Genetics and Bioengineering

Lateral flow assay (LFA), or the immunochromatographic strip test, is popular to use for rapid and sensitive immunoassays. Gold nanoparticles (GNPs), due to tunable optical characteristics and easy manipulation of size or shape, represent an attractive approach for LFA technology. Since most enterohemorrhagic infections result from water and food contaminations of Escherichia coli O157:H7, selective and rapid detection of this organism in environmental and biological complexes is necessary. In this study, optimized parameters of antibody (Ab)-based LFA for rapid detection of pathogenic E. coli O157:H7 are described. GNPs were used as visualizing agents. The measuring parameters include the Ab concentration on the capture lines, the concentration of gold conjugate, and flow rate. M180 and 36 nm were the ideal membrane and GNP size, respectively, for bacterial detection of LFA. The target, E. coli O157:H7, could be detected with a visual limit of detection of 10 5 cfu/mL in 3-5 min. Selectivity of the system was very high and the target was recognized by developed strips, regardless of its presence singly or in mixed bacterial samples.

Surface-enhanced Raman scattering (SERS) is a technique capable of identifying each component in a mixture because of its intrinsically narrow spectral bands. In a clinical setting, the identification of bacteria from its initial culture by collecting the colonies on the culture plate significantly ...

Salmonella is among the very important pathogens threating human and animal health. It is a common food pathogen transmitted from animals to humans via contaminated food, drinking water, and air. It invades the intestinal tract of hosts and causes salmonellosis leading to death. S. enteritidis was the most common species accounted for all salmonellosis cases. S. typhimurium is also another significant species causing the serious cases worldwide. To ensure public health, early detection of pathogens is crucial. Lateral flow assay (LFA), immunochromatographic assay, is a simple and rapid diagnostic test kits used in various fields and can be developed by, aptamers, antibodies (Abs), and nucleic acids. They are also being continued to develop different capture reagents coming from the recombinant technology. It has many advantages such as having mature technology, market presence, low cost, easy to use for end users without education, and stable shelf life. Gold nanoparticles (GNPs) are the most commonly used labels in the LFAs for the naked-eye analysis. Therefore, Salmonella detection by LFA based on GNPs in a rapid and simple way is always open to be developed by new reagents and methods.

A fast, sensitive and ratiometric biosensor strategy for small molecule detection was developed through nanopore actuation. The new platform engineers together, a highly selective molecular recognition element, aptamers, and a novel signal amplification mechanism, gated nanopores. As a proof of concept, aptamer gated silica nanoparticles have been successfully used as a sensing platform for the detection of ATP concentrations at a wide linear range from 100 μM up to 2 mM.

The interaction of nanoparticles with living systems is under intense investigations due to their increasing use. The unknown effects on human health and vague understanding of their interactions with living systems are the fundamental issues before their use in products consumed by humans. The gold nanoparticles (GNPs) are mostly assumed safe and their use is considered in applications such as drug and gene delivery, photothermal therapy to selectively kill the cancerous cells, and nanoprobes for cellular investigations. This review provides the recent developments in the use of GNPs in medicine and their interactions with living cells along with the basic concepts and techniques used to monitor the interactions such as surface-enhanced Raman scattering (SERS).

Salmonella and E.coli, food pathogens, are among the very important pathogens threating the health. Rapid and easy detection of these pathogens is crucial. In this context, lateral flow assay (LFA) platform prepared by gold nanoparticles (GNPs) based on antibody for Salmonella and E.coli O157:H7 was developed in the first part of this study. In second part, single chain variable fragments (scFv) and SNAP-Tagged full IgG (fusion protein) of se155-4 antibody specific to Salmonella were genetically produced. Target capturing potential of the antibodies was examined by conventional and immunomagnetic ELISA. HF180 and 36nm GNPs were ideal membrane and dimension, respectively in the developed LFA. The best recognized species were S. enteritidis and S. infantis. E.coli were also specifically captured by developed strips. 5x105 S. typhimurium and 3x104 E.coli cells were determined as limit of detection by strips. scFv captured 2.8x104 and 2.8x105 target cells alone and in bacterial mixture,...

Salmonella is among the very important pathogens threating the human and animal health. Rapid and easy detection of these pathogens is crucial. In this context, antibody (Ab) based lateral flow assays (LFAs) which are simple immunochromatographic point of care test kits were developed by gold nanoparticles (GNPs) as labelling agent for Salmonella detection. For that purpose some critical parameters such as reagent concentrations on the capture zones, conjugate concentrations and ideal membrane type needed for LFAs for whole cell detection were tested for naked eye analysis. Therefore, prepared LFAs were applied to the live and heat inactivated cells when they were used alone or included in different bacterial mixtures. Among the test platforms, membrane 180 (M180) was found as an ideal membrane and 36 nm GNPs showed highly good labelling in the developed LFAs. Diluted conjugates and low concentrations of reagents affected the test signal negatively. Salmonella was detected in different bacterial mixtures, selectively in 4-5 min. The best recognized species by used Ab were S. enteritidis and S. infantis. 5 9 10 5 S. typhimurium cells were also determined as a limit of detection of this study with mentioned parameters.

Surface-enhanced Raman scattering (SERS) is a technique capable of identifying each component in a mixture because of its intrinsically narrow spectral bands. In a clinical setting, the identification of bacteria from its initial culture by collecting the colonies on the culture plate significantly decreases the analysis time and the cost. The identification of bacteria from their mixtures is attempted using SERS. A simple mixing procedure of bacterial samples and concentrated colloidal suspension is proven to be mostly satisfactory for the generation of the reproducible SERS spectra that can be used for bacterial identification. The mixture of three different but related bacterial species Shigella sonnei, Proteus vulgaris, and Erwinia amylovara and three Escherichia coli strains (BFK13, BHK7, DH5 α) are used as model systems to test the feasibility of the approach. The results indicate that it is possible to identify the composition of a bacterial mixture. This approach can easily be utilized for the bacteria originating from the same source with similar growth profiles.

Lateral flow assay (LFA), or the immunochromatographic strip test, is popular to use for rapid and sensitive immunoassays. Gold nanoparticles (GNPs), due to tunable optical characteristics and easy manipulation of size or shape, represent an attractive approach for LFA technology. Since most enterohemorrhagic infections result from water and food contaminations of Escherichia coli O157:H7, selective and rapid detection of this organism in environmental and biological complexes is necessary. In this study, optimized parameters of antibody (Ab)-based LFA for rapid detection of pathogenic E. coli O157:H7 are described. GNPs were used as visualizing agents. The measuring parameters include the Ab concentration on the capture lines, the concentration of gold conjugate, and flow rate. M180 and 36 nm were the ideal membrane and GNP size, respectively, for bacterial detection of LFA. The target, E. coli O157:H7, could be detected with a visual limit of detection of 10 5 cfu/mL in 3-5 min. Selectivity of the system was very high and the target was recognized by developed strips, regardless of its presence singly or in mixed bacterial samples.

The interaction of nanoparticles with living systems is under intense investigations due to their increasing use. The unknown effects on human health and vague understanding of their interactions with living systems are the fundamental issues before their use in products consumed by humans. The gold nanoparticles (GNPs) are mostly assumed safe and their use is considered in applications such as drug and gene delivery, photothermal therapy to selectively kill the cancerous cells, and nanoprobes for cellular investigations. This review provides the recent developments in the use of GNPs in medicine and their interactions with living cells along with the basic concepts and techniques used to monitor the interactions such as surface-enhanced Raman scattering (SERS).

Sequence data from cDNA and genomic clones, coupled with analyses of expressed sequence tag databases, indicate that the CesA (cellulose synthase) gene family from barley (Hordeum vulgare) has at least eight members, which are distributed across the genome. Quantitative polymerase chain reaction has been used to determine the relative abundance of mRNA transcripts for individual HvCesA genes in vegetative and floral tissues, at different stages of development. To ensure accurate expression profiling, geometric averaging of multiple internal control gene transcripts has been applied for the normalization of transcript abundance. Total HvCesA mRNA levels are highest in coleoptiles, roots, and stems and much lower in floral tissues, early developing grain, and in the elongation zone of leaves. In most tissues, HvCesA1, HvCesA2, and HvCesA6 predominate, and their relative abundance is very similar; these genes appear to be coordinately transcribed. A second group, comprising HvCesA4, HvCesA7, and HvCesA8, also appears to be coordinately transcribed, most obviously in maturing stem and root tissues. The HvCesA3 expression pattern does not fall into either of these two groups, and HvCesA5 transcript levels are extremely low in all tissues. Thus, the HvCesA genes fall into two general groups of three genes with respect to mRNA abundance, and the co-expression of the groups identifies their products as candidates for the rosettes that are involved in cellulose biosynthesis at the plasma membrane. Phylogenetic analysis allows the two groups of genes to be linked with orthologous Arabidopsis CesA genes that have been implicated in primary and secondary wall synthesis. . Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/

Two ␤-glucan exohydrolases of apparent molecular masses 69,000 and 71,000 Da have been purified from extracts of 8-day germinated barley grains and are designated isoenzymes ExoI and ExoII, respectively. The sequences of their first 52 NH 2 -terminal amino acids show 64% positional identity. Both enzymes hydrolyze the (1,3)-␤-glucan, laminarin, but also hydrolyze (1,3; 1,4)-␤-glucan and 4-nitrophenyl ␤-D-glucoside. The complete sequence of 602 amino acid residues of the mature ␤-glucan exohydrolase isoenzyme ExoII has been deduced by nucleotide sequence analysis of a near fulllength cDNA. Two other enzymes of apparent molecular mass 62,000 Da, designated ␤I and ␤II, were also purified from the extracts. Their amino acid sequences are similar to enzymes classified as ␤-glucosidases and although they hydrolyze 4-nitrophenyl ␤-glucoside, their substrate specificities and action patterns are more typical of polysaccharide exohydrolases of the (1,4)-␤-glucan glucohydrolase type. Both the ␤-glucan exohydrolase isoenzyme ExoI and the ␤-glucosidase isoenzyme ␤II release single glucosyl residues from the nonreducing ends of substrates and proton-NMR shows that anomeric configurations are retained during hydrolysis by both classes of enzyme. These results raise general questions regarding the distinction between polysaccharide exohydrolases and glucosidases, together with more specific questions regarding the functional roles of the two classes of enzyme in germinating barley grain.

Cellulose synthase-like CslF genes have been implicated in the biosynthesis of (1,3;1,4)-b-D-glucans, which are major cell wall constituents in grasses and cereals. Seven CslF genes from barley (Hordeum vulgare) can be divided into two classes on the basis of intron-exon arrangements. Four of the HvCslF genes have been mapped to a single locus on barley chromosome 2H, in a region corresponding to a major quantitative trait locus for grain (1,3;1,4)-b-D-glucan content. The other HvCslF genes map to chromosomes 1H, 5H, and 7H, and in two cases the genes are close to other quantitative trait loci for grain (1,3;1,4)-b-D-glucan content. Spatial and temporal patterns of transcription of the seven genes have been defined through quantitative polymerase chain reaction. In developing barley coleoptiles HvCslF6 mRNA is most abundant. Transcript levels are maximal in 4-to 5-d coleoptiles, at a time when (1,3;1,4)-b-D-glucan content of coleoptile cell walls also reaches maximal levels. In the starchy endosperm of developing grain, HvCslF6 and HvCslF9 transcripts predominate. Two peaks of transcription are apparent. One occurs just after endosperm cellularization, 4 to 8 d after pollination, while the second occurs much later in grain development, more than 20 d after pollination. Marked varietal differences in transcription of the HvCslF genes are observed during endosperm development. Given the commercial importance of cereal (1,3;1,4)-b-D-glucans in human nutrition, in stock feed, and in malting and brewing, the observation that only two genes, HvCslF6 and HvCslF9, are transcribed at high levels in developing grain is of potential relevance for the future manipulation of grain (1,3;1,4)-b-D-glucan levels.

A putative barley (1→3)-β-D-glucan synthase cDNA of 6.1 kb, which is homologous to the yeast FKS gene, was assembled from DNA fragments obtained through screening of barley cDNA and BAC libraries, and by PCR amplification. The corresponding gene, designated HvGSL1, is a member of a family of at least six genes in barley. Gene transcripts are detected at relatively high levels in early developing grain, florets, coleoptiles and roots, but not in leaves infected with a fungal pathogen. A (1→3)-β-D-glucan synthase has been purified more than 60-fold from barley suspension-cultured cells by detergent extraction, CaCl 2 treatment, sucrose density gradient centrifugation and non-denaturing gel electrophoresis. The enzyme synthesizes (1→3)-β-D-glucan in vitro and is recognized by antibodies raised against a 17 kDa protein generated by heterologous expression of a fragment of the HvGSL1 cDNA. Furthermore, mass spectrometric analyses show that tryptic peptides produced by in-gel digestion of the active enzyme match peptides predicted from the gene sequence. Thus, the amino acid sequence predicted from the HvGSL1 gene has been linked with the actual amino acid sequence of an active (1→3)-β-D-glucan synthase fraction from barley.

Type I1 antifreeze proteins (AFP), which inhibit the growth of seed ice crystals in the blood of certain fishes (sea raven, herring, and smelt), are the largest known fish AFPs and the only class for which detailed structural information is not yet available. However, a sequence homology has been recognized between these proteins and the carbohydrate recognition domain of C-type lectins. The structure of this domain from rat mannose-binding protein (MBP-A) has been solved by X-ray crystallography (Weis WI, Drickamer K, Hendrickson WA, 1992, Nature 360:127-134) and provided the coordinates for constructing the three-dimensional model of the 129-amino acid Type I1 AFP from sea raven, to which it shows 19% sequence identity. Multiple sequence alignments between Type I1 AFPs, pancreatic stone protein, MBP-A, and as many as 50 carbohydrate-recognition domain sequences from various lectins were performed to determine reliably aligned sequence regions. Successive molecular dynamics and energy minimization calculations were used to relax bond lengths and angles and to identify flexible regions. The derived structure contains two a-helices, two o-sheets, and a high proportion of amino acids in loops and turns. The model is in good agreement with preliminary NMR spectroscopic analyses. It explains the observed differences in calcium binding between sea raven Type I1 AFP and MBP-A. Furthermore, the model proposes the formation of five disulfide bridges between Cys 7 and Cys 18, Cys 35 and Cys 125, Cys 69 and Cys 100, Cys 89 and Cys 11 1, and Cys 101 and Cys 117. Based on the predicted features of this model, a site for proteinice interaction is proposed.

Family GH16 glycoside hydrolases can be assigned to five subgroups according to their substrate specificities, including xyloglucan transglucosylases/hydrolases (XTHs), (1,3)-β-galactanases, (1,4)-β-galactanases/κ-carrageenases, “nonspecific” (1,3/1,3;1,4)- β-d-glucan endohydrolases, and (1,3;1,4)-β-d-glucan endohydrolases. A structured family GH16 glycoside hydrolase database has been constructed (http://www.ghdb.uni-stuttgart.de) and provides multiple sequence alignments with functionally annotated amino acid residues and phylogenetic trees. The database has been used for homology modeling of seven glycoside hydrolases from the GH16 family with various substrate specificities, based on structural coordinates for (1,3;1,4)-β-d-glucan endohydrolases and a κ-carrageenase. In combination with multiple sequence alignments, the models predict the three-dimensional (3D) dispositions of amino acid residues in the substrate-binding and catalytic sites of XTHs and (1,3/1,3;1,4)-β-d-glucan endohydrolases; there is no structural information available in the databases for the latter group of enzymes. Models of the XTHs, compared with the recently determined structure of a Populus tremulos × tremuloides XTH, reveal similarities with the active sites of family GH11 (1,4)-β-d-xylan endohydrolases. From a biological viewpoint, the classification, molecular modeling and a new 3D structure of the P. tremulos × tremuloides XTH establish structural and evolutionary connections between XTHs, (1,3;1,4)-β-d-glucan endohydrolases and xylan endohydrolases. These findings raise the possibility that XTHs from higher plants could be active not only on cell wall xyloglucans, but also on (1,3;1,4)-β-d-glucans and arabinoxylans, which are major components of walls in grasses. A role for XTHs in (1,3;1,4)-β-d-glucan and arabinoxylan modification would be consistent with the apparent overrepresentation of XTH sequences in cereal expressed sequence tags databases.

Cell wall degradation is an important event during endosperm mobilization in the germinated barley grain. A battery of polysaccharide and oligosaccharide hydrolases is required for the complete depolymerization of the arabinoxylans and (13,14)-i-glucans which comprise in excess of 90% by weight of these walls. The (1 3,1 4)-i-glucan endohydrolases release oligosaccharides from their substrate and are probably of central importance for the initial solubilization of the (13,1 4)-i-glucans, but i-glucan exohydrolases and i-glucosidases may be important additional enzymes for the conversion of released oligosaccharides to glucose. The latter enzymes have recently been purified from germinated barley and characterized. There is an increasing body of evidence to support the notion that the (13,14)-i-glucan endohydrolases from germinated barley evolved from the pathogenesis-related (1 3)-i-glucanases which are widely distributed in plants and which hydrolyse polysaccharides that are abundant in fungal cell walls. Arabinoxylan depolymerization is also mediated by a family of enzymes, but these are less well characterized. (1 4)-i-Xylan endohydrolases have been purified and the corresponding cDNAs and genes isolated. While the presence of (1 4)-i-xylan exohydrolases and h-L-arabinofuranosidases has been reported many times, the enzymes have not yet been studied in detail. Here, recent advances in the enzymology and physiology of cell wall degradation in the germinated barley grain are briefly reviewed. © 1997 Elsevier Science B.V.