis.

    Directory of Open Access Journals (Sweden)

    Andi Tarigan

    2011-03-01

    Full Text Available Twenty weaned male goats (F1 of Boer x Kacang with initial weight of 9 to 12 kg and ages ranging between 3.0 and 4.0 months were used in a study to evaluate the increasing inclusion of Indigofera sp foliage as a source of protein in diets based on chopped Brachiaria ruziziensis for growing goats. Five goats were allocated to one of four treatments in a randomised block design. The diet treatments were: T0 (control diets: B. ruziziensis (100%, T1 (85% B. ruziziensis + 15% Indigofera sp., T2 (70% B. ruziziensis + 30% Indigofera sp. T3 (55% B. ruziziensis + 45% Indigofera sp. all on DM basis. Feed (DM was offered daily at 3.5% BW. The content of CP in Indigofera sp is relatively high (258 g/kg DM, while the NDF (350.7 g/kg DM and ADF (232.2 g/ kg DM concentrations were low. The content of secondary compounds such as total phenol (8.9 g/kg DM, total tannin (0.8 g/kg DM and condensed tannin (0,5 g/kg DM were considerably low. The inclusion of Indigofera sp foliage in diets increased (P 0.05 among the T0,T1 and T2 diets and ranged from 0.08 to 0.09. It is concluded that the foliage of Indigofera sp could be used as feed supplement to supply proteins with low tannin contents. In a grass-based diets Indigofera sp colud be used at the level of 30 to 45% (DM for growing kids.

  7. Identification, characterization, and in vitro culture of highly divergent arenaviruses from boa constrictors and annulated tree boas: candidate etiological agents for snake inclusion body disease.

    Science.gov (United States)

    Stenglein, Mark D; Sanders, Chris; Kistler, Amy L; Ruby, J Graham; Franco, Jessica Y; Reavill, Drury R; Dunker, Freeland; Derisi, Joseph L

    2012-01-01

    Inclusion body disease (IBD) is an infectious fatal disease of snakes typified by behavioral abnormalities, wasting, and secondary infections. At a histopathological level, the disease is identified by the presence of large eosinophilic cytoplasmic inclusions in multiple tissues. To date, no virus or other pathogen has been definitively characterized or associated with the disease. Using a metagenomic approach to search for candidate etiologic agents in snakes with confirmed IBD, we identified and de novo assembled the complete genomic sequences of two viruses related to arenaviruses, and a third arenavirus-like sequence was discovered by screening an additional set of samples. A continuous boa constrictor cell line was established and used to propagate and isolate one of the viruses in culture. Viral nucleoprotein was localized and concentrated within large cytoplasmic inclusions in infected cells in culture and tissues from diseased snakes. In total, viral RNA was detected in 6/8 confirmed IBD cases and 0/18 controls. These viruses have a typical arenavirus genome organization but are highly divergent, belonging to a lineage separate from that of the Old and New World arenaviruses. Furthermore, these viruses encode envelope glycoproteins that are more similar to those of filoviruses than to those of other arenaviruses. These findings implicate these viruses as candidate etiologic agents of IBD. The presence of arenaviruses outside mammals reveals that these viruses infect an unexpectedly broad range of species and represent a new reservoir of potential human pathogens. Inclusion body disease (IBD) is a common infectious disease of captive snakes. IBD is fatal and can cause the loss of entire animal collections. The cause of the disease has remained elusive, and no treatment exists. In addition to being important to pet owners, veterinarians, breeders, zoological parks, and aquariums, the study of animal disease is significant since animals are the source of

  8. Silica-coated magnetic nanoparticles impair proteasome activity and increase the formation of cytoplasmic inclusion bodies in vitro

    Science.gov (United States)

    Phukan, Geetika; Shin, Tae Hwan; Shim, Jeom Soon; Paik, Man Jeong; Lee, Jin-Kyu; Choi, Sangdun; Kim, Yong Man; Kang, Seong Ho; Kim, Hyung Sik; Kang, Yup; Lee, Soo Hwan; Mouradian, M. Maral; Lee, Gwang

    2016-01-01

    The potential toxicity of nanoparticles, particularly to neurons, is a major concern. In this study, we assessed the cytotoxicity of silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate dye (MNPs@SiO2(RITC)) in HEK293 cells, SH-SY5Y cells, and rat primary cortical and dopaminergic neurons. In cells treated with 1.0 μg/μl MNPs@SiO2(RITC), the expression of several genes related to the proteasome pathway was altered, and proteasome activity was significantly reduced, compared with control and with 0.1 μg/μl MNPs@SiO2(RITC)-treated cells. Due to the reduction of proteasome activity, formation of cytoplasmic inclusions increased significantly in HEK293 cells over-expressing the α–synuclein interacting protein synphilin-1 as well as in primary cortical and dopaminergic neurons. Primary neurons, particularly dopaminergic neurons, were more vulnerable to MNPs@SiO2(RITC) than SH-SY5Y cells. Cellular polyamines, which are associated with protein aggregation, were significantly altered in SH-SY5Y cells treated with MNPs@SiO2(RITC). These findings highlight the mechanisms of neurotoxicity incurred by nanoparticles. PMID:27378605

  9. Active protein aggregates induced by terminally attached self-assembling peptide ELK16 in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Zhou Bihong

    2011-02-01

    Full Text Available Abstract Background In recent years, it has been gradually realized that bacterial inclusion bodies (IBs could be biologically active. In particular, several proteins including green fluorescent protein, β-galactosidase, β-lactamase, alkaline phosphatase, D-amino acid oxidase, polyphosphate kinase 3, maltodextrin phosphorylase, and sialic acid aldolase have been successfully produced as active IBs when fused to an appropriate partner such as the foot-and-mouth disease virus capsid protein VP1, or the human β-amyloid peptide Aβ42(F19D. As active IBs may have many attractive advantages in enzyme production and industrial applications, it is of considerable interest to explore them further. Results In this paper, we report that an ionic self-assembling peptide ELK16 (LELELKLK2 was able to effectively induce the formation of cytoplasmic inclusion bodies in Escherichia coli (E. coli when attached to the carboxyl termini of four model proteins including lipase A, amadoriase II, β-xylosidase, and green fluorescent protein. These aggregates had a general appearance similar to the usually reported cytoplasmic inclusion bodies (IBs under transmission electron microscopy or fluorescence confocal microscopy. Except for lipase A-ELK16 fusion, the three other fusion protein aggregates retained comparable specific activities with the native counterparts. Conformational analyses by Fourier transform infrared spectroscopy revealed the existence of newly formed antiparallel beta-sheet structures in these ELK16 peptide-induced inclusion bodies, which is consistent with the reported assembly of the ELK16 peptide. Conclusions This has been the first report where a terminally attached self-assembling β peptide ELK16 can promote the formation of active inclusion bodies or active protein aggregates in E. coli. It has the potential to render E. coli and other recombinant hosts more efficient as microbial cell factories for protein production. Our observation might

  10. Importin-α7 Is Involved in the Formation of Ebola Virus Inclusion Bodies but Is Not Essential for Pathogenicity in Mice.

    Science.gov (United States)

    Gabriel, Gülsah; Feldmann, Friederike; Reimer, Rudolph; Thiele, Swantje; Fischer, Meike; Hartmann, Enno; Bader, Michael; Ebihara, Hideki; Hoenen, Thomas; Feldmann, Heinz

    2015-10-01

    Ebola virus (EBOV) protein 24 antagonizes the host interferon (IFN) response by hijacking select nuclear importin-α isoforms. Thereby, it blocks STAT1-mediated IFN-α/β and IFN-γ synthesis. However, owing to the lack of importin-α knockout animal models in the past, their role in EBOV pathogenesis remained largely unknown. Here, we demonstrate that importin-α7 is involved in the formation of EBOV inclusion bodies and replication. However, deletion of the gene encoding importin-α7 was not sufficient to increase survival rates among mice infected with EBOV. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  11. A Drosophila model of dominant inclusion body myopathy type 3 shows diminished myosin kinetics that reduce muscle power and yield myofibrillar defects.

    Science.gov (United States)

    Suggs, Jennifer A; Melkani, Girish C; Glasheen, Bernadette M; Detor, Mia M; Melkani, Anju; Marsan, Nathan P; Swank, Douglas M; Bernstein, Sanford I

    2017-06-01

    Individuals with inclusion body myopathy type 3 (IBM3) display congenital joint contractures with early-onset muscle weakness that becomes more severe in adulthood. The disease arises from an autosomal dominant point mutation causing an E706K substitution in myosin heavy chain type IIa. We have previously expressed the corresponding myosin mutation (E701K) in homozygous Drosophila indirect flight muscles and recapitulated the myofibrillar degeneration and inclusion bodies observed in the human disease. We have also found that purified E701K myosin has dramatically reduced actin-sliding velocity and ATPase levels. Since IBM3 is a dominant condition, we now examine the disease state in heterozygote Drosophila in order to gain a mechanistic understanding of E701K pathogenicity. Myosin ATPase activities in heterozygotes suggest that approximately equimolar levels of myosin accumulate from each allele. In vitro actin sliding velocity rates for myosin isolated from the heterozygotes were lower than the control, but higher than for the pure mutant isoform. Although sarcomeric ultrastructure was nearly wild type in young adults, mechanical analysis of skinned indirect flight muscle fibers revealed a 59% decrease in maximum oscillatory power generation and an approximately 20% reduction in the frequency at which maximum power was produced. Rate constant analyses suggest a decrease in the rate of myosin attachment to actin, with myosin spending decreased time in the strongly bound state. These mechanical alterations result in a one-third decrease in wing beat frequency and marginal flight ability. With aging, muscle ultrastructure and function progressively declined. Aged myofibrils showed Z-line streaming, consistent with the human heterozygote phenotype. Based upon the mechanical studies, we hypothesize that the mutation decreases the probability of the power stroke occurring and/or alters the degree of movement of the myosin lever arm, resulting in decreased in vitro

  12. A Drosophila model of dominant inclusion body myopathy type 3 shows diminished myosin kinetics that reduce muscle power and yield myofibrillar defects

    Directory of Open Access Journals (Sweden)

    Jennifer A. Suggs

    2017-06-01

    Full Text Available Individuals with inclusion body myopathy type 3 (IBM3 display congenital joint contractures with early-onset muscle weakness that becomes more severe in adulthood. The disease arises from an autosomal dominant point mutation causing an E706K substitution in myosin heavy chain type IIa. We have previously expressed the corresponding myosin mutation (E701K in homozygous Drosophila indirect flight muscles and recapitulated the myofibrillar degeneration and inclusion bodies observed in the human disease. We have also found that purified E701K myosin has dramatically reduced actin-sliding velocity and ATPase levels. Since IBM3 is a dominant condition, we now examine the disease state in heterozygote Drosophila in order to gain a mechanistic understanding of E701K pathogenicity. Myosin ATPase activities in heterozygotes suggest that approximately equimolar levels of myosin accumulate from each allele. In vitro actin sliding velocity rates for myosin isolated from the heterozygotes were lower than the control, but higher than for the pure mutant isoform. Although sarcomeric ultrastructure was nearly wild type in young adults, mechanical analysis of skinned indirect flight muscle fibers revealed a 59% decrease in maximum oscillatory power generation and an approximately 20% reduction in the frequency at which maximum power was produced. Rate constant analyses suggest a decrease in the rate of myosin attachment to actin, with myosin spending decreased time in the strongly bound state. These mechanical alterations result in a one-third decrease in wing beat frequency and marginal flight ability. With aging, muscle ultrastructure and function progressively declined. Aged myofibrils showed Z-line streaming, consistent with the human heterozygote phenotype. Based upon the mechanical studies, we hypothesize that the mutation decreases the probability of the power stroke occurring and/or alters the degree of movement of the myosin lever arm, resulting in

  13. Effects of exposure to thin-ideal media images on body dissatisfaction: testing the inclusion of a disclaimer versus warning label.

    Science.gov (United States)

    Ata, Rheanna N; Thompson, J Kevin; Small, Brent J

    2013-09-01

    The current study was designed to determine whether the inclusion of a disclaimer (i.e., "Retouched photograph aimed at changing a person's physical appearance.") or warning (i.e., "Warning: Trying to look as thin as this model may be dangerous to your health.") added to images of thin/attractive models would affect body dissatisfaction and intent to diet in female undergraduate students (n=342). Participants were randomly assigned to one of four groups: (a) disclaimer, (b) warning, (c) model control, or (d) car control. Results revealed a significant interaction between group and time, whereby only the car control group reported a significant change (i.e., decrease) in body dissatisfaction over time. Groups did not differ on intent to diet measured at post-exposure. The results largely replicate other findings in this area and call into question advocacy efforts to label media images as a strategy to decrease women's identification with the stimuli. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. E. Coli

    Science.gov (United States)

    ... for the bacteria's medical name Escherichia coli . The strange thing about these bacteria — and lots of other ... In some cases, E. coli poisoning can cause life-threatening kidney problems. What Can Kids Do? Adults ...

  15. Frequency and circumstances of falls in people with inclusion body myositis: a questionnaire survey to explore falls management and physiotherapy provision.

    Science.gov (United States)

    Hiscock, A; Dewar, L; Parton, M; Machado, P; Hanna, M; Ramdharry, G

    2014-03-01

    To survey the incidence and circumstances of falls for people with inclusion body myositis (IBM) in the UK, and to investigate the provision of physiotherapy and falls management. Postal questionnaire survey. Participants completed questionnaires at home. Ninety-four people diagnosed with IBM were screened against the inclusion criteria. Seventy-two potential participants were sent a questionnaire, and 62 were completed and returned. Invited participants were sent an adapted Falls Event Questionnaire pertaining to falls, perceived causes of falls and the provision of physiotherapy. Questionnaires were returned anonymously. The proportions of respondents who reported a fall or a near fall, along with the frequencies of falls and near falls were calculated. Descriptive data of falls were collected pertaining to location and cause. Data analysis was performed to investigate provision of physiotherapy services. The response rate was 86% [62/72, mean (standard deviation) age 68 (8) years]. Falls were reported by 98% (61/62) of respondents, with 60% (37/62) falling frequently. In this study, age was not found to be an indicator of falls risk or frequency. Twenty-one percent (13/62) of respondents had not seen a physiotherapist in relation to their IBM symptoms, and of those that had, 31% (15/49) had not seen a physiotherapist until more than 12 months after IBM was diagnosed. Only 18% (11/61) of fallers reported that they had received falls management input. Falls are a common occurrence for people with IBM, independent of age and years since symptoms first presented, and are poorly addressed by appropriate physiotherapy management. National falls guidelines are not being followed, and referral rates to physiotherapy need to improve. Copyright © 2013 Chartered Society of Physiotherapy. Published by Elsevier Ltd. All rights reserved.

  16. Body mass index was associated with upstaging and upgrading in patients with low-risk prostate cancer who met the inclusion criteria for active surveillance.

    Science.gov (United States)

    de Cobelli, Ottavio; Terracciano, Daniela; Tagliabue, Elena; Raimondi, Sara; Galasso, Giacomo; Cioffi, Antonio; Cordima, Giovanni; Musi, Gennaro; Damiano, Rocco; Cantiello, Francesco; Detti, Serena; Victor Matei, Deliu; Bottero, Danilo; Renne, Giuseppe; Ferro, Matteo

    2015-05-01

    Obesity is associated with an increased risk of high-grade prostate cancer (PCa). The effect of body mass index (BMI) as a predictor of progression in men with low-risk PCa has been only poorly assessed. In this study, we evaluated the association of BMI with progression in patients with low-risk PCa who met the inclusion criteria for the active surveillance (AS) protocol. We assessed 311 patients who underwent radical prostatectomy and were eligible for AS according to the following criteria: clinical stage T2a or less, prostate-specific antigen level pT2) and upgraded (Gleason score ≥ 7; primary Gleason pattern 4) disease. Seminal vesicle invasion, positive lymph nodes, and tumor volume ≥ 0.5 ml were also recorded. We found that high BMI was significantly associated with upgrading, upstaging, and seminal vesicle invasion, whereas it was not associated with positive lymph nodes or large tumor volume. At multivariate analysis, 1 unit increase of BMI significantly increased the risk of upgrading, upstaging, seminal vesicle invasion, and any outcome by 21%, 23%, 27%, and 20%, respectively. The differences between areas under the receiver operating characteristics curves comparing models with and without BMI were statistically significant for upgrading (P = 0.0002), upstaging (P = 0.0007), and any outcome (P = 0.0001). BMI should be a selection criterion for inclusion of patients with low-risk PCa in AS programs. Our results support the idea that obesity is associated with worse prognosis and suggest that a close AS program is an appropriate treatment option for obese subjects. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Nonlinear elastic inclusions in isotropic solids

    KAUST Repository

    Yavari, A.; Goriely, A.

    2013-01-01

    We introduce a geometric framework to calculate the residual stress fields and deformations of nonlinear solids with inclusions and eigenstrains. Inclusions are regions in a body with different reference configurations from the body itself and can

  18. Prevention of inclusion body hepatitis/hydropericardium syndrome in progeny chickens by vaccination of breeders with fowl adenovirus and chicken anemia virus.

    Science.gov (United States)

    Toro, H; González, C; Cerda, L; Morales, M A; Dooner, P; Salamero, M

    2002-01-01

    The hypothesis that an effective protection of progeny chickens against inclusion body hepatitis/hydropericardium syndrome (IBH/HP) can be achieved by dual vaccination of breeders with fowl adenovirus (FAV) serotype 4 and chicken anemia virus (CAV) was tested. Thus, 17-wk-old brown leghorn pullet groups were vaccinated by different schemes including single FAV (inactivated), single CAV (attenuated), FAV and CAV dually, or were not vaccinated (controls). Subsequent progenies of these breeders were challenged with the virulent strains FAV-341 and CAV-10343 following three strategies: 1) FAV-341 intramuscularly (i.m.) at day 10 of age (only FAV-vaccinated and control progenies); 2) FAV + CAV i.m. simultaneously at day 10 of age (all progenies); 3) CAV i.m. at day 1 and FAV orally at day 10 of age (all progenies). The induction of IBH/HP in these progenies was evaluated throughout a 10-day period. Both breeder groups vaccinated against FAV and those vaccinated against CAV increased virus neutralizing specific antibodies. Challenge strategy 1 showed 26.6% mortality in control progeny chickens and 13.3% in the progeny of FAV-vaccinated breeders. Presence of lesions in the liver of these groups showed no significant differences (P > 0.05), suggesting a discreet protective effect of the vaccine. Challenge strategy 2 showed 29.4% mortality in controls and 94% of chickens showed hepatic inclusion bodies (HIB). Single CAV vaccination of breeders did not demonstrate a beneficial effect, with both mortality and liver lesions resembling the nonvaccinated controls. FAV vaccination of breeders significantly reduced both mortality (7.4%) and liver lesions (26% HIB) (P vaccination of breeders with FAV and CAV proved to be necessary to achieve maximum protection of the progeny (no mortality and 7% HIB). Challenge strategy 3 produced no mortality but consistent liver damage in controls (96% HIB). In this case, both CAV and FAV + CAV-vaccinated breeders showed best protection results

  19. Sporadic inclusion body myositis: pilot study on the effects of a home exercise program on muscle function, histopathology and inflammatory reaction.

    Science.gov (United States)

    Arnardottir, Snjolaug; Alexanderson, Helene; Lundberg, Ingrid E; Borg, Kristian

    2003-01-01

    To evaluate the safety and effect of a home training program on muscle function in 7 patients with sporadic inclusion body myositis. The patients performed exercise 5 days a week over a 12-week period. Safety was assessed by clinical examination, repeated muscle biopsies and serum levels of creatine kinase. Muscle strength was evaluated by clinical examination, dynamic dynamometer and by a functional index in myositis. Strength was not significantly improved after the exercise, however none of the patients deteriorated concerning muscle function. The histopathology was unchanged and there were no signs of increased muscle inflammation or of expression of cytokines and adhesion molecules in the muscle biopsies. Creatine kinase levels were unchanged. A significant decrease was found in the areas that were positively stained for EN-4 (a marker for endothelial cells) in the muscle biopsies after training. The home exercise program was considered as not harmful to the muscles regarding muscle inflammation and function. Exercise may prevent loss of muscle strength due to disease and/or inactivity.

  20. Comparison of Serum rAAV Serotype-Specific Antibodies in Patients with Duchenne Muscular Dystrophy, Becker Muscular Dystrophy, Inclusion Body Myositis, or GNE Myopathy.

    Science.gov (United States)

    Zygmunt, Deborah A; Crowe, Kelly E; Flanigan, Kevin M; Martin, Paul T

    2017-09-01

    Recombinant adeno-associated virus (rAAV) is a commonly used gene therapy vector for the delivery of therapeutic transgenes in a variety of human diseases, but pre-existing serum antibodies to viral capsid proteins can greatly inhibit rAAV transduction of tissues. Serum was assayed from patients with Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), inclusion body myositis (IBM), and GNE myopathy (GNE). These were compared to serum from otherwise normal human subjects to determine the extent of pre-existing serum antibodies to rAAVrh74, rAAV1, rAAV2, rAAV6, rAAV8, and rAAV9. In almost all cases, patients with measurable titers to one rAAV serotype showed titers to all other serotypes tested, with average titers to rAAV2 being highest in all instances. Twenty-six percent of all young normal subjects (18 years old). Fifty percent of all IBM and GNE patients also had antibody titers to all rAAV serotypes, while only 18% of DMD and 0% of BMD patients did. In addition, serum-naïve macaques treated systemically with rAAVrh74 could develop cross-reactive antibodies to all other serotypes tested at 24 weeks post treatment. These data demonstrate that most DMD and BMD patients should be amenable to vascular rAAV-mediated treatment without the concern of treatment blockage by pre-existing serum rAAV antibodies, and that serum antibodies to rAAVrh74 are no more common than those for rAAV6, rAAV8, or rAAV9.

  1. Identification of the CFTR c.1666A>G Mutation in Hereditary Inclusion Body Myopathy Using Next-Generation Sequencing Analysis

    Directory of Open Access Journals (Sweden)

    Yan Lu

    2018-05-01

    Full Text Available Hereditary inclusion body myopathy (HIBM is a rare autosomal recessive adult onset muscle disease which affects one to three individuals per million worldwide. This disease is autosomal dominant and occurs in adulthood. Our previous study reported a new subtype of HIBM linked to the susceptibility locus at 7q22.1-31.1. The present study is aimed to identify the candidate gene responsible for the phenotype in HIBM pedigree. After multipoint linkage analysis, we performed targeted capture sequencing on 16 members and whole-exome sequencing (WES on 5 members. Bioinformatics filtering was performed to prioritize the candidate pathogenic gene variants, which were further genotyped by Sanger sequencing. Our results showed that the highest peak of LOD score (4.70 was on chromosome 7q22.1-31.1.We identified 2 and 22 candidates using targeted capture sequencing and WES respectively, only one of which as CFTRc.1666A>G mutation was well cosegregated with the HIBM phenotype. Using transcriptome analysis, we did not detect the differences of CFTR's mRNA expression in the proband compared with healthy members. Due to low incidence of HIBM and there is no other pedigree to assess, mutation was detected in three patients with duchenne muscular dystrophyn (DMD and five patients with limb-girdle muscular dystrophy (LGMD. And we found that the frequency of mutation detected in DMD and LGMD patients was higher than that of being expected in normal population. We suggested that the CFTRc.1666A>G may be a candidate marker which has strong genetic linkage with the causative gene in the HIBM family.

  2. Newcastle disease virus-attenuated vaccine co-contaminated with fowl adenovirus and chicken infectious anemia virus results in inclusion body hepatitis-hydropericardium syndrome in poultry.

    Science.gov (United States)

    Su, Qi; Li, Yang; Meng, Fanfeng; Cui, Zhizhong; Chang, Shuang; Zhao, Peng

    2018-05-01

    Inclusion body hepatitis-hydropericardium syndrome (IBH-HPS) induced by fowl adenovirus type 4 (FAdV-4) has caused huge economic losses to the poultry industry of China, but the source of infection for different flocks, especially flocks with high biological safety conditions, has remained unclear. This study tested the pathogenicity of Newcastle disease virus (NDV)-attenuated vaccine from a large-scale poultry farm in China where IBH-HPS had appeared with high mortality. Analysis revealed that the NDV-attenuated vaccine in use from the abovementioned poultry farm was simultaneously contaminated with FAdV-4 and chicken infectious anemia virus (CIAV). The FAdV and CIAV isolated from the vaccine were purified for the artificial preparation of an NDV-attenuated vaccine singly contaminated with FAdV or CIAV, or simultaneously contaminated with both of them. Seven-day-old specific pathogen-free chicks were inoculated with the artificially prepared contaminated vaccines and tested for corresponding indices. The experiments showed that no hydropericardium syndrome (HPS) and corresponding death occurred after administering the NDV-attenuated vaccine singly contaminated with FAdV or CIAV, but a mortality of 75% with IBH-HPS was commonly found in birds after administering the NDV-attenuated vaccine co-contaminated with FAdV and CIAV. In conclusion, this study found the co-contamination of FAdV-4 and CIAV in the same attenuated vaccine and confirmed that such a contaminated attenuated vaccine was a significant source of infection for outbreaks of IBH-HPS in some flocks. Copyright © 2018 Elsevier B.V. All rights reserved.

  3. Mortality and Causes of Death in Patients with Sporadic Inclusion Body Myositis: Survey Study Based on the Clinical Experience of Specialists in Australia, Europe and the USA

    Science.gov (United States)

    Price, Mark A.; Barghout, Victoria; Benveniste, Olivier; Christopher-Stine, Lisa; Corbett, Alastair; de Visser, Marianne; Hilton-Jones, David; Kissel, John T.; Lloyd, Thomas E.; Lundberg, Ingrid E.; Mastaglia, Francis; Mozaffar, Tahseen; Needham, Merrilee; Schmidt, Jens; Sivakumar, Kumaraswamy; DeMuro, Carla; Tseng, Brian S.

    2016-01-01

    Background: There is a paucity of data on mortality and causes of death (CoDs) in patients with sporadic inclusion body myositis (sIBM), a rare, progressive, degenerative, inflammatory myopathy that typically affects those aged over 50 years. Objective: Based on patient records and expertise of clinical specialists, this study used questionnaires to evaluate physicians’ views on clinical characteristics of sIBM that may impact on premature mortality and CoDs in these patients. Methods: Thirteen physicians from seven countries completed two questionnaires online between December 20, 2012 and January 15, 2013. Responses to the first questionnaire were collated and presented in the second questionnaire to seek elaboration and identify consensus. Results: All 13 physicians completed both questionnaires, providing responses based on 585 living and 149 deceased patients under their care. Patients were reported to have experienced dysphagia (60.2%) and injurious falls (44.3%) during their disease. Over half of physicians reported that a subset of their patients with sIBM had a shortened lifespan (8/13), and agreed that bulbar dysfunction/dysphagia/oropharyngeal involvement (12/13), early-onset disease (8/13), severe symptoms (8/13), and falls (7/13) impacted lifespan. Factors related to sIBM were reported as CoDs in 40% of deceased patients. Oropharyngeal muscle dysfunction was ranked as the leading feature of sIBM that could contribute to death. The risk of premature mortality was higher than the age-matched comparison population. Conclusions: In the absence of data from traditional sources, this study suggests that features of sIBM may contribute to premature mortality and may be used to inform future studies. PMID:27854208

  4. Nuclear inclusion bodies of mutant and wild-type p53 in cancer: a hallmark of p53 inactivation and proteostasis remodelling by p53 aggregation.

    Science.gov (United States)

    De Smet, Frederik; Saiz Rubio, Mirian; Hompes, Daphne; Naus, Evelyne; De Baets, Greet; Langenberg, Tobias; Hipp, Mark S; Houben, Bert; Claes, Filip; Charbonneau, Sarah; Delgado Blanco, Javier; Plaisance, Stephane; Ramkissoon, Shakti; Ramkissoon, Lori; Simons, Colinda; van den Brandt, Piet; Weijenberg, Matty; Van England, Manon; Lambrechts, Sandrina; Amant, Frederic; D'Hoore, André; Ligon, Keith L; Sagaert, Xavier; Schymkowitz, Joost; Rousseau, Frederic

    2017-05-01

    Although p53 protein aggregates have been observed in cancer cell lines and tumour tissue, their impact in cancer remains largely unknown. Here, we extensively screened for p53 aggregation phenotypes in tumour biopsies, and identified nuclear inclusion bodies (nIBs) of transcriptionally inactive mutant or wild-type p53 as the most frequent aggregation-like phenotype across six different cancer types. p53-positive nIBs co-stained with nuclear aggregation markers, and shared molecular hallmarks of nIBs commonly found in neurodegenerative disorders. In cell culture, tumour-associated stress was a strong inducer of p53 aggregation and nIB formation. This was most prominent for mutant p53, but could also be observed in wild-type p53 cell lines, for which nIB formation correlated with the loss of p53's transcriptional activity. Importantly, protein aggregation also fuelled the dysregulation of the proteostasis network in the tumour cell by inducing a hyperactivated, oncogenic heat-shock response, to which tumours are commonly addicted, and by overloading the proteasomal degradation system, an observation that was most pronounced for structurally destabilized mutant p53. Patients showing tumours with p53-positive nIBs suffered from a poor clinical outcome, similar to those with loss of p53 expression, and tumour biopsies showed a differential proteostatic expression profile associated with p53-positive nIBs. p53-positive nIBs therefore highlight a malignant state of the tumour that results from the interplay between (1) the functional inactivation of p53 through mutation and/or aggregation, and (2) microenvironmental stress, a combination that catalyses proteostatic dysregulation. This study highlights several unexpected clinical, biological and therapeutically unexplored parallels between cancer and neurodegeneration. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2016 Pathological Society of Great

  5. Inclusive pedagogy

    DEFF Research Database (Denmark)

    Korsgaard, Morten Timmermann; Skov Mortensen, Stig

    This article will present a case for a shift in perspective in inclusive education research towards a continentally inspired approach. Drawing on the age old distinction between continental and Anglo-American educational research the aim is to flesh out what a shift to a continental approach...... will entail, and why it might be beneficial to research in inclusive education...

  6. Social inclusion and inclusive education

    Directory of Open Access Journals (Sweden)

    Marsela Robo

    2014-07-01

    In line with global debate on social inclusion and exclusion, the author brings the way this debate has now pervaded both the official and development policy discourse in Albania.Social inclusion is considered as one of the priorities of the current government, with poverty reduction as its main focus, which will be ensured not only through economic development. In the end, the article focuses on the role of education as a very important and useful tool for ensuring social inclusion.Social inclusion through education, in particular through vocational education, considered by the author as the only way towards sustainable development of Albanian society.

  7. Inclusive pedagogy

    DEFF Research Database (Denmark)

    Korsgaard, Morten Timmermann; Mortensen, Stig Skov

    of education. The article explores the relationship between Continental and Anglo-American educational theory, and why they seem to have developed in such distinct directions. Beginning with the Anglo-American perspective it is outlined how pedagogy and the so called educational interest became replaced...... and the politicisation of inclusive education, and a positive aim in the form of an argument for a move towards constructing a pedagogical ideal of inclusion....

  8. Inclusions and inhomogeneities under stress

    CSIR Research Space (South Africa)

    Nabarro, FRN

    1996-02-01

    Full Text Available Some general theorems, new and old, concerning the behaviour of elastic inclusions and inhomogeneities in bodies without or with external stress, are assembled. The principal new result is that arbitrary external tractions cannot influence the shape...

  9. Mechanisms of pressure-mediated cell death and injury in Escherichia coli: from fundamentals to food applications.

    Directory of Open Access Journals (Sweden)

    Michael eGänzle

    2015-06-01

    Full Text Available High hydrostatic pressure is commercially applied to extend the shelf life of foods, and to improve food safety. Current applications operate at ambient temperature and 600 MPa or less. However, bacteria that may resist this pressure level include the pathogens Staphylococcus aureus and strains of Escherichia coli, including shiga-toxin producing E. coli. The resistance of E. coli to pressure is variable between strains and highly dependent on the food matrix. The targeted design of processes for the safe elimination of E. coli thus necessitates deeper insights into mechanisms of interaction and matrix-strain interactions. Cellular targets of high pressure treatment in E. coli include the barrier properties of the outer membrane, the integrity of the cytoplasmic membrane as well as the activity of membrane-bound enzymes, and the integrity of ribosomes. The pressure-induced denaturation of membrane bound enzymes results in generation of reactive oxygen species and subsequent cell death caused by oxidative stress. Remarkably, pressure resistance at the single cell level relates to the disposition of misfolded proteins in inclusion bodies. While the pressure resistance E. coli can be manipulated by over-expression or deletion of (stress proteins, the mechanisms of pressure resistance in wild type strains is multi-factorial and not fully understood. This review aims to provide an overview on mechanisms of pressure-mediated cell death in E. coli, and the use of this information for optimization of high pressure processing of foods.

  10. Mechanisms of pressure-mediated cell death and injury in Escherichia coli: from fundamentals to food applications.

    Science.gov (United States)

    Gänzle, Michael; Liu, Yang

    2015-01-01

    High hydrostatic pressure is commercially applied to extend the shelf life of foods, and to improve food safety. Current applications operate at ambient temperature and 600 MPa or less. However, bacteria that may resist this pressure level include the pathogens Staphylococcus aureus and strains of Escherichia coli, including shiga-toxin producing E. coli. The resistance of E. coli to pressure is variable between strains and highly dependent on the food matrix. The targeted design of processes for the safe elimination of E. coli thus necessitates deeper insights into mechanisms of interaction and matrix-strain interactions. Cellular targets of high pressure treatment in E. coli include the barrier properties of the outer membrane, the integrity of the cytoplasmic membrane as well as the activity of membrane-bound enzymes, and the integrity of ribosomes. The pressure-induced denaturation of membrane bound enzymes results in generation of reactive oxygen species and subsequent cell death caused by oxidative stress. Remarkably, pressure resistance at the single cell level relates to the disposition of misfolded proteins in inclusion bodies. While the pressure resistance E. coli can be manipulated by over-expression or deletion of (stress) proteins, the mechanisms of pressure resistance in wild type strains is multi-factorial and not fully understood. This review aims to provide an overview on mechanisms of pressure-mediated cell death in E. coli, and the use of this information for optimization of high pressure processing of foods.

  11. inclusions revisited

    Directory of Open Access Journals (Sweden)

    S. Migórski

    1995-01-01

    Full Text Available In this paper we confirm the validity of some recent results of Hu, Lakshmikantham, Papageorgiou [4] and Papageorgiou [13] concerning the existence and relaxation for nonlinear evolution inclusions. We fill a gap in the proofs of these results due to the use of incorrect Nagy's compactness embedding theorem.

  12. Translating Inclusion

    DEFF Research Database (Denmark)

    Fallov, Mia Arp; Birk, Rasmus

    2018-01-01

    The purpose of this paper is to explore how practices of translation shape particular paths of inclusion for people living in marginalized residential areas in Denmark. Inclusion, we argue, is not an end-state, but rather something which must be constantly performed. Active citizenship, today......, is not merely a question of participation, but of learning to become active in all spheres of life. The paper draws on empirical examples from a multi-sited field work in 6 different sites of local community work in Denmark, to demonstrate how different dimensions of translation are involved in shaping active...... citizenship. We propose the following different dimensions of translation: translating authority, translating language, translating social problems. The paper takes its theoretical point of departure from assemblage urbanism, arguing that cities are heterogeneous assemblages of socio-material interactions...

  13. Engineering the growth pattern and cell morphology for enhanced PHB production by Escherichia coli.

    Science.gov (United States)

    Wu, Hong; Chen, Jinchun; Chen, Guo-Qiang

    2016-12-01

    E. coli JM109∆envC∆nlpD deleted with genes envC and nlpD responsible for degrading peptidoglycan (PG) led to long filamentous cell shapes. When cell fission ring location genes minC and minD of Escherichia coli were deleted, E. coli JM109∆minCD changed the cell growth pattern from binary division to multiple fissions. Bacterial morphology can be further engineered by overexpressing sulA gene resulting in inhibition on FtsZ, thus generating very long cellular filaments. By overexpressing sulA in E. coli JM109∆envC∆nlpD and E. coli JM109∆minCD harboring poly(3-hydroxybutyrate) (PHB) synthesis operon phbCAB encoded in plasmid pBHR68, respectively, both engineered cells became long filaments and accumulated more PHB compared with the wild-type. Under same shake flask growth conditions, E. coli JM109∆minCD (pBHR68) overexpressing sulA grown in multiple fission pattern accumulated approximately 70 % PHB in 9 g/L cell dry mass (CDM), which was significantly higher than E. coli JM109∆envC∆nlpD and the wild type, that produced 7.6 g/L and 8 g/L CDM containing 64 % and 51 % PHB, respectively. Results demonstrated that a combination of the new division pattern with elongated shape of E. coli improved PHB production. This provided a new vision on the enhanced production of inclusion bodies.

  14. A study of the cytoplasmic expression of a form of human prolactin and of its solubilization and renaturation from bacterial inclusion bodies

    International Nuclear Information System (INIS)

    Affonso, Regina

    2000-01-01

    Different vector elements, that can determine a high expression level of a form of human prolactin (taghPrl) in bacterial cytoplasm, were studied. Expression conditions were first optimized for a reference vector, which was used to transform different strains of E. coli: HB2151, RRI and RB791. The highest expression level (113 ±16 μg/mL.A 600 ) was obtained in HB2151, after activation with only 0.1 mM IPTG. At this point the influence of the transcription terminator (g32 from bacteriophage T4), of the translation enhancer (g10 from bacteriophage T7), of the promoter (λP L or tac) and of the antibiotic resistance gene (amp r or kan r ) were studied. The first three elements did not show any significant influence, at least in our systems. On the contrary, the analysis of the influence of amp r and kan r genes showed, unexpectedly, that the presence of the last one provides an approximately 5-fold higher expression for taghPrl in E. coli cytoplasm. Finally, an appropriate extraction, solubilization, renaturation and purification process, able to provide a monomeric form of taghPrl, was studied. A method utilizing urea and mercaptoethanol as solubilizing agents and a dialysis as a renaturation procedure, provided with some modifications, one of the highest yields ever reported in the literature: 35.4 ± 4.5% of total recovery. Moreover, the biological activity of the taghPrl obtained, when tested in the Nb2 cell proliferation assay, was of the same order of that shown by the International Standard of human prolactin of pituitary origin. These data show that the cytoplasmic expression system here described, which can provide an expression efficiency 50-100 - fold higher than the periplasmic expression, can represent a valid alternative for the production of this and of other hormones of pharmaceutical interest and grade. (author)

  15. Escherichia Coli

    Science.gov (United States)

    Goodsell, David S.

    2009-01-01

    Diverse biological data may be used to create illustrations of molecules in their cellular context. I describe the scientific results that support a recent textbook illustration of an "Escherichia coli cell". The image magnifies a portion of the bacterium at one million times, showing the location and form of individual macromolecules. Results…

  16. Production of recombinant proteins from Plasmodium falciparum in Escherichia coli.

    Science.gov (United States)

    Guerra, Ángela Patricia; Calvo, Eliana Patricia; Wasserman, Moisés; Chaparro-Olaya, Jacqueline

    2016-02-23

    The production of recombinant proteins is essential for the characterization and functional study of proteins from Plasmodium falciparum. However, the proteins of P. falciparum are among the most challenging to express, and when expression is achieved, the recombinant proteins usually fold incorrectly and lead to the formation of inclusion bodies.  To obtain and purify four recombinant proteins and to use them as antigens to produce polyclonal antibodies. The production efficiency and solubility were evaluated as the proteins were expressed in two genetically modified strains of Escherichia coli to favor the production of heterologous proteins (BL21-CodonPlus (DE3)-RIL and BL21-pG-KJE8).  The four recombinant P. falciparum proteins corresponding to partial sequences of PfMyoA (Myosin A) and PfGAP50 (gliding associated protein 50), and the complete sequences of PfMTIP (myosin tail interacting protein) and PfGAP45 (gliding associated protein 45), were produced as glutathione S-transferase-fusion proteins, purified and used for immunizing mice.  The protein expression was much more efficient in BL21-CodonPlus, the strain that contains tRNAs that are rare in wild-type E. coli, compared to the expression in BL21-pG-KJE8. In spite of the fact that BL21-pG-KJE8 overexpresses chaperones, this strain did not minimize the formation of inclusion bodies.  The use of genetically modified strains of E. coli was essential to achieve high expression levels of the four evaluated P. falciparum proteins and lead to improved solubility of two of them. The approach used here allowed us to obtain and purify four P. falciparum proteins in enough quantity to produce polyclonal antibodies in mice, and a fair amount of two pure and soluble recombinant proteins for future assays.

  17. Engineering cell wall synthesis mechanism for enhanced PHB accumulation in E. coli.

    Science.gov (United States)

    Zhang, Xing-Chen; Guo, Yingying; Liu, Xu; Chen, Xin-Guang; Wu, Qiong; Chen, Guo-Qiang

    2018-01-01

    The rigidity of bacterial cell walls synthesized by a complicated pathway limit the cell shapes as coccus, bar or ellipse or even fibers. A less rigid bacterium could be beneficial for intracellular accumulation of poly-3-hydroxybutyrate (PHB) as granular inclusion bodies. To understand how cell rigidity affects PHB accumulation, E. coli cell wall synthesis pathway was reinforced and weakened, respectively. Cell rigidity was achieved by thickening the cell walls via insertion of a constitutive gltA (encoding citrate synthase) promoter in front of a series of cell wall synthesis genes on the chromosome of several E. coli derivatives, resulting in 1.32-1.60 folds increase of Young's modulus in mechanical strength for longer E. coli cells over-expressing fission ring FtsZ protein inhibiting gene sulA. Cell rigidity was weakened by down regulating expressions of ten genes in the cell wall synthesis pathway using CRISPRi, leading to elastic cells with more spaces for PHB accumulation. The regulation on cell wall synthesis changes the cell rigidity: E. coli with thickened cell walls accumulated only 25% PHB while cell wall weakened E. coli produced 93% PHB. Manipulation on cell wall synthesis mechanism adds another possibility to morphology engineering of microorganisms. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  18. Evaluation of a Novel Tc-99m Labelled Vitamin B12 Derivative for Targeting Escherichia coli and Staphylococcus aureus In Vitro and in an Experimental Foreign-Body Infection Model.

    Science.gov (United States)

    Baldoni, Daniela; Waibel, Robert; Bläuenstein, Peter; Galli, Filippo; Iodice, Violetta; Signore, Alberto; Schibli, Roger; Trampuz, Andrej

    2015-12-01

    Vitamin B12 (cyanocobalamin, Cbl) is accumulated by rapidly replicating prokaryotic and eukaryotic cells. We investigated the potential of a Tc-99m labelled Cbl derivative ([(99m)Tc]PAMA(4)-Cbl) for targeting infections caused by Escherichia coli and Staphylococcus aureus. In vitro binding assays were followed by biodistribution studies in a mouse model of foreign body infection. E. coli (ATCC 25922) and S. aureus (ATCC 43335) were used as test strains. [(57)Co]Cbl, [(67)Ga]citrate and [(99m)Tc]DTPA served as reference compounds. The in vitro competitive binding of [(57)Co]Cbl or [(99m)Tc]PAMA(4)-Cbl, and unlabeled Cbl, to viable or killed bacteria, was evaluated at 37 and 4 °C. A cage mouse model of infection was used for biodistribution of intravenous [(57)Co]Cbl and [(99m)Tc]PAMA(4)-Cbl in cage and dissected tissues of infected and non-infected mice. Maximum binding (mean ± SD) of [(57)Co]Cbl to viable E. coli was 81.7 ± 2.6 % and to S. aureus 34.0 ± 6.7 %, at 37 °C; no binding occurred to heat-killed bacteria. Binding to both test strains was displaced by 100- to 1000-fold excess of unlabeled Cbl. The in vitro binding of [(99m)Tc]PAMA(4)-Cbl was 100-fold and 3-fold lower than the one of [(57)Co]Cbl for E. coli and S. aureus, respectively. In vivo, [(99m)Tc]PAMA(4)-Cbl showed peak percentage of injected dose (% ID) values between 1.33 and 2.3, at 30 min post-injection (p.i.). Significantly higher retention occurred in cage fluids infected with S. aureus at 4 h and with E. coli at 8 h p.i. than in non-infected animals. Accumulation into infected cages was also higher than the one of [(99m)Tc]DTPA, which showed similar biodistribution in infected and sterile mice. [(57)Co]Cbl gradually accumulated in cages with peaks % ID between 3.58 and 4.83 % achieved from 24 to 48 h. Discrimination for infection occurred only in E. coli-infected mice, at 72 h p.i. [(67)Ga]citrate, which showed a gradual accumulation into cage fluids during 12 h, was

  19. E. Coli and Pregnancy

    Science.gov (United States)

    ... chat Live Help Fact Sheets Share Escherichia coli (E. coli) Friday, 01 September 2017 In every pregnancy, a ... risk. This sheet talks about whether exposure to E. coli may increase the risk for birth defects over ...

  20. E coli enteritis

    Science.gov (United States)

    ... coli; Food poisoning - E. coli; E. coli diarrhea; Hamburger disease ... coleslaw or potato salad) that have been out of the refrigerator too ... reheated Fish or oysters Raw fruits or vegetables that have ...

  1. Expression of human mag-1 gene in E. coli and preparation of its antibody

    International Nuclear Information System (INIS)

    Lin Huiyun; Xu Yuanji; Wang Yan; Chen Huihua; Du Zhiyan; Tan Xiaogang; Lu Yinglin

    2006-01-01

    Objective: To further investigate the new metastasis associated gene, mag-1 expressed in E. coli and its anti-body was prepared in rabbit. Methods: mag-1 was amplified by PCR from pcDNA3-mag-1 and directly cloned into pET-28a vector. The fusion protein was expressed in BL21 and identified by Western blot using anti-His monoclonal antibody. Rabbit was immunized with partially purified fusion protein subcutaneously. Results: Sequence analysis revealed identity of the sequence obtained to the previous report. The recombinant His-mag-1 could be expressed in E. coli as a fusion protein of 18 x 10 3 . The recombinant protein was mostly expressed in the inclusion bodies on the induction by 0.1 mmol/L IPTG at 37 degree C for 6 hours. Western blot analysis showed that the recombinant protein could be recognized by His monoclonal anti-body. The titer of polyclonal antibody against mag-1 was 1:160000. Conclusion: The mag-1 gene is expressed in E. coli highly and its antibody is prepared successfully. (authors)

  2. Recombinant protein production data after expression in the bacterium Escherichia coli

    Directory of Open Access Journals (Sweden)

    J. Enrique Cantu-Bustos

    2016-06-01

    Full Text Available Fusion proteins have become essential for the expression and purification of recombinant proteins in Escherichia coli. The metal-binding protein CusF has shown several features that make it an attractive fusion protein and affinity tag: "Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF" (Cantu-Bustos et al., 2016 [1]. Here we present accompanying data from protein expression experiments; we tested different protein tags, temperatures, expression times, cellular compartments, and concentrations of inducer in order to obtain soluble protein and low formation of inclusion bodies. Additionally, we present data from the purification of the green fluorescent protein (GFP tagged with CusF, using Ag(I metal affinity chromatography.

  3. A Comparative Analysis of Recombinant Expression and Solubility Screening of Two Phytases in E. coli

    Directory of Open Access Journals (Sweden)

    Ashok Pandey

    2011-01-01

    Full Text Available Microbial phytases, especially from fungal and bacterial sources, have received much attention as food additives in human nutrition and as feed supplements for monogastric animals. An effective expression screening method for recombinant production of this enzyme on a small scale is industrially desirable. An effort has been made in this work to clone and express phytase genes from Aspergillus sp. and Escherichia coli with the selected host, vector and inducer combination. Albeit the formation of insoluble inclusion bodies by fungal phytase, recombinant E. coli appA was effectively expressed in a cost-effective manner in the periplasm of BL21plysS using an inducer concentration of 0.01 mM in 4 h of growth. Enzyme was purified in three consecutive steps and functional studies were carried out.

  4. Difference, inclusion, and mathematics education

    DEFF Research Database (Denmark)

    Figueiras, Lourdes; Healy, Lulu; Skovsmose, Ole

    2016-01-01

    The round-table discussion on Difference, Inclusion and Mathematics Education was in included in the scientific programme of VI SIPEM in recognition and celebration of the emerging body of research into the challenges of building a culture of mathematics education which values and respects the di...

  5. Confronting New Demands : Inclusive Growth, Inclusive Trade ...

    International Development Research Centre (IDRC) Digital Library (Canada)

    Confronting New Demands : Inclusive Growth, Inclusive Trade. Policymakers, businesspeople and civil society advocates need evidence-based research to react ... understood implications, such as labour standards and intellectual property; ...

  6. Escherichia coli pathotypes

    Science.gov (United States)

    Escherichia coli strains are important commensals of the intestinal tract of humans and animals; however, pathogenic strains, including diarrhea-inducing E. coli and extraintestinal pathogenic E. coli. Intestinal E. coli pathotypes may cause a dehydrating watery diarrhea, or more severe diseases su...

  7. Escherichia coli Protein Expression System for Acetylcholine Binding Proteins (AChBPs.

    Directory of Open Access Journals (Sweden)

    Nikita Abraham

    Full Text Available Nicotinic acetylcholine receptors (nAChR are ligand gated ion channels, identified as therapeutic targets for a range of human diseases. Drug design for nAChR related disorders is increasingly using structure-based approaches. Many of these structural insights for therapeutic lead development have been obtained from co-crystal structures of nAChR agonists and antagonists with the acetylcholine binding protein (AChBP. AChBP is a water soluble, structural and functional homolog of the extracellular, ligand-binding domain of nAChRs. Currently, AChBPs are recombinantly expressed in eukaryotic expression systems for structural and biophysical studies. Here, we report the establishment of an Escherichia coli (E. coli expression system that significantly reduces the cost and time of production compared to the existing expression systems. E. coli can efficiently express unglycosylated AChBP for crystallography and makes the expression of isotopically labelled forms feasible for NMR. We used a pHUE vector containing an N-terminal His-tagged ubiquitin fusion protein to facilitate AChBP expression in the soluble fractions, and thus avoid the need to recover protein from inclusion bodies. The purified protein yield obtained from the E. coli expression system is comparable to that obtained from existing AChBP expression systems. E. coli expressed AChBP bound nAChR agonists and antagonists with affinities matching those previously reported. Thus, the E. coli expression system significantly simplifies the expression and purification of functional AChBP for structural and biophysical studies.

  8. Diversity ? Inclusion: Promoting Integration in Higher Education

    Science.gov (United States)

    Tienda, Marta

    2013-01-01

    I argue that enrollment of a diverse student body is but a pragmatic first step toward the broader social goal of inclusion and ask whether motives for campus diversification are aligned with pedagogic goals. I address this question by focusing on inclusion, namely, organizational strategies and practices that promote meaningful social and…

  9. Singing and social inclusion

    Science.gov (United States)

    Welch, Graham F.; Himonides, Evangelos; Saunders, Jo; Papageorgi, Ioulia; Sarazin, Marc

    2014-01-01

    There is a growing body of neurological, cognitive, and social psychological research to suggest the possibility of positive transfer effects from structured musical engagement. In particular, there is evidence to suggest that engagement in musical activities may impact on social inclusion (sense of self and of being socially integrated). Tackling social exclusion and promoting social inclusion are common concerns internationally, such as in the UK and the EC, and there are many diverse Government ministries and agencies globally that see the arts in general and music in particular as a key means by which social needs can be addressed. As part of a wider evaluation of a national, Government-sponsored music education initiative for Primary-aged children in England (“Sing Up”), opportunity was taken by the authors, at the request of the funders, to assess any possible relationship between (a) children's developing singing behavior and development and (b) their social inclusion (sense of self and of being socially integrated). Subsequently, it was possible to match data from n = 6087 participants, drawn from the final 3 years of data collection (2008–2011), in terms of each child's individually assessed singing ability (based on their singing behavior of two well-known songs to create a “normalized singing score”) and their written responses to a specially-designed questionnaire that included a set of statements related to children's sense of being socially included to which the children indicated their level of agreement on a seven-point Likert scale. Data analyses suggested that the higher the normalized singing development rating, the more positive the child's self-concept and sense of being socially included, irrespective of singer age, sex and ethnicity. PMID:25120514

  10. Singing and social inclusion.

    Science.gov (United States)

    Welch, Graham F; Himonides, Evangelos; Saunders, Jo; Papageorgi, Ioulia; Sarazin, Marc

    2014-01-01

    There is a growing body of neurological, cognitive, and social psychological research to suggest the possibility of positive transfer effects from structured musical engagement. In particular, there is evidence to suggest that engagement in musical activities may impact on social inclusion (sense of self and of being socially integrated). Tackling social exclusion and promoting social inclusion are common concerns internationally, such as in the UK and the EC, and there are many diverse Government ministries and agencies globally that see the arts in general and music in particular as a key means by which social needs can be addressed. As part of a wider evaluation of a national, Government-sponsored music education initiative for Primary-aged children in England ("Sing Up"), opportunity was taken by the authors, at the request of the funders, to assess any possible relationship between (a) children's developing singing behavior and development and (b) their social inclusion (sense of self and of being socially integrated). Subsequently, it was possible to match data from n = 6087 participants, drawn from the final 3 years of data collection (2008-2011), in terms of each child's individually assessed singing ability (based on their singing behavior of two well-known songs to create a "normalized singing score") and their written responses to a specially-designed questionnaire that included a set of statements related to children's sense of being socially included to which the children indicated their level of agreement on a seven-point Likert scale. Data analyses suggested that the higher the normalized singing development rating, the more positive the child's self-concept and sense of being socially included, irrespective of singer age, sex and ethnicity.

  11. Singing and social inclusion

    Directory of Open Access Journals (Sweden)

    Graham Frederick Welch

    2014-07-01

    Full Text Available There is a growing body of neurological, cognitive and social psychological research to suggest the possibility of positive transfer effects from structured musical engagement. In particular, there is evidence to suggest that engagement in musical activities may impact on social inclusion (sense of self and of being socially integrated. Tackling social exclusion and promoting social inclusion are common concerns internationally, such as in the UK and the EC, and there are many diverse Government ministries and agencies globally that see the arts in general and music in particular as a key means by which social needs can be addressed. As part of a wider evaluation of a national, Government-sponsored music education initiative for Primary-aged children in England (‘Sing Up’, opportunity was taken by the authors, at the request of the funders, to assess any possible relationship between (a children’s developing singing behaviour and development and (b their social inclusion (sense of self and of being socially integrated. Subsequently, it was possible to match data from n=6087 participants, drawn from the final three years of data collection (2008-2011, in terms of each child’s individually assessed singing ability (based on their singing behaviour of two well-known songs to create a 'normalised singing score' and their written responses to a specially-designed questionnaire that included a set of statements related to children’s sense of being socially included to which the children indicated their level of agreement on a seven-point Likert scale. Data analyses suggested that the higher the normalized singing development rating, the more positive the child’s self-concept and sense of being socially included, irrespective of singer age, sex and ethnicity.

  12. Genetics Home Reference: inclusion body myopathy 2

    Science.gov (United States)

    ... molecules, sialic acid influences a wide variety of cellular functions including cell movement (migration), attaching cells to one another (adhesion), signaling between cells, and inflammation. The mutations responsible ...

  13. Limits to Inclusion

    Science.gov (United States)

    Hansen, Janne Hedegaard

    2012-01-01

    In this article, I will argue that a theoretical identification of the limit to inclusion is needed in the conceptual identification of inclusion. On the one hand, inclusion is formulated as a vision that is, in principle, limitless. On the other hand, there seems to be an agreement that inclusion has a limit in the pedagogical practice. However,…

  14. Sporadic inclusion-body myositis: conformational multifactorial ageing-related degenerative muscle disease associated with proteasomal and lysosomal inhibition, endoplasmic reticulum stress, and accumulation of amyloid-β42 oligomers and phosphorylated tau.

    Science.gov (United States)

    Askanas, Valerie; Engel, W King

    2011-04-01

    The pathogenesis of sporadic inclusion-body myositis (s-IBM), the most common muscle disease of older persons, is complex and multifactorial. Both the muscle fiber degeneration and the mononuclear-cell inflammation are components of the s-IBM pathology, but how each relates to the pathogenesis remains unsettled. We consider that the intramuscle fiber degenerative component plays the primary and the major pathogenic role leading to muscle fiber destruction and clinical weakness. In this article we review the newest research advances that provide a better understanding of the s-IBM pathogenesis. Cellular abnormalities occurring in s-IBM muscle fibers are discussed, including: several proteins that are accumulated in the form of aggregates within muscle fibers, including amyloid-β42 and its oligomers, and phosphorylated tau in the form of paired helical filaments, and we consider their putative detrimental influence; cellular mechanisms leading to protein misfolding and aggregation, including evidence of their inadequate disposal; pathogenic importance of endoplasmic reticulum stress and the unfolded protein response demonstrated in s-IBM muscle fibers; and decreased deacetylase activity of SIRT1. All these factors are combined with, and perhaps provoked by, an ageing intracellular milieu. Also discussed are the intriguing phenotypic similarities between s-IBM muscle fibers and the brains of Alzheimer and Parkinson's disease patients, the two most common neurodegenerative diseases associated with ageing. Muscle biopsy diagnostic criteria are also described and illustrated. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  15. Direct evidence for a chronic CD8+-T-cell-mediated immune reaction to tax within the muscle of a human T-cell leukemia/lymphoma virus type 1-infected patient with sporadic inclusion body myositis.

    Science.gov (United States)

    Ozden, Simona; Cochet, Madeleine; Mikol, Jacqueline; Teixeira, Antonio; Gessain, Antoine; Pique, Claudine

    2004-10-01

    Human T-cell leukemia/lymphoma virus type 1 (HTLV-1) infection can lead to the development of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), concomitantly with or without other inflammatory disorders such as myositis. These pathologies are considered immune-mediated diseases, and it is assumed that migration within tissues of both HTLV-1-infected CD4(+) T cells and anti-HTLV-1 cytotoxic T cells represents a pivotal event. However, although HTLV-1-infected T cells were found in inflamed lesions, the antigenic specificity of coinfiltrated CD8(+) T cells remains to be determined. In this study, we performed both ex vivo and in situ analyses using muscle biopsies obtained from an HTLV-1-infected patient with HAM/TSP and sporadic inclusion body myositis. We found that both HTLV-1-infected CD4(+) T cells and CD8(+) T cells directed to the dominant Tax antigen can be amplified from muscle cell cultures. Moreover, we were able to detect in two successive muscle biopsies both tax mRNA-positive mononuclear cells and T cells recognized by the Tax11-19/HLA-A*02 tetramer and positive for perforin. These findings provide the first direct demonstration that anti-Tax cytotoxic T cells are chronically recruited within inflamed tissues of an HTLV-1 infected patient, which validates the cytotoxic immune reaction model for the pathogenesis of HTLV-1-associated inflammatory disease.

  16. E. Coli Infections

    Science.gov (United States)

    E. coli is the name of a type of bacteria that lives in your intestines. Most types of E. coli are harmless. However, some types can make you ... type causes travelers' diarrhea. The worst type of E. coli causes bloody diarrhea, and can sometimes cause kidney ...

  17. Social imaginaries and inclusion

    DEFF Research Database (Denmark)

    Hansen, Janne Hedegaard

    2016-01-01

    that inclusion on the one hand may be seen to be about human rights, solidarity, and democracy, and on the other hand, it is about ensuring the cohesion of neoliberal society by means of every person’s obligation to realize one’s potential through learning, development, and education regardless of one’s needs......, the development of inclusive schools and inclusive learning environments will involve both inclusion and exclusion processes. With this starting point, international educational research knowledge about inclusive schools and inclusive learning environments in general will be related to the fundamental dilemma...

  18. Roadmap for Inclusive Innovation

    Data.gov (United States)

    Allegheny County / City of Pittsburgh / Western PA Regional Data Center — The Roadmap for Inclusive Innovation data set shares the status of action items under the Roadmap for Inclusive Innovation. Each action or project has been assigned...

  19. Teachers becoming inclusive practitioners

    African Journals Online (AJOL)

    , construct their identities in the light of inclusive education, and how they negotiate the tensions and contradictions emerging from the processof becoming inclusive practitioners. Central to this discussion is the understanding that teachers' ...

  20. Galectin-1 as a fusion partner for the production of soluble and folded human {beta}-1,4-galactosyltransferase-T7 in E. coli

    Energy Technology Data Exchange (ETDEWEB)

    Pasek, Marta [Structural Glycobiology Section, SAIC-Frederick, Inc., Center for Cancer Research Nanobiology Program, Center for Cancer Research, NCI-Frederick, Frederick, MD 2170 (United States); Boeggeman, Elizabeth; Ramakrishnan, Boopathy [Structural Glycobiology Section, SAIC-Frederick, Inc., Center for Cancer Research Nanobiology Program, Center for Cancer Research, NCI-Frederick, Frederick, MD 2170 (United States); Basic Science Program, SAIC-Frederick, Inc., Center for Cancer Research Nanobiology Program, Center for Cancer Research, NCI-Frederick, Frederick, MD 2170 (United States); Qasba, Pradman K., E-mail: qasba@helix.nih.gov [Structural Glycobiology Section, SAIC-Frederick, Inc., Center for Cancer Research Nanobiology Program, Center for Cancer Research, NCI-Frederick, Frederick, MD 2170 (United States)

    2010-04-09

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

  1. Mycobacterium tuberculosis HspX/EsxS Fusion Protein: Gene Cloning, Protein Expression, and Purification in Escherichia coli.

    Science.gov (United States)

    Khademi, Farzad; Yousefi-Avarvand, Arshid; Derakhshan, Mohammad; Meshkat, Zahra; Tafaghodi, Mohsen; Ghazvini, Kiarash; Aryan, Ehsan; Sankian, Mojtaba

    2017-10-01

    The purpose of this study was to clone, express, and purify a novel multidomain fusion protein of Micobacterium tuberculosis (Mtb) in a prokaryotic system. An hspX/esxS gene construct was synthesized and ligated into a pGH plasmid, E. coli TOP10 cells were transformed, and the vector was purified. The vector containing the construct and pET-21b (+) plasmid were digested with the same enzymes and the construct was ligated into pET-21b (+). The accuracy of cloning was confirmed by colony PCR and sequencing. E. coli BL21 cells were transformed with the pET-21b (+)/hspX/esxS expression vector and protein expression was evaluated. Finally, the expressed fusion protein was purified on a Ni-IDA column and verified by SDS-PAGE and western blotting. The hspX/esxS gene construct was inserted into pET-21b (+) and recombinant protein expression was induced with IPTG in E. coli BL21 cells. Various concentrations of IPTG were tested to determine the optimum concentration for expression induction. The recombinant protein was expressed in insoluble inclusion bodies. Three molar guanidine HCl was used to solubilize the insoluble protein. An HspX/EsxS Mtb fusion protein was expressed in E. coli and the recombinant protein was purified. After immunological analysis, the HspX/EsxS fusion protein might be an anti-tuberculosis vaccine candidate in future clinical trial studies.

  2. Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF.

    Science.gov (United States)

    Cantu-Bustos, J Enrique; Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Galbraith, David W; McEvoy, Megan M; Zarate, Xristo

    2016-05-01

    Production of recombinant proteins in Escherichia coli has been improved considerably through the use of fusion proteins, because they increase protein solubility and facilitate purification via affinity chromatography. In this article, we propose the use of CusF as a new fusion partner for expression and purification of recombinant proteins in E. coli. Using a cell-free protein expression system, based on the E. coli S30 extract, Green Fluorescent Protein (GFP) was expressed with a series of different N-terminal tags, immobilized on self-assembled protein microarrays, and its fluorescence quantified. GFP tagged with CusF showed the highest fluorescence intensity, and this was greater than the intensities from corresponding GFP constructs that contained MBP or GST tags. Analysis of protein production in vivo showed that CusF produces large amounts of soluble protein with low levels of inclusion bodies. Furthermore, fusion proteins can be exported to the cellular periplasm, if CusF contains the signal sequence. Taking advantage of its ability to bind copper ions, recombinant proteins can be purified with readily available IMAC resins charged with this metal ion, producing pure proteins after purification and tag removal. We therefore recommend the use of CusF as a viable alternative to MBP or GST as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Expression and purification of recombinant proteins in Escherichia coli tagged with a small metal-binding protein from Nitrosomonas europaea.

    Science.gov (United States)

    Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Zarate, Xristo

    2016-02-01

    Escherichia coli is still the preferred organism for large-scale production of recombinant proteins. The use of fusion proteins has helped considerably in enhancing the solubility of heterologous proteins and their purification with affinity chromatography. Here, the use of a small metal-binding protein (SmbP) from Nitrosomonas europaea is described as a new fusion protein for protein expression and purification in E. coli. Fluorescent proteins tagged at the N-terminal with SmbP showed high levels of solubility, compared with those of maltose-binding protein and glutathione S-transferase, and low formation of inclusion bodies. Using commercially available IMAC resins charged with Ni(II), highly pure recombinant proteins were obtained after just one chromatography step. Proteins may be purified from the periplasm of E. coli if SmbP contains the signal sequence at the N-terminal. After removal of the SmbP tag from the protein of interest, high-yields are obtained since SmbP is a protein of just 9.9 kDa. The results here obtained suggest that SmbP is a good alternative as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Delimiting Inclusive Design

    DEFF Research Database (Denmark)

    Herriott, Richard

    2014-01-01

    This paper was written as an answer to the question raised by my PhD dissertation on accessibility through user-centred and Inclusive Design (ID) methods: can Inclusive Design be delimited? The literature on Inclusive Design deals almost entrirely with consumer product design and assistive...

  5. Footstep towards Inclusive Education

    Science.gov (United States)

    Abbas, Faiza; Zafar, Aneeka; Naz, Tayyaba

    2016-01-01

    Inclusive education is a rising trend in the world. The first step towards inclusive education is providing the awareness to the general education teachers. This study focused to investigate the general education teachers of primary and secondary level awareness about the special education and inclusive education. This study is descriptive method…

  6. Inclusion: Conceptualization and measurement

    NARCIS (Netherlands)

    Jansen, W.S.; Otten, S.; Van der Zee, K.I.; Jans, L.

    2014-01-01

    n the present research, we introduced a conceptual framework of inclusion and subsequently used this as a starting point to develop and validate a scale to measure perceptions of inclusion. Departing from existing work on inclusion and complementing this with theoretical insights from optimal

  7. Inclusion : Conceptualization and measurement

    NARCIS (Netherlands)

    Jansen, Wiebren S.; Otten, Sabine; van der Zee, Karen I.; Jans, Lise

    In the present research, we introduced a conceptual framework of inclusion and subsequently used this as a starting point to develop and validate a scale to measure perceptions of inclusion. Departing from existing work on inclusion and complementing this with theoretical insights from optimal

  8. Inclusion: Conceptualization and measurement

    NARCIS (Netherlands)

    Jansens, Wiebren S.; Otten, Sabine; van der Zee, Karen; Jans, Lise

    2014-01-01

    In the present research, we introduced a conceptual framework of inclusion and subsequently used this as a starting point to develop and validate a scale to measure perceptions of inclusion. Departing from existing work on inclusion and complementing this with theoretical insights from optimal

  9. Functional expression of a valencene dioxygenase from Pleurotus sapidus in E. coli.

    Science.gov (United States)

    Zelena, Kateryna; Krings, Ulrich; Berger, Ralf G

    2012-03-01

    Valencene dioxygenase (ValOx) from the edible basidiomycete Pleurotus sapidus converted the sesquiterpene (+)-valencene to the valuable grapefruit flavour (+)-nootkatone and to nootkatols through intermediate hydroperoxides. Expression of the enzyme was carried out in the cytosol and periplasm of Escherichia coli. The heterologous production led to high yields of inclusion bodies. The poor yield of soluble recombinant protein was improved by various strategies including cold shock expression, chaperone co-expression, and employment of mutant E. coli strains. Up to 60 mg of the biologically active, soluble ValOx was produced by cold shock under control of the cspA promoter at 8 °C in the BL21(DE3)Star strain and co-expression of the E. coli trigger factor. The recombinant enzyme, purified using the N-terminal His tag, showed the catalytic properties of the wild-type enzyme, as was confirmed by the LC-MS analysis of hydroperoxide intermediates and GC-MS analysis of the volatile products. Copyright © 2011 Elsevier Ltd. All rights reserved.

  10. Secretory expression of nattokinase from Bacillus subtilis YF38 in Escherichia coli.

    Science.gov (United States)

    Liang, Xiaobo; Jia, Shifang; Sun, Yufang; Chen, Meiling; Chen, Xiuzhu; Zhong, Jin; Huan, Liandong

    2007-11-01

    Nattokinase producing bacterium, B. subtilis YF38, was isolated from douchi, using the fibrin plate method. The gene encoding this enzyme was cloned by polymerase chain reaction (PCR). Cytoplasmic expression of this enzyme in E. coli resulted in inactive inclusion bodies. But with the help of two different signal peptides, the native signal peptide of nattokinase and the signal peptide of PelB, active nattokinase was successfully expressed in E. coli with periplasmic secretion, and the nattokinase in culture medium displayed high fibrinolytic activity. The fibrinolytic activity of the expressed enzyme in the culture was determined to reach 260 urokinase units per micro-liter when the recombinant strain was induced by 0.7 mmol l(-1) isopropyl-beta-D- thiogalactopyranoside (IPTG) at 20 degrees C for 20 h, resulting 49.3 mg active enzyme per liter culture. The characteristic of this recombinant nattokinase is comparable to the native nattokinase from B. subtilis YF38. Secretory expression of nattokinase in E. coli would facilitate the development of this enzyme into a therapeutic product for the control and prevention of thrombosis diseases.

  11. Expression, crystallization and preliminary X-ray crystallographic studies of the outer membrane protein OmpW from Escherichia coli

    International Nuclear Information System (INIS)

    Albrecht, Reinhard; Zeth, Kornelius; Söding, Johannes; Lupas, Andrei; Linke, Dirk

    2006-01-01

    The outer membrane protein OmpW from E. coli was overexpressed in inclusion bodies and refolded with the help of detergent. The protein has been crystallized and the crystals diffract to 3.5 Å resolution. OmpW is an eight-stranded 21 kDa molecular-weight β-barrel protein from the outer membrane of Gram-negative bacteria. It is a major antigen in bacterial infections and has implications in antibiotic resistance and in the oxidative degradation of organic compounds. OmpW from Escherichia coli was cloned and the protein was expressed in inclusion bodies. A method for refolding and purification was developed which yields properly folded protein according to circular-dichroism measurements. The protein has been crystallized and crystals were obtained that diffracted to a resolution limit of 3.5 Å. The crystals belong to space group P422, with unit-cell parameters a = 122.5, c = 105.7 Å. A homology model of OmpW is presented based on known structures of eight-stranded β-barrels, intended for use in molecular-replacement trials

  12. Deploying and implementing Inclusive Physical Education

    Directory of Open Access Journals (Sweden)

    Eliana Lúcia Ferreira

    2014-04-01

    Full Text Available Physical Education, as a curricular component of basic education, is not indifferent to the movement of Inclusive physical education. Differentiated bodies are conquering new social spaces. Our aim through this investigation is to identify the main historical practices regarding Brazilian Policy of Inclusive Education and to point out proposals to implement inclusive Physical education. Our methodology consists of a descriptive study based on two main axes. The first axis is related to a historical discussion whose source was national documents of reference about Inclusive Physical Education. The second axis is related to the experience of 2000 teachers; we have the occasion to analyze their practices on the subject of Inclusive physical education. As a result, this investigation also develops a proposal to inclusive physical education; this proposal is established in another dimension and understanding of work and movement. Concerning the final considerations we suggest a disruption with already crystallized bodily practices and we suggest actions respecting individual differences. Moreover, we point out the fact that Inclusive Physical Education has advocated another meaning for the body, in favor of a more collective physical education and searching for activities in which individuality prevails.

  13. ANALISIS CEMARAN BAKTERI Escherichia coli ANALISIS CEMARAN BAKTERI Escherichia coli ANALISIS CEMARAN BAKTERI Escherichia coli

    OpenAIRE

    ANGGREINI, RAHAYU

    2015-01-01

    2015 RAHAYU ANGGREINI coli Penelitian ini bertujuan untuk melakukan identifikasi cemaran bakteri E. coli O157:H7 pada daging sapi di kota Makassar. Sampel pada penelitian ini sebanyak 72 sampel Kata Kunci : Daging sapi, pasar tradisional, E. coli, E. coli O157:H7, kontaminasi bakteri, identifikasi E. coli O157:H7.

  14. Foresighting for Inclusive Development

    DEFF Research Database (Denmark)

    Andersen, Allan Dahl; Andersen, Per Dannemand

    2017-01-01

    of policymaking processes affects the actual process with a focus on inclusion, and we discuss how it affects policy effectiveness and innovation system transformation. Our argument is that processes of policymaking must be inclusive to affect and transform innovation systems because a set of distributed actors...... in foresight cases in two emerging economies: Brazil and South Korea. We conclude that better systemic and innovation oriented foresight is needed to enhance inclusive development....

  15. INCLUSIVE EDUCATION IN ITALY

    OpenAIRE

    Voitkāne, Vita

    2017-01-01

    European member States implement Inclusive Education policies thus contributing to a sustainable, inclusive society, although each country is at a different stage in this process. Italy, one of the first countries to launch integrative learning, has set an example since the 1970s, although the quality of inclusive education is unpredictable due to many issues. Authors Cantoni and Panetta (2006) emphasize that, although the culture of integration in Italy exists, much needs to be done to impro...

  16. SUPPORT IN INCLUSIVE EDUCATION

    OpenAIRE

    Belma Čičkušić; Ševala Tulumović; Selma Bakić; Salem Bakić

    2016-01-01

    In order for inclusive class to be successful, associates are of great help to teachers. Besides associates, teachers' specialization can be accomplished through educational seminars on the inclusion topic. However, information about inclusion, working with children with special needs, can also be found in scientific journals that offer more information on methods of working with children with special needs, didactic materials customized according to abilities of children. Aim of ...

  17. “Inclonals”: IgGs and IgG-enzyme fusion proteins produced in an E. coli expression-refolding system

    OpenAIRE

    Hakim, Rahely; Benhar, Itai

    2009-01-01

    Full-length antibodies and antibodies that ferry a cargo to target cells are desired biopharmaceuticals. We describe the production of full-length IgGs and IgG-toxin fusion proteins in E. coli. In the presented examples of anti CD30 and anti EGF-receptor antibodies, the antibody heavy and light chains or toxin fusions thereof were expressed in separate bacterial cultures, where they accumulated as insoluble inclusion bodies. Following refolding and purification, high yields (up to 50 mg/L of ...

  18. Conjugation in Escherichia coli

    Science.gov (United States)

    Boyer, Herbert

    1966-01-01

    Boyer, Herbert (Yale University, New Haven, Conn.). Conjugation in Escherichia coli. J. Bacteriol. 91:1767–1772. 1966.—The sex factor of Escherichia coli K-12 was introduced into an E. coli B/r strain by circumventing the host-controlled modification and restriction incompatibilities known to exist between these closely related strains. The sexual properties of the constructed F+ B strain and its Hfr derivatives were examined. These studies showed that the E. coli strain B/r F+ and Hfr derivatives are similar to the E. coli strain K-12 F+ and Hfr derivatives. However, the site of sex factor integration was found to be dependent on the host genome. PMID:5327905

  19. Production of recombinant cholesterol oxidase containing covalently bound FAD in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Molla Gianluca

    2010-04-01

    Full Text Available Abstract Background Cholesterol oxidase is an alcohol dehydrogenase/oxidase flavoprotein that catalyzes the dehydrogenation of C(3-OH of cholesterol. It has two major biotechnological applications, i.e. in the determination of serum (and food cholesterol levels and as biocatalyst providing valuable intermediates for industrial steroid drug production. Cholesterol oxidases of type I are those containing the FAD cofactor tightly but not covalently bound to the protein moiety, whereas type II members contain covalently bound FAD. This is the first report on the over-expression in Escherichia coli of type II cholesterol oxidase from Brevibacterium sterolicum (BCO. Results Design of the plasmid construct encoding the mature BCO, optimization of medium composition and identification of the best cultivation/induction conditions for growing and expressing the active protein in recombinant E. coli cells, concurred to achieve a valuable improvement: BCO volumetric productivity was increased from ~500 up to ~25000 U/L and its crude extract specific activity from 0.5 up to 7.0 U/mg protein. Interestingly, under optimal expression conditions, nearly 55% of the soluble recombinant BCO is produced as covalently FAD bound form, whereas the protein containing non-covalently bound FAD is preferentially accumulated in insoluble inclusion bodies. Conclusions Comparison of our results with those published on non-covalent (type I COs expressed in recombinant form (either in E. coli or Streptomyces spp., shows that the fully active type II BCO can be produced in E. coli at valuable expression levels. The improved over-production of the FAD-bound cholesterol oxidase will support its development as a novel biotool to be exploited in biotechnological applications.

  20. Efficient production of xylitol from hemicellulosic hydrolysate using engineered Escherichia coli.

    Science.gov (United States)

    Su, Buli; Wu, Mianbin; Zhang, Zhe; Lin, Jianping; Yang, Lirong

    2015-09-01

    A metabolically engineered Escherichia coli has been constructed for the production of xylitol, one of the top 12 platform chemicals from agricultural sources identified by the US Department of Energy. An optimal plasmid was constructed to express xylose reductase from Neurospora crassa with almost no inclusion bodies at relatively high temperature. The phosphoenolpyruvate-dependent glucose phosphotransferase system (ptsG) was disrupted to eliminate catabolite repression and allow simultaneous uptake of glucose and xylose. The native pathway for D-xylose catabolism in E. coli W3110 was blocked by deleting the xylose isomerase (xylA) and xylulose kinase (xylB) genes. The putative pathway for xylitol phosphorylation was also blocked by disrupting the phosphoenolpyruvate-dependent fructose phosphotransferase system (ptsF). The xylitol producing recombinant E. coli allowed production of 172.4 g L(-1) xylitol after 110 h of fed-batch cultivation with an average productivity of 1.57 g L(-1) h(-1). The molar yield of xylitol to glucose reached approximately 2.2 (mol xylitol mol(-1) glucose). Furthermore, the recombinant strain also produced about 150 g L(-1) xylitol from hemicellulosic sugars in modified M9 minimal medium and the overall productivity was 1.40 g L(-1) h(-1), representing the highest xylitol concentration and productivity reported to date from hemicellulosic sugars using bacteria. Thus, this engineered E. coli is a candidate for the development of efficient industrial-scale production of xylitol from hemicellulosic hydrolysate. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  1. Inclusive Services Innovation Configuration

    Science.gov (United States)

    Holdheide, Lynn R.; Reschly, Daniel J.

    2011-01-01

    Teacher preparation to deliver inclusive services to students with disabilities is increasingly important because of changes in law and policy emphasizing student access to, and achievement in, the general education curriculum. This innovation configuration identifies the components of inclusive services that should be incorporated in teacher…

  2. Testing and Inclusive Schooling

    DEFF Research Database (Denmark)

    Morin, Anne; Hamre, Bjørn; Ydesen, Christian

    Testing and Inclusive Schooling provides a comparative on seemingly incompatible global agendas and efforts to include all children in the general school system, Thus reducing exclusion. With an examination of the international testing culture and the politics of inclusion currently permeating...

  3. Understanding Inclusion in Cyprus

    Science.gov (United States)

    Mamas, Christoforos

    2013-01-01

    This paper provides a framework for understanding inclusion in Cyprus. The evidence base is the result of a six-month qualitative research study in five Cypriot mainstream primary schools. Despite the rhetoric in favour of inclusion, it seems that the Cypriot educational system is still highly segregating in its philosophy and does not fully…

  4. Jet inclusive cross sections

    International Nuclear Information System (INIS)

    Del Duca, V.

    1992-11-01

    Minijet production in jet inclusive cross sections at hadron colliders, with large rapidity intervals between the tagged jets, is evaluated by using the BFKL pomeron. We describe the jet inclusive cross section for an arbitrary number of tagged jets, and show that it behaves like a system of coupled pomerons

  5. Production of recombinant proteins in Escherichia coli tagged with the fusion protein CusF3H.

    Science.gov (United States)

    Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Zarate, Xristo

    2017-04-01

    Recombinant protein expression in the bacterium Escherichia coli still is the number one choice for large-scale protein production. Nevertheless, many complications can arise using this microorganism, such as low yields, the formation of inclusion bodies, and the requirement for difficult purification steps. Most of these problems can be solved with the use of fusion proteins. Here, the use of the metal-binding protein CusF3H+ is described as a new fusion protein for recombinant protein expression and purification in E. coli. We have previously shown that CusF produces large amounts of soluble protein, with low levels of formation of inclusion bodies, and that proteins can be purified using IMAC resins charged with Cu(II) ions. CusF3H+ is an enhanced variant of CusF, formed by the addition of three histidine residues at the N-terminus. These residues then can bind Ni(II) ions allowing improved purity after affinity chromatography. Expression and purification of Green Fluorescent Protein tagged with CusF3H+ showed that the mutation did not alter the capacity of the fusion protein to increase protein expression, and purity improved considerably after affinity chromatography with immobilized nickel ions; high yields are obtained after tag-removal since CusF3H+ is a small protein of just 10 kDa. Furthermore, the results of experiments involving expression of tagged proteins having medium to large molecular weights indicate that the presence of the CusF3H+ tag improves protein solubility, as compared to a His-tag. We therefore endorse CusF3H+ as a useful alternative fusion protein/affinity tag for production of recombinant proteins in E. coli. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. GroEL-GroES assisted folding of multiple recombinant proteins simultaneously over-expressed in Escherichia coli.

    Science.gov (United States)

    Goyal, Megha; Chaudhuri, Tapan K

    2015-07-01

    Folding of aggregation prone recombinant proteins through co-expression of chaperonin GroEL and GroES has been a popular practice in the effort to optimize preparation of functional protein in Escherichia coli. Considering the demand for functional recombinant protein products, it is desirable to apply the chaperone assisted protein folding strategy for enhancing the yield of properly folded protein. Toward the same direction, it is also worth attempting folding of multiple recombinant proteins simultaneously over-expressed in E. coli through the assistance of co-expressed GroEL-ES. The genesis of this thinking was originated from the fact that cellular GroEL and GroES assist in the folding of several endogenous proteins expressed in the bacterial cell. Here we present the experimental findings from our study on co-expressed GroEL-GroES assisted folding of simultaneously over-expressed proteins maltodextrin glucosidase (MalZ) and yeast mitochondrial aconitase (mAco). Both proteins mentioned here are relatively larger and aggregation prone, mostly form inclusion bodies, and undergo GroEL-ES assisted folding in E. coli cells during over-expression. It has been reported that the relative yield of properly folded functional forms of MalZ and mAco with the exogenous GroEL-ES assistance were comparable with the results when these proteins were overexpressed alone. This observation is quite promising and highlights the fact that GroEL and GroES can assist in the folding of multiple substrate proteins simultaneously when over-expressed in E. coli. This method might be a potential tool for enhanced production of multiple functional recombinant proteins simultaneously in E. coli. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Inclusion in Elementary Schools

    Directory of Open Access Journals (Sweden)

    Susan Allan Galis

    1995-10-01

    Full Text Available This study of reform policy focused on inclusive education in the 1990s in the state of Georgia, United States of America. Program modifications including, individualizing instructional methods, adapting the instructional environment, and lowering maximum class size emerged as significant issues. We found that policies related to these areas were compounded by the less experienced educators not readily accepting change strategies for serving students. Apparently younger educators are engrossed in surviving daily routine and have difficulty coping with the complex demands of change. Regular education teachers have difficulty with the idea of inclusion. Legal aspects dealing inclusion need clarification, especially for regular education teachers.

  8. Assessment of inclusive education

    DEFF Research Database (Denmark)

    Miller, Tanja

    2014-01-01

    The focus of this paper is on the way teachers, in teams can work with assessing the inclusive practice in their own classes. In 2012 a joint effort between CEPRA, teachers and school administrators from the municipality of Hjørring developed a dialog based model for continually assessing...... the quality of the learning environment in regard to inclusion – this model draws heavily on the logic and mindset of ECERS (Early child environment program). This article will relate the rationale of the assessment model called “Dialoger om Kvalitet” (dialogues on quality) to LSP’s definition of inclusion...

  9. Fluid inclusion geothermometry

    Science.gov (United States)

    Cunningham, C.G.

    1977-01-01

    Fluid inclusions trapped within crystals either during growth or at a later time provide many clues to the histories of rocks and ores. Estimates of fluid-inclusion homogenization temperature and density can be obtained using a petrographic microscope with thin sections, and they can be refined using heating and freezing stages. Fluid inclusion studies, used in conjunction with paragenetic studies, can provide direct data on the time and space variations of parameters such as temperature, pressure, density, and composition of fluids in geologic environments. Changes in these parameters directly affect the fugacity, composition, and pH of fluids, thus directly influencing localization of ore metals. ?? 1977 Ferdinand Enke Verlag Stuttgart.

  10. Can we build inclusion?

    DEFF Research Database (Denmark)

    Kirkeby, Inge Mette; Grangaard, Sidse

    2016-01-01

    of some children with special needs. Hence the title – can we build inclusion? In the literature of Universal Design, accommodation and design features seldom reflect the less visible disabilities. The paper is based on a research project initiated to investigate how more or less space influences daily......Inclusion of children with special needs in kindergartens and preschools may be approached from different angles. This paper raises the question of whether the physical framework of kindergartens makes any difference for daily life at the kindergarten at all, and whether it can support inclusion...... on the answers in the interviews, we found support for answering the question in the title in the affirmative; we can build inclusion! This is because the teachers' experience indicated that, if there was sufficient space per child, there were fewer conflicts and the children managed to stay in the same activity...

  11. Evidence on Inclusion

    DEFF Research Database (Denmark)

    Dyssegaard, Camilla Brørup; Larsen, Michael Søgaard

    The purpose of this publication is to examine existing research on inclusion to identify strategies of inclusion that have generated positive effects. To do so it is necessary to understand the effect of the applied strategies. One approach, which is being discussed, is to use evidence to determine...... which methods have proven more effective than others. The desire to gain insight into research on inclusion forms the basis of the current systematic review. The task was to determine which strategies primary research has found to be most effective for inclusion purposes. We have solved this task...... by addressing the existing research with the following question: What is the effect of including children with special needs in mainstream teaching in basic school, and which of the applied educational methods have proved to have a positive effect?...

  12. Melt inclusions: Chapter 6

    Science.gov (United States)

    ,; Lowenstern, J. B.

    2014-01-01

    Melt inclusions are small droplets of silicate melt that are trapped in minerals during their growth in a magma. Once formed, they commonly retain much of their initial composition (with some exceptions) unless they are re-opened at some later stage. Melt inclusions thus offer several key advantages over whole rock samples: (i) they record pristine concentrations of volatiles and metals that are usually lost during magma solidification and degassing, (ii) they are snapshots in time whereas whole rocks are the time-integrated end products, thus allowing a more detailed, time-resolved view into magmatic processes (iii) they are largely unaffected by subsolidus alteration. Due to these characteristics, melt inclusions are an ideal tool to study the evolution of mineralized magma systems. This chapter first discusses general aspects of melt inclusions formation and methods for their investigation, before reviewing studies performed on mineralized magma systems.

  13. Enterohemorrhagic Escherichia coli (EHEC

    Directory of Open Access Journals (Sweden)

    Abdullah Kilic

    2011-08-01

    Full Text Available Escherichia coli is a bacterium that is commonly found in the gut of humans and warm-blooded animals. Most strains of E. coli are harmless for human. E. coli O157:H7 is the most common member of a group of pathogenic E. coli strains known variously as enterohaemorrhagic, verocytotoxin-producing, or Shiga-toxin-producing organisms. EHEC bacterium is the major cause of haemorrhagic colitis and haemolytic uraemic syndrome. The reservoir of this pathogen appears to be mainly cattle and other ruminants such as camels. It is transmitted to humans primarily through consumption of contaminated foods. [TAF Prev Med Bull 2011; 10(4.000: 387-388

  14. Can E. coli fly?

    DEFF Research Database (Denmark)

    Lindeberg, Yrja Lisa; Egedal, Karen; Hossain, Zenat Zebin

    2018-01-01

    , and the numbers of flies landing on the exposed rice were counted. Following exposure, the surface of the rice was microbiologically and molecularly analysed for the presence of E. coli and genes of diarrheagenic E. coli and Shigella strains. RESULTS: Rice was at greater risk (p ... with E. coli if flies landed on the rice than if no flies landed on the rice (odds ratio 5·4 (p ...-landings, the average CFU per fly-landing was > 0·6 x 103 CFU. Genes of diarrheagenic E. coli and Shigella species were detected in 39 of 60 (65%) of exposed rice samples. Two fly species were identified; the common housefly (Musca domestica) and the oriental latrine fly (Chrysomya megacephala). CONCLUSION: Flies may...

  15. RISKS OF INCLUSIVE EDUCATION

    OpenAIRE

    M. R. Husnutdinova

    2017-01-01

    Introduction. Inclusion is a new and unfamiliar phenomenon for most of the Russians which is treated as ensuring equal access to education for all students taking into account a variety of their special educational needs and individual capabilities. Inclusive educational model began to take root in Russia without a broad public debate and today’s parents and teachers were not ready to the cardinal changes caused by transition to the new model of education. In this regard, the studying of dire...

  16. Endogenous E. coli endophthalmitis.

    Science.gov (United States)

    Shammas, H F

    1977-01-01

    A case of Escherichia coli septicemia with associated metastatic en dophthalmitis and endocarditis is presented. The ocular signs and symptoms were the initial manifestations of sepsis. Irreversible damage to the eye occurred in less than 24 hours. The pattern of metastatic bacterial endophthalmitis has changed since the introduction of potent antimicrobial agents, with an increased incidence of Gram-negative bacillemia. E. coli endophthalmitis carries a poor prognosis. Early diagnosis and systemic treatment will prevent the life-threatening complications of sepsis.

  17. Limitations of inclusive fitness.

    Science.gov (United States)

    Allen, Benjamin; Nowak, Martin A; Wilson, Edward O

    2013-12-10

    Until recently, inclusive fitness has been widely accepted as a general method to explain the evolution of social behavior. Affirming and expanding earlier criticism, we demonstrate that inclusive fitness is instead a limited concept, which exists only for a small subset of evolutionary processes. Inclusive fitness assumes that personal fitness is the sum of additive components caused by individual actions. This assumption does not hold for the majority of evolutionary processes or scenarios. To sidestep this limitation, inclusive fitness theorists have proposed a method using linear regression. On the basis of this method, it is claimed that inclusive fitness theory (i) predicts the direction of allele frequency changes, (ii) reveals the reasons for these changes, (iii) is as general as natural selection, and (iv) provides a universal design principle for evolution. In this paper we evaluate these claims, and show that all of them are unfounded. If the objective is to analyze whether mutations that modify social behavior are favored or opposed by natural selection, then no aspect of inclusive fitness theory is needed.

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

    Energy Technology Data Exchange (ETDEWEB)

    Woon, J. S. K., E-mail: jameswoon@siswa.ukm.edu.my; Murad, A. M. A., E-mail: munir@ukm.edu.my; Abu Bakar, F. D., E-mail: fabyff@ukm.edu.my [School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor (Malaysia)

    2015-09-25

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

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

    Science.gov (United States)

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

    2015-09-01

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

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

    International Nuclear Information System (INIS)

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

    2015-01-01

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

  1. Expression, purification, and refolding of active recombinant human E-selectin lectin and EGF domains in Escherichia coli.

    Science.gov (United States)

    Kawano, Susumu; Iyaguchi, Daisuke; Okada, Chiaki; Sasaki, Yusuke; Toyota, Eiko

    2013-06-01

    Attempts to obtain active E-selectin from Escherichia coli (E. coli) have not yet been successful. In this study, we succeeded in expressing the recombinant lectin and epidermal growth factor domain fragments of human E-selectin (rh-ESLE) in E. coli on a large-scale. The rh-ESLE protein was expressed as an inactive form in the inclusion bodies. The inactive form of rh-ESLE was denatured and solubilized by 6 M guanidine hydrochloride and then purified by Ni(2+) affinity chromatography under denaturing conditions. Denatured rh-ESLE was then refolded by a rapid-dilution method using a large amount of refolding buffer, which contained arginine and cysteine/cystine. The refolded rh-ESLE showed binding affinity for sLe(X) (K(d) = 321 nM, B(max) = 1.9 pmol/μg protein). This result suggests that the refolded rh-ESLE recovered its native and functional structure.

  2. RISKS OF INCLUSIVE EDUCATION

    Directory of Open Access Journals (Sweden)

    M. R. Husnutdinova

    2017-01-01

    Full Text Available Introduction. Inclusion is a new and unfamiliar phenomenon for most of the Russians which is treated as ensuring equal access to education for all students taking into account a variety of their special educational needs and individual capabilities. Inclusive educational model began to take root in Russia without a broad public debate and today’s parents and teachers were not ready to the cardinal changes caused by transition to the new model of education. In this regard, the studying of directly educational process by consequences of inclusive training and education is urgent now.The aims of the research are the following: to identify the major risks that characterize the current stage of the implementation process of inclusion in the Russian educational organizations; to consider the main causes that lead to their occurrence; to present a comparative analysis of the views of respondents in comprehensive, inclusive and correctional schools. Methodology and research methods. The Sector Monitoring Studies of Moscow State University of Psychology & Education in 2010 and 2014 implemented a sociological study on the process of introduction of inclusion in the Russian schools. 200 teachers and 244 parents were interviewed in 2010; in 2014, in addition, 178 teachers and 386 parents were interviewed; 47 senior students including those with disabilities were interviewed too.Results. According to the results, the main concerns of the parents of students of comprehensive, inclusive and correctional schools are reduced to a few basic risks: lack of individual approach while teaching children with different educational needs, increased emotional pressure on the child, and child’s perception of the complexity of disability as an equal. These risks arise primarily because of the acute shortage of especially prepared-governmental teachers and socio-cultural, psychological unpreparedness of most contemporary children with disabilities to the perception of

  3. How Can We Make Computing Lessons More Inclusive?

    OpenAIRE

    Shelton , Chris

    2017-01-01

    Part 3: Computer Science Education and Its Future Focus and Development; International audience; Whilst there is a substantial body of research that shows how Information and Communications Technologies (ICTs) can support schools and teachers to make their classrooms more inclusive, there is a need for more evidence describing how best to ensure that the teaching of computing itself is inclusive. This paper reports on a literature review of inclusive education in school computing lessons. It ...

  4. Expression of the major outer membrane protein (MOMP) of Chlamydophila abortus, Chlamydophila pecorum, and Chlamydia suis in Escherichia coli using an arabinose-inducible plasmid vector.

    Science.gov (United States)

    Hoelzle, L E; Hoelzle, K; Wittenbrink, M M

    2003-10-01

    The ompA genes encoding the 40 kDa major outer membrane protein (MOMP) of Chlamydophila (Ch.) abortus, Ch. pecorum, and Chlamydia (C.) suis were cloned into the arabinose-inducible plasmid vector pBADMycHis, and recombinant MOMPs (rMOMP) from the three chlamydial species were expressed at high levels in Escherichia (E.) coli. The proteins lacking the 22 aa N-terminal signal peptide were expressed as insoluble cytoplasmic inclusion bodies which were readily purified using immobilized metal-affinity chromatography. The rMOMPs including the N-terminal signal peptide were expressed and translocated as a surface-exposed immunoaccessible protein into the outer membrane of E. coli. Transformants expressing this full-length rMOMP were significantly reduced in viability. Purified native elementary bodies (EB) and rMOMPs of the three chlamydial species purified from the E. coli cytoplasm were used for immunization of rabbits. The resulting sera were analysed for their ability to recognize homologous and heterologous rMOMP and native EB. When testing rMOMP antisera against rMOMP and EB antigens, marked cross-reactivities were detected between the three species. Using EB antisera and rMOMPs as antigens, a significant species-specific reactivity was measured.

  5. Effective interactions between inclusions in an active bath

    Science.gov (United States)

    Zaeifi Yamchi, Mahdi; Naji, Ali

    2017-11-01

    We study effective two- and three-body interactions between non-active colloidal inclusions in an active bath of chiral or non-chiral particles, using Brownian dynamics simulations within a standard, two-dimensional model of disk-shaped inclusions and active particles. In a non-chiral active bath, we first corroborate previous findings on effective two-body repulsion mediated between the inclusions by elucidating the detailed non-monotonic features of the two-body force profiles, including a primary maximum and a secondary hump at larger separations that was not previously reported. We then show that these features arise directly from the formation, and sequential overlaps, of circular layers (or "rings") of active particles around the inclusions, as the latter are brought to small surface separations. These rings extend to radial distances of a few active-particle radii from the surface of inclusions, giving the hard-core inclusions relatively thick, soft, repulsive "shoulders," whose multiple overlaps then enable significant (non-pairwise) three-body forces in both non-chiral and chiral active baths. The resulting three-body forces can even exceed the two-body forces in magnitude and display distinct repulsive and attractive regimes at intermediate to large self-propulsion strengths. In a chiral active bath, we show that, while active particles still tend to accumulate at the immediate vicinity of the inclusions, they exhibit strong depletion from the intervening region between the inclusions and partial depletion from relatively thick, circular zones further away from the inclusions. In this case, the effective, predominantly repulsive interactions between the inclusions turn to active, chirality-induced, depletion-type attractions, acting over an extended range of separations.

  6. Enhancement of solubility in Escherichia coli and purification of an aminotransferase from Sphingopyxis sp. MTA144 for deamination of hydrolyzed fumonisin B1

    Directory of Open Access Journals (Sweden)

    Hartinger Doris

    2010-08-01

    Full Text Available Abstract Background Fumonisin B1 is a cancerogenic mycotoxin produced by Fusarium verticillioides and other fungi. Sphingopyxis sp. MTA144 can degrade fumonisin B1, and a key enzyme in the catabolic pathway is an aminotransferase which removes the C2-amino group from hydrolyzed fumonisin B1. In order to study this aminotransferase with respect to a possible future application in enzymatic fumonisin detoxification, we attempted expression of the corresponding fumI gene in E. coli and purification of the enzyme. Since the aminotransferase initially accumulated in inclusion bodies, we compared the effects of induction level, host strain, expression temperature, solubility enhancers and a fusion partner on enzyme solubility and activity. Results When expressed from a T7 promoter at 30°C, the aminotransferase accumulated invariably in inclusion bodies in DE3 lysogens of the E. coli strains BL21, HMS174, Rosetta 2, Origami 2, or Rosetta-gami. Omission of the isopropyl-beta-D-thiogalactopyranoside (IPTG used for induction caused a reduction of expression level, but no enhancement of solubility. Likewise, protein production but not solubility correlated with the IPTG concentration in E. coli Tuner(DE3. Addition of the solubility enhancers betaine and sorbitol or the co-enzyme pyridoxal phosphate showed no effect. Maltose-binding protein, used as an N-terminal fusion partner, promoted solubility at 30°C or less, but not at 37°C. Low enzyme activity and subsequent aggregation in the course of purification and cleavage indicated that the soluble fusion protein contained incorrectly folded aminotransferase. Expression in E. coli ArcticExpress(DE3, which co-expresses two cold-adapted chaperonins, at 11°C finally resulted in production of appreciable amounts of active enzyme. Since His tag-mediated affinity purification from this strain was hindered by co-elution of chaperonin, two steps of chromatography with optimized imidazole concentration in the

  7. Nonlinear elastic inclusions in isotropic solids

    KAUST Repository

    Yavari, A.

    2013-10-16

    We introduce a geometric framework to calculate the residual stress fields and deformations of nonlinear solids with inclusions and eigenstrains. Inclusions are regions in a body with different reference configurations from the body itself and can be described by distributed eigenstrains. Geometrically, the eigenstrains define a Riemannian 3-manifold in which the body is stress-free by construction. The problem of residual stress calculation is then reduced to finding a mapping from the Riemannian material manifold to the ambient Euclidean space. Using this construction, we find the residual stress fields of three model systems with spherical and cylindrical symmetries in both incompressible and compressible isotropic elastic solids. In particular, we consider a finite spherical ball with a spherical inclusion with uniform pure dilatational eigenstrain and we show that the stress in the inclusion is uniform and hydrostatic. We also show how singularities in the stress distribution emerge as a consequence of a mismatch between radial and circumferential eigenstrains at the centre of a sphere or the axis of a cylinder.

  8. Multilingualism and Social Inclusion

    DEFF Research Database (Denmark)

    2017-01-01

    This is a thematic issue on the relation between multilingualism and social inclusion. Due to globalization, Europeanization, supranational and transnational regulations linguistic diversity and multilingualism are on the rise. Migration and old and new forms of mobility play an important role...... in these processes. As a consequence, English as the only global language is spreading around the world, including Europe and the European Union. Social and linguistic inclusion was accounted for in the pre-globalization age by the nation-state ideology implementing the ‘one nation-one people-one language’ doctrine...... in governance and daily life protected by a legal framework. This does not mean that there is full equality of languages. This carries over to the fair and just social inclusion of the speakers of these weaker, dominated languages as well. There is always a power question related to multilingualism. The ten...

  9. TOWARDS AN INCLUSIVE SCHOOL

    Directory of Open Access Journals (Sweden)

    Lucía López Menéndez

    2015-06-01

    Full Text Available Nowadays schools have to direct all efforts towards the comprehensive development of all students whatever their individual characteristics and their environment; they have to give a quality educational attention a qualified educational attention to the diversity in all their schools. This article presents an approach on the possibilities of developing a self-assessment using the Guide: "Index for Inclusion": “Index for Inclusion” published in the United kingdom by Centre for Studies on Inclusive Education (CSIE Tony Booth - Mel Ainscow (2002. This guide is a simple tool intended for schools to evaluate their reality in relation to important aspects of school organization and teaching-learning from the perspective of inclusion. From this self-assessment they can design specific programs to guide their educacional practice.

  10. Thioredoxin from Escherichia coli

    International Nuclear Information System (INIS)

    Holmgren, A.; Ohlsson, I.; Grankvist, M.L.

    1978-01-01

    A competition radioimmunoassay for Escherichia coli thioredoxin using 125 I-labeled thioredoxin-S 2 and a double antibody technique was developed. The method permits determination of picomole amounts of thioredoxin in crude cell extracts and was used to study the localization of thioredoxin cell fractions. E. coli B was calculated to have approximately 10,000 copies of thioredoxin per cell mainly located in the soluble fraction after separation of the membrane and soluble fractions by gentle lysis and centrifugation. E. coli B tsnC mutants which are defective in the replication of phage T7 DNA in vivo and in vitro were examined for their content of thioredoxin. E. coli B tsnC 7004 contained no detectable level of thioredoxin in cell-free extracts examined under a variety of conditions. The results strongly suggest that tsnC 7004 is a nonsense or deletion mutant. Two other E. coli tsnC mutants, 7007 and 7008, contained detectable levels of thioredoxin in crude extracts as measured by thioredoxin reductase and gave similar immunoprecipitation reactions as the parent strain B/1. By radioimmunoassay incompletely cross-reacting material was present in both strains. These results show that tsnC 7007 and 7008 belong to a type of thioredoxin mutants with missence mutations in the thioredoxin gene affecting the function of thioredoxin as subunit in phage T7 DNA polymerase

  11. Expression and purification of functional human mu opioid receptor from E.coli.

    Directory of Open Access Journals (Sweden)

    Yanbin Ma

    Full Text Available N-terminally his-tagged human mu opioid receptor, a G protein-coupled receptor was produced in E.coli employing synthetic codon-usage optimized constructs. The receptor was expressed in inclusion bodies and membrane-inserted in different E.coli strains. By optimizing the expression conditions the expression level for the membrane-integrated receptor was raised to 0.3-0.5 mg per liter of culture. Milligram quantities of receptor could be enriched by affinity chromatography from IPTG induced cultures grown at 18°C. By size exclusion chromatography the protein fraction with the fraction of alpha-helical secondary structure expected for a 7-TM receptor was isolated, by CD-spectroscopy an alpha-helical content of ca. 45% was found for protein solubilised in the detergent Fos-12. Receptor in Fos-12 micelles was shown to bind endomorphin-1 with a K(D of 61 nM. A final yield of 0.17 mg functional protein per liter of culture was obtained.

  12. Inclusive Education in Israel

    OpenAIRE

    O. V. Mayzel

    2013-01-01

    To get acquainted with the practice of inclusive education in mainstream schools, with professionals who work with special children, to visit the specialist centers to share experiences - all of this was part of an internship program «Early Childhood Education for Children with Special Needs», held in Israel (April 8 -02 May 2013) this year. The country has been selected for an internship, because the practice of inclusive education has been used for over 20 years in Israel. Moreover, a lot ...

  13. Creative activity and inclusion

    Directory of Open Access Journals (Sweden)

    Shemanov A.Yu.

    2013-09-01

    Full Text Available The aim of the article was to analyze the inclusion potential of art creative activity, namely of theatre performance, in people with disabilities. The article provides examples of disagreements in understanding the significance of these art activities for exercising the rights of people with disabilities to contribute to culture and art and some problems arising here. The conclusion is made that theatre art performed by people with disabilities is gradually changing its function: from being a means of self-affirmation to the determination of its specific place in overall theatre process. These changes confirm the inclusion potential of theatre art activity.

  14. Lead inclusions in aluminium

    International Nuclear Information System (INIS)

    Johnson, E.; Johansen, A.; Sarholt-Kristensen, L.; Andersen, H.H.; Grabaek, L.; Bohr, J.

    1990-01-01

    Ion implantation at room temperature of lead into aluminum leads to spontaneous phase separation and formation of lead precipitates growing topotactically with the matrix. Unlike the highly pressurized (∼ 1-5 GPa) solid inclusions formed after noble gas implantations, the pressure in the lead precipitates is found to be less than 0.12 GPa. Recently the authors have observed the result that the lead inclusions in aluminum exhibit both superheating and supercooling. In this paper they review and elaborate on these results. Small implantation-induced lead precipitates embedded in an aluminum matrix were studied by x-ray diffraction

  15. Developing a financial inclusion index and inclusive growth in India

    OpenAIRE

    Susanta Kumar SETHY

    2016-01-01

    Financial inclusion is one of the systems through which Inclusive Growth can be achieved in developing countries like India where large sections are unable or hopeless to contribute in the financial system. An inclusive financial system mobilizes more resources for productive purposes leading to higher economic growth, better opportunities and reduction of poverty. This study, proposed an Index of financial inclusion – a multidimensional measure. The Financial Inclusion Index c...

  16. Irradiation history of meteoritic inclusions

    DEFF Research Database (Denmark)

    Wielandt, Daniel Kim Peel

    Understanding the formation and earliest evolution of our solar system is a longstanding goal shared by cosmochemistry, astronomy and astrophysics. Meteorites play a key role in this pursuit, providing a ground truth against which all theories must be weighed. Chondritic meteorites are in essence...... extraterrestrial sediments that contain Calcium-Aluminium-rich Inclusions (CAIs) and chondrules that formed as individual objects during the earliest stages of solar system evolution. They later accreted together to form large bodies, after spending up to several million years in individual orbit around the proto...... of presolar and protosolar materials, as well as evidence for the former presence of over 10 extinct shortlived radionuclei of varying stability and provenance that play a key role in deciphering early solar system evolution. Some shortlived radionuclei, such as 60Fe (T½ 2.5 Myr), must have formed...

  17. Difference, inclusion, and mathematics education

    DEFF Research Database (Denmark)

    Figueiras, Lourdes; Healy, Lulu; Skovsmose, Ole

    2016-01-01

    The round-table discussion on Difference, Inclusion and Mathematics Education was in included in the scientific programme of VI SIPEM in recognition and celebration of the emerging body of research into the challenges of building a culture of mathematics education which values and respects...... the diversity of learners in different educational contexts – in Brazil and beyond. This paper presents the contributions to the discussion, which focus on the problematisation of the term “inclusion”, explorations of how the practices of previously marginalized students can bring new resources to the teaching...... and learning of mathematics and reflections upon the potentially discriminatory nature of the structures which currently mould school mathematics. The paper aims to serve as material for the developing research agenda of the thirteenth working group of the Brazilian Society of Mathematics Education, which met...

  18. Linguistic Diversity and Social Inclusion

    Science.gov (United States)

    Piller, Ingrid; Takahashi, Kimie

    2011-01-01

    This introduction provides the framework for the special issue by describing the social inclusion agenda of neoliberal market democracies. While the social inclusion agenda has been widely adopted, social inclusion policies are often blind to the ways in which language proficiency and language ideologies mediate social inclusion in linguistically…

  19. escherichia coli serotypes confirmed in experimental mammary ...

    African Journals Online (AJOL)

    DJFLEX

    VARIATIONS IN VIRULENCE OF THREE (3) ESCHERICHIA COLI. SEROTYPES CONFIRMED IN ... ows are susceptible to E. coli infection because. E. coli exist in the .... Coli infections in mice: A laboratory animal model for research in.

  20. Relationships in Inclusive Classrooms

    Science.gov (United States)

    Santos, Graça Duarte; Sardinha, Susana; Reis, Silvia

    2016-01-01

    Climate in the classroom is one of the determining factors in the development of practices in Inclusive Education. Many factors contribute to the climate in the classroom. However, there are predominance on affective-relational factors, with impact on action, norms and values, social interactions and learning processes. In this paper, the authors…

  1. Inclusion on the Bookshelf

    Science.gov (United States)

    Jackson, Camille

    2009-01-01

    Three decades have passed since federal law mandated inclusion--ending, officially at least, a system that segregated students with disabilities from the rest of the student population. The publishing world has yet to catch up. In children's books, characters with disabilities often inhabit their own separate world, where disability is the only…

  2. Mathematics Teaching and Inclusion

    DEFF Research Database (Denmark)

    This volume contains the proceedings of the 3rd Nordic Research Conference on Special Needs Education in Mathematics, which took place in Rebild organised by Aalborg University in November 23-25, 2005. The theme of the conference was Mathematics Education and Inclusion. The conference theme...

  3. Multilingualism and social inclusion

    NARCIS (Netherlands)

    Marácz, L.; Adamo, S.

    2017-01-01

    This is a thematic issue on the relation between multilingualism and social inclusion. Due to globalization, Europeanization, supranational and transnational regulations linguistic diversity and multilingualism are on the rise. Migration and old and new forms of mobility play an important role in

  4. Inclusion's Confusion in Alberta

    Science.gov (United States)

    Gilham, Chris; Williamson, W. John

    2014-01-01

    This hermeneutic paper interprets a recent series of reforms to inclusive education policy undertaken by the ministry of education in the province of Alberta, Canada. A 2007 Alberta Education review of the 16,000 student files in the province that school boards had claimed met the criteria for severe disability codification status -- the level of…

  5. Reptured Epidermal Inclusion Cyst in the Axilla: A Case Report

    International Nuclear Information System (INIS)

    Kim, Kyu Soon; Kim, Hak Hee; Shin, Hee Jeong; Yang, Hye Rin; Sohn, Jeong Hee; Kwon, Gui Young; Gong, Gyung Yub

    2006-01-01

    Epidermal inclusion cysts, the most common type of simple epithelial cyst, are typically well-encapsulated, subepidermal and mobile nodules. They may occur anywhere, but are mostly found on the scalp, face, neck, trunk, and back. Less than 10% of epidermal inclusion cysts occur on the extremities, and even fewer are found on the palms, soles, and breasts. If epidermal inclusion cysts rupture, foreign body reaction, granulomatous reaction or abscess formation could follow. We described here the sonographic findings of ruptured epidermal inclusion cyst of the right axilla in a 33-year-old woman who presented with a palpable axillary mass forming an inflammatory abscess

  6. COMPOSITION OF FOWLPOX VIRUS AND INCLUSION MATRIX.

    Science.gov (United States)

    RANDALL, C C; GAFFORD, L G; DARLINGTON, R W; HYDE, J

    1964-04-01

    Randall, Charles C. (University of Mississippi School of Medicine, Jackson), Lanelle G. Gafford, Robert W. Darlington, and James M. Hyde. Composition of fowlpox virus and inclusion matrix. J. Bacteriol. 87:939-944. 1964.-Inclusion bodies of fowlpox virus infection are especially favorable starting material for the isolation of virus and inclusion matrix. Electron micrographs of viral particles and matrix indicated a high degree of purification. Density-gradient centrifugation of virus in cesium chloride and potassium tartrate was unsatisfactory because of inactivation, and clumping or disintegration. Chemical analyses of virus and matrix revealed significant amounts of lipid, protein, and deoxyribonucleic acid, but no ribonucleic acid or carbohydrate. Approximately 47% of the weight of the virus and 83% of the matrix were extractable in chloroform-methanol. The lipid partitions of the petroleum ether extracts were similar, except that the phospholipid content of the matrix was 2.2 times that of the virus. Viral particles were sensitive to diethyl ether and chloroform.

  7. [Expression, purification and immunogenicity of human papillomavirus type 11 virus-like particles from Escherichia coli].

    Science.gov (United States)

    Yan, Chunyan; Li, Shaowei; Wang, Jin; Wei, Minxi; Huang, Bo; Zhuang, Yudi; Li, Zhongyi; Pan, Huirong; Zhang, Jun; Xia, Ningshao

    2009-11-01

    To produce human papillomavirus type 11 virus-like particles (HPV11 VLPs) from Escherichia coli and to investigate its immunogenicity and type cross neutralization nature. We expressed the major capsid protein of HPV11 (HPV11-L1) in Escherichia coli ER2566 in non fusion fashion and purified by amino sulfate precipitation, ion-exchange chromatography and hydrophobic interaction chromatography, sequentially. Then we removed the reductant DTT to have the purified HPV11-L1 self-assemble into VLPs in vitro. We investigated the morphology of these VLPs with dynamic light scattering and transmission electron microscopy. We assayed the immunogenicity of the resultant HPV11 VLPs by vaccinations on mice and evaluated by HPV6/11/16/18 pseudovirion neutralization cell models. We expressed HPV11 L1 in Escherichia coli with two forms, soluble and inclusion body. The soluble HPV11 L1 with over 95% purity can self assemble to VLPs in high efficiency. Morphologically, these VLPs were globular, homogeneous and with a diameter of - 50 nm, which is quite similar with native HPV11 virions. The half effective dosage (ED50) of HPV11 VLPs is 0.031 microg, and the maximum titer of neutralizing antibody elicited is averaged to 10(6). The cross neutralization activity (against HPV6/16/18) of the anti-HPV11 serum was found to have exact correlation to the inter-type homology in amino acid alignment. We can provide HPV11 VLPs with highly immunogenicity from prokaryote expression system, which may pave a new way for research and development of prophylactic vaccine for HPV11.

  8. High-level expression and purification of soluble recombinant FGF21 protein by SUMO fusion in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Huang Yadong

    2010-02-01

    Full Text Available Abstract Background Fibroblast growth factor 21 (FGF21 is a promising drug candidate to combat metabolic diseases. However, high-level expression and purification of recombinant FGF21 (rFGF21 in Escherichia coli (E. coli is difficult because rFGF21 forms inclusion bodies in the bacteria making it difficult to purify and obtain high concentrations of bioactive rFGF21. To overcome this problem, we fused the FGF21 with SUMO (Small ubiquitin-related modifier by polymerase chain reaction (PCR, and expressed the fused gene in E. coli BL21(DE3. Results By inducing with IPTG, SUMO-FGF21 was expressed at a high level. Its concentration reached 30% of total protein, and exceeded 95% of all soluble proteins. The fused protein was purified by DEAE sepharose FF and Ni-NTA affinity chromatography. Once cleaved by the SUMO protease, the purity of rFGF21 by high performance liquid chromatography (HPLC was shown to be higher than 96% with low endotoxin level (in vivo animal experiments showed that rFGF21 produced by using this method, could decrease the concentration of plasma glucose in diabetic rats by streptozotocin (STZ injection. Conclusions This study demonstrated that SUMO, when fused with FGF21, was able to promote its soluble expression of the latter in E. coli, making it more convenient to purify rFGF21 than previously. This may be a better method to produce rFGF21 for pharmaceutical research and development.

  9. The E. coli pET expression system revisited-mechanistic correlation between glucose and lactose uptake.

    Science.gov (United States)

    Wurm, David Johannes; Veiter, Lukas; Ulonska, Sophia; Eggenreich, Britta; Herwig, Christoph; Spadiut, Oliver

    2016-10-01

    Therapeutic monoclonal antibodies are mainly produced in mammalian cells to date. However, unglycosylated antibody fragments can also be produced in the bacterium Escherichia coli which brings several advantages, like growth on cheap media and high productivity. One of the most popular E. coli strains for recombinant protein production is E. coli BL21(DE3) which is usually used in combination with the pET expression system. However, it is well known that induction by isopropyl β-D-1-thiogalactopyranoside (IPTG) stresses the cells and can lead to the formation of insoluble inclusion bodies. In this study, we revisited the pET expression system for the production of a novel antibody single-chain variable fragment (scFv) with the goal of maximizing the amount of soluble product. Thus, we (1) investigated whether lactose favors the recombinant production of soluble scFv compared to IPTG, (2) investigated whether the formation of soluble product can be influenced by the specific glucose uptake rate (q s,glu) during lactose induction, and (3) determined the mechanistic correlation between the specific lactose uptake rate (q s,lac) and q s,glu. We found that lactose induction gave a much greater amount of soluble scFv compared to IPTG, even when the growth rate was increased. Furthermore, we showed that the production of soluble protein could be tuned by varying q s,glu during lactose induction. Finally, we established a simple model describing the mechanistic correlation between q s,lac and q s,glu allowing tailored feeding and prevention of sugar accumulation. We believe that this mechanistic model might serve as platform knowledge for E. coli.

  10. Inclusive Physical Education

    DEFF Research Database (Denmark)

    Østergaard, Charlotte; Rostbøll, Solveig Fogh

    2015-01-01

    EN317 - Inclusive Physical Education - with a focus on active and successful participation Charlotte Østergaard, Solveig Fogh Rostbøll, Department of School and Learning, Metropolitan University College (DK) chao@phmetropol.dk The Danish School Reform 2014 intends to raise the amount and intensity...... and is often a bad experience for students who do not have the required skills or the necessary competitive mentality. The purpose of the study is to generate increased knowledge of how to work with inclusive education in PE in schools. The aims of the study are to identify groups of “outsiders” and to find...... and ability to participate in PE must be understood in specific socio-cultural and socio-economic conditions. The hypothesis of the study is that the experience of being acknowledged for your efforts in physical education by significant others can form the basis for the construction of physical capital. EN323...

  11. Inclusive differentiated instruction

    Directory of Open Access Journals (Sweden)

    Jerković Ljiljana S.

    2017-01-01

    Full Text Available Inclusive differentiated instruction is a new model of didactic instruction, theoretically described and established in this paper for the first time, after being experimentally verified through teaching of the mother tongue (instruction in reading and literature. Inclusive individually planned instruction is based on a phenomenological and constructivist didactic instructional paradigm. This type of teaching is essentially developmental and person-oriented. The key stages of inclusive differentiated instruction of literature are: 1 recognition of individual students' potential and educational needs regarding reading and work on literary texts; 2 planning and preparation of inclusive individually planned instruction in reading and literature; 3 actual class teaching of lessons thus prepared; and 4 evaluation of the student achievement following inclusive differentiated instruction in reading and literature. A highly important element of the planning and preparation of inclusive differentiated instruction is the creation of student profiles and inclusive individualized syllabi. Individualized syllabi specify the following: 1. a brief student profile; 2. the student position on the continuum of the learning outcomes of instruction in the Serbian language; 3. reverse-engineered macro-plan stages of instruction in the Serbian language (3.1. identifying expected outcomes and fundamental qualities of learners' work, 3.2. defining acceptable proofs of their realisation, 3.3. planning learning and teaching experiences, and 3.4. providing material and technical requisites for teaching; 4 the contents and procedure of individualized lessons targeting the student; 5 a plan of syllabus implementation monitoring and evaluation. The continuum of the learning outcomes of inclusive differentiated instruction in literature exists at three main levels, A, B and C. The three levels are: A reading techniques and learning about the main literary theory concepts; B

  12. Inclusive Flavour Tagging Algorithm

    International Nuclear Information System (INIS)

    Likhomanenko, Tatiana; Derkach, Denis; Rogozhnikov, Alex

    2016-01-01

    Identifying the flavour of neutral B mesons production is one of the most important components needed in the study of time-dependent CP violation. The harsh environment of the Large Hadron Collider makes it particularly hard to succeed in this task. We present an inclusive flavour-tagging algorithm as an upgrade of the algorithms currently used by the LHCb experiment. Specifically, a probabilistic model which efficiently combines information from reconstructed vertices and tracks using machine learning is proposed. The algorithm does not use information about underlying physics process. It reduces the dependence on the performance of lower level identification capacities and thus increases the overall performance. The proposed inclusive flavour-tagging algorithm is applicable to tag the flavour of B mesons in any proton-proton experiment. (paper)

  13. Can We Build Inclusion?

    Science.gov (United States)

    Kirkeby, Inge Mette; Grangaard, Sidse

    2016-01-01

    Inclusion of children with special needs in kindergartens and preschools may be approached from different angles. This paper raises the question of whether the physical framework of kindergartens makes any difference for daily life at the kindergarten at all, and whether it can support inclusion of some children with special needs. Hence the title - can we build inclusion? In the literature of Universal Design, accommodation and design features seldom reflect the less visible disabilities. The paper is based on a research project initiated to investigate how more or less space influences daily pedagogical practice in general. Twelve interviews were conducted with experienced teachers from twelve different kindergartens with different amounts of space, varying from a ratio of 2.1 m2 play area per child to 5.5 m2. The results indicated that, for a group of children with special needs in particular, the amount of space is crucial. This group consisted of children who were socially very extrovert, and who maybe were noisy, easily provoked, and quick to get involved in arguments with other children. Alternatively, children in the group were very restrained and withdrawn in social interaction. Based on the answers in the interviews, we found support for answering the question in the title in the affirmative; we can build inclusion! This is because the teachers' experience indicated that, if there was sufficient space per child, there were fewer conflicts and the children managed to stay in the same activity for a much longer period. Sufficient space made it possible to divide the children into smaller groups, and use any secluded space. Therefore, it was much easier for other children to include some children with special needs. Accordingly, we can say that, sufficient space per child and an adequate layout and furnishing of the kindergarten is an advantage for all children. This is a clear example of Universal Design in which architectural solutions that are good for

  14. Foresighting for Inclusive Development

    DEFF Research Database (Denmark)

    Dahl Andersen, Allan; Andersen, Per Dannemand

    and transform innovation systems because a set of distributed actors, rather than ministries and innovation agencies, is the gatekeepers of change. From this perspective, inclusion is a precondition rather than an obstacle for transformation. We develop a conceptual framework and use it to study design...... and processes in two foresight cases in two emerging economies - Brazil and South Korea. Although the research is exploratory and the results tentative, the empirical studies support our main propositions....

  15. Modification in Media Composition to Obtain Secretory Production of STxB-based Vaccines using Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    Mohammad Sadraeian; Mohammad Bagher Ghoshoon; Milad Mohkam; Zeinab Karimi; Sara Rasoul-Amini; Younes Ghasemi

    2013-01-01

    Shiga toxin B-subunit (STxB) from Shigella dysenteriae targets in vivo antigen to cancer cells,dendritic cells (DC) and B cells,which preferentially express the globotriaosylceramide (Gb3) receptor.This pivotal role has encouraged scientists to investigate fusing STxB with other clinical antigens.Due to the challenges of obtaining a functional soluble form of the recombinant STxB,such as formation of inclusion bodies during protein expression,scientists tend to combine STxB with vaccine candidates rather than using their genetically fused forms.In this work,we fused HPV16 E7 as a vaccine candidate to the recombinantly-produced STxB.To minimize the formation of inclusion bodies,we investigated a number of conditions during the expression procedure.Then various strategies were used in order to obtain high yield of soluble recombinant protein from E.coli which included the use of different host strains,reduction of cultivation temperature,as well as using different concentrations of IPTG and different additives (Glycin,Triton X-100,ZnC12).Our study demonstrated the importance of optimizing incubation parameters for recombinant protein expression in E.coli; also showed that the secretion production can be achieved over the course of a few hours when using additives such as glycine and Triton X-100.Interestingly,it was shown that when the culture mediums were supplemented by additives,there was an inverse ratio between time of induction (TOI) and the level of secreted protein at louver temperatures.This study determines the optimal conditions for high yield soluble E7-STxB expression and subsequently facilitates reaching a functionally soluble form of STxB-based vaccines,which can be considered as a potent vaccine candidate for cervical cancer.

  16. How to find soluble proteins: a comprehensive analysis of alpha/beta hydrolases for recombinant expression in E. coli

    Directory of Open Access Journals (Sweden)

    Barth Sandra

    2005-04-01

    Full Text Available Abstract Background In screening of libraries derived by expression cloning, expression of active proteins in E. coli can be limited by formation of inclusion bodies. In these cases it would be desirable to enrich gene libraries for coding sequences with soluble gene products in E. coli and thus to improve the efficiency of screening. Previously Wilkinson and Harrison showed that solubility can be predicted from amino acid composition (Biotechnology 1991, 9(5:443–448. We have applied this analysis to members of the alpha/beta hydrolase fold family to predict their solubility in E. coli. alpha/beta hydrolases are a highly diverse family with more than 1800 proteins which have been grouped into homologous families and superfamilies. Results The predicted solubility in E. coli depends on hydrolase size, phylogenetic origin of the host organism, the homologous family and the superfamily, to which the hydrolase belongs. In general small hydrolases are predicted to be more soluble than large hydrolases, and eukaryotic hydrolases are predicted to be less soluble in E. coli than prokaryotic ones. However, combining phylogenetic origin and size leads to more complex conclusions. Hydrolases from prokaryotic, fungal and metazoan origin are predicted to be most soluble if they are of small, medium and large size, respectively. We observed large variations of predicted solubility between hydrolases from different homologous families and from different taxa. Conclusion A comprehensive analysis of all alpha/beta hydrolase sequences allows more efficient screenings for new soluble alpha/beta hydrolases by the use of libraries which contain more soluble gene products. Screening of hydrolases from families whose members are hard to express as soluble proteins in E. coli should first be done in coding sequences of organisms from phylogenetic groups with the highest average of predicted solubility for proteins of this family. The tools developed here can be used

  17. Inclusive Education in Italy: Description and Reflections on Full Inclusion

    Science.gov (United States)

    Anastasiou, Dimitris; Kauffman, James M.; Di Nuovo, Santo

    2015-01-01

    Inclusion of students with disabilities when appropriate is an important goal of special education for students with special needs. Full inclusion, meaning no education for any child in a separate setting, is held to be desirable by some, and Italy is likely the nation with an education system most closely approximating full inclusion on the…

  18. Financial inclusion: Policies and practices

    OpenAIRE

    Thankom Arun; Rajalaxmi Kamath

    2015-01-01

    As a key enabler for development, financial inclusion is firmly placed on the agenda of most governments as a key policy priority. Against this background, this round table provides a global and regional perspective on the policies and practices of financial inclusion. Using macro data, the collection reveals the diversity in the efforts towards achieving financial inclusion and the need for a progressive approach in financial inclusion. Further to this, the round table provides the regional ...

  19. Removal of inclusions from silicon

    Science.gov (United States)

    Ciftja, Arjan; Engh, Thorvald Abel; Tangstad, Merete; Kvithyld, Anne; Øvrelid, Eivind Johannes

    2009-11-01

    The removal of inclusions from molten silicon is necessary to satisfy the purity requirements for solar grade silicon. This paper summarizes two methods that are investigated: (i) settling of the inclusions followed by subsequent directional solidification and (infiltration by ceramic foam filters. Settling of inclusions followed by directional solidification is of industrial importance for production of low-cost solar grade silicon. Filtration is reported as the most efficient method for removal of inclusions from the top-cut silicon scrap.

  20. Supporting Teachers in Inclusive Education

    Directory of Open Access Journals (Sweden)

    Alekhina S.V.

    2015-03-01

    Full Text Available The article regards the issues of support provision to teachers involved in inclusive education as the main requirement for successful realization of inclusion. The methodological framework used in the study is a resource approach. The article describes the ways of extending the means of supporting teachers. The article also arguments for consolidating all the educators of inclusive schools into inclusive teams equally interested in joint work of administration and educators of intervention programs.

  1. Designing Inclusive Spaces

    DEFF Research Database (Denmark)

    Colfelt, Solvej

    2012-01-01

    CWUAAT -6.TH CAMBRIDGE WORKSHOP – 2012 Designing inclusive systems for real-world applications Abstracht: Denmark has planned huge investments in development in healthcare systems. Nearly 50 billion danish krones has been set aside on the stately budget for this purpose to be spent over the next 10...... hospital complexes ? The article will explore the fundament of wayshowing on the basis of prior research as well as on the basis of the results of a case study in a large existing danish hospital complex. The result points to signage being an inevitable factor but also that it is a factor that is not very...

  2. Designing Inclusive Spaces

    DEFF Research Database (Denmark)

    Colfelt, Solvej

    2012-01-01

    CWUAAT -6.TH CAMBRIDGE WORKSHOP – 2012 Designing inclusive systems for real-world applications Abstracht: Denmark has planned huge investments in development in healthcare systems. Nearly 50 billion danish krones has been set aside on the stately budget for this purpose to be spent over the next 10...... in existing hospital complexes only half the size of these new ones is already recognized as a big problem: How can we avoid the wayfinding-problem of the new complexes to grow to the double with the doubling of the complex size ? What kind of design application can improve the accessibility of future...

  3. Inclusive Education in South Korea

    Science.gov (United States)

    Kim, Yong-Wook

    2014-01-01

    The purpose of this paper is to examine the current implementation of inclusive education in South Korea and discuss its challenges. The history of special education is first described followed by an introduction to policies relevant to special and inclusive education. Next, a critical discussion of the state of inclusive education follows built…

  4. Inclusive Education under Collectivistic Culture

    Science.gov (United States)

    Futaba, Yasuko

    2016-01-01

    This paper addresses how inclusive education under collective culture is possible. Inclusive education, which more-or-less involves changing the current schools, has been denied, doubted or distorted by both policy-makers and practitioners of general and special education in Japan. Main reason for the setback in inclusive education can be…

  5. PART I. ESCHERICHIA COLI

    Directory of Open Access Journals (Sweden)

    Sanaa Mahdi Oraibi

    2016-11-01

    Full Text Available The presence of Escherichia coli in the air of facilities involved in management and composting of post-slaughter poultry wastes in selected plants of West Western Pomerania region was studied. Measurements were made on four dates in a variety of weather conditions during the year. The study was conducted at 5 objects that differ in the type of waste and the degree of preparation for composting. These were: chemical treatment and preliminary processing plant, liquid wastes reservoir, platform for preparation of materials for composting, storage of biological sediments, and composting facility. Measurement of bacteria count was carried out in accordance with the applicable procedures on selective chromogenic TBX medium. The assays revealed the presence of E. coli at all test objects, but not always on all measurement dates. It has been shown that the presence of E. coli was from 20 to 3047 CFU∙m-3 of air, although the largest quantities were most frequently detected in the air of the building for post-slaughter waste pre-treatment in chemical treatment plant.

  6. Financial inclusion: Policies and practices

    Directory of Open Access Journals (Sweden)

    Thankom Arun

    2015-12-01

    Full Text Available As a key enabler for development, financial inclusion is firmly placed on the agenda of most governments as a key policy priority. Against this background, this round table provides a global and regional perspective on the policies and practices of financial inclusion. Using macro data, the collection reveals the diversity in the efforts towards achieving financial inclusion and the need for a progressive approach in financial inclusion. Further to this, the round table provides the regional perspectives on the policies and practices of financial inclusion in India, South Africa, and Australia.

  7. IPads in Inclusive Classrooms

    DEFF Research Database (Denmark)

    Meyer, Bente Tobiesen

    2015-01-01

    This paper builds on data from a research project where iPads were used in a lower secondary school in Denmark to support school development and inclusive learning environments. The paper explores how iPads enter into and work as part of an ecology of learning resources in five classes in lower...... secondary school. I conceptualize the systems of related technologies observed in this school as ecologies of learning resources as they present themselves as carefully balanced systems in which educational resources circulate in different ways that make sense for learners’ needs. Inspired by Actor...... in by pupils the paper argues that we should disengage approaches to the iPad in education from ideas of what the properties of these technologies are, and see the device as a more relational and situated actor, avoiding the definition of properties of technologies outside the contexts specific to their use....

  8. Between psychopathology and inclusion

    DEFF Research Database (Denmark)

    Hedegaard-Sørensen, Lotte; Hamre, Bjørn Frithiof

    2017-01-01

    and social pedagogy. We thus enquire into how the rise of diagnostics and medicalisation affects our understanding of children’s difficulties. We discuss a paradox that is present in Denmark and other countries. As educational policies emphasise inclusion, the field of schooling experiences a huge rise......This article reports on a Danish study on interprofessional collaboration between child psychiatrists and educational psychologists concerning children who are categorised as being at risk. Methodologically, the analysis is grounded in qualitative interviews with psychologists. A Foucauldian...... approach is applied to narratives and experiences that occur within these interviews concerning external collaboration with child psychiatrists. The article is informed by the research tradition that has problematised the significance of psychiatry and diagnoses in the field of special needs education...

  9. Patterns of inclusion

    DEFF Research Database (Denmark)

    Pedersen, Alex Young; Nørgård, Rikke Toft; Köppe, Christian

    2018-01-01

    Reconsidering the concept of digital citizenship and the essential component of education the authors propose that the concept of Hybrid Education may serve both as a guideline for the utilization of digital technologies in education and as a methodology for fostering new forms of participation......, inclusion and engagement in society. Following T.H. Marshall’s conception of citizenship the authors suggest that becoming, belonging and the capabilities to do so is essential to digital citizenship in a culturally diverse and digitally mediated world. The paper presents a theory-based, value driven...... for Hybrid Education that are directly applicable in relation to the concept of digital citizenship. The process introduces a value-based and vision-driven design pattern approach to innovation in education by framing and aligning values and visions of the participants. This work resulted in approximately 85...

  10. Autonomy, Independence, Inclusion

    Directory of Open Access Journals (Sweden)

    Filippo Angelucci

    2015-04-01

    Full Text Available The living environment must not only meet the primary needs of living, but also the expectations of improvement of life and social relations and people’s work. The need for a living environment that responds to the needs of users with their different abilities, outside of standardizations, is increasingly felt as autonomy, independence and well-being are the result of real usability and adaptability of the spaces. The project to improve the inclusivity of living space and to promote the rehabilitation of fragile users need to be characterized as an interdisciplinary process in which the integration of specialized contributions leads to adaptive customization of space solutions and technological that evolve with the changing needs, functional capacities and abilities of individuals.

  11. Optimizing HIV-1 protease production in Escherichia coli as fusion protein

    Directory of Open Access Journals (Sweden)

    Piubelli Luciano

    2011-06-01

    Full Text Available Abstract Background Human immunodeficiency virus (HIV is the etiological agent in AIDS and related diseases. The aspartyl protease encoded by the 5' portion of the pol gene is responsible for proteolytic processing of the gag-pol polyprotein precursor to yield the mature capsid protein and the reverse transcriptase and integrase enzymes. The HIV protease (HIV-1Pr is considered an attractive target for designing inhibitors which could be used to tackle AIDS and therefore it is still the object of a number of investigations. Results A recombinant human immunodeficiency virus type 1 protease (HIV-1Pr was overexpressed in Escherichia coli cells as a fusion protein with bacterial periplasmic protein dithiol oxidase (DsbA or glutathione S-transferase (GST, also containing a six-histidine tag sequence. Protein expression was optimized by designing a suitable HIV-1Pr cDNA (for E. coli expression and to avoid autoproteolysis and by screening six different E. coli strains and five growth media. The best expression yields were achieved in E. coli BL21-Codon Plus(DE3-RIL host and in TB or M9 medium to which 1% (w/v glucose was added to minimize basal expression. Among the different parameters assayed, the presence of a buffer system (based on phosphate salts and a growth temperature of 37°C after adding IPTG played the main role in enhancing protease expression (up to 10 mg of chimeric DsbA:HIV-1Pr/L fermentation broth. GST:HIVPr was in part (50% produced as soluble protein while the overexpressed DsbA:HIV-1Pr chimeric protein largely accumulated in inclusion bodies as unprocessed fusion protein. A simple refolding procedure was developed on HiTrap Chelating column that yielded a refolded DsbA:HIV-1Pr with a > 80% recovery. Finally, enterokinase digestion of resolubilized DsbA:HIV-1Pr gave more than 2 mg of HIV-1Pr per liter of fermentation broth with a purity ≤ 80%, while PreScission protease cleavage of soluble GST:HIVPr yielded ~ 0.15 mg of pure HIV-1

  12. Approaching Inclusion as Social Practice: Processes of Inclusion and Exclusion

    DEFF Research Database (Denmark)

    Molbæk, Mette; Hansen, Janne Hedegaard; Lassen, Mikkel

    2018-01-01

    The article presents the results of a review of international research investigating mechanisms and processes of inclusion and exclusion as an ongoing part of social practice in a school context. The review forms part of a research project investigating the social practices of inclusive education...... in primary and lower-secondary education (age 6–16) in public schools as constituted by processes of inclusion and exclusion. The project aims to shift the scientific focus of research in inclusive education from the development of pedagogical and didactic practice to the importance of community construction...... through inclusion and exclusion processes. The project arises in context of Danish education policy, while the review looked for international research findings on the limits between inclusion and exclusion: how they are drawn, by whom, for what reasons, and for whose benefit? On the background...

  13. Inclusive Business - What It Is All About? Managing Inclusive Companies

    Directory of Open Access Journals (Sweden)

    Tea Golja

    2012-01-01

    Full Text Available Following the challenges we face today, the inclusive business models are future business models through which the Millennium Development Goals can be fostered and strengthen. These are the models which, through their strategic orientation on inclusivity, include low income communities in their value chain. This can be done through combining variety of strategies which all have two common points – recognition of stakeholders and adjustment of the product to the target market. The paper presents the analysis of inclusive markets. Hence, the research results show the dispersion of inclusive businesses worldwide, type of the organization, sector coverage, and contribution to MDGs as well as the particular way of inclusion of low income communities in their value chain. The aim is to present how inclusive business benefits not only the low income societies, but the companies that operate in this way as well.

  14. Application of a Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes Expressed with Activity in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Bechard Matthew E.

    2003-01-01

    Full Text Available Tetrahydromethanopterin (H4MPT is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H4MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H4MPT and tetrahydrofolate is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase. Given the importance of RFAP synthase in H4MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide an indication of the presence of H4MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies in Escherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression, RFAP synthase from Archaeoglobus fulgidus was produced in E. coli and purified to homogeneity. The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase.

  15. Application of a Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes Expressed with Activity in Escherichia coli.

    Science.gov (United States)

    Bechard, Matthew E.; Chhatwal, Sonya; Garcia, Rosemarie E.; Rasche, Madeline E.

    2003-01-01

    Tetrahydromethanopterin (H(4)MPT) is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H(4)MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H(4)MPT and tetrahydrofolate is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase). Given the importance of RFAP synthase in H(4)MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide an indication of the presence of H(4)MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies in Escherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression, RFAP synthase from Archaeoglobus fulgidus was produced in E. coli and purified to homogeneity. The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase.

  16. Study of Meteoritic Inclusion

    DEFF Research Database (Denmark)

    Olsen, Mia Bjørg Stolberg

    by small, potentially terrestrial-like planets. Given the tantalizing perspective of discovering an Earth-like world, understanding the sequence of events leading to the formation our solar system and planetary bodies has never been so relevant. Theoretical and computational astrophysics as well...... of meteorite samples that date back to the birth of the solar system. In this thesis, we have taken advantage of novel methods for the high-precision analysis of various radiogenic and stable isotope systems by plasma source and thermal ionization mass spectrometry (ICPMS and TIMS) as well as by secondary....... The manuscripts presented in this thesis have provided critical insights into the origin and distribution of short-lived radioisotopes as well as the formation and transport history of chondrules and, by extension, the precursor material to asteroidal and planetary bodies. The proposal of 26Al heterogeneity...

  17. ANIMAL ENTEROTOXIGENIC ESCHERICHIA COLI

    Science.gov (United States)

    Dubreuil, J. Daniel; Isaacson, Richard E.; Schifferli, Dieter M.

    2016-01-01

    Enterotoxigenic Escherichia coli (ETEC) is the most common cause of E. coli diarrhea in farm animals. ETEC are characterized by the ability to produce two types of virulence factors; adhesins that promote binding to specific enterocyte receptors for intestinal colonization and enterotoxins responsible for fluid secretion. The best-characterized adhesins are expressed in the context of fimbriae, such as the F4 (also designated K88), F5 (K99), F6 (987P), F17 and F18 fimbriae. Once established in the animal small intestine, ETEC produces enterotoxin(s) that lead to diarrhea. The enterotoxins belong to two major classes; heat-labile toxin that consist of one active and five binding subunits (LT), and heat-stable toxins that are small polypeptides (STa, STb, and EAST1). This chapter describes the disease and pathogenesis of animal ETEC, the corresponding virulence genes and protein products of these bacteria, their regulation and targets in animal hosts, as well as mechanisms of action. Furthermore, vaccines, inhibitors, probiotics and the identification of potential new targets identified by genomics are presented in the context of animal ETEC. PMID:27735786

  18. Inclusion in a Polarised World

    OpenAIRE

    Freeman, Alan

    2005-01-01

    This paper on inclusion was presented to the at the 2005 summer school of DEEEP (Development Education Exchange in Europe Project), Härnösand - Sweden, 5 - 12 June 2005. It addresses the significance of the concept of world civilisation. It assesses how meaning may be attached to the concept of inclusion in an economically polarised world. It develops a critique of the conception of economic inclusion, by means of an exploration of linguistic inclusion and the notion of ‘disability’. ‘...

  19. Accessibility and inclusion informational

    Directory of Open Access Journals (Sweden)

    Mônica Sena de Souza

    2013-06-01

    Full Text Available Introduction: Discusses the role of information professionals in meeting the informational demands of people with disabilities in the information society. The librarian is crucial for the effectiveness and success in the informational inclusion of people with disabilities, considering also continuing education for their professional qualification.Objective: To provide reflections on the role of the librarian in serving users with disabilities, highlighting the need for improvement in information units, identified in the scientific literature with regard to accessibility.Methodology: Literature search, based on a review of literature in books and scientific papers, highlighting the main authors: Adams (2000, Mazzoni (2001 and Sassaki (1997, 2002, 2005.Results: The lack of informational access for people with disabilities hampers their social and political participation, hence, reduces its condition of citizenship.Conclusion: The librarian responsible for seeking continuing education, greater involvement in the events of the area and the constant search for job training, which will reflect on the best service the information needs of users with disabilities.

  20. Inclusion in the East: Chinese Students' Attitudes towards Inclusive Education

    Science.gov (United States)

    Malinen, Olli-Pekka; Savolainen, Hannu

    2008-01-01

    A sample of 523 Chinese university students was given a questionnaire on their attitudes towards the inclusion of children with disabilities into regular classrooms. Factor analysis, analysis of variance, t-test and correlations were used to assess the respondents' general attitude towards inclusion, the factor structure of the attitudes, the…

  1. Neutral lipid biosynthesis in engineered Escherichia coli: jojoba oil-like wax esters and fatty acid butyl esters.

    Science.gov (United States)

    Kalscheuer, Rainer; Stöveken, Tim; Luftmann, Heinrich; Malkus, Ursula; Reichelt, Rudolf; Steinbüchel, Alexander

    2006-02-01

    Wax esters are esters of long-chain fatty acids and long-chain fatty alcohols which are of considerable commercial importance and are produced on a scale of 3 million tons per year. The oil from the jojoba plant (Simmondsia chinensis) is the main biological source of wax esters. Although it has a multitude of potential applications, the use of jojoba oil is restricted, due to its high price. In this study, we describe the establishment of heterologous wax ester biosynthesis in a recombinant Escherichia coli strain by coexpression of a fatty alcohol-producing bifunctional acyl-coenzyme A reductase from the jojoba plant and a bacterial wax ester synthase from Acinetobacter baylyi strain ADP1, catalyzing the esterification of fatty alcohols and coenzyme A thioesters of fatty acids. In the presence of oleate, jojoba oil-like wax esters such as palmityl oleate, palmityl palmitoleate, and oleyl oleate were produced, amounting to up to ca. 1% of the cellular dry weight. In addition to wax esters, fatty acid butyl esters were unexpectedly observed in the presence of oleate. The latter could be attributed to solvent residues of 1-butanol present in the medium component, Bacto tryptone. Neutral lipids produced in recombinant E. coli were accumulated as intracytoplasmic inclusions, demonstrating that the formation and structural integrity of bacterial lipid bodies do not require specific structural proteins. This is the first report on substantial biosynthesis and accumulation of neutral lipids in E. coli, which might open new perspectives for the biotechnological production of cheap jojoba oil equivalents from inexpensive resources employing recombinant microorganisms.

  2. Identification of a heterologous cellulase and its N-terminus that can guide recombinant proteins out of Escherichia coli.

    Science.gov (United States)

    Gao, Dongfang; Wang, Shengjun; Li, Haoran; Yu, Huili; Qi, Qingsheng

    2015-04-10

    The Gram-negative bacterium Escherichia coli has been widely used as a cell factory for the production of proteins and specialty chemicals because it is the best characterized host with many available expression and regulation systems. However, recombinant proteins produced in Escherichia coli are generally intracellular and often found in the form of inclusion bodies. Extracellular production of proteins is advantageous compared with intracellular production because extracellular proteins can be purified more easily and can avoid protease attack, which results in higher product quality. In this study, we found a catalytic domain of a cellulase (Cel-CD) and its N-terminus can be employed as carriers for extracellular production of recombinant proteins. In this report, we identified the catalytic domain of a cellulase (Cel-CD) from Bacillus sp. that can be secreted into the medium from recombinant E. coli BL21 (DE3) in large quantities without its native signal peptide. By subcellular location analysis, we proved that the secretion was a two-step process and the N-terminal sequence of the full length Cel-CD played a crucial function in secretion. Both the Cel-CD and its N-terminal sequence can serve as carriers for efficient extracellular production of select target proteins. Fusion of heterologous proteins with N20 from Cel-CD can carry the target proteins out of the cells with a concentration from 101 to 691 mg/L in flask cultivation. The extracellular recombinant proteins with a relative high purity. The results suggested that this system has a potential application in plant biomass conversion and industrial production of enzymes and therapeutic proteins.

  3. Efficient soluble expression of disulfide bonded proteins in the cytoplasm of Escherichia coli in fed-batch fermentations on chemically defined minimal media.

    Science.gov (United States)

    Gąciarz, Anna; Khatri, Narendar Kumar; Velez-Suberbie, M Lourdes; Saaranen, Mirva J; Uchida, Yuko; Keshavarz-Moore, Eli; Ruddock, Lloyd W

    2017-06-15

    The production of recombinant proteins containing disulfide bonds in Escherichia coli is challenging. In most cases the protein of interest needs to be either targeted to the oxidizing periplasm or expressed in the cytoplasm in the form of inclusion bodies, then solubilized and re-folded in vitro. Both of these approaches have limitations. Previously we showed that soluble expression of disulfide bonded proteins in the cytoplasm of E. coli is possible at shake flask scale with a system, known as CyDisCo, which is based on co-expression of a protein of interest along with a sulfhydryl oxidase and a disulfide bond isomerase. With CyDisCo it is possible to produce disulfide bonded proteins in the presence of intact reducing pathways in the cytoplasm. Here we scaled up production of four disulfide bonded proteins to stirred tank bioreactors and achieved high cell densities and protein yields in glucose fed-batch fermentations, using an E. coli strain (BW25113) with the cytoplasmic reducing pathways intact. Even without process optimization production of purified human single chain IgA 1 antibody fragment reached 139 mg/L and hen avidin 71 mg/L, while purified yields of human growth hormone 1 and interleukin 6 were around 1 g/L. Preliminary results show that human growth hormone 1 was also efficiently produced in fermentations of W3110 strain and when glucose was replaced with glycerol as the carbon source. Our results show for the first time that efficient production of high yields of soluble disulfide bonded proteins in the cytoplasm of E. coli with the reducing pathways intact is feasible to scale-up to bioreactor cultivations on chemically defined minimal media.

  4. Designing Inclusive Systems Designing Inclusion for Real-world Applications

    CERN Document Server

    Clarkson, John; Robinson, Peter; Lazar, Jonathan; Heylighen, Ann

    2012-01-01

    The Cambridge Workshops on Universal Access and Assistive Technology (CWUAAT) are a series of workshops held at a Cambridge University College every two years. The workshop theme: “Designing inclusion for real-world applications” refers to the emerging potential and relevance of the latest generations of inclusive design thinking, tools, techniques, and data, to mainstream project applications such as healthcare and the design of working environments. Inclusive Design Research involves developing tools and guidance enabling product designers to design for the widest possible population, for a given range of capabilities. There are five main themes: •Designing for the Real-World •Measuring Demand And Capabilities •Designing Cognitive Interaction with Emerging Technologies •Design for Inclusion •Designing Inclusive Architecture In the tradition of CWUAAT, we have solicited and accepted contributions over a wide range of topics, both within individual themes and also across the workshop’s scope. ...

  5. Study of Meteoritic Inclusion

    DEFF Research Database (Denmark)

    Olsen, Mia Bjørg Stolberg

    of meteorite samples that date back to the birth of the solar system. In this thesis, we have taken advantage of novel methods for the high-precision analysis of various radiogenic and stable isotope systems by plasma source and thermal ionization mass spectrometry (ICPMS and TIMS) as well as by secondary....... The manuscripts presented in this thesis have provided critical insights into the origin and distribution of short-lived radioisotopes as well as the formation and transport history of chondrules and, by extension, the precursor material to asteroidal and planetary bodies. The proposal of 26Al heterogeneity...

  6. Teacher preparedness for inclusive education

    African Journals Online (AJOL)

    lynette

    Department of Information Systems, Faculty of Economic & Management ... paredness of teachers for this new policy of inclusion. ... of inclusive classrooms, if viewed in global perspective (Dyson & .... teachers experience stress when including learners with special needs. ..... Stress areas and coping skills of South African.

  7. The Inclusive Classroom. Professional's Guide.

    Science.gov (United States)

    Grenot-Scheyer, Marquita; And Others

    Inclusive education reflects the changing culture of contemporary schools with emphasis on active learning, authentic assessment practices, applied curriculum, multi-level instructional approaches, and increased attention to diverse student needs and individualization. This guide is intended to help teachers implement inclusive educational…

  8. Measuring Attitudes toward Inclusive Education.

    Science.gov (United States)

    Wilczenski, Felicia L.

    1992-01-01

    Developed scale to measure attitudes toward inclusive education, in which disabled students are responsibility of regular teacher supported by specialists. Administered scale to 301 elementary and secondary teachers and to 144 undergraduate elementary education majors. Analysis yielded four discrete dimensions of inclusive education with…

  9. IDEA and Early Childhood Inclusion.

    Science.gov (United States)

    Smith, Barbara J.; Rapport, Mary Jane K.

    This paper discusses 1997 amendments to the Individuals with Disabilities Education Act (IDEA) that promote the inclusion of children with disabilities in general early childhood education settings. The evolution of inclusion policy is explored and changes in disability terminology are described. Amended provisions are then explained and include:…

  10. The Evolution of Secondary Inclusion.

    Science.gov (United States)

    Thousand, Jacqueline; Rosenberg, Richard L.; Bishop, Kathryn D.; Villa, Richard A.

    1997-01-01

    Offers an alternative "Circle of Courage" model of education, derived from Native American culture, for creating inclusive high schools that welcome, value, support, and facilitate the learning of adolescents with differing abilities. Best practices related to curriculum, instruction, assessment, and campus life for effective inclusion are…

  11. Inclusion in Malaysian Integrated Preschools

    Science.gov (United States)

    Sukumaran, Sailajah; Loveridge, Judith; Green, Vanessa A.

    2015-01-01

    Inclusive education has been introduced through a number of policy developments in Malaysia over the last 10 years but there is little research investigating the extent and nature of inclusive education for preschoolers with special educational needs (SEN). This study surveyed both regular and special education teachers in Malaysian integrated…

  12. Early Childhood Inclusion in Turkey

    Science.gov (United States)

    Diken, Ibrahim H.; Rakap, Salih; Diken, Ozlem; Tomris, Gozde; Celik, Secil

    2016-01-01

    Inclusion of young children with disabilities into regular preschool classrooms is a common practice that has been implemented for several decades in industrialized nations around the world, and many developing countries including Turkey have been developing and implementing laws, regulation, and services to support inclusion and teaching in…

  13. Early Childhood Inclusion in Spain

    Science.gov (United States)

    Giné, Climent; Balcells-Balcells, Anna; Cañadas, Margarita; Paniagua, Gema

    2016-01-01

    This article describes early childhood inclusion in educational settings in Spain. First, we address the legislative framework of preschool education in Spain and offer a brief analysis of some relevant issues, including the current situation of early childhood education and inclusion at this stage. Second, current policies and practices relating…

  14. Social Inclusion and Metrolingual Practices

    Science.gov (United States)

    Otsuji, Emi; Pennycook, Alastair

    2011-01-01

    In this paper, we explore the implications of metrolingual language practices for how we understand social inclusion. A vision of social inclusion that includes bi- and multilingual capacities may comprise an appreciation of a diversity of languages other than English, and the skills and capabilities of multilingual language users, yet it is all…

  15. Stiffening solids with liquid inclusions

    Science.gov (United States)

    Style, Robert W.; Boltyanskiy, Rostislav; Allen, Benjamin; Jensen, Katharine E.; Foote, Henry P.; Wettlaufer, John S.; Dufresne, Eric R.

    2015-01-01

    From bone and wood to concrete and carbon fibre, composites are ubiquitous natural and synthetic materials. Eshelby’s inclusion theory describes how macroscopic stress fields couple to isolated microscopic inclusions, allowing prediction of a composite’s bulk mechanical properties from a knowledge of its microstructure. It has been extended to describe a wide variety of phenomena from solid fracture to cell adhesion. Here, we show experimentally and theoretically that Eshelby’s theory breaks down for small liquid inclusions in a soft solid. In this limit, an isolated droplet’s deformation is strongly size-dependent, with the smallest droplets mimicking the behaviour of solid inclusions. Furthermore, in opposition to the predictions of conventional composite theory, we find that finite concentrations of small liquid inclusions enhance the stiffness of soft solids. A straightforward extension of Eshelby’s theory, accounting for the surface tension of the solid-liquid interface, explains our experimental observations. The counterintuitive stiffening of solids by fluid inclusions is expected whenever inclusion radii are smaller than an elastocapillary length, given by the ratio of the surface tension to Young’s modulus of the solid matrix. These results suggest that surface tension can be a simple and effective mechanism to cloak the far-field elastic signature of inclusions.

  16. Efficient secretory expression of recombinant proteins in Escherichia coli with a novel actinomycete signal peptide.

    Science.gov (United States)

    Cui, Yanbing; Meng, Yiwei; Zhang, Juan; Cheng, Bin; Yin, Huijia; Gao, Chao; Xu, Ping; Yang, Chunyu

    2017-01-01

    In well-established heterologous hosts, such as Escherichia coli, recombinant proteins are usually intracellular and frequently found as inclusion bodies-especially proteins possessing high rare codon content. In this study, successful secretory expression of three hydrolases, in a constructed inducible or constitutive system, was achieved by fusion with a novel signal peptide (Kp-SP) from an actinomycete. The signal peptide efficiently enabled extracellular protein secretion and also contributed to the active expression of the intracellular recombinant proteins. The thermophilic α-amylase gene of Bacillus licheniformis was fused with Kp-SP. Both recombinants, carrying inducible and constitutive plasmids, showed remarkable increases in extracellular and intracellular amylolytic activity. Amylase activity was observed to be > 10-fold in recombinant cultures with the constitutive plasmid, pBSPPc, compared to that in recombinants lacking Kp-SP. Further, the signal peptide enabled efficient secretion of a thermophilic cellulase into the culture medium, as demonstrated by larger halo zones and increased enzymatic activities detected in both constructs from different plasmids. For heterologous proteins with a high proportion of rare codons, it is difficult to obtain high expression in E. coli owing to the codon bias. Here, the fusion of an archaeal homologue of the amylase encoding gene, FSA, with Kp-SP resulted in > 5-fold higher extracellular activity. The successful extracellular expression of the amylase indicated that the signal peptide also contributed significantly to its active expression and signified the potential value of this novel and versatile signal peptide in recombinant protein production. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Heterologous overexpression of Glomerella cingulata FAD-dependent glucose dehydrogenase in Escherichia coli and Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Sygmund Christoph

    2011-12-01

    Full Text Available Abstract Background FAD dependent glucose dehydrogenase (GDH currently raises enormous interest in the field of glucose biosensors. Due to its superior properties such as high turnover rate, substrate specificity and oxygen independence, GDH makes its way into glucose biosensing. The recently discovered GDH from the ascomycete Glomerella cingulata is a novel candidate for such an electrochemical application, but also of interest to study the plant-pathogen interaction of a family of wide-spread, crop destroying fungi. Heterologous expression is a necessity to facilitate the production of GDH for biotechnological applications and to study its physiological role in the outbreak of anthracnose caused by Glomerella (anamorph Colletotrichum spp. Results Heterologous expression of active G. cingulata GDH has been achieved in both Escherichia coli and Pichia pastoris, however, the expressed volumetric activity was about 4800-fold higher in P. pastoris. Expression in E. coli resulted mainly in the formation of inclusion bodies and only after co-expression with molecular chaperones enzymatic activity was detected. The fed-batch cultivation of a P. pastoris transformant resulted in an expression of 48,000 U L-1 of GDH activity (57 mg L-1. Recombinant GDH was purified by a two-step purification procedure with a yield of 71%. Comparative characterization of molecular and catalytic properties shows identical features for the GDH expressed in P. pastoris and the wild-type enzyme from its natural fungal source. Conclusions The heterologous expression of active GDH was greatly favoured in the eukaryotic host. The efficient expression in P. pastoris facilitates the production of genetically engineered GDH variants for electrochemical-, physiological- and structural studies.

  18. Heterologous overexpression of Glomerella cingulata FAD-dependent glucose dehydrogenase in Escherichia coli and Pichia pastoris.

    Science.gov (United States)

    Sygmund, Christoph; Staudigl, Petra; Klausberger, Miriam; Pinotsis, Nikos; Djinović-Carugo, Kristina; Gorton, Lo; Haltrich, Dietmar; Ludwig, Roland

    2011-12-12

    FAD dependent glucose dehydrogenase (GDH) currently raises enormous interest in the field of glucose biosensors. Due to its superior properties such as high turnover rate, substrate specificity and oxygen independence, GDH makes its way into glucose biosensing. The recently discovered GDH from the ascomycete Glomerella cingulata is a novel candidate for such an electrochemical application, but also of interest to study the plant-pathogen interaction of a family of wide-spread, crop destroying fungi. Heterologous expression is a necessity to facilitate the production of GDH for biotechnological applications and to study its physiological role in the outbreak of anthracnose caused by Glomerella (anamorph Colletotrichum) spp. Heterologous expression of active G. cingulata GDH has been achieved in both Escherichia coli and Pichia pastoris, however, the expressed volumetric activity was about 4800-fold higher in P. pastoris. Expression in E. coli resulted mainly in the formation of inclusion bodies and only after co-expression with molecular chaperones enzymatic activity was detected. The fed-batch cultivation of a P. pastoris transformant resulted in an expression of 48,000 U L⁻¹ of GDH activity (57 mg L⁻¹). Recombinant GDH was purified by a two-step purification procedure with a yield of 71%. Comparative characterization of molecular and catalytic properties shows identical features for the GDH expressed in P. pastoris and the wild-type enzyme from its natural fungal source. The heterologous expression of active GDH was greatly favoured in the eukaryotic host. The efficient expression in P. pastoris facilitates the production of genetically engineered GDH variants for electrochemical-, physiological- and structural studies.

  19. Politics of Inclusion and Empowerment

    DEFF Research Database (Denmark)

    Andersen, John; Siim, Birte

    2004-01-01

    identities. Politics of empowerment has to do with the agency and mobilisation dimension of social and political change. The title of the book "Politics of Inclusion and Empowerment" address the leitmotiv: namely to discuss plussumgame between politics of inclusion and politics of empowerment......The objective of the book is to analyse different politics of inclusion and empowerment and the different paradigms of inclusion/exclusion in order to underline the close link between politics of scoial equality and politics of recognition of ciultural difference. Politics of inclusion is thus...... theproductive/innovative linkage of politics of redistributuin and politics og resognition, whnich over a longer time span creates sustainable paths of democratic and social development, which increases the capacity to handle both conflicts about economic resources and life-chances and conflicts about...

  20. Ileal adhesion of virulent E. coli LF82 is not enhanced in Crohn’s disease

    DEFF Research Database (Denmark)

    Jensen, Rikke S.; Fink, Lisbeth Nielsen; Pedersen, Susanne Brix

    2011-01-01

    Adherent-invasive Escherichia coli (AIEC) comprise a new group of E. coli species named from their distinctive ability to adhere to and invade the intestinal epithelium. The AIEC strains have been associated to the ileal mucosa in Crohn’s disease (CD), and the impact of AIEC in the pathogenesis...... of CD has been further strengthened from the evidence that the ileum in CD harbors an abnormally high number of E. coli species. S16 2010 IBD Abstracts The aim of this study was to examine the adhesion of the AIEC reference strain, LF82, to tissue samples from ileum and colon in CD and healthy controls...... and comprised: 1) incubation of tissue (inclusive of mucous) with 107 bacteria or buffer for 1 hour, 2) removal of non-adhered bacteria by extensive washing, and 3) absolute quantification of tissue-adhered LF82 and indigenous E. coli by a pre-validated assay including quantitative real-time PCR. Selective...

  1. Asymptomatic bacteriuria Escherichia coli strains

    DEFF Research Database (Denmark)

    Hancock, Viktoria; Nielsen, E.M.; Klemm, Per

    2006-01-01

    Urinary tract infections (UTIs) affect millions of people each year. Escherichia coli is the most common organism associated with asymptomatic bacteriuria (ABU) in humans. Persons affected by ABU may carry a particular E. coli strain for extended periods of time without any symptoms. In contrast...... to uropathogenic E. coli (UPEC) that cause symptomatic UTI, very little is known about the mechanisms by which these strains colonize the urinary tract. Here, we have investigated the growth characteristics in human urine as well as adhesin repertoire of nine ABU strains; the ability of ABU strains to compete...

  2. Genotypic Diversity of Escherichia coli in the Water and Soil of Tropical Watersheds in Hawaii ▿

    Science.gov (United States)

    Goto, Dustin K.; Yan, Tao

    2011-01-01

    High levels of Escherichia coli were frequently detected in tropical soils in Hawaii, which present important environmental sources of E. coli to water bodies. This study systematically examined E. coli isolates from water and soil of several watersheds in Hawaii and observed high overall genotypic diversity (35.5% unique genotypes). In the Manoa watershed, fewer than 9.3% of the observed E. coli genotypes in water and 6.6% in soil were shared between different sampling sites, suggesting the lack of dominant fecal sources in the watershed. High temporal variability of E. coli genotypes in soil was also observed, which suggests a dynamic E. coli population corresponding with the frequently observed high concentrations in tropical soils. When E. coli genotypes detected from the same sampling events were compared, limited sharing between the soil and water samples was observed in the majority of comparisons (73.5%). However, several comparisons reported up to 33.3% overlap of E. coli genotypes between soil and water, illustrating the potential for soil-water interactions under favorable environmental conditions. In addition, genotype accumulation curves for E. coli from water and soil indicated that the sampling efforts in the Manoa watershed could not exhaust the overall genotypic diversity. Comparisons of E. coli genotypes from other watersheds on Oahu, Hawaii, identified no apparent grouping according to sampling locations. The results of the present study demonstrate the complexity of using E. coli as a fecal indicator bacterium in tropical watersheds and highlight the need to differentiate environmental sources of E. coli from fecal sources in water quality monitoring. PMID:21515724

  3. Molecular basis for dominantly inherited inclusion body β-thalassemia

    International Nuclear Information System (INIS)

    Thein, S.L.; Hesketh, C.; Wood, W.G.; Clegg, J.B.; Old, J.M.; Weatherall, D.J.; Taylor, P.; Temperley, I.J.; Hutchinson, R.M.

    1990-01-01

    Analysis of the molecular basis of dominantly inherited β-thalassemia in four families has revealed different mutations involving exon 3 of the β-globin gene. It is suggested that the phenotypic difference between this condition and the more common recessive forms of β-thalassemia lies mainly in the length and stability of the abnormal translation products that are synthesized and, in particular, whether they are capable of binding heme and producing aggregations that are relatively resistant to proteolytic degradation

  4. Drug Resistance Patterns of Escherichia coli in Ethiopia: A Meta-Analysis.

    Science.gov (United States)

    Tuem, Kald Beshir; Gebre, Abadi Kahsu; Atey, Tesfay Mehari; Bitew, Helen; Yimer, Ebrahim M; Berhe, Derbew Fikadu

    2018-01-01

    Antimicrobial drug resistance is a global threat for treatment of infectious diseases and costs life and money and threatens health delivery system's effectiveness. The resistance of E. coli to frequently utilized antimicrobial drugs is becoming a major challenge in Ethiopia. However, there is no inclusive countrywide study. Therefore, this study intended to assess the prevalence of E. coli resistance and antimicrobial-specific resistance pattern among E. coli clinical isolates in Ethiopia. Articles were retrieved from PubMed, Embase, and grey literature from 2007 to 2017. The main outcome measures were overall E. coli and drug-specific resistance patterns. A random-effects model was used to determine pooled prevalence with 95% confidence interval (CI), using DerSimonian and Laird method. In addition, subgroup analysis was conducted to improve the outcome. The study bias was assessed by Begg's funnel plot. This study was registered in PROSPERO as follows: PROSPERO 2017: CRD42017070106. Of 164 articles retrieved, 35 articles were included. A total of 19,235 study samples participated in the studies and 2,635 E. coli strains were isolated. Overall, E. coli antibacterial resistance was 45.38% (95% confidence interval (CI): 33.50 to 57.27). The resistance pattern ranges from 62.55% in Addis Ababa to 27.51% in Tigray region. The highest resistance of E. coli reported was to ampicillin (83.81%) and amoxicillin (75.79%), whereas only 13.55% of E. coli isolates showed resistance to nitrofurantoin. E. coli antimicrobial resistance remains high with disparities observed among regions. The bacterium was found to be highly resistant to aminopenicillins. The finding implies the need for effective prevention strategies for the E. coli drug resistance and calls for multifaceted approaches with full involvement of all stakeholders.

  5. Escherichia coli ST131, an Intriguing Clonal Group

    Science.gov (United States)

    Bertrand, Xavier; Madec, Jean-Yves

    2014-01-01

    SUMMARY In 2008, a previously unknown Escherichia coli clonal group, sequence type 131 (ST131), was identified on three continents. Today, ST131 is the predominant E. coli lineage among extraintestinal pathogenic E. coli (ExPEC) isolates worldwide. Retrospective studies have suggested that it may originally have risen to prominence as early as 2003. Unlike other classical group B2 ExPEC isolates, ST131 isolates are commonly reported to produce extended-spectrum β-lactamases, such as CTX-M-15, and almost all are resistant to fluoroquinolones. Moreover, ST131 E. coli isolates are considered to be truly pathogenic, due to the spectrum of infections they cause in both community and hospital settings and the large number of virulence-associated genes they contain. ST131 isolates therefore seem to contradict the widely held view that high levels of antimicrobial resistance are necessarily associated with a fitness cost leading to a decrease in pathogenesis. Six years after the first description of E. coli ST131, this review outlines the principal traits of ST131 clonal group isolates, based on the growing body of published data, and highlights what is currently known and what we need to find out to provide public health authorities with better information to help combat ST131. PMID:24982321

  6. Chromosomal features of Escherichia coli serotype O2:K2, an avian pathogenic E. coli

    DEFF Research Database (Denmark)

    Jørgensen, Steffen L; Kudirkiene, Egle; Li, Lili

    2017-01-01

    Escherichia coli causing infection outside the gastrointestinal system are referred to as extra-intestinal pathogenic E. coli. Avian pathogenic E. coli is a subgroup of extra-intestinal pathogenic E. coli and infections due to avian pathogenic E. coli have major impact on poultry production econo...

  7. Ca isotopes in refractory inclusions

    International Nuclear Information System (INIS)

    Niederer, F.R.; Papanastassiou, D.A.

    1984-01-01

    We report measurements of the absolute isotope abundance of Ca in Ca-Al-rich inclusions from the Allende and Leoville meteorites. Improved high precision measurements are reported also for 46 Ca. We find that nonlinear isotope effects in Ca are extremely rare in these inclusions. The absence of nonlinear effects in Ca, except for the effects in FUN inclusions, is in sharp contrast to the endemic effects in Ti. One fine-grained inclusion shows an excess of 46 Ca of (7 +- 1) per mille, which is consistent with addition of only 46 Ca or of an exotic (*) component with 46 Ca* approx. 48 Ca*. FUN inclusion EK-1-4-1 shows a small 46 Ca excess of (3.3 +- 1.0) per mille; this confirms that the exotic Ca components in EK-1-4-1 were even more deficient in 46 Ca relative to 48 Ca than is the case for normal Ca. The Ca in the Ca-Al-rich inclusions shows mass dependent isotope fractionation effects which have a range from -3.8 to +6.7 per mille per mass unit difference. This range is a factor of 20 wider than the range previously established for bulk meteorites and for terrestrial and lunar samples. Ca and Mg isotope fractionation effects in the Ca-Al-rich inclusions are common and attributed to kinetic isotope effects. (author)

  8. Inclusive education and social exclusion

    Directory of Open Access Journals (Sweden)

    Maria Luisa Bissoto

    2013-01-01

    Full Text Available The aim of this paper is critically examining assumptions underlying the Inclusive Education concept, arguing that this can only be effectively considered when understood in a broader context of social inclusion and exclusion. Methodologically, this article relies on international documents and bibliographic references about Inclusive Education, that have been chosen by systematize and characterize different social and educational inclusive practices, encouraging the elaboration of a general overview on this topic. The results of this analysis conclude that it is essential for Inclusive Education that educational institutions review their goals and reasons of social existence. In the concluding remarks it is argued that education is better understood as the act of encouraging and welcoming the efforts of individuals in their attempts to engage in social networking, which sustains life. This includes the acceptance of other reality interpretations and understanding that educational action cannot be restricted by the walls of institutions. It requires the participation of the whole community. Action perspectives likely to promote social inclusion and inclusive education are suggested.

  9. Inclusive education: Ideas vs reality

    Directory of Open Access Journals (Sweden)

    Jovanović-Popadić Ana M.

    2016-01-01

    Full Text Available This paper discusses some of the key factors significant for the process of implementation of inclusive education in Serbia. After a brief review of the legal provisions regulating inclusive education in our country, the results of the research of attitudes towards inclusive education of teachers from six primary schools and one school for students with disabilities, as well as their experience, competencies, working conditions and opinions on consequences of inclusive education and education of children with developmental disabilities in specialized educational institutions are shown. Research was conducted in Krusevac, on a random sample of 60 teachers (51 teachers from primary schools and 9 special education teachers from schools for students with disabilities. We used two forms of questionnaires with twenty questions, and the results show predominantly negative attitudes towards inclusion in the majority of teachers in the sample. A significant percentage of respondents in both sub-samples considered that education of children with developmental disorders in specialized institutions (schools may give better results, primarily due to a lack of systematic support to teachers of primary schools in the process of implementing educational inclusion. The conclusion provides a critical overview of the current situation and presents the potential solutions to the problems that were identified during the research, and refer to the unsustainability of the current practice of inclusive education in Serbia.

  10. Effects of in-feed Chlortetracycline Prophylaxis of beef cattle on animal health and antimicrobial-resistant Escherichia coli

    Science.gov (United States)

    Objective: The objective of this study was to evaluate the effect of a one-time, five-day in-feed CTC prophylaxis on animal health (morbidity and body weight gain), occurrence of TETr E. coli, and occurrence of 3GCr E. coli over a four-month follow-up period. Experimental Design & Analysis: We eval...

  11. Visualized and precise design of artificial small RNAs for regulating T7 RNA polymerase and enhancing recombinant protein folding in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Yujia Zhao

    2016-12-01

    Full Text Available Small non-coding RNAs (sRNAs have received much attention in recent years due to their unique biological properties, which can efficiently and specifically tune target gene expressions in bacteria. Inspired by natural sRNAs, recent works have proposed the use of artificial sRNAs (asRNAs as genetic tools to regulate desired gene that has been applied in several fields, such as metabolic engineering and bacterial physiology studies. However, the rational design of asRNAs is still a challenge. In this study, we proposed structure and length as two criteria to implement rational visualized and precise design of asRNAs. T7 expression system was one of the most useful recombinant protein expression systems. However, it was deeply limited by the formation of inclusion body. To settle this problem, we designed a series of asRNAs to inhibit the T7 RNA polymerase (Gene1 expression to balance the rate between transcription and folding of recombinant protein. Based on the heterologous expression of Aspergillus oryzae Li-3 glucuronidase in E. coli, the asRNA-antigene1-17bp can effectively decrease the inclusion body and increase the enzyme activity by 169.9%.

  12. How Bureaucracy Promotes Inclusive Organizing

    DEFF Research Database (Denmark)

    Holck, Lotte

    Diversity literature in general and Feminist in particular have long promoted alternatives to bureaucracy on the premise that this form of governance is far from gender- and race-neutral, and that inclusive organizing necessitate a flatter, decentralized and more ‘organic’ set-up (Ferguson 1984...... and opportunities conducive to their inclusion. Guided by Ashcraft (2001) concept of organized dissonance, this paper explores how the combination of apparent incongruent elements of stability/flexibility and formality/informality might offer a passage for inclusive organizing....

  13. Controlling inclusions through filtration in investment casting process

    International Nuclear Information System (INIS)

    Ahmad, R.; Marshall, R.I.

    2004-01-01

    A technique for the placement of a ceramic foam filter in the feeding up of investment mould was developed which proved quite efficient in removing smaller and major inclusions through various filtration modes. Contaminated old aluminum scrap was used to prepare the melt without the addition of any cleansing and covering fluxes and the main reason was to produce more and more inclusions. Vigorous stirring was also intentionally carried out to form as much oxides as possible. During present research work effective filtration was observed. No leakage through sides of the filter occurred and similarly no choking was seen during feeding of molten metal. Microstructural studies showed the maximum retention of inclusions not only on the surface of filters but also within the various channels of the main body of the filter. The microstructures taken from the filtered test pieces were free from inclusions, which showed the effectiveness and proper placement of the filter. (author)

  14. Wholistic and Ethical: Social Inclusion with Indigenous Peoples

    Directory of Open Access Journals (Sweden)

    Kathleen E. Absolon

    2016-02-01

    Full Text Available This paper begins with a poem and is inclusive of my voice as Anishinaabekwe (Ojibway woman and is authored from my spirit, heart, mind and body. The idea of social inclusion and Indigenous peoples leave more to the imagination and vision than what is the reality and actuality in Canada. This article begins with my location followed with skepticism and hope. Skepticism deals with the exclusion of Indigenous peoples since colonial contact and the subsequent challenges and impacts. Hope begins to affirm the possibilities, strengths and Indigenous knowledge that guides wholistic cultural frameworks and ethics of social inclusion. A wholistic cultural framework is presented; guided by seven sacred teachings and from each element thoughts for consideration are guided by Indigenous values and principles. From each element this paper presents a wholistic and ethical perspective in approaching social inclusion and Indigenous peoples.

  15. Measuring Nurse Educators' Willingness to Adopt Inclusive Teaching Strategies.

    Science.gov (United States)

    Levey, Janet A

    The purpose of the study was to examine the characteristics and relationships of nurse educators' teaching practices, knowledge, support, and willingness to adopt inclusive teaching strategies (WillAdITS). Adopting more inclusive teaching strategies based on universal design for instruction is an innovative way for educators to reach today's diverse student body. However, the pedagogy has not diffused into nursing education. Descriptive statistics and hierarchical multiple regression were used for analyzing data from 311 nurse educators in prelicensure and RN to BSN programs. The model explained 44.8 percent of the variance in WillAdITS. The best indicators for this pedagogy were knowledge of universal design for instruction, social system support for inclusive teaching strategies, multiple instructional formats, and years of teaching. Knowing factors influencing the adoption of inclusive teaching strategies can inform schools of nursing of areas needing further development in the preparation of novice to experienced educators to teach diverse learners.

  16. Body Hair

    Science.gov (United States)

    ... girlshealth.gov/ Home Body Puberty Body hair Body hair Even before you get your first period , you ... removing pubic hair Ways to get rid of hair top Removing body hair can cause skin irritation, ...

  17. Inclusões intracitoplasmáticas hialinas na medular da adrenal de bovinos

    Directory of Open Access Journals (Sweden)

    L.P Mesquita

    2011-02-01

    Full Text Available Cytoplasmic inclusion bodies in adrenal medullary chromaffin cells have been described in various species including humans. These inclusions are believed to be related to certain infectious, toxic and neurodegenerative diseases. No reports concerning such adrenal inclusions have been described in bovines. Adrenal glands from twenty bovines were evaluated in a retrospective study. Seven of these exhibited inclusions - three cases of rabies, two cases of chronic suppurative bronchopneumonia, one case of chronic suppurative peritonitis, and one case of gangrenous mastitis. The inclusions were present in higher numbers especially in cases of rabies and also in one case of chronic suppurative bronchopneumonia. The inclusions were intracytoplasmic, eosinophilic, rounded, single or multiple, of various sizes, strongly stained by PAS and were present in higher numbers in the external layer of the adrenal medulla. The inclusions were negative when subjected to immunohistochemistry for detection of viral antigens in the cases of rabies. Although inclusion bodies were present in adrenal glands devoid of other histological alterations, they were more abundant in cases in which the adrenal gland had other alterations. The correlation between certain diseases and the development of inclusion bodies is not known, which highlights the importance of further studies on these inclusions in adrenal glands of bovines.

  18. Inclusive approach to particle production

    International Nuclear Information System (INIS)

    Bialas, A.

    1977-01-01

    The problems are reviewed of inclusive spectra. The data show strong disagreement with short-range order picture which is fundamental for most of the existing descriptions of inclusive spectra. There are two physical effects which should be taken into account and which give hopes to restore the agreement with data: compositeness of hadrons and unitarity correlations. The data on diffraction dissociation and cross section rise seem to indicate that hadrons are made of well-separated objects of rather small dimensions

  19. Forum, Dedicated to Inclusive Education

    Directory of Open Access Journals (Sweden)

    Vachkov I.V.

    2015-06-01

    Full Text Available 26 – 27 of February 2015 in Kazan, in the University of Management “TISBI” been held National (All-Russian forum of promotion of ideas and principles of inclusive education (with international participants “Study and live together: open space of inclusion”. During the work of Forum the most topical questions of inclusive education implement in Russian Federation been discussed.

  20. Preparation and Characterization of a Novel Chimeric Protein VEGI-CTT in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Jiping Cai

    2008-01-01

    Full Text Available Vascular endothelial cell growth inhibitor (VEGI is a recently identified antiangiogenic cytokine that belongs to the TNF superfamily, and could effectively inhibit endothelial cell proliferation and angiogenesis. Synthetic peptide CTT (CTTHWGFTLC has been found to suppress invasion and migration of both tumor and endothelial cells by potent and selective inhibition of MMP-2 and MMP-9. To prepare chimeric protein VEGI-CTT for more potent antitumor therapy, the recombinant expression vector pET-VEGI-CTT was constructed. This fusion protein was expressed in inclusion bodies in E. coli BL21 (DE3, and was refolded and purified by immobilized metal affinity chromatography using His-tag. Purified VEGI-CTT protein was characterized by proliferation assays of the endothelial cells and casein degradation assay in vitro. The results demonstrated that chimeric protein VEGI-CTT had a potent activity of antiangiogenesis through inhibiting the proliferation of endothelial cells, and could effectively reduce the activity of MMP-2 and MMP-9. The preliminarily in vivo study demonstrated that chimeric protein VEGI-CTT had more potent antitumor activity than VEGI and/or CTT peptide against CA46 human lymphoma xenografts in nude mice. Thus, these facts that are derived from the present study suggest that the chimeric protein VEGI-CTT may be used for tumor therapy in the future.

  1. Effect of signal peptide on stability and folding of Escherichia coli thioredoxin.

    Directory of Open Access Journals (Sweden)

    Pranveer Singh

    Full Text Available The signal peptide plays a key role in targeting and membrane insertion of secretory and membrane proteins in both prokaryotes and eukaryotes. In E. coli, recombinant proteins can be targeted to the periplasmic space by fusing naturally occurring signal sequences to their N-terminus. The model protein thioredoxin was fused at its N-terminus with malE and pelB signal sequences. While WT and the pelB fusion are soluble when expressed, the malE fusion was targeted to inclusion bodies and was refolded in vitro to yield a monomeric product with identical secondary structure to WT thioredoxin. The purified recombinant proteins were studied with respect to their thermodynamic stability, aggregation propensity and activity, and compared with wild type thioredoxin, without a signal sequence. The presence of signal sequences leads to thermodynamic destabilization, reduces the activity and increases the aggregation propensity, with malE having much larger effects than pelB. These studies show that besides acting as address labels, signal sequences can modulate protein stability and aggregation in a sequence dependent manner.

  2. Effect of signal peptide on stability and folding of Escherichia coli thioredoxin.

    Science.gov (United States)

    Singh, Pranveer; Sharma, Likhesh; Kulothungan, S Rajendra; Adkar, Bharat V; Prajapati, Ravindra Singh; Ali, P Shaik Syed; Krishnan, Beena; Varadarajan, Raghavan

    2013-01-01

    The signal peptide plays a key role in targeting and membrane insertion of secretory and membrane proteins in both prokaryotes and eukaryotes. In E. coli, recombinant proteins can be targeted to the periplasmic space by fusing naturally occurring signal sequences to their N-terminus. The model protein thioredoxin was fused at its N-terminus with malE and pelB signal sequences. While WT and the pelB fusion are soluble when expressed, the malE fusion was targeted to inclusion bodies and was refolded in vitro to yield a monomeric product with identical secondary structure to WT thioredoxin. The purified recombinant proteins were studied with respect to their thermodynamic stability, aggregation propensity and activity, and compared with wild type thioredoxin, without a signal sequence. The presence of signal sequences leads to thermodynamic destabilization, reduces the activity and increases the aggregation propensity, with malE having much larger effects than pelB. These studies show that besides acting as address labels, signal sequences can modulate protein stability and aggregation in a sequence dependent manner.

  3. Sex-related effects of nutritional supplementation of Escherichia coli: relevance to eating disorders.

    Science.gov (United States)

    Tennoune, Naouel; Legrand, Romain; Ouelaa, Wassila; Breton, Jonathan; Lucas, Nicolas; Bole-Feysot, Christine; do Rego, Jean-Claude; Déchelotte, Pierre; Fetissov, Sergueï O

    2015-03-01

    The biological background of sex-related differences in the development of eating disorders (EDs) is unknown. Recent data showed that gut bacteria Escherichia coli induce autoantibodies against anorexigenic α-melanocyte-stimulating hormone (α-MSH) associated with psychopathology in ED. The aim of this study was to compare the effects of E. coli on feeding and autoantibodies against α-MSH and adrenocorticotropic hormone (ACTH), between female and male rats. Commensal E. coli K12 were given in a culture medium daily to adult Wistar rats by intragastric gavage over a 3-wk period; control rats received culture medium only. Before gavage, E. coli K12 DNA was detected in feces of female but not male rats. E. coli provision was accompanied by an increase in body weight gain in females, but a decrease in body weight gain and food intake in males. Independent of E. coli treatment, plasma levels of anti-α-MSH and ACTH immunoglobulin (Ig)G were higher in female than male rats. Females responded to E. coli by increasing α-MSH IgG levels and affinity, but males by increasing α-MSH IgM levels. Affinity of IgG for ACTH was increased in both E. coli-treated females and males, although with different kinetics. IgG from females stimulated more efficiently α-MSH-induced cyclic adenosine monophosphate production by melanocortin 4 receptor-expressing cells compared with IgG from males. Sex-related response to how E. coli affects feeding and anti-melanocortin hormone antibody production may depend on the presence of these bacteria in the gut before E. coli supplementation. These data suggest that sex-related presence of certain gut bacteria may represent a risk factor for ED development. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Antimicrobial activity of Lycoperdon perlatum whole fruit body on ...

    African Journals Online (AJOL)

    Antimicrobial activities of extracts of fruit bodies of Lycoperdon perlatum against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Bacillus cereus, Candida albicans and Candida glabrata were investigated. Antimicrobial components from the mushrooms were extracted using ethanol, methanol and ...

  5. Diarrheagenic Escherichia coli Markers and Phenotypes among Fecal E. coli Isolates Collected from Nicaraguan Infants ▿

    OpenAIRE

    Reyes, Daniel; Vilchez, Samuel; Paniagua, Margarita; Colque-Navarro, Patricia; Weintraub, Andrej; Möllby, Roland; Kühn, Inger

    2010-01-01

    We analyzed the prevalence of diarrheagenic Escherichia coli (DEC) markers and common phenotypes in 2,164 E. coli isolates from 282 DEC-positive samples. Enteropathogenic E. coli (EPEC) and enteroaggregative E. coli (EAEC) were very diverse and were not correlated with diarrhea. Enterotoxigenic E. coli (ETEC) estA and enterohemorrhagic E. coli (EHEC) belonged to a few phenotypes and were significantly correlated with diarrhea.

  6. SENSITIVITY TEST OF Escherichia coli AGAINST EXTRACT Tinospora crispa

    OpenAIRE

    Lucia Ratna Winata Muslimin; abdul wahid jamaluddin

    2017-01-01

    In general, a bacterium such as Escherichia coli produces a kind of toxic protein which can disrupt intestinal wall. Livestock reacts to these toxins by pumping lots of water into the intestine in order to rinse or flush these toxins. As a result, the livestocks have diarrhea as a body response to remove the toxin in the digestive system. In the presence of these problems, breeders take a measure such as using antibiotics freely. Among breeders, antibiotics are often used freely ...

  7. Becoming Inclusive: A Code of Conduct for Inclusion and Diversity.

    Science.gov (United States)

    Schmidt, Bonnie J; MacWilliams, Brent R; Neal-Boylan, Leslie

    There are increasing concerns about exclusionary behaviors and lack of diversity in the nursing profession. Exclusionary behaviors, which may include incivility, bullying, and workplace violence, discriminate and isolate individuals and groups who are different, whereas inclusive behaviors encourage diversity. To address inclusion and diversity in nursing, this article offers a code of conduct. This code of conduct builds on existing nursing codes of ethics and applies to nursing students and nurses in both educational and practice settings. Inclusive behaviors that are demonstrated in nurses' relationships with patients, colleagues, the profession, and society are described. This code of conduct provides a basis for measureable change, empowerment, and unification of the profession. Recommendations, implications, and a pledge to action are discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Demands from the school inclusion

    Directory of Open Access Journals (Sweden)

    Selma Norberto Matos

    2014-04-01

    Full Text Available From the implementation of public policies on school inclusion, mainly those directed to the target audience of special education, the number of students with special educational needs in common classes has increased. This fact has helped to compose the picture in schools where the limitations and contradictions of the Brazilian educational system have appeared. Educational actors and authors are challenged to build knowledge able of responding to demands of daily school, concerning living and learning in diversity. Whereas this inclusive process is new in the schools, the study aimed to analyze the demands of teachers from the school inclusion. The research was qualitative and exploratory, and six teachers, their students with special educational needs and three professionals in the Nucleus of Inclusive Education from the Municipal Department of Education took in it. Technique of participant observation, field diary, semi-structured interview and questionnaire were used for data collection, while analysis of content was used for discussion of the data. The results indicate that there are achievements and contradictions in the reality of schools that themselves propose inclusive; advances and limitations resulting from the municipal politics; that the model of performance of the group of special education, in the context analyzed, may be revised or expanded; and that the teachers has demands with regard to public policy, training, and the psychologist.

  9. Social inclusion and mental health.

    Science.gov (United States)

    Cobigo, Virginie; Stuart, Heather

    2010-09-01

    Recent research on approaches to improving social inclusion for people with mental disabilities is reviewed. We describe four approaches (or tools) that can be used to improve social inclusion for people with mental disabilities: legislation, community-based supports and services, antistigma/antidiscrimination initiatives, and system monitoring and evaluation. While legislative solutions are the most prevalent, and provide an important framework to support social inclusion, research shows that their full implementation remains problematic. Community-based supports and services that are person-centered and recovery-oriented hold considerable promise, but they are not widely available nor have they been widely evaluated. Antistigma and antidiscrimination strategies are gaining in popularity and offer important avenues for eliminating social barriers and promoting adequate and equitable access to care. Finally, in the context of the current human rights and evidence-based health paradigms, systematic evidence will be needed to support efforts to promote social inclusion for people with mental disabilities, highlight social inequities, and develop best practice approaches. Tools that promote social inclusion of persons with mental disabilities are available, though not yet implemented in a way to fully realize the goals of current disability discourse.

  10. The Extracellular Domain of Human High Affinity Copper Transporter (hNdCTR1), Synthesized by E. coli Cells, Chelates Silver and Copper Ions In Vivo.

    Science.gov (United States)

    Sankova, Tatiana P; Orlov, Iurii A; Saveliev, Andrey N; Kirilenko, Demid A; Babich, Polina S; Brunkov, Pavel N; Puchkova, Ludmila V

    2017-11-03

    There is much interest in effective copper chelators to correct copper dyshomeostasis in neurodegenerative and oncological diseases. In this study, a recombinant fusion protein for expression in Escherichia coli cells was constructed from glutathione-S-transferase (GST) and the N-terminal domain (ectodomain) of human high affinity copper transporter CTR1 (hNdCTR1), which has three metal-bound motifs. Several biological properties of the GST-hNdCTR1 fusion protein were assessed. It was demonstrated that in cells, the protein was prone to oligomerization, formed inclusion bodies and displayed no toxicity. Treatment of E. coli cells with copper and silver ions reduced cell viability in a dose- and time-dependent manner. Cells expressing GST-hNdCTR1 protein demonstrated resistance to the metal treatments. These cells accumulated silver ions and formed nanoparticles that contained AgCl and metallic silver. In this bacterial population, filamentous bacteria with a length of about 10 µm were often observed. The possibility for the fusion protein carrying extracellular metal binding motifs to integrate into the cell's copper metabolism and its chelating properties are discussed.

  11. The Extracellular Domain of Human High Affinity Copper Transporter (hNdCTR1, Synthesized by E. coli Cells, Chelates Silver and Copper Ions In Vivo

    Directory of Open Access Journals (Sweden)

    Tatiana P. Sankova

    2017-11-01

    Full Text Available There is much interest in effective copper chelators to correct copper dyshomeostasis in neurodegenerative and oncological diseases. In this study, a recombinant fusion protein for expression in Escherichia coli cells was constructed from glutathione-S-transferase (GST and the N-terminal domain (ectodomain of human high affinity copper transporter CTR1 (hNdCTR1, which has three metal-bound motifs. Several biological properties of the GST-hNdCTR1 fusion protein were assessed. It was demonstrated that in cells, the protein was prone to oligomerization, formed inclusion bodies and displayed no toxicity. Treatment of E. coli cells with copper and silver ions reduced cell viability in a dose- and time-dependent manner. Cells expressing GST-hNdCTR1 protein demonstrated resistance to the metal treatments. These cells accumulated silver ions and formed nanoparticles that contained AgCl and metallic silver. In this bacterial population, filamentous bacteria with a length of about 10 µm were often observed. The possibility for the fusion protein carrying extracellular metal binding motifs to integrate into the cell’s copper metabolism and its chelating properties are discussed.

  12. Inclusive production at LHC energies

    International Nuclear Information System (INIS)

    Merino, C.; Pajares, C.; Shabelski, Yu.M.

    2011-01-01

    We consider the first LHC data for pp collisions in the framework of Regge theory. The integral cross sections and inclusive densities of secondaries are determined by the Pomeron exchange, and we present the corresponding predictions for them. The first measurements of inclusive densities in the midrapidity region are in agreement with these predictions. The contribution of the baryon-number transfer due to String Junction diffusion in the rapidity space is at the origin of the differences in the inclusive spectra of particle and antiparticle in the central region, and this effect could be significant at LHC energies. We discuss the first data of ALICE and LHCb collaborations on the baryon/antibaryon asymmetry at LHC. (orig.)

  13. Photoreactivating enzyme from Escherichia coli

    International Nuclear Information System (INIS)

    Snapka, R.M.; Fuselier, C.O.

    1977-01-01

    Escherichia coli photoreactivating enzyme (PRE) has been purified in large amounts from an E.coli strain lysogenic for a defective lambda bacteriophage carrying the phr gene. The resulting enzyme had a pH optimum of 7.2 and an ionic strength optimum of 0.18. It consisted of an apoprotein and cofactor, both of which were necessary for catalytic activity. The apoprotein had a monomer molecular weight of 35,200 and showed stable aggregates under denaturing conditions. The amino acid analysis of the E.coli enzyme was very similar to that of the photoreactivating enzyme from orchid seedlings (Cattelya aurantiaca). Both had arginine at the amino terminus. The cofactor, like the holoenzyme, showed absorption, magnetic circular dichroism, and emission properties indicative of an adenine moiety. Although the isolated enzyme had an action spectrum which peaked at about 360 nm, neither the cofactor, apoenzyme nor holoenzyme showed any detectable absorption between 300 and 400 nm. (author)

  14. Photoreactivating enzyme from Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Snapka, R M; Fuselier, C O [California Univ., Irvine (USA)

    1977-05-01

    Escherichia coli photoreactivating enzyme (PRE) has been purified in large amounts from an E.coli strain lysogenic for a defective lambda bacteriophage carrying the phr gene. The resulting enzyme had a pH optimum of 7.2 and an ionic strength optimum of 0.18. It consisted of an apoprotein and cofactor, both of which were necessary for catalytic activity. The apoprotein had a monomer molecular weight of 35,200 and showed stable aggregates under denaturing conditions. The amino acid analysis of the E.coli enzyme was very similar to that of the photoreactivating enzyme from orchid seedlings (Cattelya aurantiaca). Both had arginine at the amino terminus. The cofactor, like the holoenzyme, showed absorption, magnetic circular dichroism, and emission properties indicative of an adenine moiety. Although the isolated enzyme had an action spectrum which peaked at about 360 nm, neither the cofactor, apoenzyme nor holoenzyme showed any detectable absorption between 300 and 400 nm.

  15. Doing Research Inclusively: Bridges to Multiple Possibilities in Inclusive Research

    Science.gov (United States)

    Nind, Melanie; Vinha, Hilra

    2014-01-01

    This article reports on a study of how people do research that matters to people with learning disabilities and that involves them and their views and experiences. The study was an attempt to bring together people doing inclusive research so that, collectively, we could take stock of our practices. This would add to the individual reports and…

  16. The Inclusion of Music/the Music of Inclusion

    Science.gov (United States)

    Lubet, Alex

    2009-01-01

    The intention of this paper is to situate music within inclusive education. Intersections of music--widely regarded as a "talent" or hyperability--and disability provide unique perspectives on social organisation in general and human valuation in particular. Music is a ubiquitous and an essential component of learning beginning in infancy.…

  17. Foreign Body

    Science.gov (United States)

    ... SearchingPediatrics.com Pediatrics Common Questions, Quick Answers Foreign Body Donna D'Alessandro, M.D. Lindsay Huth, B. ... I call the doctor? What is a foreign body? A foreign body is when an object is ...

  18. Inclusiveness program - a SWOT analysis

    Science.gov (United States)

    Dósa, M.; Szegő, K.

    2017-09-01

    The Inclusiveness Program was created with the aim to integrate currently under-represented countries into the mainstream of European planetary research. Main stages of the working plan include setting up a database containing all the research institutes and universities where astronomical or geophysical research is carried out. It is necessary to identify their problems and needs. Challenging part of the project is to find exact means that help their work in a sustainable way. Strengths, weaknesses, opportunities and threats of the program were identified based on feedback from the inclusiveness community. Our conclusions, further suggestions are presented.

  19. Expression in E. coli systems

    DEFF Research Database (Denmark)

    Krogsdam, Anne-M; Kristiansen, Karsten; Nøhr, Jane

    2003-01-01

    intracellularly in soluble form. In E. coli, proteins containing disulfide bonds are best produced by secretion because the disulfide forming foldases reside in the periplasm. Likewise, a correct N-terminus is more likely to be obtained upon secretion. Moreover, potentially toxic proteins are more likely......Owing to cost advantage, speed of production, and often high product yield (up to 50% of total cell protein), expression in Escherichia coli is generally the first choice when attempting to express a recombinant protein. Expression systems exist to produce recombinant protein intracellularly...

  20. Experimental evolution of E. coli

    Science.gov (United States)

    Zhang, Mengshi

    The evolution from unicellular to multicellular behavior is an essential step in the history of life. Our aim is to investigate the emergence of collective behavior in the model organism Escherichia coli (E. coli) and its selection advantages, such as better utilization of public goods. Our preliminary results suggest that the evolution of collective behavior may be a natural response to stressed conditions. Mailing address: Room 306 Science Centre North Block, The Chinese University of Hong Kong, Shatin, N.T. Hong Kong SAR. Phone: +852-3943-6354. Fax: +852-2603-5204. E-mail: mengshi0928@gmail.com.

  1. The oxygen effect in E. coli cells

    International Nuclear Information System (INIS)

    Myasnik, M.N.; Skvortsov, V.G.; Sokolov, V.A.

    1982-01-01

    In experiments on E. coli strains deficient in some stages of DNA repair from radiation damages, it was demonstrated that the value of the oxygen effect, under optimal conditions for manifestation thereof, decreases in the following order: E. coli WP2 (the wild type) → E. coli WP2 exr - and E. coli B → E. coli WP2 uvr A6 → E. coli WP2 rec Al and E. coli WP2 hcr - exr - . It was detected that 0.14 M NaCl solution sensitizes the anoxic cells of some E. coli strains to the effect of γ-radiation. It was established that mutation of the uvr A-gene increases sharply the sensitivity of cells to iradiation under the anoxic conditions in the presence of NaCl, the reverse'' oxygen effect being observed

  2. Immunoconcentration of Shiga toxin-producing Escherichia coli O157 from animal faeces and raw meats by using Dynabeads anti-E. coli O157 and the VIDAS system

    NARCIS (Netherlands)

    Islam, M.A.; Heuvelink, A.E.; Talukder, K.A.; Boer, de E.

    2006-01-01

    To identify the reservoirs and routes of transmission of Shiga toxin-producing Escherichia coli (STEC) O157, sensitive detection and isolation methods are necessary. The sensitivity of traditional culture methods can be improved significantly by the inclusion of an immunoconcentration step,

  3. Diverse Perspectives on Inclusive School Communities

    Science.gov (United States)

    Tsokova, Diana; Tarr, Jane

    2012-01-01

    What is an inclusive school community? How do stakeholders perceive their roles and responsibilities towards inclusive school communities? How can school communities become more inclusive through engagement with individual perspectives? "Diverse Perspectives on Inclusive School Communities" captures and presents the voices of a wide…

  4. Inclusion in the Workplace - Text Version | NREL

    Science.gov (United States)

    Careers » Inclusion in the Workplace - Text Version Inclusion in the Workplace - Text Version This is the text version for the Inclusion: Leading by Example video. I'm Martin Keller. I'm the NREL of the laboratory. Another very important element in inclusion is diversity. Because if we have a

  5. Early Childhood Inclusion in Croatia

    Science.gov (United States)

    Ljubešic, Marta; Šimleša, Sanja

    2016-01-01

    This article explains early childhood inclusion in Croatia from its beginnings up to challenges in current policy and practice. The first preschool education for children with disabilities dates back to the 1980s and was provided in special institutions. In the last 10 years, mainstream kindergartens have been enrolling children with disabilities…

  6. Inclusive Education and the Arts

    Science.gov (United States)

    Allan, Julie

    2014-01-01

    This paper addresses the troubled, problematic and contested field of inclusive education, characterised by antagonisms between so-called inclusionists and special educationists; frustration, particularly among disability activists caused by the abstraction of the social model of disability and the expansion of the special educational needs…

  7. Inclusive Education: Lessons from History

    Science.gov (United States)

    Boroson, Barbara

    2017-01-01

    How has education evolved from exclusion to inclusion, from judgment to acceptance, and from disability to difference? This is the question that frames Barbara Boroson's article in the theme issue, "Differences, Not Disabilities." Boroson begins by taking a historical view of how schools have treated those who were perceived to be…

  8. Nonlocal quasilinear damped differential inclusions

    Directory of Open Access Journals (Sweden)

    Mouffak Benchohra

    2002-01-01

    Full Text Available In this paper we investigate the existence of mild solutions to second order initial value problems for a class of damped differential inclusions with nonlocal conditions. By using suitable fixed point theorems, we study the case when the multivalued map has convex and nonconvex values.

  9. Evolution: From Isolation to Inclusion.

    Science.gov (United States)

    Kehoe, Sharon M.

    1998-01-01

    Today, school district leaders must comply with multiple federal statutes (Individuals with Disabilities Act, Section 504 of the Rehabilitation Act, and Americans with Disabilities Act). The trend is toward full inclusion, despite judicial overrulings, clarifications of "least restrictive environment," and emergence of opposing groups.…

  10. Tracing Inclusion: Determining Teacher Attitudes

    Science.gov (United States)

    Logan, Brenda E.; Wimer, Gregory

    2013-01-01

    Though there appears to be an onslaught of No Child Left Behind, there is still more emphasis on testing than ever before. With the new implementation of national common-core standards, many school districts have moved towards full inclusive classrooms. However, it is rare that teachers have any input on whether such major decisions are apropos…

  11. Early Childhood Inclusion in Israel

    Science.gov (United States)

    Al-Yagon, Michal; Aram, Dorit; Margalit, Malka

    2016-01-01

    This article describes conceptual aspects, current policies and practices, and research representing the Israeli perspective regarding early childhood inclusion (ECI) at preschool ages (3-6 years). We review legislative, historical, attitudinal, philosophical, practical, empirical, and cultural issues regarding ECI in Israel. Finally, we focus on…

  12. Early Childhood Inclusion in Australia

    Science.gov (United States)

    Kemp, Coral R.

    2016-01-01

    From the introduction of early intervention services in Australian in the mid-1970s, the families of children with intellectual and multiple disabilities have been encouraged to enroll their children in local preschools and childcare centers. Children with disabilities have also accessed a range of alternatives to full inclusion, such as reverse…

  13. Inclusive fitness theory and eusociality

    DEFF Research Database (Denmark)

    Abbot, P.; Wrangham, R.; Abe, J.

    2011-01-01

    Arising from M. A. Nowak, C. E. Tarnita & E. O. Wilson 466, 1057-1062 (2010); Nowak et al. reply. Nowak et al. argue that inclusive fitness theory has been of little value in explaining the natural world, and that it has led to negligible progress in explaining the evolution of eusociality. However...

  14. Inclusion

    DEFF Research Database (Denmark)

    Jørgensen, Stine Kaplan

    2017-01-01

    Paperet sætter fokus på inklusion i en dans sammenhæng, med særligt fokus på mobning. Der tages udgangspunkt i et fællesskabsorienteret mobbesyn, der udpeger særlige opmærksomhedspunkter til såvel forebyggelse som intervention....

  15. INCLUSIVE CULTURE IN PRE-SCHOOL INSTITUTIONS

    Directory of Open Access Journals (Sweden)

    Irena NOVACHEVSKA

    2011-04-01

    Full Text Available Inclusive education is a rational concept that refers to the overall and long-term transformation of institutional systems in society, especially in education. Along with the transformation, a number of important and unresolved issues still appear in both theory and practice, as the duty of pre-school institutions and schools is to educate every student in the mainstream education system. One of the most important aspects of inclusion is the inclusive culture. Regardless of the good inclusive policy and practice, one cannot talk about successful inclusion without a properly developed inclusive institutional culture.This paper is a contribution to the research considering the development of inclusive culture in three preschool institutions. It is based on the thinking and attitudes of the pre­school staff toward the necessity of developing and nurturing an inclusive culture. Successful inclusion of pupils with special needs in the mainstream school system cannot be conceived without an inclusive culture.

  16. Inclusion control in high-performance steels

    International Nuclear Information System (INIS)

    Holappa, L.E.K.; Helle, A.S.

    1995-01-01

    Progress of clean steel production, fundamentals of oxide and sulphide inclusions as well as inclusion morphology in normal and calcium treated steels are described. Effects of cleanliness and inclusion control on steel properties are discussed. In many damaging constructional and engineering applications the nonmetallic inclusions have a quite decisive role in steel performance. An example of combination of good mechanical properties and superior machinability by applying inclusion control is presented. (author)

  17. Actions and advice in coli

    DEFF Research Database (Denmark)

    Knoche, Hendrik; Jamadagni, HS; Rao, PR Sheshagiri

    2015-01-01

    To improve their agricultural output, farmers require timely and contextualized information and advice. Relevant information and advice provided by trusted peers represents a promising approach. We present the considerations for the design of coli, an agricultural information network on touch scr...

  18. Infektionen mit darmpathogenen Escherichia coli.

    NARCIS (Netherlands)

    Friedrich, Alexander; Stein, Jürgen; Dignass, Axel

    2001-01-01

    E. coli ist ein wesentlicher Bestandteil der physiologischen Darmflora des Menschen. Die üblicherweise im Darm vorkommenden Kolibakterien sind apathogen und für den Menschen eher nützlich (Sonnenborn u. Greinwald 1990). Allerdings kennen wir bei dieser Bakterienspezies auch ein breites Spektrum von

  19. 76 FR 20542 - Escherichia coli

    Science.gov (United States)

    2011-04-13

    ... beef, Escherichia coli and coliphages were found in chicken, fresh pork, fresh oyster, fresh mushrooms, lettuce, chicken pot pie, biscuit dough, deli loaf, deli roasted turkey, and package roasted chicken... surfaces, and in foods such as ground beef, pork sausage, chicken, oysters, cheese, fresh mushrooms, and...

  20. ESCHERICHIA COLI AND STAPHYLOCOCCUS AUREUS

    African Journals Online (AJOL)

    DR. AMINU

    ABSTRACT. The bio-effects of the ethanol extracts from the leaf and stem of Momordica charantia were studied with the view to ascertain the medical usefulness ascribed to the plant by the locals. The plant parts, stem and leaf, revealed remarkable activity against Escherichia coli and Staphlococcus aureus. The leaves ...

  1. Conjugal Pairing in Escherichia Coli

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 13; Issue 8. Conjugal Pairing in Escherichia Coli. Joshua Lederberg. Classics Volume 13 Issue 8 August 2008 pp 793-794. Fulltext. Click here to view fulltext PDF. Permanent link: https://www.ias.ac.in/article/fulltext/reso/013/08/0793-0794 ...

  2. Escherichia coli as a probiotic?

    NARCIS (Netherlands)

    Jansen, GJ; Wildeboer-Veloo, ACM; van der Waaij, D; Degener, JE

    1998-01-01

    The influence of oral treatment with a suspension of non-pathogenic Escherichia coli cells (commercially available as: Symbioflor II(R)) on the morphological composition of the gut microflora and on the systemic humoral immune response (the IgG-, IgA- and IgM-isotype) against the bacterial cells in

  3. Expression in E. coli systems

    DEFF Research Database (Denmark)

    Krogsdam, Anne-M; Kristiansen, Karsten; Nøhr, Jane

    2003-01-01

    intracellularly in soluble form. In E. coli, proteins containing disulfide bonds are best produced by secretion because the disulfide forming foldases reside in the periplasm. Likewise, a correct N-terminus is more likely to be obtained upon secretion. Moreover, potentially toxic proteins are more likely...

  4. Microvillus Inclusion Disease Associated with Necrotizing Enterocolitis in a Premature Infant

    Directory of Open Access Journals (Sweden)

    Ersin Sayar

    2014-11-01

    Full Text Available Microvillus inclusion disease is one of the congenital diarrheal disorders characterized by the appearance of inclusion bodies on the intestinal epithelium. To date there are a few cases and also a few other associated finding reports related to this life-threatening disease in literature. In this report, we present a premature infant with microvillus inclusion disease that was associated with necrotizing enterocolitis. Thus, we should be aware of the appearance of necrotizing enterocolitis in patients with microvillus inclusion disease, especially when contributing factors are present.

  5. A fluorescent-based HPLC assay for quantification of cysteine and cysteamine adducts in Escherichia coli-derived proteins.

    Science.gov (United States)

    Soriano, Brian D; Tam, Lei-Ting T; Lu, Hsieng S; Valladares, Violeta G

    2012-01-01

    Recombinant proteins expressed in Escherichia coli are often produced as unfolded, inactive forms accumulated in inclusion bodies. Redox-coupled thiols are typically employed in the refolding process in order to catalyze the formation of correct disulfide bonds at maximal folding efficiency. These thiols and the recombinant proteins can form mixed disulfide bonds to generate thiol-protein adducts. In this work, we apply a fluorescent-based assay for the quantification of cysteine and cysteamine adducts as observed in E. coli-derived proteins. The thiols are released by reduction of the adducted protein, collected and labeled with a fluorescent reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. The derivatized thiols are separated by reversed-phase HPLC and can be accurately quantified after method optimization. The estimated thiol content represents total amount of adducted forms present in the analyzed samples. The limit of quantification (LOQ) was established; specifically, the lowest amount of quantifiable cysteine adduction is 30 picograms and the lowest amount of quantifiable cysteamine adduction is 60 picograms. The assay is useful for quantification of adducts in final purified products as well as in-process samples from various purification steps. The assay indicates that the purification process accomplishes a decrease in cysteine adduction from 0.19 nmol adduct/nmol protein to 0.03 nmol adduct/nmol protein as well as a decrease in cysteamine adduction from 0.24 nmol adduct/nmol protein to 0.14 nmol adduct/nmol protein. Copyright © 2011. Published by Elsevier B.V.

  6. 99mTechnetium labelled Escherichia coli

    International Nuclear Information System (INIS)

    Diniz, S.O.F.; Cardoso, V.N.; Resende, B.M.; Nunan, E.A.; Simal, C.J.R.

    1999-01-01

    Samples of a culture of unlabeled Escherichia coli were incubated with different concentrations of stannous chloride for various time periods. 99m Tc (26.0 MBq) was added to each preparation and the results showed a labelling yield of 98% for E. coli. Since the bacterial viability of 99m Tc-E. coli and E. coli did not show any statistical differences, these results demonstrate that labelling of E. coli with 99m Tc does not modify the bacterial viability, and the radiolabelled bacteria may be a good model to study bacterial translocation

  7. Human Meningitis-Associated Escherichia coli

    Science.gov (United States)

    KIM, KWANG SIK

    2016-01-01

    E. coli is the most common Gram-negative bacillary organism causing meningitis and E. coli meningitis continues to be an important cause of mortality and morbidity throughout the world. Our incomplete knowledge of its pathogenesis contributes to such mortality and morbidity. Recent reports of E. coli strains producing CTX-M-type or TEM-type extended-spectrum β-lactamases create a challenge. Studies using in vitro and in vivo models of the blood-brain barrier have shown that E. coli meningitis follows a high-degree of bacteremia and invasion of the blood-brain barrier. E. coli invasion of the blood-brain barrier, the essentials step in the development of E. coli meningitis, requires specific microbial and host factors as well as microbe- and host-specific signaling molecules. Blockade of such microbial and host factors contributing to E. coli invasion of the blood-brain barrier is shown to be efficient in preventing E. coli penetration into the brain. The basis for requiring a high-degree of bacteremia for E. coli penetration of the blood-brain barrier, however, remains unclear. Continued investigation on the microbial and host factors contributing to a high-degree of bacteremia and E. coli invasion of the blood-brain barrier is likely to identify new targets for prevention and therapy of E. coli meningitis. PMID:27223820

  8. β-Cyclodextrin Inclusion Complex to Improve Physicochemical Properties of Pipemidic Acid: Characterization and Bioactivity Evaluation

    Directory of Open Access Journals (Sweden)

    Gaetano Malgieri

    2013-06-01

    Full Text Available The aptitude of cyclodextrins (CDs to form host-guest complexes has prompted an increase in the development of new drug formulations. In this study, the inclusion complexes of pipemidic acid (HPPA, a therapeutic agent for urinary tract infections, with native β-CD were prepared in solid state by kneading method and confirmed by FT-IR and 1H NMR. The inclusion complex formation was also characterized in aqueous solution at different pH via UV-Vis titration and phase solubility studies obtaining the stability constant. The 1:1 stoichiometry was established by a Job plot and the inclusion mechanism was clarified using docking experiments. Finally, the antibacterial activity of HPPA and its inclusion complex was tested on P. aeruginosa, E. coli and S. aureus to determine the respective EC50s and EC90s. The results showed that the antibacterial activity of HPPA:β-CD against E. coli and S. aureus is higher than that of HPPA. Furthermore, HPPA and HPPA:β-CD, tested on human hepatoblastoma HepG2 and MCF-7 cell lines by MTT assay, exhibited, for the first time, antitumor activities, and the complex revealed a higher activity than that of HPPA. The use of β-CD allows an increase in the aqueous solubility of the drug, its bioavailability and then its bioactivity.

  9. Friendship in inclusive physical education.

    Science.gov (United States)

    Seymour, Helena; Reid, Greg; Bloom, Gordon A

    2009-07-01

    Social interaction and development of friendships between children with and without a disability are often proposed as potential outcomes of inclusive education. Physical activity specialists assert that exercise and sport environments may be conducive to social and friendship outcomes. This study investigated friendship in inclusive physical education from the perspective of students with (n = 8) and without (n = 8) physical disabilities. All participants attended a reversely integrated school and were interviewed using a semistructured, open-ended format. An adapted version of Weiss, Smith, and Theeboom's (1996) interview guide exploring perceptions of peer relationships in the sport domain was used. Four conceptual categories emerged from the analysis: development of friendship, best friend, preferred physical activities and outcomes, and dealing with disability. The results demonstrated the key characteristics of best friends and the influential role they play.

  10. Does financial inclusion affect monetary policy in SAARC countries?

    Directory of Open Access Journals (Sweden)

    Sanjaya Kumar Lenka

    2016-12-01

    Full Text Available Alike the role of heart for human body, finance is the focal point of an economy, whereas savings and investment are its tubes and vessels. Hence, a solid financial system is a fundamental character of an enduring economy. The frozen financial system endures longer if its foundation is concrete and subsists in the people of grass-root level. They are those, who live in villages and small towns, earn meager income, work in primary sector, spend more on food, and have lesser social securities. In this setting, the process of bringing these people into the main stream of financial activities is called financial inclusion. This study describes the impact of financial inclusion on monetary policy of South Asian Association for Regional Cooperation (SAARC countries from 2004–2013. The study uses principal component analysis (PCA to construct a Financial Inclusion Index that serves as a proxy variable for the accessibility of financial inclusion in the SAARC countries. Adding to it, three different models like FEM, REM, and Panel-corrected standard errors are used for the analysis. In this study, an empirical result of generalized least square(GLS estimation shows that financial inclusion, exchange rate, and interest rate are negatively associated with inflation in SAARC countries.

  11. EDPS 265: The Inclusive Classroom

    OpenAIRE

    Begeske, Jasmine

    2014-01-01

    EDPS 265: The Inclusive Classroom is a foundational, large enrollment lecture course and is taught in a lecture hall with a stadium style seating arraignment. This configuration results in a course that is not student-centered, promotes one-way communication and hinders cooperative learning. Education courses should be structured so that the course in itself is instructive. This course teaches interventions for reaching all students, using techniques that engage students in the learning proce...

  12. Student' responses to inclusive design

    DEFF Research Database (Denmark)

    Herriott, Richard; Geert Jensen, Birgitte

    2013-01-01

    interact with our environment and technology. Specifically this involved welfare technology, equipment and tools that can help with daily activities. This technology is designed help to citizens to be 'master of his or her own life' while off-setting capability changes due to declining physical ability...... was originally written for the DRTS 2012 conference "Articulating Design Thinking" and proposed for inclusion in a special edition of Design Issues....

  13. Student Engagement In Inclusive Classrooms

    DEFF Research Database (Denmark)

    Rangvid, Beatrice Schindler

    There is general agreement that to thrive and learn at their best, students must be engaged. However, schools face a particular challenge to provide a suitable and engaging learning environment for SEN (special educational needs) students who are educated in general education classes. Using data......-students as for other students. This highlights the need for better inclusion initiatives aimed at strengthening engagement of SEN-students in regular classrooms....

  14. Perceived and actual social discrimination: the case of overweight and social inclusion.

    Science.gov (United States)

    Hartung, Freda-Marie; Renner, Britta

    2013-01-01

    The present study examined the correspondence between perceived and actual social discrimination of overweight people. In total, 77 first-year students provided self-ratings about their height, weight, and perceived social inclusion. To capture actual social inclusion, each participant nominated those fellow students (a) she/he likes and dislikes and (b) about whom she/he is likely to hear social news. Students with lower Body Mass Index (BMI) felt socially included, irrespective of their actual social inclusion. In contrast, students with higher BMI felt socially included depending on the degree of their actual social inclusion. Specifically, their felt social inclusion accurately reflected whether they were actually liked/disliked, but only when they were part of social news. When not part of social news, they also showed insensitivity to their actual social inclusion status. Thus, students with a lower BMI tended to be insensitive, while students with a higher BMI showed a differential sensitivity to actual social discrimination.

  15. Impact of cranberry on Escherichia coli cellular surface characteristics

    International Nuclear Information System (INIS)

    Johnson, Brandy J.; Lin Baochuan; Dinderman, Michael A.; Rubin, Robert A.; Malanoski, Anthony P.; Ligler, Frances S.

    2008-01-01

    The anti-adhesive effects of cranberry have been attributed to both interactions of its components with the surface of bacterial cells and to inhibition of p-fimbriae expression. Previous reports also suggested that the presence of cranberry juice changed the Gram stain characteristics of Escherichia coli. Here, we show that the morphology of E. coli is changed when grown in the presence of juice or extract from Vaccinium macrocarpon (cranberry). Gene expression analysis indicates the down regulation of flagellar basal body rod and motor proteins. Consistent with this finding and previous reports, the SEM images indicate a decrease in the visible p-fimbriae. The iodine used in Gram-staining protocols was found to interact differently with the bacterial membrane when cells were cultured in spiked media. Slight alterations in the Gram stain protocol demonstrated that culturing in the presence of cranberry juice does not change the Gram stain characteristics contradicting other reports.

  16. Triglyceride kinetics in fasted and fed E.coli septic rats

    International Nuclear Information System (INIS)

    Lanza-Jacoby, S.; Tabares, A.

    1990-01-01

    The mechanism for the development of hypertriglyceridemia during gram-negative sepsis was studies by examining the liver production and clearance of very-low-density lipoprotein (VLDL) triglyceride (TG). To assess the liver output and peripheral clearance the kinetics of VLDL-TG were determined by a constant intravenous infusion of [2- 3 H] glycerol-labeled VLDL in fasted control, fasted E. coli-treated, fed control, and fed E.coli-treated rats. Lewis inbred rats, 275-300 g, were made septic with 8 x 10 7 live E.coli colonies per 100 g body weight. Twenty-four hours following E.coli injection serum TG of fasted E.coli-treated rats was elevated by 170% which was attributed to a 67% decrease in the clearance rate of VLDL-TG in fasted E.coli-treated rats compared with their fasted controls. The secretion of VLDL-TG declined by 31% in the livers of the fasted E.coli-treated rats which was accompanied by a 2-fold increase in the composition of liver TG. In a second series of experiments control and E.coli-treated rats were fed intragastrically (IG) a balanced solution containing glucose plus fat as the sources of nonprotein calories. Serum TG were 26% lower in the fed E.coli-treated rats because the clearance rate increased by 86%. The secretion of TG in the fed septic rats increased by 40% but this difference was not significant. In the septic rat the ability to clear triglycerides from the plasma depends upon the nutritional state

  17. Pneumatosis Coli Mimicking Colorectal Cancer

    Directory of Open Access Journals (Sweden)

    Teresa Jacob

    2014-01-01

    Full Text Available Pneumatosis coli (PC is a rare condition of the gastrointestinal tract involving extraluminal gas confined within the bowel wall. We report the case of a 40-year-old gentleman presenting clinically and endoscopically with suspected colorectal cancer. In light of the patient’s red flag symptoms, and carpet of polyps seen endoscopically, surgical management by an anterior resection was performed with the patient making a successful recovery. Histological analysis of the resected specimen confirmed pneumatosis coli with no evidence of colonic neoplasia. Although PC can be an incidental finding in asymptomatic patients and considered a benign condition, it can also present as a life-threatening emergency with bowel necrosis and obstruction requiring emergency surgical intervention. Also, when PC mimics malignancy, surgical management is the most appropriate step to ensure that the diagnosis of cancer is not missed.

  18. Multiplex PCR Assay for Identification of Human Diarrheagenic Escherichia coli

    OpenAIRE

    Toma, Claudia; Lu, Yan; Higa, Naomi; Nakasone, Noboru; Chinen, Isabel; Baschkier, Ariela; Rivas, Marta; Iwanaga, Masaaki

    2003-01-01

    A multiplex PCR assay for the identification of human diarrheagenic Escherichia coli was developed. The targets selected for each category were eae for enteropathogenic E. coli, stx for Shiga toxin-producing E. coli, elt and est for enterotoxigenic E. coli, ipaH for enteroinvasive E. coli, and aggR for enteroaggregative E. coli. This assay allowed the categorization of a diarrheagenic E. coli strain in a single reaction tube.

  19. The adenomatous polyposis coli protein.

    OpenAIRE

    Näthke, I S

    1999-01-01

    Mutations in the adenomatous polyposis coli (APC) gene are associated with most colorectal cancers. The APC protein has been implicated in many aspects of tumour development. This article will discuss recent data suggesting that APC may have multiple functions in the cell. First, APC is a component of the Wnt signalling pathway; second, APC may have a role in cell migration; finally, APC may regulate proliferation and apoptosis.

  20. Body Lice

    Science.gov (United States)

    What are body lice? Body lice (also called clothes lice) are tiny insects which live and lay nits (lice eggs) on clothing. They are parasites, ... usually only move to the skin to feed. Body lice are one of the three types of ...

  1. Inclusive outreach practices in Palaeontology: Inclusive-Coworking

    Science.gov (United States)

    García-Frank, Alejandra; Gomez-Heras, Miguel; Fesharaki, Omid

    2017-04-01

    Previous experiences with people with both physical and intellectual functional diversity around palaeontological issues have demonstrated the important value of science outreach directed to people with disabilities. The aforementioned practices act twofold: as a learning tool and also improving the quality of life of the participants and thus, their self-image. All these pioneer experiences were the first step in a process of developing new attitudes contributing the 2030 Agenda for Sustainable Development of United Nations, where among the 17 goals proposed an effective social inclusion of people with disabilities is required. For this, real inclusive practices in geological outreach are imperious. A close cooperation with all the parts (researchers and participants), in a kind of coworking attitude is needed. This Inclusive-Coworking is considered in the sense of social gathering in order to share equal values and look for the synergy that this different outlook implies. And what is more important: the change of role of the previously learners into an active part of the scientific outreach, providing the adequate methodology for that. The offer of non-formal learning activities normally includes the participation of university professors and researchers in Science Week editions. During the 2016 session in Madrid, four adults with intellectual disability who were participants in the previous edition, contributed in the palaeontological workshop. They were in charge of four of the eight modules explaining the origin of fossils and how to collect them, the evolution of equids' limbs, and the main dentition types in vertebrates to the twenty 16 year old secondary students who attended the workshop. During the development of the experience all the students were pleased with the inclusive approach, and the interaction of all participants was fruitful. Although the explanations took a bit more time when made by our functional diverse fellows, all the abstracts concepts

  2. Inhibition of initial adhesion of oral bacteria through a lectin from Bauhinia variegata L. var. variegata expressed in Escherichia coli.

    Science.gov (United States)

    Klafke, G B; Borsuk, S; Gonçales, R A; Arruda, F V S; Carneiro, V A; Teixeira, E H; Coelho da Silva, A L; Cavada, B S; Dellagostin, O A; Pinto, L S

    2013-11-01

    The aim of the present work was to study the in vitro effect of native and recombinant Bauhinia variegata var. variegata lectins in inhibiting early adhesion of Streptococcus mutans, Streptococcus sanguis and Streptococcus sobrinus to experimentally acquired pellicle. Native lectin from B. variegata (BVL) was purified by affinity chromatography of extract of seeds. The recombinant lectin (rBVL-I) was expressed in E. coli strain BL21 (DE3) from a genomic clone encoding the mature B. variegata lectin gene using the vector pAE-bvlI. Recombinant protein deposited in inclusion bodies was solubilized and subsequently purified by affinity chromatography. The rBVL-I was compared to BVL for agglutination of erythrocytes and initial adherence of oral bacteria on a saliva-coated surface. The results revealed that rBVL-I acts similarly to BVL for agglutination of erythrocytes. Both lectins showed adhesion inhibition effect on Step. sanguis, Step. mutans and Step. sobrinus. We report, for the first time, the inhibition of early adhesion of oral bacteria by a recombinant lectin. Our results support the proposed biotechnological application of lectins in a strategy to reduce development of dental caries by inhibiting the initial adhesion and biofilm formation. © 2013 The Society for Applied Microbiology.

  3. High-yield expression in Escherichia coli, purification and application of budding yeast K2 killer protein.

    Science.gov (United States)

    Podoliankaitė, Monika; Lukša, Juliana; Vyšniauskis, Gintautas; Sereikaitė, Jolanta; Melvydas, Vytautas; Serva, Saulius; Servienė, Elena

    2014-07-01

    Saccharomyces cerevisiae K2 toxin is a highly active extracellular protein, important as a biocontrol agent for biotechnological applications in the wine industry. This protein is produced at negligible levels in yeast, making difficult to isolate it in amounts sufficient for investigation and generation of analysis tools. In this work, we demonstrate the use of a bacterial system for expression of the recombinant K2 protein, suitable for generation of antibodies specific for toxin of the yeast origin. Synthesis of the full-length S. cerevisiae K2 preprotoxin in Escherichia coli was found to be toxic to the host cell, resulting in diminished growth. Such effect was abolished by the introduction of the C-terminal truncation into K2 protein, directing it into non-toxic inclusion body fraction. The obtained protein is of limited solubility thus, facilitating the purification by simple and efficient chromatography-free procedure. The protein aggregates were successfully refolded into a soluble form yielding sufficient amounts of a tag-less truncated K2 protein suitable for polyclonal antibody production. Antibodies were raised in rabbit and found to be specific for detection of both antigen and native S. cerevisiae K2 toxin.

  4. High-level expression of human stem cell factor fused with erythropoietin mimetic peptide in Escherichia coli.

    Science.gov (United States)

    Su, Lin; Chen, Song-Sen; Yang, Ke-Gong; Liu, Chang-Zheng; Zhang, Yan-Li; Liang, Zhi-Quan

    2006-06-01

    Stem cell factor (SCF) and erythropoietin are essential for normal erythropoiesis and induce proliferation and differentiation synergistically for erythroid progenitor cells. Here, we report our work on construction of SCF/erythropoietin mimetic peptide (EMP) fusion protein gene, in which human SCF cDNA (1-165aa) and EMP sequence (20aa) were connected using a short (GGGGS) or long (GGGGSGGGGGS) linker sequence. The SCF/EMP gene was cloned into the pBV220 vector and expressed in the Escherichia coli DH5alpha strain. The expression level of the fusion protein was about 30% of total cell protein. The resulting inclusion bodies were solubilized with 8 M urea, followed by dilution refolding. The renatured protein was subsequently purified by Q-Sepharose FF column. The final product was >95% pure by SDS-PAGE and the yield of fusion protein was about 40 mg/L of culture. UT-7 cell proliferation and human cord blood cell colony-forming assays showed that the fusion proteins exhibited more potent activity than recombinant human SCF, suggesting a new strategy to enhance biological activities of growth factors.

  5. A novel multimodal chromatography based single step purification process for efficient manufacturing of an E. coli based biotherapeutic protein product.

    Science.gov (United States)

    Bhambure, Rahul; Gupta, Darpan; Rathore, Anurag S

    2013-11-01

    Methionine oxidized, reduced and fMet forms of a native recombinant protein product are often the critical product variants which are associated with proteins expressed as bacterial inclusion bodies in E. coli. Such product variants differ from native protein in their structural and functional aspects, and may lead to loss of biological activity and immunogenic response in patients. This investigation focuses on evaluation of multimodal chromatography for selective removal of these product variants using recombinant human granulocyte colony stimulating factor (GCSF) as the model protein. Unique selectivity in separation of closely related product variants was obtained using combined pH and salt based elution gradients in hydrophobic charge induction chromatography. Simultaneous removal of process related impurities was also achieved in flow-through leading to single step purification process for the GCSF. Results indicate that the product recovery of up to 90.0% can be obtained with purity levels of greater than 99.0%. Binding the target protein at pHproduct variants using the combined pH and salt based elution gradient and removal of the host cell impurities in flow-through are the key novel features of the developed multimodal chromatographic purification step. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. On Lipschitzian quantum stochastic differential inclusions

    International Nuclear Information System (INIS)

    Ekhaguere, G.O.S.

    1990-12-01

    Quantum stochastic differential inclusions are introduced and studied within the framework of the Hudson-Parthasarathy formulation of quantum stochastic calculus. Results concerning the existence of solutions of a Lipschitzian quantum stochastic differential inclusion and the relationship between the solutions of such an inclusion and those of its convexification are presented. These generalize the Filippov existence theorem and the Filippov-Wazewski Relaxation Theorem for classical differential inclusions to the present noncommutative setting. (author). 9 refs

  7. Regge cuts in inclusive reactions

    International Nuclear Information System (INIS)

    Paige, F.E.; Trueman, T.L.

    1975-01-01

    The contribution of Regge cuts to single-particle inclusive processes is analyzed using the techniques of Gribov. The dependence of these contributions on the polarization state of the target is emphasized. A general formula is obtained and certain contributions to it are calculated. It is not possible, however, to reduce this to a simple, powerful formula expressing the total cut contribution in terms of other measurable quantities, as can be done for the cut contribution to the total cross section. The reasons for this are discussed in detail. The single-particle intermediate states, analogous to the absorption model for elastic scattering, are explicitly calculated as an illustration

  8. Teaching Competences and Inclusive Education

    Directory of Open Access Journals (Sweden)

    José María Fernández Batanero

    2013-08-01

    Full Text Available This paper focuses on teaching competencies that are conducive to good educational practices in relation to inclusion, from the perspective of teachers. The methodology employed in the study is descriptive/comprehensive, and of an exploratory nature. By means of four case studies, the perceptions of teachers from two secondary schools—characterized by the Spanish Educational Administration as having “good practices”— are examined. The techniques used for information collection in this study include documentary analysis, in-depth interviews and focus groups. The findings emphasize the importance of strategic skills, combined with innovation and creativity, among others.

  9. Theory of inclusive pionic reactions

    International Nuclear Information System (INIS)

    Oset, E.; Salcedo, L.L.; Strottman, D.

    1985-01-01

    A theory is developed for all the inclusive pion nuclear reactions, quasielastic, single charge exchange, double charge exchange and absorption, around the resonance region. The theory is based on the isobar hole model and makes an expansion in the number of particle-hole excitations. Up to 3p3h for pion absorption and 2p2h for quasielastic or charge exchange, where good convergence is found, are considered. The results obtained with this theory agree remarkably well with experiment for the different reactions and different nuclei in a wide region of energies around resonance

  10. Detecting Anisotropic Inclusions Through EIT

    Science.gov (United States)

    Cristina, Jan; Päivärinta, Lassi

    2017-12-01

    We study the evolution equation {partialtu=-Λtu} where {Λt} is the Dirichlet-Neumann operator of a decreasing family of Riemannian manifolds with boundary {Σt}. We derive a lower bound for the solution of such an equation, and apply it to a quantitative density estimate for the restriction of harmonic functions on M}=Σ_{0 to the boundaries of {partialΣt}. Consequently we are able to derive a lower bound for the difference of the Dirichlet-Neumann maps in terms of the difference of a background metrics g and an inclusion metric {g+χ_{Σ}(h-g)} on a manifold M.

  11. Expression of an Acid Urease with Urethanase Activity in E. coli and Analysis of Urease Gene.

    Science.gov (United States)

    Liu, Xiaofeng; Zhang, Qian; Zhou, Nandi; Tian, Yaping

    2017-03-01

    Urea in alcoholic beverage is a precursor of ethyl carbamate (EC), which is carcinogenic. Enzymatic elimination of urea has attracted much research interest. Acid urease with good tolerance toward ethanol and acid is ideal enzyme for such applications. In the present work, the structural genes of urease from Providencia rettgeri JN-B815, ureABC were efficiently expressed in E. coli BL21(DE3) in an active form (apourease) exhibiting both urease and urethanase (hydrolyze EC) activities. The specific activities of the purified apourease were comparatively low, which were 2.1 U/mg for urease and 0.6 U/mg for urethanase, respectively. However, apourease exhibited good resistance toward ethanol and acidic conditions. The relative activities of urease and urethanase remained over 80% in the buffers within pH 4-7. And the recoveries of both urease and urethanase activities were more than 50% in 5-25% ethanol solution. Apourease was utilized to eliminate urea in wine, and the residual urea in model wine was less than 50% after treatment with apourease for 30 h. Then 3D structure of UreC was predicted, and it was docked with urea and EC, respectively. The docking result revealed that three hydrogen bonds were formed between urea and amino acid residues in the active site of urease, whereas only one hydrogen bond can be formed between EC and the active center. Moreover, EC exhibited greater steric hindrance than urea when combined with the active site. Due to the low specific activities of apourease, both structural genes and accessory genes of urease were co-expressed in E. coli BL21(DE3). The holoenzyme was expressed as inclusion body. After renaturation and purification, the specific activities of urease and urethanase reached 10.7 and 3.8 U/mg, which were 5.62-fold and 6.33-fold of those of apourease, respectively. Therefore, accessory subunits of urease play an important role in enhancing urease and urethanase activities.

  12. Expression of the phycoerythrin gene of Gracilaria lemaneiformis (Rhodophyta) in E. coli and evaluation of the bioactivity of recombinant PE

    Science.gov (United States)

    Wen, Ruobing; Sui, Zhenghong; Zhang, Xuecheng; Zhang, Shuang; Qin, Song

    2007-10-01

    Phycoerythrin (PE) is one of the most important proteins involved in light capturing during photosynthesis in red algae. Its potential biological activities had gained wide concerns. In the present study, tumor cytotoxic and hydroxyl radical assay were preformed to detect the bioactivity of recombinant PE. Recombinant plasmids pGEX-PE and pBGL were transformed into E. coli BL21 to make two recombinant strains BEX (pGEX-PE) and BGL (pBGL). PE expressing in BEX (pGEX-PE) was validated by SDS-PAGE and Western blotting analysis. SDS-PAGE analysis indicated that the PE-GST fusion protein was mostly inclusion bodies. Specific expression of PE was confirmed by Western blotting analysis. The recombinant E. coli BEX (pGEX-PE) cells were collected and sonicated. The supernatants were reserved for the tumor cytotoxic experiments. The result of tumor cytotoxic assay indicated that the supernatants containing PE had the activity of inhibiting the growth of Hela cells and with the increase of protein concentration, the inhibiting rate increased from 37.31% to 63.26%, which showed significant difference from the control. Hydroxyl radical scavenging effect was tested with supernatants of BEX (pGEX-PE) and BGL (pBGL) cell lysates treated with sonication and heating. For the sonication samples, the scavenging rates of the supernatants of BEX (pGEX-PE) and BGL (pBGL) cell lysates were significantly higher than the negative control BL21(pGEX-4T) ( Pprotein content. For the heating samples, except for the 0.2 mg mL-1 BGL (pBGL) products, the scavenging effects of the supernatants of BEX (pGEX-PE) and BGL (pBGL) cell lysates were stronger than that of negative control BL21(pGEX-4T). However, the effect intensity was not positively correlated with the increase of the protein concentration. Though a partially decreased hydroxyl radical scavenging activity was led by heating, the biological activity was still retained and conspicuous. This research showed that phycoerythrin protein

  13. Forging Inclusive Solutions: Experiential Earth Charter Education

    Science.gov (United States)

    Hill, Linda D.

    2010-01-01

    Forging Inclusive Solutions describes the aims, methodology and outcomes of Inclusive Leadership Adventures, an experiential education curriculum for exploring the Earth Charter. Experiential education builds meaningful relationships, skills, awareness and an inclusive community based on the Earth Charter principles. When we meet people where they…

  14. Measuring Inclusive Education Outcomes in Alberta, Canada

    Science.gov (United States)

    Loreman, Tim

    2014-01-01

    This study details the results of a review of the academic and public sector literature on measuring inclusive education in large systems. It highlights some outcomes drawn from the international literature on inclusion that might be indicative of the presence and quality of inclusive education in an effort to develop a set of outcomes for…

  15. 75 FR 81395 - Minority and Women Inclusion

    Science.gov (United States)

    2010-12-28

    ... 2590-AA28 Minority and Women Inclusion AGENCIES: Federal Housing Finance Board; Federal Housing Finance... and the inclusion of women and minorities in all activities. The final rule implements the provisions.... It also requires each regulated entity to establish an Office of Minority and Women Inclusion, or...

  16. Diversity, Equity, & Inclusion at Berkeley Lab

    Science.gov (United States)

    Berkeley Lab A-Z Index Directory Search Diversity, Equity, & Inclusion at Berkeley Lab Home Diversity & Inclusion Council Women Scientists & Engineers Council Employee Resource Groups -and culture of inclusion are key to attracting and engaging the brightest minds and furthering our

  17. 75 FR 10446 - Minority and Women Inclusion

    Science.gov (United States)

    2010-03-08

    ... 2590-AA28 Minority and Women Inclusion AGENCIES: Federal Housing Finance Board; Federal Housing Finance... minority and women inclusion. Section 1116 of the Housing and Economic Recovery Act of 2008 amended section... Loan Banks to promote diversity and the inclusion of women and minorities in all activities...

  18. Transport Policy and Social Inclusion

    Directory of Open Access Journals (Sweden)

    Miriam Ricci

    2016-06-01

    Full Text Available ‘Transport-related Social inclusion’ is a specific naming of the complex set of interrelationships within which accessibility plays an important role in whether a citizen achieves the level of participation in socioeconomic life that he or she seeks. It has its origins in the United Kingdom of the early 2000s, but the diversity of theoretical perspectives, research methods and practical focus shown by the contributions to the present issue on this theme bears witness to the evolution and translation this concept and term has undergone over more than a decade. Nine papers are presented, concerning applications of the concept in three continents, and including some of the poorest and richest per capita income countries on the globe. As well as developing and applying the multi-faceted theories of the processes of exclusion and techniques for the quantitative identification of inclusion, they consider important topics such as the treatment of the less abled and more frail members of society when on the move and the potential for new technological design methods and practical solutions either to enhance inclusion or deepen inequality in our societies. Collectively their conclusions reinforce the message that social exclusion remains multi-dimensional, relational and dynamic, located both in the circumstances of the excluded individual as well as in the processes, institutions and structures that permeate wider society.

  19. Silicate Inclusions in IAB Irons: Correlations Between Metal Composition and Inclusion Properties, and Inferences for Their Origin

    Science.gov (United States)

    Benedix, G. K.; McCoy, T. J.; Keil, K.

    1995-09-01

    IAB irons are the largest group of iron meteorites, exhibit a large range of siderophile element concentrations in their metal, and commonly contain silicate inclusions with roughly chondritic composition. They are closely related to IIICD irons [1,2] and their inclusions resemble winonaites [3]. It has been suggested that IAB's and IIICD's formed in individual impact melt pools [4,2] on a common parent body. However, it has also been suggested that fractional crystallization [5,6] of a S-saturated core could produce the observed siderophile element trends. Metal composition is correlated with silicate inclusion mineralogy in IIICD's [1], indicating reactions between solid silicates and the metallic magma in a core. These trends observed in IIICD's differ from those in IAB's, suggesting different parent bodies. A bi-modal grouping, based primarily on mineralogy and mineral abundances, was suggested for IAB inclusions [7]. However, recent recoveries of several new silicate-bearing IAB's, along with the emergence of new ideas on their origins, prompted a comprehensive study to document more fully the range of inclusions within IAB irons, to examine possible correlations between the compositions of the metallic host and the silicate inclusions, and to elucidate the origin of IAB irons. We are studying troilite-graphite-silicate inclusions in 24 IAB irons with Ni concentrations ranging from 6.6-25.0%. These include Odessa and Copiapo types [7], newly recovered meteorites (e.g., Lueders [8]) and meteorites with extreme Ni contents (e.g., Jenny's Creek, 6.8%; San Cristobal, 25.0% [9]). The inclusions exhibit a range of textures from recrystallized to partial melts (e.g., Caddo County [10]). Rigorous classification [7] is hampered by heterogeneities between group meteorites, between different samples of distinct meteorites, and within individual inclusions. While intergroup heterogeneities make comparisons between the suite of IAB's somewhat difficult, some general trends

  20. Annual Surveillance Summary: Escherichia coli (E. coli) Infections in the Military Health System (MHS), 2015

    Science.gov (United States)

    2017-03-01

    Annual Surveillance Summary: Escherichia coli ( E . coli ) Infections in the Military Health System (MHS...or position of the Department of the Navy, Department of Defense, nor the U.S. Government. i i E . coli in the MHS: Annual Summary 2015 Prepared...March 2017 EpiData Center Department NMCPHC-EDC-TR-187-2017 ii ii E . coli in the MHS: Annual Summary 2015 Prepared March 2017 EpiData

  1. Chromosomal features of Escherichia coli serotype O2:K2, an avian pathogenic E. coli.

    Science.gov (United States)

    Jørgensen, Steffen L; Kudirkiene, Egle; Li, Lili; Christensen, Jens P; Olsen, John E; Nolan, Lisa; Olsen, Rikke H

    2017-01-01

    Escherichia coli causing infection outside the gastrointestinal system are referred to as extra-intestinal pathogenic E. coli. Avian pathogenic E. coli is a subgroup of extra-intestinal pathogenic E. coli and infections due to avian pathogenic E. coli have major impact on poultry production economy and welfare worldwide. An almost defining characteristic of avian pathogenic E. coli is the carriage of plasmids, which may encode virulence factors and antibiotic resistance determinates. For the same reason, plasmids of avian pathogenic E. coli have been intensively studied. However, genes encoded by the chromosome may also be important for disease manifestation and antimicrobial resistance. For the E. coli strain APEC_O2 the plasmids have been sequenced and analyzed in several studies, and E. coli APEC_O2 may therefore serve as a reference strain in future studies. Here we describe the chromosomal features of E. coli APEC_O2. E. coli APEC_O2 is a sequence type ST135, has a chromosome of 4,908,820 bp (plasmid removed), comprising 4672 protein-coding genes, 110 RNA genes, and 156 pseudogenes, with an average G + C content of 50.69%. We identified 82 insertion sequences as well as 4672 protein coding sequences, 12 predicated genomic islands, three prophage-related sequences, and two clustered regularly interspaced short palindromic repeats regions on the chromosome, suggesting the possible occurrence of horizontal gene transfer in this strain. The wildtype strain of E. coli APEC_O2 is resistant towards multiple antimicrobials, however, no (complete) antibiotic resistance genes were present on the chromosome, but a number of genes associated with extra-intestinal disease were identified. Together, the information provided here on E. coli APEC_O2 will assist in future studies of avian pathogenic E. coli strains, in particular regarding strain of E. coli APEC_O2, and aid in the general understanding of the pathogenesis of avian pathogenic E. coli .

  2. Effect of rigid inclusions on sintering

    International Nuclear Information System (INIS)

    Rahaman, M.N.; De Jonghe, L.C.

    1988-01-01

    The predictions of recent theoretical studies on the effect of inert, rigid inclusions on the sintering of ceramic powder matrices are examined and compared with experimental data. The densification of glass matrix composites with inclusion volume fractions of ≤0.15 can be adequately explained by Scherer's theory for viscous sintering with rigid inclusions. Inclusions cause a vast reduction in the densification rates of polycrystalline matrix composites even at low inclusion volume fractions. Models put forward to explain the sintering of polycrystalline matrix composites are discussed

  3. The Politics of Inclusion and Empowerment

    DEFF Research Database (Denmark)

    Andersen, John; Siim, Birte

    The objective of the book is to analyse different politics of inclusion and empowerment and the different paradigms of inclusion/exclusion in order to underline the close link between politics of scoial equality and politics of recognition of ciultural difference. Politics of inclusion is thus...... identities. Politics of empowerment has to do with the agency and mobilisation dimension of social and political change. The title of the book "Politics of Inclusion and Empowerment" address the leitmotiv: namely to discuss plussumgame between politics of inclusion and politics of empowerment...

  4. Inclusion-initiated fracture model for ceramics

    International Nuclear Information System (INIS)

    Sung, J.; Nicholson, P.S.

    1990-01-01

    The fracture of ceramics initiating from a typical inclusion is analyzed. The inclusion is considered to have a thermal expansion coefficient and fracture toughness lower than those of the matrix and a Young's modulus higher than that of the matrix. Inclusion-initiated fracture is modeled for a spherical inclusion using a weight function method to compute the residual stress intensity factor for a part-through elliptical crack. The results are applied to an α-Al 2 O 3 inclusion embedded in a tetragonal ZrO 2 ceramic. The strength predictions agree well with experimental data

  5. Axonal inclusions in the crab Hemigrapsus nudus.

    Science.gov (United States)

    Smith, R S

    1978-10-01

    Light microscopic examination of living giant axons from the walking legs of Hemigrapsus nudus revealed intra-axonal inclusions which were usually several tens of micrometers long and about 5 micron wide. The inclusions were filled with small light-scattering particles. The inclusions were shown, by thin section electron microscopy, to be composed largely 68% by volume) of mitochondria. Each inclusion was surrounded by membrane bounded spaces which are presumed to represent a part of the smooth endoplasmic reticulum. Similar inclusions were not found in the leg axons of a variety of other decapod crustaceans.

  6. Functional properties of the recombinant kringle-2 domain of tissue plasminogen activator produced in Escherichia coli

    International Nuclear Information System (INIS)

    Wilhelm, O.G.; Jaskunas, S.R.; Vlahos, C.J.; Bang, N.U.

    1990-01-01

    The kringle-2 domain (residues 176-262) of tissue-type plasminogen activator (t-PA) was cloned and expressed in Escherichia coli. The recombinant peptide, which concentrated in cytoplasmic inclusion bodies, was isolated, solubilized, chemically refolded, and purified by affinity chromatography on lysine-Sepharose to apparent homogeneity. [35S]Cysteine-methionine-labeled polypeptide was used to study the interactions of kringle-2 with lysine, fibrin, and plasminogen activator inhibitor-1. The kringle-2 domain bound to lysine-Sepharose and to preformed fibrin with a Kd = 104 +/- 6.2 microM (0.86 +/- 0.012 binding site) and a Kd = 4.2 +/- 1.05 microM (0.80 +/- 0.081 binding site), respectively. Competition experiments and direct binding studies showed that the kringle-2 domain is required for the formation of the ternary t-PA-plasminogen-intact fibrin complex and that the association between the t-PA kringle-2 domain and fibrin does not require plasmin degradation of fibrin and exposure of new COOH-terminal lysine residues. We also observed that kringle-2 forms a complex with highly purified guanidine-activated plasminogen activator inhibitor-1, dissociable by 0.2 M epsilon-aminocaproic acid. The kringle-2 polypeptide significantly inhibited tissue plasminogen activator/plasminogen activator inhibitor-1 interaction. The kringle-2 domain bound to plasminogen activator inhibitor-1 in a specific and saturable manner with a Kd = 0.51 +/- 0.055 microM (0.35 +/- 0.026 binding site). Therefore, the t-PA kringle-2 domain is important for the interaction of t-PA not only with fibrin, but also with plasminogen activator inhibitor-1 and thus represents a key structure in the regulation of fibrinolysis

  7. A New Player in the Biorefineries Field: Phasin PhaP Enhances Tolerance to Solvents and Boosts Ethanol and 1,3-Propanediol Synthesis in Escherichia coli.

    Science.gov (United States)

    Mezzina, Mariela P; Álvarez, Daniela S; Egoburo, Diego E; Díaz Peña, Rocío; Nikel, Pablo I; Pettinari, M Julia

    2017-07-15

    The microbial production of biofuels and other added-value chemicals is often limited by the intrinsic toxicity of these compounds. The phasin PhaP from the soil bacterium Azotobacter sp. strain FA8 is a polyhydroxyalkanoate granule-associated protein that protects recombinant Escherichia coli against several kinds of stress. PhaP enhances growth and poly(3-hydroxybutyrate) synthesis in polymer-producing recombinant strains and reduces the formation of inclusion bodies during overproduction of heterologous proteins. In this work, the heterologous expression of this phasin in E. coli was used as a strategy to increase tolerance to several biotechnologically relevant chemicals. PhaP was observed to enhance bacterial fitness in the presence of biofuels, such as ethanol and butanol, and other chemicals, such as 1,3-propanediol. The effect of PhaP was also studied in a groELS mutant strain, in which both GroELS and PhaP were observed to exert a beneficial effect that varied depending on the chemical tested. Lastly, the potential of PhaP and GroEL to enhance the accumulation of ethanol or 1,3-propanediol was analyzed in recombinant E. coli Strains that overexpressed either groEL or phaP had increased growth, reflected in a higher final biomass and product titer than the control strain. Taken together, these results add a novel application to the already multifaceted phasin protein group, suggesting that expression of these proteins or other chaperones can be used to improve the production of biofuels and other chemicals. IMPORTANCE This work has both basic and applied aspects. Our results demonstrate that a phasin with chaperone-like properties can increase bacterial tolerance to several biochemicals, providing further evidence of the diverse properties of these proteins. Additionally, both the PhaP phasin and the well-known chaperone GroEL were used to increase the biosynthesis of the biotechnologically relevant compounds ethanol and 1,3-propanediol in recombinant E

  8. Production of a recombinant phospholipase A2 in Escherichia coli using resonant acoustic mixing that improves oxygen transfer in shake flasks.

    Science.gov (United States)

    Valdez-Cruz, Norma A; Reynoso-Cereceda, Greta I; Pérez-Rodriguez, Saumel; Restrepo-Pineda, Sara; González-Santana, Jesus; Olvera, Alejandro; Zavala, Guadalupe; Alagón, Alejandro; Trujillo-Roldán, Mauricio A

    2017-07-25

    Shake flasks are widely used during the development of bioprocesses for recombinant proteins. Cultures of recombinant Escherichia coli with orbital mixing (OM) have an oxygen limitation negatively affecting biomass growth and recombinant-protein production. With the aim to improve mixing and aeration in shake flask cultures, we analyzed cultures subjected to OM and the novel resonant acoustic mixing (RAM) by applying acoustic energy to E. coli BL21-Gold (DE3): a producer of recombinant phospholipase A2 (rPLA2) from Micrurus laticollaris snake venom. Comparing OM with RAM (200 rpm vs. 7.5g) at the same initial volumetric oxygen transfer coefficient (k L a ≈ 80 h -1 ) ~69% less biomass was obtained with OM compared with RAM. We analyzed two more conditions increasing agitation until maximal speed (12.5 and 20g), and ~1.6- and ~1.4-fold greater biomass was obtained as compared with cultures at 7.5g. Moreover, the specific growth rate was statistically similar in all cultures carried out in RAM, but ~1.5-fold higher than that in cultures carried out under OM. Almost half of the glucose was consumed in OM, whereas between 80 and 100% of the glucose was consumed in RAM cultures, doubling biomass per glucose yields. Differential organic acid production was observed, but acetate production was prevented at the maximal RAM (20g). The amount of rPLA2 in both, OM and RAM cultures, represented 38 ± 5% of the insoluble protein. A smaller proportion of α-helices and β-sheet of purified inclusion bodies (IBs) were appreciated by ATR-FTIR from cultures carried out under OM, than those from RAM. At maximal agitation by RAM, internal E. coli localization patterns of protein aggregation changed, as well as, IBs proteolytic degradation, in conjunction with the formation of small external vesicles, although these changes did not significantly affect the cell survival response. In moderate-cell-density recombinant E. coli BL21-Gold (DE3) cultures, the agitation increases in

  9. Occurrence of Escherichia coli in the Cuyahoga River in the Cuyahoga Valley National Park, Ohio

    Science.gov (United States)

    Brady, Amie M.G.; Plona, Meg B.

    2010-01-01

    There are several measures of the 'cleanliness' of a natural body of water, including concentrations of indicator bacteria, anthropogenic chemicals (chemicals derived from human activities), and nutrients, such as nitrogen and phosphorous. Escherichia coli (E. coli) is a bacterium that lives in the intestinal tract of warm-blooded animals, such as humans, deer, cows, and dogs. Most strains of E. coli are not harmful and are in fact beneficial to humans by aiding in the digestive process. A few strains, such as the O157 strain, produce toxins that can cause gastrointestinal illness, but occurrence of toxic strains in the environment is not common. E. coli is considered a good indicator bacterium because its occurrence in the environment indicates the presence of fecal contamination and therefore the possible presence of pathogenic organisms associated with feces. The U.S. Environmental Protection Agency (USEPA) recommends using measurements of E. coli to monitor freshwaters and set criteria for the concentration of bacteria that can be present in the water with minimal adverse human-health effects. Typically, a State's waters are assigned a recreational-use designation, such as bathing, primary-contact, or secondary contact waters, which is used to set the State's water-quality standards based on the USEPA criteria. The Cuyahoga River in the Cuyahoga Valley National Park is designated for primary-contact recreation; therefore, when concentrations of E. coli exceed 298 CFU/100mL, the river would be considered potentially unsafe for recreation.

  10. Modern space/time geostatistics using river distances: data integration of turbidity and E. coli measurements to assess fecal contamination along the Raritan River in New Jersey.

    Science.gov (United States)

    Money, Eric S; Carter, Gail P; Serre, Marc L

    2009-05-15

    Escherichia coli (E. coli) is a widely used indicator of fecal contamination in water bodies. External contact and subsequent ingestion of bacteria coming from fecal contamination can lead to harmful health effects. Since E. coli data are sometimes limited, the objective of this study is to use secondary information in the form of turbidity to improve the assessment of E. coli at unmonitored locations. We obtained all E. coli and turbidity monitoring data available from existing monitoring networks for the 2000-2006 time period for the Raritan River Basin, New Jersey. Using collocated measurements, we developed a predictive model of E. coli from turbidity data. Using this model, soft data are constructed for E. coli given turbidity measurements at 739 space/time locations where only turbidity was measured. Finally, the Bayesian Maximum Entropy (BME) method of modern space/time geostatistics was used for the data integration of monitored and predicted E. coli data to produce maps showing E. coli concentration estimated daily across the river basin. The addition of soft data in conjunction with the use of river distances reduced estimation error by about 30%. Furthermore, based on these maps, up to 35% of river miles in the Raritan Basin had a probability of E coli impairment greater than 90% on the most polluted day of the study period.

  11. Hemolytic porcine intestinal Escherichia coli without virulence-associated genes typical of intestinal pathogenic E. coli.

    Science.gov (United States)

    Schierack, Peter; Weinreich, Joerg; Ewers, Christa; Tachu, Babila; Nicholson, Bryon; Barth, Stefanie

    2011-12-01

    Testing 1,666 fecal or intestinal samples from healthy and diarrheic pigs, we obtained hemolytic Escherichia coli isolates from 593 samples. Focusing on hemolytic E. coli isolates without virulence-associated genes (VAGs) typical for enteropathogens, we found that such isolates carried a broad variety of VAGs typical for extraintestinal pathogenic E. coli.

  12. An Arendtian perspective on inclusive education

    DEFF Research Database (Denmark)

    Korsgaard, Morten Timmermann

    2016-01-01

    Inclusive education currently appears to be undergoing a crisis and re- examination. This paper presents a new approach to thinking about inclusiveness in the school context. Many positions within inclusive education seem to take political, social and ethical perspectives as a starting point, which...... has allowed inclusive movements and initiatives around the world to succumb to neo-liberal policy-making and has neglected the development of an educational vocabulary that is theoretically and conceptually appropriate for confronting teachers’ central concerns regarding inclusive practices....... The concepts of suspension, bearing with strangers and enlarged thought inspired by Hannah Arendt provide a basis for a re-imagining of inclusive education and for outlining a future school in which inclusiveness is embedded in the very way we think and position ourselves as teachers and pupils...

  13. A review of social inclusion measures.

    Science.gov (United States)

    Coombs, Tim; Nicholas, Angela; Pirkis, Jane

    2013-10-01

    Social inclusion is crucial to mental health and well-being and is emphasised in Australia's Fourth National Mental Health Plan. There is a recognition that a measure of social inclusion would complement the suite of outcome measures that is currently used in public sector mental health services. This paper is an initial scope of candidate measures of social inclusion and considers their suitability for this purpose. We identified potential measures through searches of PsycINFO and Medline and a more general Internet search. We extracted descriptive and evaluative information on each measure identified and compared this information with a set of eight criteria. The criteria related to the measure's inclusion of four domains of social inclusion outlined in Australia's Fourth National Mental Health Plan, its usability within the public mental health sector and its psychometric properties. We identified 10 candidate measures of social inclusion: the Activity and Participation Questionnaire (APQ-6); the Australian Community Participation Questionnaire (ACPQ); the Composite Measure of Social Inclusion (CMSI); the EMILIA Project Questionnaire (EPQ); the Evaluating Social Inclusion Questionnaire (ESIQ); the Inclusion Web (IW); the Social and Community Opportunities Profile (SCOPE); the Social Inclusion Measure (SIM); the Social Inclusion Questionnaire (SIQ); and the Staff Survey of Social Inclusion (SSSI). After comparison with the eight review criteria, we determined that the APQ-6 and the SCOPE-short form show the most potential for further testing. Social inclusion is too important not to measure. This discussion of individual-level measures of social inclusion provides a springboard for selecting an appropriate measure for use in public sector mental health services. It suggests that there are two primary candidates, but neither of these is quite fit-for-purpose in their current form. Further exploration will reveal whether one of these is suitable, whether another

  14. Inclusion of liver in meat products from sheep

    Directory of Open Access Journals (Sweden)

    T. H. Borghi

    2017-03-01

    Full Text Available The objective of this study was to evaluate the physicochemical (pH, color, water retention capacity, cooking losses, shear force, iron content, and lipid oxidation and sensory characteristics of koftas and sausages prepared from lamb meat and enriched with sheep liver. Meat from eight lambs weaned at 60 days and slaughtered at a body weight of 32.0 ± 0.2 kg was used. Percentages of 10 and 20% sheep liver were included in koftas and of 15 and 30% in sausages. A completely randomized experimental design was used and the data were submitted to analysis of variance and regression. The inclusion of liver in koftas linearly increased pH (P=0.001 and shear force (P=0.028, and reduced luminosity (P=0.005 and lipid oxidation at the time of (P=0.001 and 24 hours after (P=0.004 fabrication of the products. In sausages, the inclusion of liver linearly increased shear force (P=0.03. The inclusion of liver exerted decreasing linear effects on the scores related to the flavor of kofta and sausage products (P=0.001 and 0.033, respectively and a quadratic effect (P=0.008 on the overall acceptance of sausages. Although the inclusion of liver altered some relevant quality-related attributes of sheep koftas and sausages, the physicochemical and sensory characteristics were not compromised.

  15. Enteroaggregative Escherichia coli in Daycare

    DEFF Research Database (Denmark)

    Hebbelstrup Jensen, Betina; Stensvold, Christen R.; Struve, Carsten

    2016-01-01

    Enteroaggregative Escherichia coli (EAEC) has been associated with persistent diarrhea, reduced growth acceleration, and failure to thrive in children living in developing countries and with childhood diarrhea in general in industrialized countries. The clinical implications of an EAEC carrier...... and answered a questionnaire regarding gastrointestinal symptoms and exposures. Exposures included foreign travel, consumption of antibiotics, and contact with a diseased animal. In the capital area of Denmark, a total of 179 children aged 0-6 years were followed in a cohort study, in the period between 2009...

  16. Inclusive breakup of three-fragment weakly bound nuclei

    International Nuclear Information System (INIS)

    Carlson, B.V.; Frederico, T.; Hussein, M.S.

    2017-01-01

    The inclusive breakup of three-fragment projectiles is discussed within a four-body spectator model. Both the elastic breakup and the non-elastic breakup are obtained in a unified framework. Originally developed in the 80's for two-fragment projectiles such as the deuteron, in this paper the theory is successfully generalized to three-fragment projectiles. The expression obtained for the inclusive cross section allows the extraction of the incomplete fusion cross section, and accordingly generalizes the surrogate method to cases such as (t, p) and (t, n) reactions. It is found that two-fragment correlations inside the projectile affect in a conspicuous way the elastic breakup cross section. The inclusive non-elastic breakup cross section is calculated and is found to contain the contribution of a three-body absorption term that is also strongly influenced by the two-fragment correlations. This latter cross section contains the so-called incomplete fusion where more than one compound nuclei are formed. Our theory describes both stable weakly bound three-fragment projectiles and unstable ones such as the Borromean nuclei.

  17. Inclusive breakup of three-fragment weakly bound nuclei

    Energy Technology Data Exchange (ETDEWEB)

    Carlson, B.V.; Frederico, T. [Instituto Tecnológico de Aeronáutica, DCTA, 12.228-900 São José dos Campos, SP (Brazil); Hussein, M.S., E-mail: hussein@if.usp.br [Instituto Tecnológico de Aeronáutica, DCTA, 12.228-900 São José dos Campos, SP (Brazil); Instituto de Estudos Avançados, Universidade de São Paulo, C.P. 72012, 05508-970 São Paulo, SP (Brazil); Instituto de Física, Universidade de São Paulo, C.P. 66318, 05314-970 São Paulo, SP (Brazil)

    2017-04-10

    The inclusive breakup of three-fragment projectiles is discussed within a four-body spectator model. Both the elastic breakup and the non-elastic breakup are obtained in a unified framework. Originally developed in the 80's for two-fragment projectiles such as the deuteron, in this paper the theory is successfully generalized to three-fragment projectiles. The expression obtained for the inclusive cross section allows the extraction of the incomplete fusion cross section, and accordingly generalizes the surrogate method to cases such as (t, p) and (t, n) reactions. It is found that two-fragment correlations inside the projectile affect in a conspicuous way the elastic breakup cross section. The inclusive non-elastic breakup cross section is calculated and is found to contain the contribution of a three-body absorption term that is also strongly influenced by the two-fragment correlations. This latter cross section contains the so-called incomplete fusion where more than one compound nuclei are formed. Our theory describes both stable weakly bound three-fragment projectiles and unstable ones such as the Borromean nuclei.

  18. Inclusive radiative J/ψ decays

    International Nuclear Information System (INIS)

    Besson, D.; Pedlar, T. K.; Cronin-Hennessy, D.; Gao, K. Y.; Hietala, J.; Kubota, Y.; Klein, T.; Lang, B. W.; Poling, R.; Scott, A. W.; Zweber, P.; Dobbs, S.; Metreveli, Z.; Seth, K. K.; Tomaradze, A.; Libby, J.; Powell, A.; Wilkinson, G.; Ecklund, K. M.; Love, W.

    2008-01-01

    Using data taken with the CLEO-c detector at the Cornell Electron Storage Ring, we have investigated the direct-photon momentum spectrum in the decay J/ψ(1S)→γgg, via the ''tagged'' process: e + e - →ψ(2S); ψ(2S)→J/ψπ + π - ; J/ψ→γ+X. Including contributions from two-body radiative decay processes, we find the ratio of the inclusive direct-photon branching fraction to that of the dominant three-gluon branching fraction [R γ =B(ggγ)/B(ggg)] to be R γ =0.137±0.001±0.016±0.004, where the errors shown are statistical, systematic, and the model-dependent uncertainty related to the extrapolation to zero photon energy. The shape of the scaled photon energy spectrum in J/ψ→ggγ is observed to be very similar to that of Υ→ggγ. The R γ value obtained is roughly consistent with that expected by a simple quark-charge scaling [R γ ∼(q c /q b ) 2 ] of the value determined at the Υ(1S), but somewhat higher than the value expected from the running of the strong coupling constant.

  19. Inclusion, children's groups, music therapy

    DEFF Research Database (Denmark)

    Holck, Ulla; Jacobsen, Stine Lindahl

    2016-01-01

    portrayal of the qualities of musical interplay that promotes well-being in group settings and, thus, the inclusion of vulnerable students. Therefore, we open the chapter with a focus on musicality and on the importance of applying a musical approach in relation to the children.......Music has a rare ability to affect us directly. Pulse and rhythms make us move, and notes and harmonies inspire and express our inner emotions in a direct and immediate way that goes beyond what words or even other art forms can rarely achieve (Panksepp & Trevarthen, 2009). Music creates...... a delightful build-up of tension or soothes us, and its narrative character gives rise to mental imagery or memories. Music brings people together and helps build communities across languages and common divides. And – not least – music captures children’s immediate attention, so when the music starts, so do...

  20. Interprofessional development in inclusive schools

    DEFF Research Database (Denmark)

    Hansen, Britt Blaabjerg; Højholdt, Andy; Arndal, Lars Stubbe

    educators, both initiatives of which support the subject-oriented education at school. These changes have challenged the way [education/school] professionals work and how they see their own and each other's roles in the school system (EVA 2013). The changes have also increased the need for collaborative...... in the classroom.4. Changing teaching methods: Students with weak school affiliation or special needs would benefit from a change in teaching methods that takes some of the competencies possessed by child and youth educators and uses them in the classroom. As a further step, it is relevant to open up students......Recent political reforms in the Danish school system have lengthened the school day and integrated child and youth educators into aspects of the teaching programme as part of a common European strategy of inclusion. The school day now contains homework cafes and lessons with child and youth...

  1. Inclusive Design for Assistive Technology

    DEFF Research Database (Denmark)

    Herriott, Richard

    2014-01-01

    Objectives/focus/background: Design for Assistive Technology (AT) requires a special focus on user-requirements during product development. Inclusive Design theory and methodology thus has been relevant to AT design processes. Research in AT design has both drawn from and added to the ID knowledge...... the question of whether AT design processes fit neatly into a model created for a different context . Semi-structured interviews were carried out with eight/nine designers of assistive technology. This provided raw data concerning the processes used by the designers in a range of products including...... a wheelchair for children, a washbasin system, a rollator and breathing apparatus for patients with chronic obstructive pulmonary disorder. The interviews conducted showed that there was required a different emphasis in the means by which users´ requirements were incorporated into the design process...

  2. STUDENTS’ RESPONSES TO INCLUSIVE DESIGN

    DEFF Research Database (Denmark)

    Herriott, Richard; Geert Jensen, Birgitte

    2012-01-01

    raw data on how students broke down the design problem into a variety of tasks: problem solving, data gathering and ideation among others. The work thus provides insight into how the problem is resolved into design solutions. The results provide some quantitative insight into how time is allocated......This paper looks at how students’ design process responds to the requirements of inclusive design. The background to the students´ brief was the concept of welfare technology. People wish to retain their customary life-style even as ageing brings with it a reduction in physical capability: loss...... of muscle strength and manual dexterity or deterioration of eyesight and hearing. They wish to remain in their familiar home and to be able to equip it with such necessary enhancements that help them cope with the minimum of outside support. At the Aarhus School of Architecture, Institute of Design...

  3. Body contact and body language

    DEFF Research Database (Denmark)

    Winther, Helle

    2008-01-01

    and the boundaries between self and world. In western societies, the modern premises for contact are in some ways developing from close contact to virtual communication. With this breadth of perspective in mind, the ques­tion is whether conscious and experimental work with body contact and body language in move......­ment psychology and education provide potential for intense personal develop­ment as well as for social and cultural learning processes. This performative research project originates from the research project entitled, Movement Psy­chol­ogy: The Language of the Body and the Psy­chol­ogy of Movement based......Body contact and body language are unique and existential and, although culturally dependent and socially embodied, they are also universal communication forms. For small children all over the world, warm, close and nourishing body contact is fundamental to their embodied experi­ence of themselves...

  4. Penile epidermal inclusion cyst: a late complication of penile girth enhancement surgery.

    Science.gov (United States)

    Park, Hyun Jun; Park, Nam Cheol; Park, Sung Woo; Jern, Tae Kyung; Choi, Kyung-Un

    2008-09-01

    Epidermal inclusion cysts are benign lesions that can develop in any part of the body. However, the finding of an epidermal inclusion cyst in the penis is rare. The aim of this article was to present the management of a case of a penile epidermal inclusion cyst that occurred because of late complications of a penile girth enhancement surgery. A 52-year-old man presented with a painless, slowly growing mass in the penis, which was first noted after a penile girth enhancement surgery 20 years ago. A cystic mobile mass about 2 cm in depth was found surrounding the coronal sulcus. Excision of the mass was performed for diagnosis and treatment. There was no communication with the urethra. The pathological diagnosis was an epidermal inclusion cyst of the penis. A penile epidermal inclusion cyst in adult men is rare. It can develop after an inadequate procedure for penile girth enhancement, and should be treated by complete resection.

  5. Body punk

    DEFF Research Database (Denmark)

    Mogensen, Kevin

    BODYPUNK - A Treatise on male body builders and the meaning of the body in the shadow of an Anti Doping Campaign Based on a qualitative study, the thesis investigates the visual representation of the male bodybuilder found in the national anti doping campaign: ‗ "The hunt has begun" along...

  6. Peptidoglycan Hydrolases of Escherichia coli

    Science.gov (United States)

    van Heijenoort, Jean

    2011-01-01

    Summary: The review summarizes the abundant information on the 35 identified peptidoglycan (PG) hydrolases of Escherichia coli classified into 12 distinct families, including mainly glycosidases, peptidases, and amidases. An attempt is also made to critically assess their functions in PG maturation, turnover, elongation, septation, and recycling as well as in cell autolysis. There is at least one hydrolytic activity for each bond linking PG components, and most hydrolase genes were identified. Few hydrolases appear to be individually essential. The crystal structures and reaction mechanisms of certain hydrolases having defined functions were investigated. However, our knowledge of the biochemical properties of most hydrolases still remains fragmentary, and that of their cellular functions remains elusive. Owing to redundancy, PG hydrolases far outnumber the enzymes of PG biosynthesis. The presence of the two sets of enzymes acting on the PG bonds raises the question of their functional correlations. It is difficult to understand why E. coli keeps such a large set of PG hydrolases. The subtle differences in substrate specificities between the isoenzymes of each family certainly reflect a variety of as-yet-unidentified physiological functions. Their study will be a far more difficult challenge than that of the steps of the PG biosynthesis pathway. PMID:22126997

  7. Third International E. coli genome meeting

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1994-12-31

    Proceedings of the Third E. Coli Genome Meeting are provided. Presentations were divided into sessions entitled (1) Large Scale Sequencing, Sequence Analysis; (2) Databases; (3) Sequence Analysis; (4) Sequence Divergence in E. coli Strains; (5) Repeated Sequences and Regulatory Motifs; (6) Mutations, Rearrangements and Stress Responses; and (7) Origins of New Genes. The document provides a collection of abstracts of oral and poster presentations.

  8. Escherichia Coli Removal from Water Using Electrophotocatalytic ...

    African Journals Online (AJOL)

    Michael Horsfall

    inactivation of bacterial microorganisms in areas with low ... disinfection of water contaminated with fecal indicators such as E. coli ... media, brain heart infusion, sodium chloride, sodium hydroxide ... furnace at temperature 105 and 320°C f0r 60 min. For 2- and .... charge of E. coli logarithmic growth phase might affect the ...

  9. Antimicrobial resistance among commensal Escherichia coli from ...

    African Journals Online (AJOL)

    Commensal bacteria contribute to the distribution and persistence of antimicrobial resistance in the environment. This study monitored antimicrobial resistance in commensal Escherichia coli from the faeces of on-farm and slaughter cattle and beef. A total of 342 (89.5%) E. coli isolates were obtained from 382 samples.

  10. Characterization of Escherichia coli Phylogenetic Groups ...

    African Journals Online (AJOL)

    Background: Escherichia coli strains mainly fall into four phylogenetic groups (A, B1, B2, and D) and that virulent extra‑intestinal strains mainly belong to groups B2 and D. Aim: The aim was to determine the association between phylogenetic groups of E. coli causing extraintestinal infections (ExPEC) regarding the site of ...

  11. Biochemical and serological characterization of Escherichia coli ...

    African Journals Online (AJOL)

    This study was designed to determine the isolation rate, serotypes and biochemical profiles of E. coli from colibacillosis and dead-in-shell embryos in Zaria, Northern-Nigeria. The isolation rate of E. coli from hatcheries studied were 4.67% and 7.50% from farms of Simtu Agricultural Company and National Animal Production ...

  12. PATHOGENIC POTENTIALS OF ESCHERICHIA COLI ISOLATED ...

    African Journals Online (AJOL)

    Electrolyte and haematological parameters in rabbits infected with pathogenic isolates of Escherichia coli from rural water supplies in Rivers State, Nigeria, where monitored. Rabbits were orally infected with suspension containing 3x107 cfu /ml of Escherichia coli to induce diarrhoea, and the electrolyte (sodium, potassium ...

  13. Fosfomycin Resistance in Escherichia coli, Pennsylvania, USA.

    Science.gov (United States)

    Alrowais, Hind; McElheny, Christi L; Spychala, Caressa N; Sastry, Sangeeta; Guo, Qinglan; Butt, Adeel A; Doi, Yohei

    2015-11-01

    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.

  14. Strategies for Protein Overproduction in Escherichia coli.

    Science.gov (United States)

    Mott, John E.

    1984-01-01

    Examines heterologous expression in Escherichia coli and the role of regulatory sequences which control gene expression at transcription resulting in abundant production of messenger RNA and regulatory sequences in mRNA which promote efficient translation. Also examines the role of E. coli cells in stabilizing mRNA and protein that is…

  15. Escherichia coli survival in waters: Temperature dependence

    Science.gov (United States)

    Knowing the survival rates of water-borne Escherichia coli is important in evaluating microbial contamination and making appropriate management decisions. E. coli survival rates are dependent on temperature, a dependency that is routinely expressed using an analogue of the Q10 mo...

  16. Comparison of 61 Sequenced Escherichia coli Genomes

    DEFF Research Database (Denmark)

    Lukjancenko, Oksana; Wassenaar, T. M.; Ussery, David

    2010-01-01

    Escherichia coli is an important component of the biosphere and is an ideal model for studies of processes involved in bacterial genome evolution. Sixty-one publically available E. coli and Shigella spp. sequenced genomes are compared, using basic methods to produce phylogenetic and proteomics...

  17. Antimicrobial resistance among commensal Escherichia coli from ...

    African Journals Online (AJOL)

    user1

    2012-07-19

    Jul 19, 2012 ... Commensal bacteria contribute to the distribution and persistence of antimicrobial resistance in the environment. This study monitored antimicrobial resistance in commensal Escherichia coli from the faeces of on-farm and slaughter cattle and beef. A total of 342 (89.5%) E. coli isolates were obtained.

  18. Fimbrial adhesins from extraintestinal Escherichia coli

    DEFF Research Database (Denmark)

    Klemm, Per; Hancock, Viktoria; Schembri, Mark A.

    2010-01-01

    Extraintestinal pathogenic Escherichia coli (ExPEC) represent an important subclass of E. coli that cause a wide spectrum of diseases in human and animal hosts. Fimbriae are key virulence factors of ExPEC strains. These long surface located rod-shaped organelles mediate receptor-specific attachment...

  19. lactamase in clinical isolates of Escherichia coli

    African Journals Online (AJOL)

    Jane

    2011-08-22

    Aug 22, 2011 ... The beta lactamase enzyme producing E. coli, resistant to β-lactam antibiotics, created many problems ... Key words: Escherichia coli, β-lactamase enzymes, TEM-type extended spectrum ... difficulties in treatment using antibiotics that are currently ... and chloramphenicol (30 µg) (Mast Diagnostics Ltd., UK).

  20. Production of the {sup 14}C-labeled insecticidal protein Cry1Ab for soil metabolic studies using a recombinant Escherichia coli in small-scale batch fermentations

    Energy Technology Data Exchange (ETDEWEB)

    Valldor, Petra; Miethling-Graff, Rona; Dockhorn, Susanne; Martens, Rainer; Tebbe, Christoph C. [Federal Research Institute for Rural Areas, Forestry and Fisheries, Braunschweig (Germany). Thuenen Institute (vTI) for Biodiversity

    2012-10-15

    Insecticidal Cry proteins naturally produced by Bacillus thuringiensis are a major recombinant trait expressed by genetically modified crops. They are released into the soil during and after cropping. The objective of this study was to produce {sup 14}C-labeled Cry1Ab proteins for soil metabolic studies in scope of their environmental risk assessment. Cry1Ab was synthesized as a protoxin by Escherichia coli HB101 pMP in 200-mL liquid batch culture fermentations and purified from inclusion bodies after trypsin digestion. For cultivation, U-{sup 14}C-glycerol was the main carbon source. Inclusion bodies were smaller and Cry1Ab yield was lower when the initial amount of total organic carbon in the cultivation broth was below 6.4 mg C L{sup -1}. Concentrations of 12.6 g {sup 14}C-labeled glycerol L{sup -1} (1 % v/v) resulted in the production of 17.1 mg {sup 14}C-Cry1Ab L{sup -1} cultivation medium. {sup 14}C mass balances showed that approx. 50 % of the label was lost by respiration and 20 % remained in the growth media, while the residual activity was associated with biomass. Depending on the production batch, 0.01 to 0.05 % of the total {sup 14}C originated from Cry1Ab. In the presence of 2.04 MBq {sup 14}C-labeled carbon sources, a specific activity of up to 268 Bq mg{sup -1} {sup 14}C-Cry1Ab was obtained. A more than threefold higher specific activity was achieved with 4.63 MBq and an extended cultivation period of 144 h. This study demonstrates that {sup 14}C-labeled Cry1Ab can be obtained from batch fermentations with E. coli in the presence of a simple {sup 14}C-labeled carbon source. It also provides a general strategy to produce {sup 14}C-labeled proteins useful for soil metabolic studies. (orig.)

  1. Recovery of inclusions in 2D and 3D domains for Poisson’s equation

    International Nuclear Information System (INIS)

    Ito, Kazufumi; Liu, Ji-Chuan

    2013-01-01

    In this paper, we consider the recovery problem of inclusions in three-dimensional and three-dimensional domains for Poisson’s equation from noisy observation data. We propose effective reconstruction algorithms to recover hidden inclusions within a body when one can only make measurements of voltage and current on the external boundary. Our motivation is to detect the number, the location, the size and the shape of inclusions. This problem is nonlinear and severely ill posed, thus we should apply regularization techniques in our approaches in order to improve the corresponding approximation. We give several examples to show the viability of our proposed methods. (paper)

  2. Characterization of a Fe inclusion in beryllium-matrix using auger electron spectroscopy

    International Nuclear Information System (INIS)

    Arkusk, R.; Moreno, D.; Simca, F.; Yeheskel, O.; Utzmoni, U.

    1991-04-01

    The auger electron spectroscopy techniques was employed to investigate the nature of an inclusion that had been revealed by radiography in a beryllium body produced by the hot isostatic press technique. The investigation's are that the inclusion is composed of several different iron-beryllium intermetallic compounds (BeFe 3 , BeFe 5 , Be 7 Fe). The conclusion drawn is that iron metal impurity was imbedded in the Be powder and that interdiffusion under the process's conditions gave rise to the enlarged inclusion. (author)

  3. Indonesian teachers' epistemological beliefs and inclusive education.

    Science.gov (United States)

    Sheehy, Kieron; Budiyanto; Kaye, Helen; Rofiah, Khofidotur

    2017-01-01

    A growing number of children with intellectual disabilities attend inclusive schools in Indonesia. Previous research has suggested that teachers' type of school and experience influences their beliefs about inclusive education. This research collected questionnaire data from 267 Indonesian teachers and compared the responses from those working in inclusive, special and regular schools regarding their epistemological and pedagogical beliefs. The results showed that teachers in inclusive schools expressed stronger social constructivist beliefs than those in other schools. However, it was teachers' epistemological beliefs, rather than their type of school or experience, which were the significant predictor of their beliefs about inclusive education. The findings suggest that international epistemological research needs to have a more nuanced view of constructivist models of learning to better understand and inform how inclusive pedagogy is being enacted in different contexts.

  4. Peningkatan Performa Ayam Broiler dengan Suplementasi Daun Salam [Syzygium polyanthum (Wight Walp] Sebagai Antibakteri Escherichia coli

    Directory of Open Access Journals (Sweden)

    K.G. Wiryawan

    2007-04-01

    Full Text Available The objective of this experiment was to determine the effect of bay leaves (S. polyanthum (Wight Walp used in the diet as E. coli antibacteria in improving broiler performances. This experiment used 180 day old chicks (DOC of Cobb strain which were kept in litter system for five weeks. The experiment used completely randomized design with six treatments and three replications consisting of 10 broilers in each replication. The treatment diets were R0 = control diet, R1 = R0 infected with E. coli, R2 = R1 + 1% bay leaves, R3 = R1 + 2% bay leaves, R4 = R1 + 3% bay leaves, R5 = R1 + antibiotic. The data were analyzed with analysis of variance. The variables observed were feed consumption, body weight gain, final body weight, feed conversion ratio, mortality, and total colony of E. coli in broiler excreta. The results showed that the use of bay leaves up to 3% in the ration increased broiler performance by increasing feed consumption and body weight gain, depressing the number of E. coli in excreta, and reducing mortality compared to the other treatments, but it did not affect the feed conversion ratio.

  5. Signifying Bodies

    DEFF Research Database (Denmark)

    of biosemiosis connect signifying bodies with their natural surroundings, cultural activities and subjective experiences. Health stretches all the way from the ecosocial surroundings, through the skin and into the self-organizing processes of every living cell. Signifying Bodies lays out a new approach to health...... and health care. Eschewing all forms of dualism, the authors emphasise the interdependency of how we act, think, feel and function. They advocate a relational turn in health care, in which bodies live and learn from suffering and care. In this view, health is inseparable from both living beings...

  6. DEMONETIZATION A STEP TOWARDS MODERN FINANCIAL INCLUSION

    OpenAIRE

    Dharini Raje Sisodia; Akanksha Kapoor

    2017-01-01

    India creating a high expectation for the world to become economic leader. From quite a while Indian took Financial Inclusion as serious course of Business as a development tool and encouraging result are been observed. In order to have strong financial inclusion serious action are required. In this era of technology with largest youth population in India, this is the best time to strike the chords of modern financial inclusion. This paper will take up the issues related to demonetization and...

  7. Inclusive Fitness Maximization:An Axiomatic Approach

    OpenAIRE

    Okasha, Samir; Weymark, John; Bossert, Walter

    2014-01-01

    Kin selection theorists argue that evolution in social contexts will lead organisms to behave as if maximizing their inclusive, as opposed to personal, fitness. The inclusive fitness concept allows biologists to treat organisms as akin to rational agents seeking to maximize a utility function. Here we develop this idea and place it on a firm footing by employing a standard decision-theoretic methodology. We show how the principle of inclusive fitness maximization and a related principle of qu...

  8. Examining inclusive practices in Nicaraguan schools

    OpenAIRE

    Delkamiller, Julie; Swain, Kristine; Leader-Janssen, Elizabeth M.; Ritzman, Mitzi J.

    2013-01-01

    Inkliuzinis praktikos nagrinėjimas Nikaragvos mokyklose. The purpose of this study was to examine Nicaraguan teachers’ efficacy for inclusive practices and current teaching practices in Nicaraguan schools as the first step in developing a special education training program. Sixty-one teachers in 15 schools completed the Teacher Efficacy of Inclusive Practice (TEIP) survey to determine their confidence in inclusive practices, collaboration and dealing with disruptive behaviors. Classroom ob...

  9. Inclusive central region in perturbative Reggeon calculus

    International Nuclear Information System (INIS)

    Pajares, C.; Pascual, R.

    1976-01-01

    The single-particle inclusive cross section and the correlation function are studied in the perturbative approach to Gribov's Reggeon calculus; the leading contributions to both functions are evaluated. The large energy rise of the inclusive cross section appears as a consequence of the Pomerons having an intercept larger than 1. The same set of parameters which describes correctly the cross-section data and the triple-Regge region also describes the inclusive data in the central region

  10. The problems of Russian inclusive education

    Directory of Open Access Journals (Sweden)

    Kurmyshova O.A.

    2017-02-01

    Full Text Available the article is devoted to problems of Russian inclusive education. Based on the results of theoretical analysis the author raises some questions and their solutions may help to increase the effectiveness of the activity of inclusive educational organizations, provide harmonization of relations of inclusive education members and that will guarantee a successful integration of children with disabilities and special needs in the society.

  11. Is Urban Economic Growth Inclusive in India?

    OpenAIRE

    Tripathi, Sabyasachi

    2013-01-01

    This paper measures the overall inclusive growth of a city by considering changing trends in the key economic variables based on ‘Borda ranking’ and establishes a relationship between city economic growth and overall city inclusive growth. By using data of 52 large cities in India, this paper finds that higher urban economic growth is associated with an increase in urban inequality, a reduction in urban poverty, and a lower level of overall inclusive growth of a city.

  12. Diversity at work the practice of inclusion

    CERN Document Server

    Deane, Barbara R

    2014-01-01

    This book outlines the key issues involved in framing, designing, and implementing inclusion initiatives for organizations and groups. It offers ideas for helping individuals develop competencies for inclusion. It shows how to apply the practices of inclusion and provides a unified model by employing diverse voices to address a range of related topics in multiple contexts. It also contains examples of how diversity and inclusion has worked in a variety of settings. The book is includes information from topic experts, including internal and external change agents and academics.

  13. Inclusive education foreign experience and Russian realities

    Directory of Open Access Journals (Sweden)

    Natalya V. Borodina

    2011-04-01

    Full Text Available The article analyses social processes, effecting inclusive education development in countries with liberal welfare model and problems of integrated education development in Russia.

  14. Towards a More Inclusive Democracy

    Directory of Open Access Journals (Sweden)

    Peter Emerson

    2008-08-01

    Full Text Available For reasons both historical and psychological, many have come to believe that ‘democracy is based upon a decision taken by the majority’. This basic principle has been subject to considerable abuse, as many politicians have interpreted it to turn what should have been pluralist debates into simple dichotomies: in 1804 France, for example, any sane and sober adult could have been a candidate for the post of Emperor, but the question was only ‘Napoleon, yes or no?’. Some of the other methodologies by which “the will of the people” can be determined are regarded by many social choice scientists as being more accurate, especially those multi-option preferential procedures in which all preferences cast by all voters are taken into account. After a brief historical note, this article offers a critique of majoritarianism before outlining that which could be the three-pronged basis of a more consensual polity, namely: multi-option preference voting in decision-making; multi-candidate preference voting in elections; and, as the basic system of inclusive governance, an elected all-party coalition government.

  15. Bog bodies

    DEFF Research Database (Denmark)

    Lynnerup, Niels

    2015-01-01

    In northern Europe during the Iron Age, many corpses were deposited in bogs. The cold, wet and anaerobic environment leads in many cases to the preservation of soft tissues, so that the bodies, when found and excavated several thousand years later, are remarkably intact. Since the 19th century...... the bog bodies have been studied using medical and natural scientific methods, and recently many bog bodies have been re-examined using especially modern, medical imaging techniques. Because of the preservation of soft tissue, especially the skin, it has been possible to determine lesions and trauma....... Conversely, the preservation of bones is less good, as the mineral component has been leached out by the acidic bog. Together with water-logging of collagenous tissue, this means that if the bog body is simply left to dry out when found, as was the case pre-19th century, the bones may literally warp...

  16. BODY CONDITION

    African Journals Online (AJOL)

    Andrew Taylor

    African antelope have both advantages and disadvantages in terms of meat production when compared with domestic .... Because juveniles can be differentiated from adults using BW, age differences in body ..... Meat and carcass by-products.

  17. D10 value determination for Escherichia coli O157:H7 in different cultivations

    International Nuclear Information System (INIS)

    Oliveira, Sergio Eduardo M. de; Pires, Luis Fernando G.; Vital, Helio de C.

    2002-01-01

    Escherichia coli serum type O157:H7 is a highly pathogenic bacterium. Inside the human body, that microorganism causes a disease that leads to bloody diarrhea, stoppage of kidney functions and clots in the brain. That type of infection has been related to the consumption of different varieties of foods, mainly meat and other products of animal origin. Irradiation is an efficient method for elimination of pathogenic and spoiling microorganisms in foods. Thus, this work investigates the use of gamma irradiation for elimination of Escherichia coli O157:H7. For that purpose, inoculated samples in trypticase soy broth and saline solution 0,85% media were exposed to several gamma radiation doses. Counting the number of surviving bacteria yielded the following D 10 values for Escherichia coli O157:H7: 98±7 Gy, in trypticase soy broth and 49±4 Gy in saline solution 0,85% medium. (author)

  18. Effect of bile on growth, peritoneal absorption, and blood clearance of Escherichia coli in E coli peritonitis

    International Nuclear Information System (INIS)

    Andersson, R.; Schalen, C.; Tranberg, K.G.

    1991-01-01

    The effect of intraperitoneal bile on growth, peritoneal absorption, and clearance of Escherichia coli was determined in E coli peritonitis in the rat. In E coli peritonitis, intraperitoneal bacterial counts gradually decreased, whereas they increased (after 2 hours) with subsequent development of bacteremia in E coli plus bile peritonitis. After an intraperitoneal injection of labeled bacteria, blood radioactivity was only initially lower in E coli plus bile peritonitis compared with E coli peritonitis. Clearance from blood was lower in E coli plus bile peritonitis than in E coli peritonitis. Organ localization was similar in E coli peritonitis and E coli plus bile peritonitis with decreased splenic, increased pulmonary, and unchanged hepatic uptakes compared with controls. Impaired peritoneal absorption of bacteria, together with impaired local host defense, is likely to enhance the noxious effect of bile in E coli peritonitis

  19. Determination of neutral current couplings from neutrino-induced semi-inclusive pion and inclusive reactions

    International Nuclear Information System (INIS)

    Hung, P.Q.

    1977-01-01

    It is shown that by looking at data from neutrino-induced semi-inclusive pion and inclusive reactions on isoscalar targets along, one can determine completely the neutral current couplings. Predictions for various models are also presented. (Auth.)

  20. Guided Reading in Inclusive Middle Years Classrooms

    Science.gov (United States)

    Lyons, Wanda; Thompson, Scott Anthony

    2012-01-01

    Teachers in inclusive classrooms are challenged to provide reading instruction for students with a wide range of instructional levels. This article reports on the implementation of guided reading in four middle years inclusive classrooms, the impact on student engagement and reading progress, and teacher perspectives on the guided reading…