65 results on '"Ruff AJ"'
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2. Effects of a single large dose of vitamin A, given during the postpartum period to HIV-positive women and their infants, on child HIV infection, HIV-free survival, and mortality.
- Author
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Humphrey JH, Iliff PJ, Marinda ET, Mutasa K, Moulton LH, Chidawanyika H, Ward BJ, Nathoo KJ, Malaba LC, Zijenah LS, Zvandasara P, Ntozini R, Mzengeza F, Mahomva AI, Ruff AJ, Mbizvo MT, Zunguza CD, and ZVITAMBO Study Group
- Abstract
BACKGROUND: Low maternal serum retinol level is a risk factor for mother-to-child transmission (MTCT) of human immunodeficiency virus (HIV). Multiple-large-dose vitamin A supplementation of HIV-positive children reduces mortality. The World Health Organization recommends single-large-dose vitamin A supplementation for postpartum women in areas of prevalent vitamin A deficiency; neonatal dosing is under consideration. We investigated the effect that single-large-dose maternal/neonatal vitamin A supplementation has on MTCT, HIV-free survival, and mortality in HIV-exposed infants. METHODS: A total of 14,110 mother-infant pairs were enrolled < or =96 h after delivery, and both mother and infant, mother only, infant only, or neither received vitamin A supplementation in a randomized, placebo-controlled trial with a 2 x 2 factorial design. All but 4 mothers initiated breast-feeding. A total of 4495 infants born to HIV-positive women were included in the present analysis. RESULTS: Neither maternal nor neonatal vitamin A supplementation significantly affected postnatal MTCT or overall mortality between baseline and 24 months. However, the timing of infant HIV infection modified the effect that supplementation had on mortality. Vitamin A supplementation had no effect in infants who were polymerase chain reaction (PCR) negative for HIV at baseline. In infants who were PCR negative at baseline and PCR positive at 6 weeks, neonatal supplementation reduced mortality by 28% (P=.01), but maternal supplementation had no effect. In infants who were PCR negative at 6 weeks, all 3 vitamin A regimens were associated with 2-fold higher mortality (P< or =.05). CONCLUSIONS: Targeted vitamin A supplementation of HIV-positive children prolongs their survival. However, postpartum maternal and neonatal vitamin A supplementation may hasten progression to death in breast-fed children who are PCR negative at 6 weeks. These findings raise concern about universal maternal or neonatal vitamin A supplementation in HIV-endemic areas. Copyright © 2006 Infectious Diseases Society of America [ABSTRACT FROM AUTHOR]
- Published
- 2006
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3. Cultural feasibility studies in preparation for clinical trials to reduce maternal-infant HIV transmission in Haiti.
- Author
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Coreil J, Losikoff P, Pincu R, Mayard G, Ruff AJ, Hausler HP, Desormeau J, Davis H, Boulos R, and Halsey NA
- Abstract
A cultural feasibility study is defined as one that investigates scientific as well as ethical, behavioral, and social issues in the design of clinical trials. The value of such a broadly defined assessment is illustrated through the presentation of two case studies conducted to prepare for clinical trials to reduce maternal-infant HIV transmission on Cite Soleil, Haiti. The first study addressed issues surrounding a trial of breast-feeding and exclusive bottle-feeding among HIV seropositive mothers. The second study focused on the implementation of a double-blind trial of HIV immune globulin and standard immune globulin to be administered to infants of seropositive mothers shortly after birth. Both cases used focus group interviews with mothers and in-depth interviews with key informants to investigate AIDS-related beliefs, acceptability of trial participation, risks to subjects, and community reactions and repercussions to the trial. Findings point to the difficulties posed by attempts to conduct trial involving complex research designs in socially disadvantaged populations. Recommendations highlight the need to consider the community-wide impact of a trial, and the need to undertake extensive educational preparation of participants to ensure informed consent and adherence to protocols. [ABSTRACT FROM AUTHOR]
- Published
- 1998
4. Food industry side streams: an unexploited source for biotechnological phosphorus upcycling.
- Author
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Ruff AJ
- Subjects
- Phytic Acid chemistry, Phytic Acid metabolism, 6-Phytase metabolism, 6-Phytase chemistry, Biotechnology methods, Phosphorus chemistry, Phosphorus metabolism, Food Industry
- Abstract
The phosphorus shortage is an unavoidable challenge that requires strategies to replace phosphorus sourced from ores. Food industry by-products are an unscoped resource for sustainable phosphorus recovery. Recent advances include biotechnological phosphorus upcycling from phytate-rich plant residues to polyphosphate as a food additive. The valorization of by-products such as deoiled seeds or brans additionally provides low-phosphorus feed and thereby minimizes the environmental burden. Phytate reduction in a cereal-rich diet by adding enzyme formulation is a further strategy that limits its antinutritive effect. However, sustainable P-management depends on phytases that have been customized and enhanced for thermostability and specific activity. The circular phosphorus economy is driven by emerging value chains and maturing phosphorus recovery technologies for market entry., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2024
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5. An up-scaled biotechnological approach for phosphorus-depleted rye bran as animal feed.
- Author
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Widderich N, Stotz J, Lohkamp F, Visscher C, Schwaneberg U, Liese A, Bubenheim P, and Ruff AJ
- Abstract
Side streams from the milling industry offer excellent nutritional properties for animal feed; yet their use is constrained by the elevated phosphorus (P) content, mainly in the form of phytate. Biotechnological P recovery fosters sustainable P management, transforming these streams into P-depleted animal feed through enzymatic hydrolysis. The enzymatic P mobilization not only enables P recovery from milling by-products but also supports the valorization of these streams into P-depleted animal feeds. Our study presents the scalability and applicability of the process and characterizes the resulting P-depleted rye bran as animal feed component. Batch mode investigations were conducted to mobilize P from 100 g to 37.1 kg of rye bran using bioreactors up to 400 L. P reductions of 89% to 92% (reducing from 12.7 g
P /kg to 1.41-1.28 gP /kg) were achieved. In addition, High Performance Ion Chromatography (HPIC) analysis showed complete depletion of phytate. The successful recovery of the enzymatically mobilized P from the process wastewater by precipitation as struvite and calcium hydrogen phosphate is presented as well, achieving up to 99% removal efficiency. Our study demonstrates a versatile process that is easily adaptable, allowing for a seamless implementation on a larger scale., (© 2024. The Author(s).)- Published
- 2024
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6. A Screening Method for P450 BM3 Mutant Libraries Using Multiplexed Capillary Electrophoresis for Detection of Enzymatically Converted Compounds.
- Author
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Gärtner A, de Almeida Santos G, Ruff AJ, and Schwaneberg U
- Subjects
- Electrophoresis, Capillary methods, High-Throughput Screening Assays methods
- Abstract
Capillary electrophoresis (CE) is an analytical method in which charged species are separated by attraction or repulsion performed in submillimeter diameter capillaries or micro- and nanofluidic channels through the application of a high voltage electric field. When capillary electrophoresis is assembled in a multicapillary instrument such as 96-well format (multiplexed), it becomes a powerful high-throughput system with the ability to simultaneously screen several types of samples like genetic mutations, metabolomes, kinase inhibitors, or enzymatic activities to name a few. The usage of a 96-multiplexed capillary electrophoresis system (96-MP-CE) represents a new platform for product-specific high-throughput screening of enzyme mutant libraries from directed evolution campaigns providing a comprehensive view on enzyme activity through the detection of all products formed. We describe the application of 96-MP-CE to screen mutant libraries of P450 BM3. MP-CE was used in directed evolution campaigns toward benzo-1,4-dioxane and α-isophorone., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2022
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7. Phytase blends for enhanced phosphorous mobilization of deoiled seeds.
- Author
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Infanzón B, Herrmann KR, Hofmann I, Willbold S, Ruff AJ, and Schwaneberg U
- Subjects
- Escherichia coli, Seeds, 6-Phytase
- Abstract
Phytases are hydrolytic enzymes capable of a stepwise phosphate release from phytate which is the main phosphorous storage in seeds, cereals and legumes. Limitations such as low enzyme activity or incomplete phytate hydrolysis to inositol are a great challenge in phytase applications in food and feed. Herein we report a phytase blend of two enzymes with additive effects on phytate (InsP6) hydrolysis and its application in the enzymatic phosphorous recovery process. Blending the fast 6-phytase rPhyXT52 with the 3-phytase from Debaryomyces castellii, which is capable of fully hydrolyzing InsP6, we achieved rapid phosphate release with higher yields compared to the individual enzymes and a rapid disappearance of InsP6-3 intermediates, monitored by HPLC. NMR data suggest a nearly complete phytate hydrolysis to inositol and phosphate. The blend was applied for phosphate mobilization from phytate-rich biomass, such as deoiled seeds. For this emerging application, an up to 43% increased phosphate mobilization yield was achieved when using 1000 U of the blend per kg biomass compared to using only the E. coli phytase. Even so, the time of enzyme treatment was decreased by more than half (6 h instead of 16 h) when using 4000 U of blend, we reached a 78-90% reduction of the total phosphorous content in the explored deoiled seeds. In summary, the phytase blend of Dc phyt/rPhyXT52 was proven very efficient to obtain inositol phosphate depleted meal which has its potential application in animal feeding and is concomitant with the production of green phosphate from renewable resources., (Copyright © 2021 Elsevier Inc. All rights reserved.)
- Published
- 2022
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8. Generation of phytase chimeras with low sequence identities and improved thermal stability.
- Author
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Herrmann KR, Hofmann I, Jungherz D, Wittwer M, Infanzón B, Hamer SN, Davari MD, Ruff AJ, and Schwaneberg U
- Subjects
- Citrobacter enzymology, Enzyme Stability, Hafnia alvei enzymology, Hydrogen-Ion Concentration, Recombinant Fusion Proteins, Yersinia enzymology, 6-Phytase genetics
- Abstract
Being able to recombine more than two genes with four or more crossover points in a sequence independent manner is still a challenge in protein engineering and limits our capabilities in tailoring enzymes for industrial applications. By computational analysis employing multiple sequence alignments and homology modeling, five fragments of six phytase genes (sequence identities 31-64 %) were identified and efficiently recombined through phosphorothioate-based cloning using the PTRec method. By combinatorial recombination, functional phytase chimeras containing fragments of up to four phytases were obtained. Two variants (PTRec 74 and PTRec 77) with up to 32 % improved residual activity (90 °C, 60 min) and retained specific activities of > 1100 U/mg were identified. Both variants are composed of fragments from the phytases of Citrobacter braakii, Hafnia alvei and Yersinia mollaretii. They exhibit sequence identities of ≤ 80 % to their parental enzymes, highlighting the great potential of DNA recombination strategies to generate new enzymes with low sequences identities that offer opportunities for property right claims., (Copyright © 2021 Elsevier B.V. All rights reserved.)
- Published
- 2021
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9. Understanding substrate binding and the role of gatekeeping residues in PigC access tunnels.
- Author
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Brands S, Sikkens JG, Davari MD, Brass HUC, Klein AS, Pietruszka J, Ruff AJ, and Schwaneberg U
- Abstract
Semi-rational redesign of the substrate binding pocket and access tunnels of prodigiosin ligase PigC enhanced the catalytic efficiency in the synthesis of pyrrolic anti-cancer agents more than 45 times. A molecular understanding was gained on residues V333 and T334 relevant to substrate binding and translocation of small pyrroles through PigC access tunnels.
- Published
- 2021
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10. A Chemical Approach for Programmable Protein Outputs Based on Engineered Cell Interactions.
- Author
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Jacome DA, Northrup JD, Ruff AJ, Reilly SW, Lee IK, Blizard GS, and Sellmyer MA
- Subjects
- Animals, Cell Communication, Coculture Techniques, Dose-Response Relationship, Drug, Luciferases, Firefly chemistry, Prodrugs chemistry, Small Molecule Libraries chemistry, Trimethoprim chemistry, Cell Engineering, Proteins chemistry
- Abstract
Cell-cell interactions and communication are crucial to the proper function of complex mammalian physiology including neurocognitive and immune system functions. While many tools are available for observing and perturbing intracellular processes, relatively few exist to probe intercellular processes. Current techniques for studying interactions often rely on direct protein contact, and few can manipulate diverse, functional outputs with tunable protein expression. To address these limitations, we have developed a small-molecule approach based on a trimethoprim prodrug-enzyme pair capable of reporting the presence of two different engineered cell populations with programmable protein outputs. The approach relies on bacterial nitroreductase enzyme catalysis, which is orthogonal to normal mammalian biology, and diffusion of trimethoprim from "activator" cells to "receiver" cells. We test this strategy, which can theoretically regulate many different types of proteins, using biochemical and in vitro culture assays with optical and cytokine protein readouts. This describes the first small-molecule approach capable of detecting and controlling engineered cell-cell outputs, and we anticipate future applications that are especially relevant to the field of immuno-oncology.
- Published
- 2021
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11. Insights on intermolecular FMN-heme domain interaction and the role of linker length in cytochrome P450cin fusion proteins.
- Author
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Belsare KD, Ruff AJ, Martinez R, and Schwaneberg U
- Subjects
- Bacterial Proteins genetics, Citrobacter genetics, Cytochrome P-450 Enzyme System genetics, Eucalyptol metabolism, Flavin Mononucleotide genetics, Heme genetics, Hydroxylation, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Bacterial Proteins metabolism, Citrobacter metabolism, Cytochrome P-450 Enzyme System metabolism, Flavin Mononucleotide metabolism, Heme metabolism
- Abstract
Cytochrome P450s are an important group of enzymes catalyzing hydroxylation, and epoxidations reactions. In this work we describe the characterization of the CinA-CinC fusion enzyme system of a previously reported P450 using genetically fused heme (CinA) and FMN (CinC) enzyme domains from Citrobacter braaki. We observed that mixing individually inactivated heme (-) with FMN (-) domain in the CinA-10aa linker - CinC fusion constructs results in recovered activity and the formation of (2S)-2β-hydroxy,1,8-cineole (174 µM), a similar amount when compared to the fully functional fusion protein (176 µM). We also studied the effect of the fusion linker length in the activity complementation assay. Our results suggests an intermolecular interaction between heme and FMN parts from different CinA-CinC fusion protein similar to proposed mechanisms for P450 BM3 on the other hand, linker length plays a crucial influence on the activity of the fusion constructs. However, complementation assays show that inactive constructs with shorter linker lengths have functional subunits, and that the lack of activity might be due to incorrect interaction between fused enzymes.
- Published
- 2020
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12. Reversal of Regioselectivity in Zinc-Dependent Medium-Chain Alcohol Dehydrogenase from Rhodococcus erythropolis toward Octanone Derivatives.
- Author
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Dhoke GV, Ensari Y, Hacibaloglu DY, Gärtner A, Ruff AJ, Bocola M, and Davari MD
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- Alcohol Dehydrogenase chemistry, Biocatalysis, Coordination Complexes chemistry, Models, Molecular, Molecular Docking Simulation, Molecular Structure, Octanes chemistry, Protein Engineering, Zinc chemistry, Alcohol Dehydrogenase metabolism, Coordination Complexes metabolism, Octanes metabolism, Rhodococcus enzymology, Zinc metabolism
- Abstract
The zinc-dependent medium-chain alcohol dehydrogenase from Rhodococcus erythropolis (ReADH) is one of the most versatile biocatalysts for the stereoselective reduction of ketones to chiral alcohols. Despite its known broad substrate scope, ReADH only accepts carbonyl substrates with either a methyl or an ethyl group adjacent to the carbonyl moiety; this limits its use in the synthesis of the chiral alcohols that serve as a building blocks for pharmaceuticals. Protein engineering to expand the substrate scope of ReADH toward bulky substitutions next to carbonyl group (ethyl 2-oxo-4-phenylbutyrate) opens up new routes in the synthesis of ethyl-2-hydroxy-4-phenylbutanoate, an important intermediate for anti-hypertension drugs like enalaprilat and lisinopril. We have performed computer-aided engineering of ReADH toward ethyl 2-oxo-4-phenylbutyrate and octanone derivatives. W296, which is located in the small binding pocket of ReADH, sterically restricts the access of ethyl 2-oxo-4-phenylbutyrate, octan-3-one or octan-4-one toward the catalytic zinc ion and thereby limits ReADH activity. Computational analysis was used to identify position W296 and site-saturation mutagenesis (SSM) yielded an improved variant W296A with a 3.6-fold improved activity toward ethyl 2-oxo-4-phenylbutyrate when compared to WT ReADH (ReADH W296A: 17.10 U/mg and ReADH WT: 4.7 U/mg). In addition, the regioselectivity of ReADH W296A is shifted toward octanone substrates. ReADH W296A has a more than 16-fold increased activity toward octan-4-one (ReADH W296A: 0.97 U/mg and ReADH WT: 0.06 U/mg) and a more than 30-fold decreased activity toward octan-2-one (ReADH W296A: 0.23 U/mg and ReADH WT: 7.69 U/mg). Computational and experimental results revealed the role of position W296 in controlling the substrate scope and regiopreference of ReADH for a variety of carbonyl substrates., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)
- Published
- 2020
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13. A colourimetric high-throughput screening system for directed evolution of prodigiosin ligase PigC.
- Author
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Brands S, Brass HUC, Klein AS, Pietruszka J, Ruff AJ, and Schwaneberg U
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- Bacterial Proteins genetics, Colorimetry, Escherichia coli metabolism, Kinetics, Ligases genetics, Mutagenesis, Site-Directed, Prodigiosin metabolism, Pseudomonas putida metabolism, Substrate Specificity, Bacterial Proteins metabolism, Directed Molecular Evolution, High-Throughput Screening Assays methods, Ligases metabolism
- Abstract
A colourimetric high-throughput screening system was established for directed evolution of prodigiosin ligase PigC. The two-step system consists of a colony prescreening test and a subsequent photometric 96-well plate assay. Screening PigC epPCR libraries in Pseudomonas putida revealed a PigC variant that achieved a 2.9× increased yield of prodiginine derivatives.
- Published
- 2020
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14. Engineered P450 BM3 and cpADH5 coupled cascade reaction for β-oxo fatty acid methyl ester production in whole cells.
- Author
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Ensari Y, de Almeida Santos G, Ruff AJ, and Schwaneberg U
- Subjects
- Alcohol Dehydrogenase genetics, Bacillus megaterium enzymology, Bacillus megaterium genetics, Bacterial Outer Membrane Proteins genetics, Bacterial Outer Membrane Proteins metabolism, Biocatalysis, Candida parapsilosis enzymology, Candida parapsilosis genetics, Caproates metabolism, Cytochrome P-450 Enzyme System chemistry, Cytochrome P-450 Enzyme System genetics, Directed Molecular Evolution, Escherichia coli genetics, Escherichia coli metabolism, Escherichia coli Proteins genetics, Escherichia coli Proteins metabolism, Esters chemistry, Fatty Acids chemistry, Hydroxylation, Operon, Substrate Specificity, Alcohol Dehydrogenase metabolism, Cytochrome P-450 Enzyme System metabolism, Esters metabolism, Fatty Acids biosynthesis
- Abstract
Hydroxy- or ketone- functionalized fatty acid methyl esters (FAMEs) are important compounds for production of pharmaceuticals, vitamins, cosmetics or dietary supplements. Biocatalysis through enzymatic cascades has drawn attention to the efficient, sustainable, and greener synthetic processes. Furthermore, whole cell catalysts offer important advantages such as cofactor regeneration by cell metabolism, omission of protein purification steps and increased enzyme stability. Here, we report the first whole cell catalysis employing an engineered P450 BM3 variant and cpADH5 coupled cascade reaction for the biosynthesis of hydroxy- and keto-FAMEs. Firstly, P450 BM3 was engineered through the KnowVolution approach yielding P450 BM3 variant YE_M1_2, (R47S/Y51W/T235S/N239R/I401 M) which exhibited boosted performance toward methyl hexanoate. The initial oxidation rate of YE_M1_2 toward methyl hexanoate was determined to be 23-fold higher than the wild type enzyme and a 1.5-fold increase in methyl 3-hydroxyhexanoate production was obtained (YE_M1_2; 2.75 mM and WT; 1.8 mM). Subsequently, the whole cell catalyst for the synthesis of methyl 3-hydroxyhexanoate and methyl 3-oxohexanoate was constructed by combining the engineered P450 BM3 and cpADH5 variants in an artificial operon. A 2.06 mM total product formation was achieved by the whole cell catalyst including co-expressed channel protein, FhuA and co-solvent addition. Moreover, the generated whole cell biocatalyst also accepted methyl valerate, methyl heptanoate as well as methyl octanoate as substrates and yielded ω-1 ketones as the main product., (Copyright © 2020. Published by Elsevier Inc.)
- Published
- 2020
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15. Rapid and Robust Coating Method to Render Polydimethylsiloxane Surfaces Cell-Adhesive.
- Author
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Gehlen DB, De Lencastre Novaes LC, Long W, Ruff AJ, Jakob F, Haraszti T, Chandorkar Y, Yang L, van Rijn P, Schwaneberg U, and De Laporte L
- Subjects
- Amino Acid Sequence, Animals, Antimicrobial Cationic Peptides chemistry, Antimicrobial Cationic Peptides genetics, Antimicrobial Cationic Peptides metabolism, Bacterial Proteins chemistry, Bacterial Proteins genetics, Bacterial Proteins metabolism, Fibroblasts cytology, Fibroblasts metabolism, Fibroblasts pathology, Green Fluorescent Proteins chemistry, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Human Umbilical Vein Endothelial Cells, Humans, Hydrophobic and Hydrophilic Interactions, Mice, Microscopy, Fluorescence, Oligopeptides genetics, Oligopeptides metabolism, Recombinant Fusion Proteins biosynthesis, Recombinant Fusion Proteins isolation & purification, Recombinant Fusion Proteins pharmacology, Surface Properties, Cell Adhesion drug effects, Dimethylpolysiloxanes chemistry, Oligopeptides chemistry
- Abstract
Polydimethylsiloxane (PDMS) is a synthetic material with excellent properties for biomedical applications because of its easy fabrication method, high flexibility, permeability to oxygen, transparency, and potential to produce high-resolution structures in the case of lithography. However, PDMS needs to be modified to support homogeneous cell attachments and spreading. Even though many physical and chemical methods, like plasma treatment or extracellular matrix coatings, have been developed over the last decades to increase cell-surface interactions, these methods are still very time-consuming, often not efficient enough, complex, and can require several treatment steps. To overcome these issues, we present a novel, robust, and fast one-step PDMS coating method using engineered anchor peptides fused to the cell-adhesive peptide sequence (glycine-arginine-glycine-aspartate-serine, GRGDS). The anchor peptide attaches to the PDMS surface predominantly by hydrophobic interactions by simply dipping PDMS in a solution containing the anchor peptide, presenting the GRGDS sequence on the surface available for cell adhesion. The binding performance and kinetics of the anchor peptide to PDMS are characterized, and the coatings are optimized for efficient cell attachment of fibroblasts and endothelial cells. Additionally, the applicability is proven using PDMS-based directional nanotopographic gradients, showing a lower threshold of 5 μm wrinkles for fibroblast alignment.
- Published
- 2019
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16. A 96-multiplex capillary electrophoresis screening platform for product based evolution of P450 BM3.
- Author
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Gärtner A, Ruff AJ, and Schwaneberg U
- Abstract
The main challenge that prevents a broader application of directed enzyme evolution is the lack of high-throughput screening systems with universal product analytics. Most directed evolution campaigns employ screening systems based on colorimetric or fluorogenic surrogate substrates or universal quantification methods such as nuclear magnetic resonance spectroscopy or mass spectrometry, which have not been advanced to achieve a high-throughput. Capillary electrophoresis with a universal UV-based product detection is a promising analytical tool to quantify product formation. Usage of a multiplex system allows the simultaneous measurement with 96 capillaries. A 96-multiplexed capillary electrophoresis (MP-CE) enables a throughput that is comparable to traditional direct evolution campaigns employing 96-well microtiter plates. Here, we report for the first time the usage of a MP-CE system for directed P450 BM3 evolution towards increased product formation (oxidation of alpha-isophorone to 4-hydroxy-isophorone; highest reached total turnover number after evolution campaign: 7120 mol
4-OH molP450 -1 ). The MP-CE platform was 3.5-fold more efficient in identification of beneficial variants than the standard cofactor (NADPH) screening system.- Published
- 2019
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17. Engineered phytases for emerging biotechnological applications beyond animal feeding.
- Author
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Herrmann KR, Ruff AJ, Infanzón B, and Schwaneberg U
- Subjects
- Humans, Hydrolysis, Protein Engineering methods, 6-Phytase genetics, 6-Phytase metabolism, Biotechnology methods, Phosphates metabolism, Phytic Acid metabolism, Recombinant Proteins genetics, Recombinant Proteins metabolism
- Abstract
Phytases are important industrial enzymes able to catalyze the release of up to six phosphates from phytate in a stepwise hydrolysis reaction. Phytases are almost exclusively used as a feed supplement. However, phytases are also used in human nutrition, food processing, non-food industrial products, and emerging applications like enzymatic phosphate recovery from renewable resources. Phytate, the main phosphorus storage form in seeds, and its hydrolysis products act as a chelator and reduce protein and mineral bioavailability in intestinal absorption. Full phosphate hydrolysis from the common storage compound phytate remains a challenge. Phytate hydrolysis patterns of tailored phytases and their protein engineering campaigns are discussed. The aim of our review is to give an overview on developed and emerging application areas (animal nutrition, food processing, and environmental resource management) and thereby generate an awareness for the importance of phosphorus stewardship in a circular bioeconomy. Emphasis will be given to processes using organic-bound phosphorus and related recycling strategy of this valuable resource. In detail, the main challenge in designing phytases to completely hydrolyze phosphate from phytate to inositol and the need for engineering campaigns to broaden their industrial use are described.
- Published
- 2019
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18. Directed Evolution of P450 BM3 towards Functionalization of Aromatic O-Heterocycles.
- Author
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Santos GA, Dhoke GV, Davari MD, Ruff AJ, and Schwaneberg U
- Subjects
- Amino Acid Substitution, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Binding Sites, Biotransformation, Catalysis, Cytochrome P-450 Enzyme System metabolism, Enzyme Activation, Hydroxylation, Models, Molecular, Molecular Conformation, Molecular Structure, Mutation, Protein Binding, Protein Engineering, Structure-Activity Relationship, Bacillus megaterium enzymology, Cytochrome P-450 Enzyme System chemistry, Heterocyclic Compounds chemistry
- Abstract
The O-heterocycles, benzo-1,4-dioxane, phthalan, isochroman, 2,3-dihydrobenzofuran, benzofuran, and dibenzofuran are important building blocks with considerable medical application for the production of pharmaceuticals. Cytochrome P450 monooxygenase (P450) Bacillus megaterium 3 (BM3) wild type (WT) from Bacillus megaterium has low to no conversion of the six O-heterocycles. Screening of in-house libraries for active variants yielded P450 BM3 CM1 (R255P/P329H), which was subjected to directed evolution and site saturation mutagenesis of four positions. The latter led to the identification of position R255, which when introduced in the P450 BM3 WT, outperformed all other variants. The initial oxidation rate of nicotinamide adenine dinucleotide phosphate (NADPH) consumption increased ≈140-fold (WT: 8.3 ± 1.3 min
-1 ; R255L: 1168 ± 163 min-1 ), total turnover number (TTN) increased ≈21-fold (WT: 40 ± 3; R255L: 860 ± 15), and coupling efficiency, ≈2.9-fold (WT: 8.8 ± 0.1%; R255L: 25.7 ± 1.0%). Computational analysis showed that substitution R255L (distant from the heme-cofactor) does not have the salt bridge formed with D217 in WT, which introduces flexibility into the I-helix and leads to a heme rearrangement allowing for efficient hydroxylation.- Published
- 2019
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19. A Semi-Rationally Engineered Bacterial Pyrrolysyl-tRNA Synthetase Genetically Encodes Phenyl Azide Chemistry.
- Author
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Fladischer P, Weingartner A, Blamauer J, Darnhofer B, Birner-Gruenberger R, Kardashliev T, Ruff AJ, Schwaneberg U, and Wiltschi B
- Subjects
- Desulfitobacterium genetics, Escherichia coli genetics, Methanosarcina genetics, Substrate Specificity genetics, Amino Acids genetics, Amino Acyl-tRNA Synthetases genetics, Azides chemistry, Azides metabolism
- Abstract
The site-specific incorporation of non-canonical amino acids (ncAAs) at amber codons requires an aminoacyl-tRNA synthetase and a cognate amber suppressor tRNA (tRNA
CUA ). The archaeal tyrosyl-tRNA synthetase from Methanocaldococcus jannaschii and the pyrrolysyl-tRNA synthetase (PylRS) from Methanosarcina mazei have been extensively engineered to accept a versatile set of ncAAs. The PylRS/tRNACUA pair from the bacterium Desulfitobacterium hafniense is functional in Escherichia coli, however, variants of this PylRS have not been reported yet. In this study, the authors describe a bacterial PylRS from Desulfitobacterium hafniense, which the authors engineered for the reactive ncAA para-azido-l-phenylalanine (DhAzFRS) using a semi-rational approach. DhAzFRS preferred para-azido-l-phenylalanine to the canonical l-phenylalanine as the substrate. In addition, the authors demonstrate the functionality in E. coli of a hybrid DhAzFRS carrying the first 190 N-terminal amino acids of the Methanosarcina mazei PylRS. These results suggest that bacterial and archaeal PylRSs can be "mixed and matched" to tune their substrate specificity., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)- Published
- 2019
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20. A hydroquinone-specific screening system for directed P450 evolution.
- Author
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Weingartner AM, Sauer DF, Dhoke GV, Davari MD, Ruff AJ, and Schwaneberg U
- Subjects
- Bacillus megaterium chemistry, Bacillus megaterium genetics, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Benzene Derivatives chemistry, Benzene Derivatives metabolism, Cytochrome P-450 Enzyme System chemistry, Cytochrome P-450 Enzyme System metabolism, Directed Molecular Evolution, Hydroquinones chemistry, Hydroxylation, Molecular Docking Simulation, Oxidation-Reduction, Protein Engineering, Bacillus megaterium enzymology, Bacterial Proteins genetics, Cytochrome P-450 Enzyme System genetics, Hydroquinones metabolism
- Abstract
The direct hydroxylation of benzene to hydroquinone (HQ) under mild reaction conditions is a challenging task for chemical catalysts. Cytochrome P450 (CYP) monooxygenases are known to catalyze the oxidation of a variety of aromatic compounds with atmospheric dioxygen. Protein engineering campaigns led to the identification of novel P450 variants, which yielded improvements in respect to activity, specificity, and stability. An effective screening strategy is crucial for the identification of improved enzymes with desired characteristics in large mutant libraries. Here, we report a first screening system designed for screening of P450 variants capable to produce hydroquinones. The hydroquinone quantification assay is based on the interaction of 4-nitrophenylacetonitrile (NpCN) with hydroquinones under alkaline conditions. In the 96-well plate format, a low detection limit (5 μM) and a broad linear detection range (5 to 250 μM) were obtained. The NpCN assay can be used for the quantification of dihydroxylated aromatic compounds such as hydroquinones, catechols, and benzoquinones. We chose the hydroxylation of pseudocumene by P450 BM3 as a target reaction and screened for improved trimethylhydroquinone (TMHQ) formation. The new P450 BM3 variant AW2 (R47Q, Y51F, I401M, A330P) was identified by screening a saturation mutagenesis library of amino acid position A330 with the NpCN assay. In summary, a 70-fold improved TMHQ formation was achieved with P450 BM3 AW2 when compared to the wild type (WT) and a 1.8-fold improved TMHQ formation compared to the recently reported P450 BM3 M3 (R47S, Y51W, A330F, I401M).
- Published
- 2018
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21. A Comparative Reengineering Study of cpADH5 through Iterative and Simultaneous Multisite Saturation Mutagenesis.
- Author
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Ensari Y, Dhoke GV, Davari MD, Ruff AJ, and Schwaneberg U
- Abstract
Positions identified in directed evolution campaigns or by (semi)rational design can be recombined iteratively or simultaneously. Iterative recombination has yielded many success stories and is beneficially used if screening capabilities are limited (four iterative SSMs generate 20×4=80 different enzyme variants). Simultaneous site saturation mutagenesis offers significantly higher diversity (20
4 =160 000 variants) and enables greater improvements to be found, especially if the selected positions are in close proximity to each other (cooperative effects). Here we report a first comprehensive comparison of iterative and simultaneous saturation of four residues in Candida parapsilosis alcohol dehydrogenase 5 (cpADH5) with methyl 3-hydroxyhexanoate as substrate. Screening of 7200 clones from 33 site saturation mutagenesis libraries (exploring 17 recombination paths) yielded the cpADH5 W286A variant, with a 82-fold improved initial activity toward methyl 3-hydroxyhexanoate. Screening 3500 clones from a single OmniChange library with four positions (C57, W116, L119, and W286; 1.8 % of the generated sequence space) yielded the cpADH5 C57V/W286S variant, with a 108-fold improvement in initial activity toward methyl 3-hydroxyhexanoate. A 1.8 % coverage of the sequence space of the simultaneous multisite saturation library was, in comparison to the investigated 17 recombination paths, sufficient to identify a cpADH5 variant with improved activity., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)- Published
- 2018
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22. Improved microscale cultivation of Pichia pastoris for clonal screening.
- Author
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Eck A, Schmidt M, Hamer S, Ruff AJ, Förster J, Schwaneberg U, Blank LM, Wiechert W, and Oldiges M
- Abstract
Background: Expanding the application of technical enzymes, e.g., in industry and agriculture, commands the acceleration and cost-reduction of bioprocess development. Microplates and shake flasks are massively employed during screenings and early phases of bioprocess development, although major drawbacks such as low oxygen transfer rates are well documented. In recent years, miniaturization and parallelization of stirred and shaken bioreactor concepts have led to the development of novel microbioreactor concepts. They combine high cultivation throughput with reproducibility and scalability, and represent promising tools for bioprocess development., Results: Parallelized microplate cultivation of the eukaryotic protein production host Pichia pastoris was applied effectively to support miniaturized phenotyping of clonal libraries in batch as well as fed-batch mode. By tailoring a chemically defined growth medium, we show that growth conditions are scalable from microliter to 0.8 L lab-scale bioreactor batch cultivation with different carbon sources. Thus, the set-up allows for a rapid physiological comparison and preselection of promising clones based on online data and simple offline analytics. This is exemplified by screening a clonal library of P. pastoris constitutively expressing AppA phytase from Escherichia coli . The protocol was further modified to establish carbon-limited conditions by employing enzymatic substrate-release to achieve screening conditions relevant for later protein production processes in fed-batch mode., Conclusion: The comparison of clonal rankings under batch and fed-batch-like conditions emphasizes the necessity to perform screenings under process-relevant conditions. Increased biomass and product concentrations achieved after fed-batch microscale cultivation facilitates the selection of top producers. By reducing the demand to conduct laborious and cost-intensive lab-scale bioreactor cultivations during process development, this study will contribute to an accelerated development of protein production processes.
- Published
- 2018
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23. An Enzymatic Route to α-Tocopherol Synthons: Aromatic Hydroxylation of Pseudocumene and Mesitylene with P450 BM3.
- Author
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Dennig A, Weingartner AM, Kardashliev T, Müller CA, Tassano E, Schürmann M, Ruff AJ, and Schwaneberg U
- Subjects
- Bacterial Proteins genetics, Benzene Derivatives chemistry, Binding Sites, Biocatalysis, Catalytic Domain, Cytochrome P-450 Enzyme System genetics, Gas Chromatography-Mass Spectrometry, Hydroxylation, Magnetic Resonance Spectroscopy, Molecular Docking Simulation, Mutagenesis, Site-Directed, NADP chemistry, NADP metabolism, NADPH-Ferrihemoprotein Reductase genetics, Protein Engineering, Substrate Specificity, alpha-Tocopherol chemistry, Bacterial Proteins metabolism, Benzene Derivatives metabolism, Cytochrome P-450 Enzyme System metabolism, NADPH-Ferrihemoprotein Reductase metabolism, alpha-Tocopherol metabolism
- Abstract
Aromatic hydroxylation of pseudocumene (1 a) and mesitylene (1 b) with P450 BM3 yields key phenolic building blocks for α-tocopherol synthesis. The P450 BM3 wild-type (WT) catalyzed selective aromatic hydroxylation of 1 b (94 %), whereas 1 a was hydroxylated to a large extent on benzylic positions (46-64 %). Site-saturation mutagenesis generated a new P450 BM3 mutant, herein named "variant M3" (R47S, Y51W, A330F, I401M), with significantly increased coupling efficiency (3- to 8-fold) and activity (75- to 230-fold) for the conversion of 1 a and 1 b. Additional π-π interactions introduced by mutation A330F improved not only productivity and coupling efficiency, but also selectivity toward aromatic hydroxylation of 1 a (61 to 75 %). Under continuous nicotinamide adenine dinucleotide phosphate recycling, the novel P450 BM3 variant M3 was able to produce the key tocopherol precursor trimethylhydroquinone (3 a; 35 % selectivity; 0.18 mg mL
-1 ) directly from 1 a. In the case of 1 b, overoxidation leads to dearomatization and the formation of a valuable p-quinol synthon that can directly serve as an educt for the synthesis of 3 a. Detailed product pattern analysis, substrate docking, and mechanistic considerations support the hypothesis that 1 a binds in an inverted orientation in the active site of P450 BM3 WT, relative to P450 BM3 variant M3, to allow this change in chemoselectivity. This study provides an enzymatic route to key phenolic synthons for α-tocopherols and the first catalytic and mechanistic insights into direct aromatic hydroxylation and dearomatization of trimethylbenzenes with O2 ., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)- Published
- 2017
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24. Inversion of cpADH5 Enantiopreference and Altered Chain Length Specificity for Methyl 3-Hydroxyalkanoates.
- Author
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Ensari Y, Dhoke GV, Davari MD, Bocola M, Ruff AJ, and Schwaneberg U
- Abstract
Expanding the substrate scope of enzymes opens up new routes for synthesis of valuable chemicals. Ketone-functionalized fatty acid derivatives and corresponding chiral alcohols are valuable building blocks for the synthesis of a variety of chemicals including pharmaceuticals. The alcohol dehydrogenase from Candida parapsilosis (cpADH5) catalyzes the reversible oxidations of chiral alcohols and has a broad substrate range; a challenge for cpADH5 is to convert alcohols with small substituents (methyl or ethyl) next to the oxidized alcohol moiety. Molecular docking studies revealed that W286 is located in the small binding pocket and limits the access to substrates that contain aliphatic chains longer than ethyl substituent. In the current manuscript, we report that positions L119 and W286 are key residues to boost oxidation of medium chain methyl 3-hydroxy fatty acids; interestingly the enantiopreference toward methyl 3-hydroxybutyrate was inverted. Kinetic characterization of W286A showed a 5.5 fold increase of V
max and a 9.6 fold decrease of Km values toward methyl 3-hydroxyhexanoate (Vmax : 2.48 U mg- and Km : 4.76 mm). Simultaneous saturation at positions 119 and 286 library yielded a double mutant (L119M/W286S) with more than 30-fold improved activity toward methyl 3-hydroxyoctanoate (WT: no conversion; L119M/W286S: 30 %) and inverted enantiopreference (S-enantiomer ≥99 % activity decrease and R-enantiomer >20-fold activity improvement) toward methyl 3-hydroxybutyrate., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)- Published
- 2017
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25. Casting epPCR (cepPCR): A simple random mutagenesis method to generate high quality mutant libraries.
- Author
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Yang J, Ruff AJ, Arlt M, and Schwaneberg U
- Subjects
- Sequence Analysis, DNA methods, Bacillus subtilis genetics, Directed Molecular Evolution methods, Gene Library, Lipase genetics, Mutagenesis, Site-Directed methods, Polymerase Chain Reaction instrumentation
- Abstract
During the last decade, directed evolution has become a standard protein engineering strategy to reengineer proteins for industrial applications under high stress conditions (e.g., high temperature, extreme pH, ionic liquids, or organic solvents). The most commonly employed method for diversity generation to improve biocatalysts for these properties is random mutagenesis by error-prone polymerase chain reaction (epPCR). However, recent reports show that epPCR often fails to produce >70% of beneficial positions/amino acid exchanges which improve enzyme properties such as organic solvent or ionic liquid resistance. In this report, bsla (543 bp, small lipase gene from Bacillus subtilis) was divided into three fragments (147, 192, 204 bp). Each fragment was subjected to an epPCR with a high mutation load (22, 31, and 33 mutations per kb) in order to increase the number of identified beneficial positions while maintaining a fraction of active population which can efficiently be screened in agar plate or microtiter plate format. The use of this "casting epPCR" process termed as (cepPCR), doubles the number of identified beneficial positions (from 14% to 29%), when compared to standard epPCR for the BSLA enzyme model. A further increase to 39% of beneficial positions is obtainable through combination of cepPCR with the transversion biased sequence saturation mutagenesis (SeSaM) method. Furthermore, sequencing of up to 600 mutations per fragment provided valuable insights into the correlation of total throughput and number of identified beneficial positions as well as how an efficient balance of screening efforts to obtainable results can be achieved in directed evolution campaigns. Biotechnol. Bioeng. 2017;114: 1921-1927. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)
- Published
- 2017
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26. Amino acid substitutions in random mutagenesis libraries: lessons from analyzing 3000 mutations.
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Zhao J, Frauenkron-Machedjou VJ, Kardashliev T, Ruff AJ, Zhu L, Bocola M, and Schwaneberg U
- Subjects
- Bacillus subtilis enzymology, DNA Mutational Analysis, Directed Molecular Evolution methods, Lipase genetics, Polymerase Chain Reaction methods, Protein Engineering methods, Amino Acid Substitution, Gene Library, Mutagenesis, Mutation
- Abstract
The quality of amino acid substitution patterns in random mutagenesis libraries is decisive for the success in directed evolution campaigns. In this manuscript, we provide a detailed analysis of the amino acid substitutions by analyzing 3000 mutations of three random mutagenesis libraries (1000 mutations each; epPCR with a low-mutation and a high-mutation frequency and SeSaM-Tv P/P) employing lipase A from Bacillus subtilis (bsla). A comparison of the obtained numbers of beneficial variants in the mentioned three random mutagenesis libraries with a site saturation mutagenesis (SSM) (covering the natural diversity at each amino acid position of BSLA) concludes the diversity analysis. Seventy-six percent of the SeSaM-Tv P/P-generated substitutions yield chemically different amino acid substitutions compared to 64% (epPCR-low) and 69% (epPCR-high). Unique substitutions from one amino acid to others are termed distinct amino acid substitutions. In the SeSaM-Tv P/P library, 35% of all theoretical distinct amino acid substitutions were found in the 1000 mutation library compared to 25% (epPCR-low) and 26% (epPCR-high). Thirty-six percent of distinct amino acid substitutions found in SeSaM-Tv P/P were unobtainable by epPCR-low. Comparison with the SSM library showed that epPCR-low covers 15%, epPCR-high 18%, and SeSaM-Tv P/P 21% of obtainable beneficial amino acid positions. In essence, this study provides first insights on the quality of epPCR and SeSaM-Tv P/P libraries in terms of amino acid substitutions, their chemical differences, and the number of obtainable beneficial amino acid positions.
- Published
- 2017
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27. Novel technique for high throughput measurement of active monooxygenase concentration.
- Author
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Welters T, Horn T, Ruff AJ, Schwaneberg U, and Büchs J
- Subjects
- Carbon Monoxide analysis, Carbon Monoxide metabolism, Equipment Design, Escherichia coli enzymology, Escherichia coli metabolism, Escherichia coli Proteins analysis, Escherichia coli Proteins metabolism, Cytochrome P-450 Enzyme System analysis, Cytochrome P-450 Enzyme System metabolism, Enzyme Assays methods, High-Throughput Screening Assays methods, Spectrum Analysis methods
- Abstract
Absorbance measurements via transmitting light spectroscopy in microtiter plates are established for high throughput screening of biological systems. These measurements allow for the determination of important process parameters within a short time. However, absorbance determination via transmitted light measurements is not always feasible. As for carbon monoxide difference absorbance spectroscopy, used for concentration measurements of active P450 monooxygenases (P450s), security standards, and consistent gassing have to be addressed. In this study, a non-invasive online measuring principle for absorbance via scattered light is proposed. Based on optical fiber measurements, a decrease in scattered light signals at 450 nm wavelength of reflecting polymer particles is observed, and P450 concentrations are calculated. In this way, high throughput determination of P450 concentrations in a secure, gas-tight environment is realized. The designed method was successfully applied to concentration measurements and carbon monoxide (CO) saturation kinetics ranging from 0.3 to 5.0 μM P450 BM3 achieving a measurement accuracy of ±0.05 μM P450. Biotechnol. Bioeng. 2017;114: 929-933. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)
- Published
- 2017
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28. Directed evolution of P450cin for mediated electron transfer.
- Author
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Belsare KD, Horn T, Ruff AJ, Martinez R, Magnusson A, Holtmann D, Schrader J, and Schwaneberg U
- Subjects
- Biocatalysis, Cyclohexanols metabolism, Cytochrome P-450 Enzyme System chemistry, Electrochemistry, Electron Transport, Eucalyptol, Hydrolysis, Kinetics, Models, Molecular, Monoterpenes metabolism, Mutagenesis, Mutation, NADP metabolism, Protein Conformation, Cytochrome P-450 Enzyme System genetics, Cytochrome P-450 Enzyme System metabolism, Directed Molecular Evolution methods
- Abstract
Directed evolution is a powerful method to optimize enzyme properties for application demands. Interesting targets are P450 monooxygenases which catalyze the stereo- and regiospecific hydroxylation of chemically inert C-H bonds. Synthesis employing P450s under cell-free reaction conditions is limited by low total turnover numbers, enzyme instability, low product yields and the requirement of the expensive co-factor NADPH. Bioelectrocatalysis is an alternative to replace NADPH in cell-free P450-catalyzed reactions. However, natural enzymes are often not suitable for using non-natural electron delivery systems. Here we report the directed evolution of a previously engineered P450 CinA-10aa-CinC fusion protein (named P450cin-ADD-CinC) to use zinc/cobalt(III)sepulchrate as electron delivery system for an increased hydroxylation activity of 1,8-cineole. Two rounds of Sequence Saturation Mutagenesis (SeSaM) each followed by one round of multiple site-saturation mutagenesis of the P450 CinA-10aa-CinC fusion protein generated a variant (Gln385His, Val386Ser, Thr77Asn, Leu88Arg; named KB8) with a 3.8-fold increase in catalytic efficiency (28 µM
-1 min-1 ) compared to P450cin-ADD-CinC (7 µM-1 min-1 ). Furthermore, variant KB8 exhibited a 1.5-fold higher product formation (500 µM µM-1 P450) compared to the equimolar mixture of CinA, CinC and Fpr using NADPH as co-factor (315 µM µM-1 P450). In addition, electrochemical experiments with the electron delivery system platinum/cobalt(III)sepulchrate showed that the KB8 variant had a 4-fold higher product formation rate (0.16 nmol (nmol) P450-1 min-1 cm-2 ) than the P450cin-ADD-CinC (0.04 nmol (nmol) P450-1 min-1 cm-2 ). In summary, the current work shows prospects of using directed evolution to generate P450 enzymes suitable for use with alternative electron delivery systems., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)- Published
- 2017
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29. Tuberculosis along the continuum of HIV care in a cohort of adolescents living with HIV in Ethiopia.
- Author
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Jerene D, Abebe W, Taye K, Suarez PG, Feleke Y, Hallström I, and Ruff AJ
- Subjects
- Adolescent, Antiretroviral Therapy, Highly Active, Antitubercular Agents therapeutic use, CD4 Lymphocyte Count, Child, Ethiopia epidemiology, Female, Follow-Up Studies, HIV Infections diagnosis, HIV Infections drug therapy, Humans, Incidence, Isoniazid therapeutic use, Male, Proportional Hazards Models, Retrospective Studies, Tuberculosis diagnosis, Tuberculosis drug therapy, Young Adult, HIV Infections epidemiology, Tuberculosis epidemiology
- Abstract
Setting: Eight health facilities in Ethiopia., Objective: To determine tuberculosis (TB) incidence rates and associated factors among adolescents living with the human immunodeficiency virus (ALHIV)., Design: This was a retrospective cohort study. Adolescents enrolled in HIV care between January 2005 and 31 December 2013 constituted the study population. The main outcome variable was TB diagnosis during follow-up. Baseline World Health Organization (WHO) clinical stage, CD4 count, previous history of TB and use of isoniazid preventive therapy (IPT) were the main independent variables. We estimated TB incidence rates as incident cases per 100 person-years of observation (PYO). Cox regression analysis was used to control for confounders., Results: Of the 1221 adolescents screened, 1072 were studied; 60.1% were girls. TB incidence rate was 16.32 per 100 PYO during pre-antiretroviral therapy (pre-ART) follow-up but declined to 2.25 per 100 PYO after initiation of ART. Advanced WHO clinical stage (adjusted hazard ratio [aHR] 2.71, 95%CI 1.69-4.33) and CD4 count <350 cells/μl (aHR 2.28, 95%CI 1.10-4.81) predicted TB incidence in the pre-ART cohort. IPT use was associated with a significant reduction in TB incidence in the ART cohort, but not in the pre-ART group., Conclusion: Although TB was a significant problem in ALHIV, timely administration of ART and IPT had a significant protective effect.
- Published
- 2017
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30. A whole cell biocatalyst for double oxidation of cyclooctane.
- Author
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Müller CA, Weingartner AM, Dennig A, Ruff AJ, Gröger H, and Schwaneberg U
- Subjects
- Alcohol Dehydrogenase biosynthesis, Bacterial Proteins metabolism, Biocatalysis, Bioreactors, Directed Molecular Evolution, Escherichia coli genetics, Escherichia coli metabolism, Levilactobacillus brevis enzymology, Mixed Function Oxygenases biosynthesis, Oxidation-Reduction, Rhodococcus enzymology, Alcohol Dehydrogenase chemistry, Bacterial Proteins chemistry, Cyclooctanes chemistry, Mixed Function Oxygenases chemistry
- Abstract
A novel whole cell cascade for double oxidation of cyclooctane to cyclooctanone was developed. The one-pot oxidation cascade requires only a minimum of reaction components: resting E. coli cells in aqueous buffered medium (=catalyst), the target substrate and oxygen as environmental friendly oxidant. Conversion of cyclooctane was catalysed with high efficiency (50% yield) and excellent selectivity (>94%) to cyclooctanone. The reported oxidation cascade represents a novel whole cell system for double oxidation of non-activated alkanes including an integrated cofactor regeneration. Notably, two alcohol dehydrogenases from Lactobacillus brevis and from Rhodococcus erythropolis with opposite cofactor selectivities and one monooxygenase P450 BM3 were produced in a coexpression system in one single host. The system represents the most efficient route with a TTN of up to 24363 being a promising process in terms of sustainability as well.
- Published
- 2016
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31. Screening through the PLICable promoter toolbox enhances protein production in Escherichia coli.
- Author
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Yang J, Ruff AJ, Hamer SN, Cheng F, and Schwaneberg U
- Subjects
- 6-Phytase genetics, 6-Phytase metabolism, Bacterial Proteins genetics, Bacterial Proteins metabolism, Cellulase genetics, Cellulase metabolism, Escherichia coli metabolism, Glutamate Dehydrogenase genetics, Glutamate Dehydrogenase metabolism, Mutation, Phosphotransferases (Alcohol Group Acceptor) genetics, Phosphotransferases (Alcohol Group Acceptor) metabolism, Recombinant Proteins genetics, Reproducibility of Results, Escherichia coli genetics, Promoter Regions, Genetic, Protein Engineering methods, Recombinant Proteins metabolism
- Abstract
Escherichia coli is a common host for recombinant protein production in which production titers are highly dependent on the employed expression system. Promoters are thereby a key element to control gene expression levels. In this study, a novel PLICable promoter toolbox was developed which enables in a single cloning step and after a screening experiment to identify out of ten IPTG-inducible promoters (T7, A3, lpp, tac, pac, Sp6, lac, npr, trc and syn) the most suitable one for high level protein production. The target gene is cloned under the control of different promoters in a single and efficient cloning step using the ligase-free cloning method PLICing (phosphorothioate-based ligase-independent gene cloning). The promoter toolbox was firstly validated using three well producible proteins (a cellulase from a metagenome library, a phytase from Yersinia mollaretii and an alcohol dehydrogenase from Pseudomonas putida) and then applied to two enzymes (3D1 DNA polymerase and glutamate dehydrogenase mutant) which are poorly produced in E. coli. By applying our PLICable pET-promoter toolbox, the authors were able to increase production by two-fold for 3D1 DNA polymerase (lac promoter) and 29-fold for glutamate dehydrogenase mutant H52Y (trc promoter)., (Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2016
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32. What's My Substrate? Computational Function Assignment of Candida parapsilosis ADH5 by Genome Database Search, Virtual Screening, and QM/MM Calculations.
- Author
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Dhoke GV, Ensari Y, Davari MD, Ruff AJ, Schwaneberg U, and Bocola M
- Subjects
- Alcohol Dehydrogenase chemistry, Alcohols metabolism, Amino Acid Sequence, Drug Evaluation, Preclinical methods, Kinetics, NAD metabolism, Protein Conformation, Quantum Theory, Substrate Specificity, User-Computer Interface, Alcohol Dehydrogenase metabolism, Candida enzymology, Candida genetics, Databases, Genetic, Genomics methods
- Abstract
Zinc-dependent medium chain reductase from Candida parapsilosis can be used in the reduction of carbonyl compounds to pharmacologically important chiral secondary alcohols. To date, the nomenclature of cpADH5 is differing (CPCR2/RCR/SADH) in the literature, and its natural substrate is not known. In this study, we utilized a substrate docking based virtual screening method combined with KEGG, MetaCyc pathway, and Candida genome databases search for the discovery of natural substrates of cpADH5. The virtual screening of 7834 carbonyl compounds from the ZINC database provided 94 aldehydes or methyl/ethyl ketones as putative carbonyl substrates. Out of which, 52 carbonyl substrates of cpADH5 with catalytically active docking pose were identified by employing mechanism based substrate docking protocol. Comparison of the virtual screening results with KEGG, MetaCyc database search, and Candida genome pathway analysis suggest that cpADH5 might be involved in the Ehrlich pathway (reduction of fusel aldehydes in leucine, isoleucine, and valine degradation). Our QM/MM calculations and experimental activity measurements affirmed that butyraldehyde substrates are the potential natural substrates of cpADH5, suggesting a carbonyl reductase role for this enzyme in butyraldehyde reduction in aliphatic amino acid degradation pathways. Phylogenetic tree analysis of known ADHs from Candida albicans shows that cpADH5 is close to caADH5. We therefore propose, according to the experimental substrate identification and sequence similarity, the common name butyraldehyde dehydrogenase cpADH5 for Candida parapsilosis CPCR2/RCR/SADH.
- Published
- 2016
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33. Whole-cell double oxidation of n-heptane.
- Author
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Müller CA, Dennig A, Welters T, Winkler T, Ruff AJ, Hummel W, Gröger H, and Schwaneberg U
- Subjects
- Alcohol Dehydrogenase metabolism, Alkanes chemistry, Alkanes metabolism, Bacillus megaterium enzymology, Catalysis, Escherichia coli, Gene Expression Regulation, Enzymologic, Mixed Function Oxygenases metabolism, NADP chemistry, Oxygen chemistry, Oxygen metabolism, Rhodococcus enzymology, Water chemistry, Water metabolism, Alcohol Dehydrogenase biosynthesis, Heptanes metabolism, Mixed Function Oxygenases biosynthesis, Oxidation-Reduction
- Abstract
Biocascades allow one-pot synthesis of chemical building blocks omitting purification of reaction intermediates and expenses for downstream processing. Here we show the first whole cell double oxidation of n-heptane to produce chiral alcohols and heptanones. The concept of an artificial operon for co-expression of a monooxygenase from Bacillus megaterium (P450 BM3) and an alcohol dehydrogenase (RE-ADH) from Rhodococcus erythropolis is reported and compared to the widely used two-plasmid or Duet-vector expression systems. Both catalysts are co-expressed on a polycistronic constructs (single mRNA) that reduces recombinant DNA content and metabolic burden for the host cell, therefore increasing growth rate and expression level. Using the artificial operon system, the expression of P450 BM3 reached 81mgg(-1) cell dry weight. In addition, in situ cofactor regeneration through the P450 BM3/RE-ADH couple was enhanced by coupling to glucose oxidation by E. coli. Under optimized reaction conditions the artificial operon system displayed a product formation of 656mgL(-1) (5.7mM) of reaction products (heptanols+heptanones), which is 3-fold higher than the previously reported values for an in vitro oxidation cascade. In conjunction with the high product concentrations it was possible to obtain ee values of >99% for (S)-3-heptanol. Coexpression of a third alcohol dehydrogenase from Lactobacillus brevis (Lb-ADH) in the same host yielded complete oxidation of all heptanol isomers. Introduction of a second ADH enabled further to utilize both cofactors in the host cell (NADH and NADPH) which illustrates the simplicity and modular character of the whole cell oxidation concept employing an artificial operon system., (Copyright © 2014 Elsevier B.V. All rights reserved.)
- Published
- 2014
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34. P-LinK: A method for generating multicomponent cytochrome P450 fusions with variable linker length.
- Author
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Belsare KD, Ruff AJ, Martinez R, Shivange AV, Mundhada H, Holtmann D, Schrader J, and Schwaneberg U
- Subjects
- Amino Acid Sequence, Cytochrome P-450 Enzyme System metabolism, Escherichia coli genetics, Gene Library, Molecular Sequence Data, Polymerase Chain Reaction methods, Recombinant Fusion Proteins metabolism, Cytochrome P-450 Enzyme System genetics, Protein Engineering methods, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins genetics
- Abstract
Fusion protein construction is a widely employed biochemical technique, especially when it comes to multi-component enzymes such as cytochrome P450s. Here we describe a novel method for generating fusion proteins with variable linker lengths, protein fusion with variable linker insertion (P-LinK), which was validated by fusing P450cin monooxygenase (CinA) to the flavodoxin shuttle protein (CinC). CinC was fused to the C terminus of CinA through a series of 16 amino acid linkers of different lengths in a single experiment employing 3 PCR amplifications. Screening for 2-β-hydroxy-1,8-cineole production by CinA-CinC fusion proteins revealed that enzymatically active variants possessed linker lengths of more than 5 amino acids, reaching optimum enzyme activity at a linker length of 10 amino acids. Our P-LinK method not only minimizes experimental effort and significantly reduces time demands but also requires only a single cloning and transformation step in order to generate multiple linker variants (1 to 16 amino acids long), making the approach technically simple and robust.
- Published
- 2014
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35. A high-throughput screening method to reengineer DNA polymerases for random mutagenesis.
- Author
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Kardashliev T, Ruff AJ, Zhao J, and Schwaneberg U
- Subjects
- Genes, Archaeal genetics, Genes, Archaeal physiology, Mutagenesis, Reproducibility of Results, Sulfolobus solfataricus enzymology, DNA Primers genetics, DNA-Directed DNA Polymerase genetics, High-Throughput Screening Assays methods
- Abstract
A screening system for directed evolution of DNA polymerases employing a fluorescent Scorpion probe as a reporter has been developed. The screening system has been validated in a directed evolution experiment of a distributive polymerase from the Y-polymerase family (Dpo4 from Sulfolobus solfataricus) which was improved in elongation efficiency of consecutive mismatches. The engineering campaign yielded improved Dpo4 polymerase variants one of which was successfully benchmarked in a sequence saturation mutagenesis experiment especially with regard to the desirable consecutive transversion mutations ([2.5-fold increase in frequency relative to a reference library prepared with Dpo4 WT). The Scorpion probe screening system enables to reengineer polymerases with low processivity and fidelity, and no secondary activities (i.e. exonuclease activity or strand displacement activity) to match demands in diversity generation for directed protein evolution.
- Published
- 2014
- Full Text
- View/download PDF
36. The Sequence Saturation Mutagenesis (SeSaM) method.
- Author
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Ruff AJ, Kardashliev T, Dennig A, and Schwaneberg U
- Subjects
- Directed Molecular Evolution methods, Polymerase Chain Reaction, Mutagenesis genetics
- Abstract
Sequence Saturation Mutagenesis (SeSaM) is a random mutagenesis method developed to overcome the limitations of existing error-prone PCR (epPCR) protocols. SeSaM is advantageous with respect to (1) elimination of mutagenic "hot spots", (2) increase in frequency of subsequent nucleotide substitutions, (3) control over the mutational bias through the utilization of universal base analogs, and, consequently, (4) the prospect of generating transversion-enriched mutant libraries. These advanced features lead to chemically diverse mutant libraries on the protein level, essentially making SeSaM a complementary technology to transition biased epPCR mutagenesis methods.
- Published
- 2014
- Full Text
- View/download PDF
37. OmniChange: simultaneous site saturation of up to five codons.
- Author
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Dennig A, Marienhagen J, Ruff AJ, and Schwaneberg U
- Subjects
- Cloning, Molecular, Codon genetics, Mutagenesis, Site-Directed methods
- Abstract
Multi-site-saturation mutagenesis allows altering of "localizable" properties such as activity and selectivity and enables the discovery of cooperative amino acid substitutions which are unlikely to be discovered by saturating single codons individually or iteratively. The herein presented method "OmniChange" does not require any DNA modifying enzyme (e.g., endonucleases or ligases), and diverse mutant libraries with up to five simultaneously saturated positions are generated in a robust and technically simple manner in four steps. The key feature of the OmniChange method is a highly efficient chemical cleavage of phosphorothiolated nucleotides by ethanol-iodine to generate 12-nucleotide-long 5' overhangs in double-stranded DNA. The generated vector and inserts can be hybridized in a one-pot assembly leading to fully functional mutagenic plasmids, and the employed E. coli host can easily ligate up to 10 DNA nicks without any further enzymatic treatment. OmniChange is furthermore a reliable and general tool for multi-DNA fragment assembly which is DNA sequence independent.
- Published
- 2014
- Full Text
- View/download PDF
38. To get what we aim for--progress in diversity generation methods.
- Author
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Ruff AJ, Dennig A, and Schwaneberg U
- Subjects
- Animals, Biomedical Research trends, Biotechnology trends, Gene Library, Goals, Humans, Mutagenesis, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Recombination, Genetic, Biocatalysis, Directed Molecular Evolution trends, Protein Engineering trends
- Abstract
Protein re-engineering by directed evolution has become a standard approach for tailoring enzymes in many fields of science and industry. Advances in screening formats and screening systems are fueling progress and enabling novel directed evolution strategies, despite the fact that the quality of mutant libraries can still be improved significantly. Diversity generation strategies in directed enzyme evolution comprise three options: (a) focused mutagenesis (selected residues are randomized); (b) random mutagenesis (mutations are randomly introduced over the whole gene); and (c) gene recombination (stretches of genes are mixed to chimeras in a random or rational manner). Either format has both advantages and limitations depending on the targeted enzyme and property. The quality of diverse mutant libraries plays a key role in finding improved mutants. In this review, we summarize methodological advancements and novel concepts (since 2009) in diversity generation for all three formats. Advancements are discussed with respect to the state of the art in diversity generation and high-throughput screening capabilities, as well as robustness and simplicity in use. Furthermore, limitations and remaining challenges are emphasized 'to get what we aim for' through 'optimal diversity' generation., (© 2013 FEBS.)
- Published
- 2013
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39. PEPFAR scale-up of pediatric HIV services: innovations, achievements, and challenges.
- Author
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Abrams EJ, Simonds RJ, Modi S, Rivadeneira E, Vaz P, Kankasa C, Tindyebwa D, Phelps BR, Bowsky S, Teasdale CA, Koumans E, and Ruff AJ
- Subjects
- Adolescent, Child, Child, Preschool, Communicable Disease Control methods, Communicable Disease Control organization & administration, Communicable Disease Control trends, Drug Utilization statistics & numerical data, Female, Global Health, HIV Infections epidemiology, HIV Infections prevention & control, Humans, Incidence, Infant, Infant, Newborn, International Cooperation, Male, National Health Programs organization & administration, National Health Programs trends, Pregnancy, Public-Private Sector Partnerships organization & administration, Public-Private Sector Partnerships trends, United States, Anti-HIV Agents administration & dosage, Antiretroviral Therapy, Highly Active methods, Antiretroviral Therapy, Highly Active trends, HIV Infections diagnosis, HIV Infections drug therapy, Infectious Disease Transmission, Vertical prevention & control
- Abstract
HIV/AIDS has had a profound impact on children around the world since the start of the epidemic. There are currently 3.4 million children under the age of 15 years living with HIV globally, and more than 450,000 children currently receiving lifesaving antiretroviral treatment. This article describes efforts supported by the President's Emergency Plan for AIDS Relief (PEPFAR) to expand access to treatment for children living with HIV in high-burden countries. The article also highlights a series of case studies that illustrate the impact that the PEPFAR initiative has had on the pediatric HIV epidemic. Through its support of host governments and partner organizations, the PEPFAR initiative has expanded HIV testing and treatment for pregnant women to reduce vertical transmission of HIV, increased access to early infant diagnosis for HIV-exposed infants, improved training and resources for clinicians who provide pediatric care and antiretroviral treatment, and, through public-private partnerships with pharmaceutical manufacturers, helped increase the number of medications available for the treatment of HIV-infected children in resource-limited settings.
- Published
- 2012
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40. MuteinDB: the mutein database linking substrates, products and enzymatic reactions directly with genetic variants of enzymes.
- Author
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Braun A, Halwachs B, Geier M, Weinhandl K, Guggemos M, Marienhagen J, Ruff AJ, Schwaneberg U, Rabin V, Torres Pazmiño DE, Thallinger GG, and Glieder A
- Subjects
- Database Management Systems, Enzymes chemistry, Humans, User-Computer Interface, Databases, Protein, Enzymes genetics, Enzymes metabolism, Mutation, Pharmaceutical Preparations chemistry, Pharmaceutical Preparations metabolism
- Abstract
Mutational events as well as the selection of the optimal variant are essential steps in the evolution of living organisms. The same principle is used in laboratory to extend the natural biodiversity to obtain better catalysts for applications in biomanufacturing or for improved biopharmaceuticals. Furthermore, single mutation in genes of drug-metabolizing enzymes can also result in dramatic changes in pharmacokinetics. These changes are a major cause of patient-specific drug responses and are, therefore, the molecular basis for personalized medicine. MuteinDB systematically links laboratory-generated enzyme variants (muteins) and natural isoforms with their biochemical properties including kinetic data of catalyzed reactions. Detailed information about kinetic characteristics of muteins is available in a systematic way and searchable for known mutations and catalyzed reactions as well as their substrates and known products. MuteinDB is broadly applicable to any known protein and their variants and makes mutagenesis and biochemical data searchable and comparable in a simple and easy-to-use manner. For the import of new mutein data, a simple, standardized, spreadsheet-based data format has been defined. To demonstrate the broad applicability of the MuteinDB, first data sets have been incorporated for selected cytochrome P450 enzymes as well as for nitrilases and peroxidases. Database URL: http://www.MuteinDB.org.
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- 2012
- Full Text
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41. Predictive value of weight loss on mortality of HIV-positive mothers in a prolonged breastfeeding setting.
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Koyanagi A, Humphrey JH, Moulton LH, Ntozini R, Mutasa K, Iliff P, and Ruff AJ
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- Adult, Antiretroviral Therapy, Highly Active methods, Body Mass Index, CD4 Lymphocyte Count, Female, HIV Seropositivity drug therapy, Humans, Income, Infant, Newborn, Postpartum Period, Predictive Value of Tests, Risk Factors, Time Factors, Young Adult, Breast Feeding adverse effects, HIV Seropositivity mortality, Weight Loss
- Abstract
HIV-positive lactating women may be at high risk of weight loss due to increased caloric requirements and postpartum physiological weight loss. Ten percent weight loss is associated with a higher risk of mortality in HIV-positive patients and this alone is a criterion for highly active antiretroviral therapy (HAART) initiation where CD4 counts are not available. However, no study has investigated this association in lactating postpartum women. We investigated whether 10% weight loss predicts death in postpartum HIV-positive women. A total of 9207 HIV-negative and 4495 HIV-positive mothers were recruited at delivery. Women were weighed at 6 weeks, 3 months, and every 3 months thereafter for up to 24 months postpartum and data on mortality up to 2 years were collected. The median duration of breastfeeding was longer than 18 months. Among HIV-positive women, the independent predictors of ≥10% weight loss were CD4 cell count, body mass index, and household income. Mortality was up to 7.12 (95% CI 3.47-14.61) times higher in HIV-positive women with ≥10% weight loss than those without weight loss. Ten percent weight loss in postpartum lactating HIV-positive women was significantly predictive of death. Our findings suggest that 10% weight loss is an appropriate criterion for HAART initiation among postpartum breastfeeding women.
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- 2011
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42. Postpartum plasma CD4 change in HIV-positive women: implications for timing of HAART initiation.
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Koyanagi A, Ruff AJ, Moulton LH, Ntozini R, Mutasa K, Iliff P, and Humphrey JH
- Subjects
- Cohort Studies, Delivery, Obstetric, Drug Administration Schedule, Female, Humans, Infant, Newborn, Time Factors, Zimbabwe, Antiretroviral Therapy, Highly Active statistics & numerical data, CD4 Lymphocyte Count, HIV Seropositivity drug therapy, HIV Seropositivity immunology, Postpartum Period immunology
- Abstract
CD4 counts increase during the postpartum period and may not correctly identify HAART-eligible HIV-positive women. HAART eligibility when defined by two CD4 cutoffs (<200 and <350 cells/microl) measured at two time points (within 96 h of delivery and 6 weeks) in postpartum HIV-positive women was compared. Among HIV-positive women who had CD4 at delivery and 6 weeks (n = 423), time to Stage 3 or 4 opportunistic infection or death was compared using Cox regression between three groups of women: (1) CD4 <200 cells/microl at delivery and 6 weeks, (2) CD4 <200 cells/microl at delivery but >or=200 cells/microl at 6 weeks, and (3) CD4 >or=200 cells/microl at delivery and at 6 weeks. The analysis was repeated using the CD4 <350 cells/microl cut-off. CD4 counts increased by a median (IQR) of 70 (1-178) cells/microl between delivery and 6 weeks and decreased thereafter to approximately delivery levels at 12 months. Only 60% and 61% who had CD4 <200 cells/microl and CD4 <350 cells/microl, respectively, at delivery also had those levels at 6 weeks. Among those with CD4 <350 cells/microl at both delivery and 6 weeks, the risk of death or Stage 3 or 4 disease was 5.27 (95% CI 1.85-14.96) times higher than those with CD4 <350 at delivery but >or=350 cells/microl at 6 weeks. The use of CD4 counts immediately postpartum to define HAART eligibility may lead to substantial misclassification.
- Published
- 2010
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43. Extended-dose nevirapine to 6 weeks of age for infants to prevent HIV transmission via breastfeeding in Ethiopia, India, and Uganda: an analysis of three randomised controlled trials.
- Author
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Bedri A, Gudetta B, Isehak A, Kumbi S, Lulseged S, Mengistu Y, Bhore AV, Bhosale R, Varadhrajan V, Gupte N, Sastry J, Suryavanshi N, Tripathy S, Mmiro F, Mubiru M, Onyango C, Taylor A, Musoke P, Nakabiito C, Abashawl A, Adamu R, Antelman G, Bollinger RC, Bright P, Chaudhary MA, Coberly J, Guay L, Fowler MG, Gupta A, Hassen E, Jackson JB, Moulton LH, Nayak U, Omer SB, Propper L, Ram M, Rexroad V, Ruff AJ, Shankar A, and Zwerski S
- Subjects
- Adult, Anti-HIV Agents administration & dosage, Anti-HIV Agents adverse effects, Drug Administration Schedule, Ethiopia, Female, HIV Infections etiology, Humans, India, Infant, Infant, Newborn, Male, Multicenter Studies as Topic, Nevirapine administration & dosage, Nevirapine adverse effects, Pregnancy, Uganda, Anti-HIV Agents therapeutic use, Breast Feeding adverse effects, HIV Infections prevention & control, Nevirapine therapeutic use, Randomized Controlled Trials as Topic
- Abstract
Background: UNICEF/WHO recommends that infants born to HIV-infected mothers who do not have access to acceptable, feasible, affordable, sustainable, and safe replacement feeding should be exclusively breastfed for at least 6 months. The aim of three trials in Ethiopia, India, and Uganda was to assess whether daily nevirapine given to breastfed infants through 6 weeks of age can decrease HIV transmission via breastfeeding., Methods: HIV-infected women breastfeeding their infants were eligible for participation. Participants were randomly assigned to receive either single-dose nevirapine (nevirapine 200 mg to women in labour and nevirapine 2 mg/kg to newborns after birth) or 6 week extended-dose nevirapine (nevirapine 200 mg to women in labour and nevirapine 2 mg/kg to newborn babies after birth plus nevirapine 5 mg daily from days 8-42 for the infant). The randomisation sequences were generated by computer at a central data coordinating centre. The primary endpoint was HIV infection at 6 months of age in infants who were HIV PCR negative at birth. Analyses were by modified intention to treat, excluding infants with missing specimens and those with indeterminate or confirmed HIV infection at birth. These studies are registered with ClinicalTrials.gov, numbers NCT00074399, NCT00061321, and NCT00639938., Findings: 2024 liveborn infants randomised in the study had at least one specimen tested before 6 months of age (1047 infants in the single-dose group and 977 infants in the extended-dose group). The modified intention-to-treat population included 986 infants in the single-dose group and 901 in the extended-dose group. At 6 months, 87 children in the single-dose group and 62 in the extended-dose group were infected with HIV (relative risk 0.80, 95% CI 0.58-1.10; p=0.16). At 6 weeks of age, 54 children in the single-dose group and 25 in the extended-dose group were HIV positive (0.54, 0.34-0.85; p=0.009). 393 infants in the single-dose group and 346 in the extended-dose group experienced grade 3 or 4 serious adverse events during the study (p=0.54)., Interpretation: Although a 6-week regimen of daily nevirapine might be associated with a reduction in the risk of HIV transmission at 6 weeks of age, the lack of a significant reduction in the primary endpoint-risk of HIV transmission at 6 months-suggests that a longer course of daily infant nevirapine to prevent HIV transmission via breast milk might be more effective where access to affordable and safe replacement feeding is not yet available and where the risks of replacement feeding are high., Funding: US National Institutes of Health; US National Institute of Allergy and Infectious Diseases; Fogarty International Center.
- Published
- 2008
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44. Pseudomonas aeruginosa pre-septal cellulitis and bacteremia in a pediatric oncology patient.
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Milstone AM, Ruff AJ, Yeamans C, and Higman MA
- Subjects
- Child, Humans, Male, Bacteremia etiology, Cellulitis etiology, Leukemia, Myeloid, Acute microbiology, Orbital Diseases etiology, Pseudomonas Infections etiology
- Published
- 2005
- Full Text
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45. Horizontal transmission of recombinant vaccinia virus in strain 13 guinea pigs.
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Holt RK, Walker BK, and Ruff AJ
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- Animals, Antibodies, Viral analysis, Enzyme-Linked Immunosorbent Assay, Female, Guinea Pigs, Housing, Animal, Injections, Subcutaneous, Male, Vaccines, Synthetic administration & dosage, Vaccinia virus immunology, Disease Transmission, Infectious veterinary, Vaccinia transmission, Vaccinia virus pathogenicity
- Abstract
At our research facility, guinea pigs subcutaneously inoculated with recombinant vaccinia viruses traditionally are isolated from other research animals because of the assumption that viral shedding and transmission may occur postvaccination. However, an extensive literature search failed to reveal any information supporting this assumption. The purpose of this study was to determine whether horizontal transmission of recombinant vaccinia virus vaccines occurs postinoculation in strain 13 guinea pigs and to what degree. We scheduled 12 strain 13 guinea pigs for three subcutaneous inoculations with 10(7) PFU recombinant vaccinia virus at 3- to 4-week intervals. An additional 36 unvaccinated or naïve strain 13 guinea pigs were either cohoused with these vaccinees, housed in cages directly below the vaccinated guinea pigs, or placed in cages located across, and downwind (relative to room airflow), from vaccinees. Pre- and postvaccination serum samples were analyzed for the presence of vaccinia-specific antibodies by enzyme-linked immunosorbent assay. All 36 of the unvaccinated guinea pigs tested negative for antibodies to the vaccinia virus at all time points. The absence of virus-specific antibodies in the nonvaccinated guinea pigs, whereas vaccinated animals seroconverted, suggests that horizontal transmission of recombinant vaccinia virus does not occur between strain 13 guinea pigs housed in the same study room, regardless of cage location.
- Published
- 2002
46. HIV and infant feeding--an ongoing challenge.
- Author
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Guay LA and Ruff AJ
- Subjects
- Adult, Developing Countries, Female, Humans, Infant, Male, Risk, Breast Feeding, HIV Infections transmission, Infant Food
- Published
- 2001
- Full Text
- View/download PDF
47. Short-term effects of large-dose vitamin A supplementation on viral load and immune response in HIV-infected women.
- Author
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Humphrey JH, Quinn T, Fine D, Lederman H, Yamini-Roodsari S, Wu LS, Moeller S, and Ruff AJ
- Subjects
- Adult, Dietary Supplements, Female, HIV Infections immunology, HIV Infections virology, Humans, Middle Aged, RNA, Viral blood, T-Lymphocyte Subsets immunology, Vitamin A adverse effects, HIV Infections drug therapy, Vitamin A administration & dosage
- Abstract
Vitamin A supplementation has been suggested for treatment and prevention of HIV infection. However, some in vitro data indicate that vitamin A may activate HIV. Randomly, 40 HIV-seropositive women of reproductive age were allocated to receive a single oral dose of 9900 micromol (300,000 IU) vitamin A or placebo. Plasma HIV-1 RNA concentration, total lymphocytes, selected lymphocyte subsets and activation markers, and in vitro lymphocyte proliferation to phytohemagglutinin (PHA) and Candida were measured before dosing and at various time points over an 8-week follow-up period. No differences were found between treatment groups in the frequency of signs or symptoms of acute vitamin A toxicity, nor were differences evident in any lymphocyte subset or activation marker at any time during follow-up. Mean and median viral load concentration at each time point and change in viral load from baseline to each follow-up point did not differ between treatment groups. No difference was measured between treatment groups in the proportion of women who responded to PHA or Candida. This study provides no evidence that high dose vitamin A supplementation of HIV-infected women is associated with significant clinical or immunologic adverse effects.
- Published
- 1999
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48. Nicoll A, et al., Infant feeding policy and practice in the presence of HIV-1 infection. AIDS 1995;9:107-19.
- Author
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Ruff AJ
- Subjects
- Acquired Immunodeficiency Syndrome prevention & control, Bottle Feeding, Developed Countries, Developing Countries, Humans, Infant, Infant, Newborn, Acquired Immunodeficiency Syndrome transmission, Breast Feeding, Infant Nutritional Physiological Phenomena, Infectious Disease Transmission, Vertical, Nutrition Policy
- Published
- 1995
49. Parasitic infections of the central nervous system in children. Part III: Space-occupying lesions.
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Lowichik A and Ruff AJ
- Subjects
- Brain pathology, Brain Diseases pathology, Brain Diseases therapy, Brain Neoplasms diagnosis, Brain Neoplasms pathology, Child, Diagnosis, Differential, Humans, Parasitic Diseases pathology, Parasitic Diseases therapy, Spinal Cord pathology, Spinal Cord Diseases pathology, Spinal Cord Diseases therapy, Spinal Cord Neoplasms diagnosis, Spinal Cord Neoplasms pathology, Brain Diseases diagnosis, Parasitic Diseases diagnosis, Spinal Cord Diseases diagnosis
- Abstract
In the last part of this three-part review of parasitic infections of the central nervous system in children, we consider parasites which due to their size, distribution, or the nature of the host response, tend to cause focal lesions in the brain and spinal cord and therefore present as space-occupying lesions which occasionally mimic malignant tumors. As in Parts I and II, infections are grouped according to their predominant geographic area. Such infections include cysticercosis, one of the more common and important infections of the central nervous system.
- Published
- 1995
- Full Text
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50. Bacillus Calmette-Guérin complications in children born to HIV-1-infected women with a review of the literature.
- Author
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O'Brien KL, Ruff AJ, Louis MA, Desormeaux J, Joseph DJ, McBrien M, Coberly J, Boulos R, and Halsey NA
- Subjects
- Adult, Cohort Studies, Female, HIV Seronegativity, Humans, Infant, Infant, Newborn, Infectious Disease Transmission, Vertical, BCG Vaccine adverse effects, HIV Infections transmission, HIV Seropositivity
- Abstract
Objective: To compare the risk of complications following Bacillus Calmette-Guérin (BCG) vaccination among children by maternal and infant HIV-1 infection status as part of an investigation of an outbreak of BCG complications., Methods: A nonconcurrent cohort study of BCG complications among 125 infants born to HIV-1 seropositive and 166 infants born to HIV-1 seronegative mothers was conducted in Cité Soleil, Haiti. Infants were examined at regular intervals until 15 months of age, and complications from BCG were documented. An investigation of BCG vaccination practices was conducted., Results: Mild or moderate complications occurred among 16 of 166 (9.6%) infants born to HIV-1 seronegative mothers compared with 4 of 13 HIV-1-infected infants (30.8%, P = .04) and 10 of 75 (13.3%, P = .39) uninfected infants born to HIV-1-infected mothers. No serious complications were noted. The outbreak of complications was associated with administration of 2.0 to 2.5 times the recommended dose of BCG vaccine., Conclusions: This and five other cohort studies indicate that there may be a small increased risk of complications following BCG vaccination among HIV-1-infected children, but the reactions are usually mild and the risk does not outweigh the benefits of BCG vaccination in populations at high risk of tuberculosis during infancy and childhood.
- Published
- 1995
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