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1.  Flavonoid engineering of flax potentiate its biotechnological application 
BMC Biotechnology  2011;11:10.
Flavonoids are a group of secondary plant metabolites important for plant growth and development. They show also a protective effect against colon and breast cancer, diabetes, hypercholesterolemic atherosclerosis, lupus nephritis, and immune and inflammatory reactions. Thus, overproduction of these compounds in flax by genetic engineering method might potentiate biotechnological application of these plant products.
Flax plants of third generation overexpressing key genes of flavonoid pathway cultivated in field were used as plant material throughout this study. The biochemical properties of seed, oil and seedcake extracts and fibre from natural and transgenic flax plants were compared. The data obtained suggests that the introduced genes were stably inherited and expressed through plant generations.
Overproduction of flavonoid compounds resulted in increase of fatty acids accumulation in oil from transgenic seeds due to protection from oxidation offered during synthesis and seed maturation. The biochemical analysis of seedcake extracts from seeds of transgenic flax revealed significant increase in flavonoids (kaempferol), phenolic acids (coumaric, ferulic, synapic acids) and lignan content. Fibres, another product of flax plant showed increase in the level of catechine and acetylvanillone and decrease in phenolic acids upon flax modification.
Biochemical analysis results were confirmed using IR spectroscopy. The integral intensities of IR bands have been used for identification of the component of phenylpropanoid pathway in oil, seedcake extract and fibre from control and transgenic flax. It was shown that levels of flavonoids, phenolic acids and lignans in oil and seedcake extract was higher in transgenic flax products compared to control. An FT-IR study of fibres confirmed the biochemical data and revealed that the arrangement of the cellulose polymer in the transgenic fibres differs from the control; in particular a significant decrease in the number of hydrogen bonds was detected.
All analysed products from generated transgenic plants were enriched with antioxidant compounds derived from phenylopropanoid pathway Thus the products provide valuable source of flavonoids, phenolic acids and lignan for biomedical application. The compounds composition and quantity from transgenic plants was confirmed by IR spectroscopy. Thus the infrared spectroscopy appeared to be suitable method for characterization of flax products.
PMCID: PMC3040132  PMID: 21276227
2.  The response of diabetic foot to a new type of dressing 
FlaxAid is a newly developed type of dressing enriched in particular flavonoids through genetic engineering of flax plants that exhibit health-promoting activities due to their strong antioxidant properties. The purpose of the current study was to assess the clinical efficacy of the FlaxAid bandage therapy for a patient affected with a diabetic foot ulcer which was unresponsive to previous treatments. The patient was treated with FlaxAid bandages for 12 weeks and the size and properties of the wound were routinely observed and recorded. Due to the the clinical picture of the wound study design was adopted whereby the comparative treatment was cotton gauze wetted with isotonic salt solution.
Following therapy, the foot ulcer decreased in size, despite the decompensation of advanced diabetes. It is believed that the beneficial nature of FlaxAid is derived from its high level and broad spectrum of antioxidants.
The FlaxAid dressing provides a novel and effective method for the treatment of diabetic foot ulcers. This study presents a preliminary pilot investigation and a larger number of subjects need to be included within the study in order to draw firm clinical conclusions. Efforts to this effect are currently under way.
PMCID: PMC3557212  PMID: 23249420
Leg ulcer; Diabetes; Linen dressing; Therapy; Genetic engineering
3.  Transformation of the flax rust fungus, Melampsora lini: selection via silencing of an avirulence gene 
Rust fungi cause devastating diseases on many important food crops, with a damaging stem rust epidemic currently affecting wheat production in Africa and the Middle East. These parasitic fungi propagate exclusively on plants, precluding the use of many biotechnological tools available for other culturable fungi. In particular the lack of a stable transformation system has been an impediment to the genetic manipulation required for molecular analysis of rust pathogenicity. We have developed an Agrobacterium-mediated genetic transformation procedure for the model flax rust fungus Melampsora lini, which infects flax (Linum usitatissimum). Selection of transgenic rust lines is based on silencing of AvrL567, which encodes a rust effector protein that is recognised by the flax L6 immune receptor. The non-transgenic rust line is unable to infect flax plants expressing L6, while silenced transgenic lines are virulent on these plants, providing an effective selection system. This directly confirms that the cloned AvrL567 gene is responsible for flax rust virulence phenotypes, and demonstrates the utility of this system to probe rust gene function.
PMCID: PMC3142615  PMID: 19874543
rust; transformation; avirulence; effector; gene silencing; Agrobacterium
4.  Genome wide SNP discovery in flax through next generation sequencing of reduced representation libraries 
BMC Genomics  2012;13:684.
Flax (Linum usitatissimum L.) is a significant fibre and oilseed crop. Current flax molecular markers, including isozymes, RAPDs, AFLPs and SSRs are of limited use in the construction of high density linkage maps and for association mapping applications due to factors such as low reproducibility, intense labour requirements and/or limited numbers. We report here on the use of a reduced representation library strategy combined with next generation Illumina sequencing for rapid and large scale discovery of SNPs in eight flax genotypes. SNP discovery was performed through in silico analysis of the sequencing data against the whole genome shotgun sequence assembly of flax genotype CDC Bethune. Genotyping-by-sequencing of an F6-derived recombinant inbred line population provided validation of the SNPs.
Reduced representation libraries of eight flax genotypes were sequenced on the Illumina sequencing platform resulting in sequence coverage ranging from 4.33 to 15.64X (genome equivalents). Depending on the relatedness of the genotypes and the number and length of the reads, between 78% and 93% of the reads mapped onto the CDC Bethune whole genome shotgun sequence assembly. A total of 55,465 SNPs were discovered with the largest number of SNPs belonging to the genotypes with the highest mapping coverage percentage. Approximately 84% of the SNPs discovered were identified in a single genotype, 13% were shared between any two genotypes and the remaining 3% in three or more. Nearly a quarter of the SNPs were found in genic regions. A total of 4,706 out of 4,863 SNPs discovered in Macbeth were validated using genotyping-by-sequencing of 96 F6 individuals from a recombinant inbred line population derived from a cross between CDC Bethune and Macbeth, corresponding to a validation rate of 96.8%.
Next generation sequencing of reduced representation libraries was successfully implemented for genome-wide SNP discovery from flax. The genotyping-by-sequencing approach proved to be efficient for validation. The SNP resources generated in this work will assist in generating high density maps of flax and facilitate QTL discovery, marker-assisted selection, phylogenetic analyses, association mapping and anchoring of the whole genome shotgun sequence.
PMCID: PMC3557168  PMID: 23216845
Single nucleotide polymorphism (SNP); Genotyping-by-sequencing (GBS); Reduced representation library (RRL); Illumina; Flax; Linum usitatissimum; AGSNP
5.  Role of histamine in the aetiology of byssinosis. II. Lung histamine concentrations in guinea pigs chronically exposed to cotton and flax dusts. 
Data presented in this study support the finding that cotton and flax dusts contain agents which potentiate the formation or accumulation of histamine or both in the lungs of guinea pigs exposed to dust, and that such agents are present at much higher levels in cotton dust than in flax dust. The potentiating effect may be through the recruitment of mast cells into the lung. Both cotton and flax dusts contain methylating enzyme inhibitory agents, whereas cotton dust also contains agents that inhibit histaminase activity; flax dust contains agents potentiating histamine activity. These agents working together result in the accumulation or depletion of histamine observed in the different groups of animals exposed to either cotton or flax dust in this study.
PMCID: PMC1009285  PMID: 6722048
6.  Pollen-mediated gene flow in flax (Linum usitatissimum L.): can genetically engineered and organic flax coexist? 
Heredity  2010;106(4):557-566.
Coexistence allows growers and consumers the choice of producing or purchasing conventional or organic crops with known standards for adventitious presence of genetically engineered (GE) seed. Flax (Linum usitatissimum L.) is multipurpose oilseed crop in which product diversity and utility could be enhanced for industrial, nutraceutical and pharmaceutical markets through genetic engineering. If GE flax were released commercially, pollen-mediated gene flow will determine in part whether GE flax could coexist without compromising other markets. As a part of pre-commercialization risk assessment, we quantified pollen-mediated gene flow between two cultivars of flax. Field experiments were conducted at four locations during 2006 and 2007 in western Canada using a concentric donor (20 × 20 m) receptor (120 × 120 m) design. Gene flow was detected through the xenia effect of dominant alleles of high α-linolenic acid (ALA; 18:3cisΔ9,12,15) to the low ALA trait. Seeds were harvested from the pollen recipient plots up to a distance of 50 m in eight directions from the pollen donor. High ALA seeds were identified using a thiobarbituric acid test and served as a marker for gene flow. Binomial distribution and power analysis were used to predict the minimum number of seeds statistically required to detect the frequency of gene flow at specific α (confidence interval) and power (1−β) values. As a result of the low frequency of gene flow, approximately 4 million seeds were screened to derive accurate quantification. Frequency of gene flow was highest near the source: averaging 0.0185 at 0.1 m but declined rapidly with distance, 0.0013 and 0.00003 at 3 and 35 m, respectively. Gene flow was reduced to 50% (O50) and 90% (O90) between 0.85 to 2.64 m, and 5.68 to 17.56 m, respectively. No gene flow was detected at any site or year >35 m distance from the pollen source, suggesting that frequency of gene flow was ⩽0.00003 (P=0.95). Although it is not possible to eliminate all adventitious presence caused by pollen-mediated gene flow, through harvest blending and the use of buffer zones between GE and conventional flax fields, it could be minimized. Managing other sources of adventitious presence including seed mixing and volunteer populations may be more problematic.
PMCID: PMC3183899  PMID: 20551976
coexistence; adventitious presence; conventional; organic; pollen-mediated gene flow; regulation
7.  Perspectives on biotechnological applications of archaea 
Archaea  2002;1(2):75-86.
Many archaea colonize extreme environments. They include hyperthermophiles, sulfur-metabolizing thermophiles, extreme halophiles and methanogens. Because extremophilic microorganisms have unusual properties, they are a potentially valuable resource in the development of novel biotechnological processes. Despite extensive research, however, there are few existing industrial applications of either archaeal biomass or archaeal enzymes. This review summarizes current knowledge about the biotechnological uses of archaea and archaeal enzymes with special attention to potential applications that are the subject of current experimental evaluation. Topics covered include cultivation methods, recent achievements in genomics, which are of key importance for the development of new biotechnological tools, and the application of wild-type biomasses, engineered microorganisms, enzymes and specific metabolites in particular bioprocesses of industrial interest.
PMCID: PMC2685559  PMID: 15803645
biotechnology; extremozymes; high density cultivation; recombinant DNA technology
8.  Gene expression analysis of flax seed development 
BMC Plant Biology  2011;11:74.
Flax, Linum usitatissimum L., is an important crop whose seed oil and stem fiber have multiple industrial applications. Flax seeds are also well-known for their nutritional attributes, viz., omega-3 fatty acids in the oil and lignans and mucilage from the seed coat. In spite of the importance of this crop, there are few molecular resources that can be utilized toward improving seed traits. Here, we describe flax embryo and seed development and generation of comprehensive genomic resources for the flax seed.
We describe a large-scale generation and analysis of expressed sequences in various tissues. Collectively, the 13 libraries we have used provide a broad representation of genes active in developing embryos (globular, heart, torpedo, cotyledon and mature stages) seed coats (globular and torpedo stages) and endosperm (pooled globular to torpedo stages) and genes expressed in flowers, etiolated seedlings, leaves, and stem tissue. A total of 261,272 expressed sequence tags (EST) (GenBank accessions LIBEST_026995 to LIBEST_027011) were generated. These EST libraries included transcription factor genes that are typically expressed at low levels, indicating that the depth is adequate for in silico expression analysis. Assembly of the ESTs resulted in 30,640 unigenes and 82% of these could be identified on the basis of homology to known and hypothetical genes from other plants. When compared with fully sequenced plant genomes, the flax unigenes resembled poplar and castor bean more than grape, sorghum, rice or Arabidopsis. Nearly one-fifth of these (5,152) had no homologs in sequences reported for any organism, suggesting that this category represents genes that are likely unique to flax. Digital analyses revealed gene expression dynamics for the biosynthesis of a number of important seed constituents during seed development.
We have developed a foundational database of expressed sequences and collection of plasmid clones that comprise even low-expressed genes such as those encoding transcription factors. This has allowed us to delineate the spatio-temporal aspects of gene expression underlying the biosynthesis of a number of important seed constituents in flax. Flax belongs to a taxonomic group of diverse plants and the large sequence database will allow for evolutionary studies as well.
PMCID: PMC3107784  PMID: 21529361
9.  Physical mapping and BAC-end sequence analysis provide initial insights into the flax (Linum usitatissimum L.) genome 
BMC Genomics  2011;12:217.
Flax (Linum usitatissimum L.) is an important source of oil rich in omega-3 fatty acids, which have proven health benefits and utility as an industrial raw material. Flax seeds also contain lignans which are associated with reducing the risk of certain types of cancer. Its bast fibres have broad industrial applications. However, genomic tools needed for molecular breeding were non existent. Hence a project, Total Utilization Flax GENomics (TUFGEN) was initiated. We report here the first genome-wide physical map of flax and the generation and analysis of BAC-end sequences (BES) from 43,776 clones, providing initial insights into the genome.
The physical map consists of 416 contigs spanning ~368 Mb, assembled from 32,025 fingerprints, representing roughly 54.5% to 99.4% of the estimated haploid genome (370-675 Mb). The N50 size of the contigs was estimated to be ~1,494 kb. The longest contig was ~5,562 kb comprising 437 clones. There were 96 contigs containing more than 100 clones. Approximately 54.6 Mb representing 8-14.8% of the genome was obtained from 80,337 BES. Annotation revealed that a large part of the genome consists of ribosomal DNA (~13.8%), followed by known transposable elements at 6.1%. Furthermore, ~7.4% of sequence was identified to harbour novel repeat elements. Homology searches against flax-ESTs and NCBI-ESTs suggested that ~5.6% of the transcriptome is unique to flax. A total of 4064 putative genomic SSRs were identified and are being developed as novel markers for their use in molecular breeding.
The first genome-wide physical map of flax constructed with BAC clones provides a framework for accessing target loci with economic importance for marker development and positional cloning. Analysis of the BES has provided insights into the uniqueness of the flax genome. Compared to other plant genomes, the proportion of rDNA was found to be very high whereas the proportion of known transposable elements was low. The SSRs identified from BES will be valuable in saturating existing linkage maps and for anchoring physical and genetic maps. The physical map and paired-end reads from BAC clones will also serve as scaffolds to build and validate the whole genome shotgun assembly.
PMCID: PMC3113786  PMID: 21554714
10.  Manipulating cinnamyl alcohol dehydrogenase (CAD) expression in flax affects fibre composition and properties 
BMC Plant Biology  2014;14:50.
In recent decades cultivation of flax and its application have dramatically decreased. One of the reasons for this is unpredictable quality and properties of flax fibre, because they depend on environmental factors, retting duration and growing conditions. These factors have contribution to the fibre composition, which consists of cellulose, hemicelluloses, lignin and pectin. By far, it is largely established that in flax, lignin reduces an accessibility of enzymes either to pectin, hemicelluloses or cellulose (during retting or in biofuel synthesis and paper production).
Therefore, in this study we evaluated composition and properties of flax fibre from plants with silenced CAD (cinnamyl alcohol dehydrogenase) gene, which is key in the lignin biosynthesis. There is evidence that CAD is a useful tool to improve lignin digestibility and/or to lower the lignin levels in plants.
Two studied lines responded differentially to the introduced modification due to the efficiency of the CAD silencing. Phylogenetic analysis revealed that flax CAD belongs to the “bona-fide” CAD family. CAD down-regulation had an effect in the reduced lignin amount in the flax fibre cell wall and as FT-IR results suggests, disturbed lignin composition and structure. Moreover introduced modification activated a compensatory mechanism which was manifested in the accumulation of cellulose and/or pectin. These changes had putative correlation with observed improved fiber’s tensile strength. Moreover, CAD down-regulation did not disturb at all or has only slight effect on flax plants’ development in vivo, however, the resistance against flax major pathogen Fusarium oxysporum decreased slightly. The modification positively affected fibre possessing; it resulted in more uniform retting.
The major finding of our paper is that the modification targeted directly to block lignin synthesis caused not only reduced lignin level in fibre, but also affected amount and organization of cellulose and pectin. However, to conclude that all observed changes are trustworthy and correlated exclusively to CAD repression, further analysis of the modified plants genome is necessary. Secondly, this is one of the first studies on the crop from the low-lignin plants from the field trail which demonstrates that such plants could be successfully cultivated in a field.
PMCID: PMC3945063  PMID: 24552628
Cinnamyl alcohol dehydrogenase (CAD); Lignin; Cell wall; Flax fibre; Linum usitatissimum; L
11.  Establishment of cell suspension culture in Marchantia linearis Lehm & Lindenb. for the optimum production of flavonoids 
3 Biotech  2013;4(1):49-56.
Bryophytes are the second largest group in the plant kingdom, but studies conducted to better understand their chemical composition are limited and scattered. Axenically grown bryophytes expressed potential in biotechnological processes. The present study was designed to investigate the in vitro cell growth, culture parameters and their effect on flavonoid synthesis. Chlorophyll-containing callus cells of Marchantia linearis Lehm & Lindenb. is able to grow under low light in the presence of organic carbon source and retain the ability to produce flavonoids. Highest flavonoid production was achieved using 2,4-dichlorophenoxyacetic acid as growth hormone. Inoculum size, light intensity, organic carbon source and cations are the culture parameters affecting flavonoid productivity. Maximum flavonoid productivity is observed under low light intensity, with a photon flux density ca. 20 μmol/m2/s. Optimal inoculum size and glucose concentration for flavonoid production are 10–14 and 2–3 %, respectively. Cations like ferrous trigger flavonoid synthesis by increasing its intracellular concentrations. Flavonoid production in the cell culture is shown to be significantly growth related. Osmotic stress is ineffective in triggering flavonoid synthesis. Methyl jasmonate and 2-(2-fluoro-6-nitrobenzylsulfanyl) pyridine-4-carbothioamide elicitors showed positive effect on intracellular flavonoid content in cultured cells. Using the standard plot of quercetin (y = 0.0148x, R2 = 0.975), the flavonoid contents of in vitro samples were found ranging from 4.0 to 17.7 mg quercetin equivalent/g tissue. Flavonoids are fractionated by HPLC-PAD revealed the presence of quercetin (182.5 μg/g), luteolin (464.5 μg/g) and apigenin (297.5 μg/g). Further studies are warranted to analyze the therapeutic potentiality of the flavonoids in the liverwort.
PMCID: PMC3909577
Cell suspension; Culture parameters; Flavonoids; Marchantia linearis
12.  Characteristics of Lignin from Flax Shives as Affected by Extraction Conditions 
Lignin, a polyphenolic molecule, is a major constituent of flax shives. This polyphenolic molecular structure renders lignin a potential source of a variety of commercially viable products such as fine chemicals. This work compares the performance of different lignin isolation methods. Lignin from flax shive was isolated using both conventional alkaline extraction method and a novel experimental pressurized low polarity water (PLPW) extraction process. The lignin yields and chemical composition of the lignin fractions were determined. The conventional alkali treatment with 1.25 M NaOH, heated at 80 °C for 5 h, extracted 92 g lignin per kg flax shives, while lignin yields from the PLPW extracts ranged from 27 to 241 g lignin per kg flax shives. The purity and monomeric composition of the lignins obtained from the different extraction conditions was assessed via UV spectroscopy and alkaline nitrobenzene oxidation. Lignin obtained from conventional alkali treatment with 1.25 M NaOH, heated at 80 °C for 5 h was of low purity and exhibited the lowest yields of nitrobenzene oxidation products. With respect to alkali assisted PLPW extractions, temperature created an opposing effect on lignin yield and nitrobenzene oxidation products. More lignin was extracted as temperature increased, yet the yield of nitrobenzene oxidation products decreased. The low yield of nitrobenzene oxidation products may be attributed to either the formation of condensed structures or the selective dissolution of condensed structures of lignin during the pressurized alkaline high temperature treatment. Analytical pyrolysis, using pyroprobe GC-MS, was used to investigate the molecular composition of the lignin samples. The total yield of pyrolysis lignin products was 13.3, 64.7, and 30.5% for the 1.25 M NaOH extracted lignin, alkaline assisted PLPW extracted lignin, and the unprocessed flax shives, respectively. Key lignin derived compounds such as guaiacol, 4-vinyl guaiacol, 4-methyl guaiacol, syringol, eugenol, isoeugenol, catechol, homocatechol, and vanillin were detected in all of the samples.
PMCID: PMC2996774  PMID: 21152318
flax shives; biomass; lignin; extraction; pressurized low polarity water; PLPW; subcritical water; nitrobenzene oxidation; UV spectroscopy; Pyrolysis-GC-MS
13.  Diffusion of synthetic biology: a challenge to biosafety 
Systems and Synthetic Biology  2008;2(1-2):1-6.
One of the main aims of synthetic biology is to make biology easier to engineer. Major efforts in synthetic biology are made to develop a toolbox to design biological systems without having to go through a massive research and technology process. With this “de-skilling” agenda, synthetic biology might finally unleash the full potential of biotechnology and spark a wave of innovation, as more and more people have the necessary skills to engineer biology. But this ultimate domestication of biology could easily lead to unprecedented safety challenges that need to be addressed: more and more people outside the traditional biotechnology community will create self-replicating machines (life) for civil and defence applications, “biohackers” will engineer new life forms at their kitchen table; and illicit substances will be produced synthetically and much cheaper. Such a scenario is a messy and dangerous one, and we need to think about appropriate safety standards now.
PMCID: PMC2671588  PMID: 19003431
Biosafety; Synthetic biology; Biohackery; Safety standards
14.  Biosynthesis of isoprenoids, polyunsaturated fatty acids and flavonoids in Saccharomyces cerevisiae 
Industrial biotechnology employs the controlled use of microorganisms for the production of synthetic chemicals or simple biomass that can further be used in a diverse array of applications that span the pharmaceutical, chemical and nutraceutical industries. Recent advances in metagenomics and in the incorporation of entire biosynthetic pathways into Saccharomyces cerevisiae have greatly expanded both the fitness and the repertoire of biochemicals that can be synthesized from this popular microorganism. Further, the availability of the S. cerevisiae entire genome sequence allows the application of systems biology approaches for improving its enormous biosynthetic potential. In this review, we will describe some of the efforts on using S. cerevisiae as a cell factory for the biosynthesis of high-value natural products that belong to the families of isoprenoids, flavonoids and long chain polyunsaturated fatty acids. As natural products are increasingly becoming the center of attention of the pharmaceutical and nutraceutical industries, the use of S. cerevisiae for their production is only expected to expand in the future, further allowing the biosynthesis of novel molecular structures with unique properties.
PMCID: PMC1533850  PMID: 16719921
15.  HLA antigen frequencies in flax byssinosis patients. 
Not all workers exposed to flax dust contract byssinosis. It is not known what determines susceptibility or insusceptibility. This study is an attempt to establish whether the incidence of histocompatibility antigens is involved in susceptibility to the disease. Forty patients suffering from flax byssinosis were tissue-typed for HLA-A and -B antigens. HLA-B27 was significantly more common in the patients (22.5%) than in the controls (5.5%); P = 0.029 after correction for the number of antigens compared. HLA-A11 was present in twelve patients (30%) compared with 14% in the controls; after correction for the number of comparisons, this is not a statistically significant increase. Because HLA-B27, though significantly more common in flax byssinosis, is not necessary for its occurrence (77.5% of our patients did not have it), it is possible that the increase in the frequency of HLA-B27 is attributable to an association with other genes, perhaps those regulating the immune response or coding for antigens at other HLA loci.
PMCID: PMC1008527  PMID: 465375
16.  Dust Diseases in Dundee Textile Workers 
A survey of respiratory symptoms and function was carried out in Dundee among 123 men and women in the jute industry and 242 in the flax industry. The selection of workers was biassed in favour of those working in the dustier departments as judged by eye and those in the older age groups. A group of 72 men in a heavy engineering firm were also selected as controls for comparison.
Chronic bronchitis, as defined by Ogilvie and Newell (1957) was recorded in 27% of those interviewed, whose average age was 49. Byssinosis of various grades was recorded in 30% of all working in flax; of these 35 had cough alone worse on Monday, 34 had other respiratory symptoms worse on Monday and in only four persons did the exacerbation persist longer in the week.
In spite of the occurrence of byssinosis, chronic bronchitis was no more prevalent among flax workers than among the others, the average ventilatory function was no worse, and radiographs of the chest revealed no differences. The characteristic fall in expiratory flow rate during the course of exposure to flax dust on Mondays is similar to that found in cotton workers, and is absent in jute workers and in flax workers not admitting to symptoms of byssinosis.
PMCID: PMC1038081  PMID: 13765497
17.  Pharmacological prevention of acute ventilatory capacity reduction in flax dust exposure 
Valić, F., and Žuškin, E. (1973).British Journal of Industrial Medicine,30, 381-384. Pharmacological prevention of acute ventilatory capacity reduction in flax dust exposure. The protective effect of the preshift application of a bronchodilator (orciprenaline), an antihistamine drug (diadril), and ascorbic acid on flax-induced acute bronchoconstriction was studied in 13 byssinotic and 7 non-byssinotic female workers exposed to airborne fibres of biologically retted flax. Orciprenaline was applied by inhalation, while diadril and ascorbic acid were given orally. All the three drugs exerted a significant preventive effect, diminishing the acute fall of ventilatory capacity during the shift. The fall in forced expiratory volume (FEV1·0) was reduced by 50% and the fall in maximal flow rate at 50% vital capacity (V̇max 50% VC) by over 65%.
PMCID: PMC1069479  PMID: 4148157
18.  Pictures of Synthetic Biology 
Systems and Synthetic Biology  2009;3(1-4):27-35.
This article is concerned with the representation of Synthetic Biology in the media and by biotechnology experts. An analysis was made of German-language media articles published between 2004 and 2008, and interviews with biotechnology-experts at the Synthetic Biology conference SB 3.0 in Zurich 2007. The results have been reflected in terms of the definition of Synthetic Biology, applications of Synthetic Biology and the perspectives of opportunities and risks. In the media, Synthetic Biology is represented as a new scientific field of biology with an engineering-like thinking, while the scientists interviewed mostly define Synthetic Biology as contrary to nature and the natural system. Media articles present Synthetic Biology broadly with positive potential and inform the publics less about the potential risks than about the benefits of Synthetic Biology. In contrast, the experts interviewed reflect more on the risks than the opportunities of Synthetic Biology. Both used metaphors to describe Synthetic Biology and its aspects.
PMCID: PMC2759430  PMID: 19816797
Synthetic Biology; Representation; Media; Experts
19.  Pictures of Synthetic Biology 
Systems and Synthetic Biology  2009;3(1-4):27-35.
This article is concerned with the representation of Synthetic Biology in the media and by biotechnology experts. An analysis was made of German-language media articles published between 2004 and 2008, and interviews with biotechnology-experts at the Synthetic Biology conference SB 3.0 in Zurich 2007. The results have been reflected in terms of the definition of Synthetic Biology, applications of Synthetic Biology and the perspectives of opportunities and risks. In the media, Synthetic Biology is represented as a new scientific field of biology with an engineering-like thinking, while the scientists interviewed mostly define Synthetic Biology as contrary to nature and the natural system. Media articles present Synthetic Biology broadly with positive potential and inform the publics less about the potential risks than about the benefits of Synthetic Biology. In contrast, the experts interviewed reflect more on the risks than the opportunities of Synthetic Biology. Both used metaphors to describe Synthetic Biology and its aspects.
PMCID: PMC2759430  PMID: 19816797
Synthetic Biology; Representation; Media; Experts
20.  Flavonoids: biosynthesis, biological functions, and biotechnological applications 
Flavonoids are widely distributed secondary metabolites with different metabolic functions in plants. The elucidation of the biosynthetic pathways, as well as their regulation by MYB, basic helix-loop-helix (bHLH), and WD40-type transcription factors, has allowed metabolic engineering of plants through the manipulation of the different final products with valuable applications. The present review describes the regulation of flavonoid biosynthesis, as well as the biological functions of flavonoids in plants, such as in defense against UV-B radiation and pathogen infection, nodulation, and pollen fertility. In addition, we discuss different strategies and achievements through the genetic engineering of flavonoid biosynthesis with implication in the industry and the combinatorial biosynthesis in microorganisms by the reconstruction of the pathway to obtain high amounts of specific compounds.
PMCID: PMC3460232  PMID: 23060891
flavonoid; transcription factors; genetic engineering; defense; combinatorial biosynthesis
21.  Applications of cell sorting in biotechnology 
Due to its unique capability to analyze a large number of single cells for several parameters simultaneously, flow cytometry has changed our understanding of the behavior of cells in culture and of the population dynamics even of clonal populations. The potential of this method for biotechnological research, which is based on populations of living cells, was soon appreciated. Sorting applications, however, are still less frequent than one would expect with regard to their potential. This review highlights important contributions where flow cytometric cell sorting was used for physiological research, protein engineering, cell engineering, specifically emphasizing selection of overproducing cell lines. Finally conclusions are drawn concerning the impact of cell sorting on inverse metabolic engineering and systems biology.
PMCID: PMC1435767  PMID: 16551353
22.  Engineering prokaryotic gene circuits 
Fems Microbiology Reviews  2008;33(1):27-37.
Engineering of synthetic gene circuits is a rapidly growing discipline, currently dominated by prokaryotic transcription networks, which can be easily rearranged or rewired to give different output behaviours. In this review, we examine both a rational and a combinatorial design of such networks and discuss progress on using in vitro evolution techniques to obtain functional systems. Moving beyond pure transcription networks, more and more networks are being implemented at the level of RNA, taking advantage of mechanisms of translational control and aptamer–small molecule complex formation. Unlike gene expression systems, metabolic components are generally not as interconnectable in any combination, and so engineering of metabolic circuits is a particularly challenging field. Nonetheless, metabolic engineering has immense potential to provide useful biosynthesis tools for biotechnology applications. Finally, although prokaryotes are mostly studied as single cell systems, cell–cell communication networks are now being developed that result in spatial pattern formation in multicellular prokaryote colonies. This represents a crossover with multicellular organisms, showing that prokaryotic systems have the potential to tackle questions traditionally associated with developmental biology. Overall, the current advances in synthetic gene synthesis, ultra-high-throughput DNA sequencing and computation are synergizing to drive synthetic gene network design at an unprecedented pace.
PMCID: PMC2704926  PMID: 19016883
synthetic biology; gene network; engineering bacteria
23.  Rational design and application of responsive α-helical peptide hydrogels 
Nature materials  2009;8(7):596-600.
Biocompatible hydrogels have a wide variety of potential applications in biotechnology and medicine, such as the controlled delivery and release of cells, cosmetics and drugs; and as supports for cell growth and tissue engineering1. Rational peptide design and engineering are emerging as promising new routes to such functional biomaterials2-4. Here we present the first examples of rationally designed and fully characterized self-assembling hydrogels based on standard linear peptides with purely α-helical structures, which we call hydrogelating self-assembling fibres (hSAFs). These form spanning networks of α-helical fibrils that interact to give self-supporting physical hydrogels of >99% water content. The peptide sequences can be engineered to alter the underlying mechanism of gelation and, consequently, the hydrogel properties. Interestingly, for example, those with hydrogen-bonded networks melt upon heating, whereas those formed via hydrophobic interactions strengthen when warmed. The hSAFs are dual-peptide systems that only gel on mixing, which gives tight control over assembly5. These properties raise possibilities for using the hSAFs as substrates in cell culture. We have tested this in comparison with the widely used Matrigel substrate, and demonstrate that, like Matrigel, hSAFs support both growth and differentiation of rat adrenal pheochromocytoma cells for sustained periods in culture.
PMCID: PMC2869032  PMID: 19543314
24.  Computational approaches for rational design of proteins with novel functionalities 
Proteins are the most multifaceted macromolecules in living systems and have various important functions, including structural, catalytic, sensory, and regulatory functions. Rational design of enzymes is a great challenge to our understanding of protein structure and physical chemistry and has numerous potential applications. Protein design algorithms have been applied to design or engineer proteins that fold, fold faster, catalyze, catalyze faster, signal, and adopt preferred conformational states. The field of de novo protein design, although only a few decades old, is beginning to produce exciting results. Developments in this field are already having a significant impact on biotechnology and chemical biology. The application of powerful computational methods for functional protein designing has recently succeeded at engineering target activities. Here, we review recently reported de novo functional proteins that were developed using various protein design approaches, including rational design, computational optimization, and selection from combinatorial libraries, highlighting recent advances and successes.
PMCID: PMC3962203
De novo protein design; computational protein design; designed therapeutic proteins; metalloproteins; ROSETTA; K* algorithm; DEZYMER; ORBIT
25.  Development and Use of Field Application Vectors To Express Nonadaptive Foreign Genes in Competitive Environments † 
Many potential applications of genetically engineered microorganisms in environmental and agricultural biotechnology involve introducing genetic capabilities into nonsterile competitive environments in which they provide no advantage to the host. Field application vectors have been designed for the purpose of creating a temporary niche for the host in such environments. This technique involves the addition to the target environment of a selective substrate readily utilizable by the host microorganism but unavailable to most indigenous species. Thirteen nonionic and anionic detergents, representing a wide range of structural complexities and molecular weights, were screened as potential selective substrates. Competition experiments in soil, using Warburg respirometry, indicated that isolates from six different detergent enrichment cultures were more active on their corresponding detergents than the indigenous microorganisms. Detergents of intermediate structural complexities and molecular weights were most effective for use as selective substrates. A field application vector that utilizes 1.0% Igepal CO-720 (detergent) as the selective substrate and Pseudomonas paucimobilis 1IGP4 as the host was tested for its ability to increase the presence of nonadaptive tetracycline resistance marker genes in soil. In soil amended with the selective substrate, strain 1IGP4 plate counts increased by three orders of magnitude and tetracycline-resistant transformant (pRK293) counts increased from 1.8 × 106/g of soil to 4.3 × 108/g in 2 days. Inoculation in the absence of substrate amendment or amendment with a nonselective substrate did not result in growth of strain 1IGP4. These results demonstrate the effectiveness of field application vectors for increasing the concentration of nonadaptive genes in competitive environments.
PMCID: PMC195298  PMID: 16348652

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