Stepwise engineering of a Pichia pastoris D-amino acid oxidase whole cell catalyst

Trigonopsis variabilis D-amino acid oxidase (TvDAO) is a well characterized enzyme used for cephalosporin C conversion on industrial scale. However, the demands on the enzyme with respect to activity, operational stability and costs also vary with the field of application. Processes that use the soluble enzyme suffer from fast inactivation of TvDAO while immobilized oxidase preparations raise issues related to expensive carriers and catalyst efficiency. Therefore, oxidase preparations that are more robust and active than those currently available would enable a much broader range of economically viable applications of this enzyme in fine chemical syntheses. A multi-step engineering approach was chosen here to develop a robust and highly active Pichia pastoris TvDAO whole-cell biocatalyst. As compared to the native T. variabilis host, a more than seven-fold enhancement of the intracellular level of oxidase activity was achieved in P. pastoris through expression optimization by codon redesign as well as efficient subcellular targeting of the enzyme to peroxisomes. Multi copy integration further doubled expression and the specific activity of the whole cell catalyst. From a multicopy production strain, about 1.3 x 103 U/g wet cell weight (wcw) were derived by standard induction conditions feeding pure methanol. A fed-batch cultivation protocol using a mixture of methanol and glycerol in the induction phase attenuated the apparent toxicity of the recombinant oxidase to yield final biomass concentrations in the bioreactor of >or= 200 g/L compared to only 117 g/L using the standard methanol feed. Permeabilization of P. pastoris using 10% isopropanol yielded a whole-cell enzyme preparation that showed 49% of the total available intracellular oxidase activity and was notably stabilized (by three times compared to a widely used TvDAO expressing Escherichia coli strain) under conditions of D-methionine conversion using vigorous aeration. Stepwise optimization using a multi-level engineering approach has delivered a new P. pastoris whole cell TvDAO biocatalyst showing substantially enhanced specific activity and stability under operational conditions as compared to previously reported preparations of the enzyme. The production of the oxidase through fed-batch bioreactor culture and subsequent cell permeabilization is high-yielding and efficient. Therefore this P. pastoris catalyst has been evaluated for industrial purposes.

The salt dependence of DNA recognition by NF-kappaB p50 : a detailed kinetic analysis of the effects on affinityand specificity

The binding kinetics of NF-kappaB p50 to the Ig-kappaB site and to a DNA duplex with no specific binding site were determined under varying conditions of potassium chloride concentration using a surface plasmonresonance biosensor. Association and dissociation rate constants were measured enabling calculation of the dissociation constants. Under previously established high affinity buffer conditions, the k a for both sequences was in the order of 10(7) M-1s-1whilst the k d values varied 600-fold in a sequence-dependent manner between 10(-1) and 10(-4 )s-1, suggesting that the selectivity of p50 for different sequences is mediated primarily through sequence-dependent dissociation rates. The calculated K D value for the Ig-kappaB sequence was 16 pM, whilst the K D for the non-specific sequence was 9.9 nM. As the ionic strength increased to levels which are closer to that of the cellular environment, the binding of p50 to the non-specific sequence was abolished whilst the specific affinity dropped to nanomolar levels. From these results, a mechanism is proposed in which p50 binds specific sequences with high affinity whilst binding non-specific sequences weakly enough to allow efficient searching of the DNA.

Analysis of expressed sequence tags from Actinidia : Applications of a cross species EST database for gene discovery in the areas of flavor, health, color and ripening

Background Kiwifruit (Actinidia spp.) are a relatively new, but economically important crop grown in many different parts of the world. Commercial success is driven by the development of new cultivars with novel consumer traits including flavor, appearance, healthful components and convenience. To increase our understanding of the genetic diversity and gene-based control of these key traits in Actinidia, we have produced a collection of 132,577 expressed sequence tags (ESTs). Results The ESTs were derived mainly from four Actinidia species (A. chinensis, A. deliciosa, A. arguta and A. eriantha) and fell into 41,858 non redundant clusters (18,070 tentative consensus sequences and 23,788 EST singletons). Analysis of flavor and fragrance-related gene families (acyltransferases and carboxylesterases) and pathways (terpenoid biosynthesis) is presented in comparison with a chemical analysis of the compounds present in Actinidia including esters, acids, alcohols and terpenes. ESTs are identified for most genes in color pathways controlling chlorophyll degradation and carotenoid biosynthesis. In the health area, data are presented on the ESTs involved in ascorbic acid and quinic acid biosynthesis showing not only that genes for many of the steps in these pathways are represented in the database, but that genes encoding some critical steps are absent. In the convenience area, genes related to different stages of fruit softening are identified. Conclusion This large EST resource will allow researchers to undertake the tremendous challenge of understanding the molecular basis of genetic diversity in the Actinidia genus as well as provide an EST resource for comparative fruit genomics. The various bioinformatics analyses we have undertaken demonstrates the extent of coverage of ESTs for genes encoding different biochemical pathways in Actinidia.

Genome-wide SNP detection, validation, and development of an 8K SNP array for apple

As high-throughput genetic marker screening systems are essential for a range of genetics studies and plant breeding applications, the International RosBREED SNP Consortium (IRSC) has utilized the Illumina Infinium® II system to develop a medium- to high-throughput SNP screening tool for genome-wide evaluation of allelic variation in apple (Malus×domestica) breeding germplasm. For genome-wide SNP discovery, 27 apple cultivars were chosen to represent worldwide breeding germplasm and re-sequenced at low coverage with the Illumina Genome Analyzer II. Following alignment of these sequences to the whole genome sequence of 'Golden Delicious', SNPs were identified using SoapSNP. A total of 2,113,120 SNPs were detected, corresponding to one SNP to every 288 bp of the genome. The Illumina GoldenGate® assay was then used to validate a subset of 144 SNPs with a range of characteristics, using a set of 160 apple accessions. This validation assay enabled fine-tuning of the final subset of SNPs for the Illumina Infinium® II system. The set of stringent filtering criteria developed allowed choice of a set of SNPs that not only exhibited an even distribution across the apple genome and a range of minor allele frequencies to ensure utility across germplasm, but also were located in putative exonic regions to maximize genotyping success rate. A total of 7867 apple SNPs was established for the IRSC apple 8K SNP array v1, of which 5554 were polymorphic after evaluation in segregating families and a germplasm collection. This publicly available genomics resource will provide an unprecedented resolution of SNP haplotypes, which will enable marker-locus-trait association discovery, description of the genetic architecture of quantitative traits, investigation of genetic variation (neutral and functional), and genomic selection in apple.

Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants

Background We describe novel plasmid vectors for transient gene expression using Agrobacterium, infiltrated into Nicotiana benthamiana leaves. We have generated a series of pGreenII cloning vectors that are ideally suited to transient gene expression, by removing elements of conventional binary vectors necessary for stable transformation such as transformation selection genes. Results We give an example of expression of heme-thiolate P450 to demonstrate effectiveness of this system. We have also designed vectors that take advantage of a dual luciferase assay system to analyse promoter sequences or post-transcriptional regulation of gene expression. We have demonstrated their utility by co-expression of putative transcription factors and the promoter sequence of potential target genes and show how orthologous promoter sequences respond to these genes. Finally, we have constructed a vector that has allowed us to investigate design features of hairpin constructs related to their ability to initiate RNA silencing, and have used these tools to study cis-regulatory effect of intron-containing gene constructs. Conclusion In developing a series of vectors ideally suited to transient expression analysis we have provided a resource that further advances the application of this technology. These minimal vectors are ideally suited to conventional cloning methods and we have used them to demonstrate their flexibility to investigate enzyme activity, transcription regulation and post-transcriptional regulatory processes in transient assays.

Human breast cancer cell metastasis to long bone and soft organs of nude mice : a quantitative assay

Bone is a common metastatic site in human breast cancer (HBC). Since bone metastasis occurs very rarely from current spontaneous or experimental metastasis models of HBC cells in nude mice, an arterial seeding model involving the direct injection of the cells into the left ventricle has been developed to better understand the mechanisms involved in this process. We present here a sensitive polymerase chain reaction (PCR) method to detect and quantitate bone and soft organ metastasis in nude mice which have been intracardially inoculated with Lac Z transduced HBC cells. Amplification of genomically incorporated Lac Z sequences in MDA-MB-231-BAG HBC cells enables us to specifically detect these cells in mouse organs and bones. We have also created a competitive template to use as an internal standard in the PCR reactions, allowing us to better quantitate levels of HBC metastasis. The results of this PCR detection method correlate well with cell culture detection from alternate long bones from the same mice, and are more sensitive than gross Lac Z staining with X-gal or routine histology. Comparable qualitative results were obtained with PCR and culture in a titration experiment in which mice were inoculated with increasing numbers of cells, but PCR is more quantifiable, less time consuming, and less expensive. This assay can be employed to study the molecular and cellular aspects of bone metastasis, and could easily be used in conjunction with RT-PCR-based analyses of gene products which may be involved with HBC metastasis.

Bone sialoprotein supports breast cancer cell adhesion proliferation and migration through differential usage of the αvβ3 and αvβ5 integrins

Bone sialoprotein (BSP), a secreted glycoprotein found in bone matrix, has been implicated in the formation of mammary microcalcifications and osteotropic metastasis of human breast cancer (HBC). BSP possesses an integrin-binding RGD (Arg-Gly-Asp) domain, which may promote interactions between HBC cells and bone extracellular matrix. Purified BSP, recombinant human BSP fragments and BSP-derived RGD peptides are shown to elicit migratory, adhesive, and proliferative responses in the MDA-MB-231 HBC cell line. Recombinant BSP fragment analysis localized a significant component of these activities to the RGD domain of the protein, and synthetic RGD peptides with BSP flanking sequences (BSPRGD) also conferred these responses. The fibronectin-derived RGD counterpart, GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro), could not support these cellular responses, emphasizing specificity of the BSP configuration. Although most of the proliferative and adhesive responses could be attributed to RGD interactions, these interactions were only partly responsible for the migrational responses. Experiments with integrin-blocking antibodies demonstrated that BSP-RGD-induced migration utilizes the αvβ3 vitronectin receptor, whereas adhesion and proliferation responses were αvβ5-mediated. Using fluorescence activated cell sorting, we selected two separate subpopulations of MDA-MB-231 cells enriched for αvβ3 or αvβ5 respectively. Although some expression of the alternate αv integrin was still retained, the αvβ5-enriched MDA-MB-231 cells showed enhanced proliferative and adhesive responses, whereas the αvβ3-enriched subpopulation was suppressed for proliferation and adhesion, but showed enhanced migratory responses to BSP-RGD. In addition, similar analysis of two other HBC cell lines showed less marked, but similar RGD-dependent trends in adhesion and proliferation to the BSP fragments. Collectively, these data demonstrate BSP effects on proliferative, migratory, and adhesive functions in HBC cells and that the RGD-mediated component differentially employs αvβ3 and αvβ5 integrin receptors.

A transcriptomic analysis of striped catfish (Pangasianodon hypophthalmus) in response to salinity adaptation: De novo assembly, gene annotation and marker discovery

The striped catfish (Pangasianodon hypophthalmus) culture industry in the Mekong Delta in Vietnam has developed rapidly over the past decade. The culture industry now however, faces some significant challenges, especially related to climate change impacts notably from predicted extensive saltwater intrusion into many low topographical coastal provinces across the Mekong Delta. This problem highlights a need for development of culture stocks that can tolerate more saline culture environments as a response to expansion of saline water-intruded land. While a traditional artificial selection program can potentially address this need, understanding the genomic basis of salinity tolerance can assist development of more productive culture lines. The current study applied a transcriptomic approach using Ion PGM technology to generate expressed sequence tag (EST) resources from the intestine and swim bladder from striped catfish reared at a salinity level of 9 ppt which showed best growth performance. Total sequence data generated was 467.8 Mbp, consisting of 4,116,424 reads with an average length of 112 bp. De novo assembly was employed that generated 51,188 contigs, and allowed identification of 16,116 putative genes based on the GenBank non-redundant database. GO annotation, KEGG pathway mapping, and functional annotation of the EST sequences recovered with a wide diversity of biological functions and processes. In addition, more than 11,600 simple sequence repeats were also detected. This is the first comprehensive analysis of a striped catfish transcriptome, and provides a valuable genomic resource for future selective breeding programs and functional or evolutionary studies of genes that influence salinity tolerance in this important culture species.

The role of vascularization in 3-D tissue engineering

The role of vascularization in 3-D tissue engineering was studied. Mouse fat, angiogenic growth factors, adult human stem cells and fat tissue have been inserted and subsequent tissue growth was monitored. Human fat grafts or human lipoaspirates in SCID mouse chambers induced mouse fat generation at 6 weeks. Tissue engineering models utilizing intrinsic vascularization have major advantages including rapid and appropriate vascularization of new tissues.

Analysis of albumin-associated peptides and proteins from ovarian cancer patients

Albumin binds low–molecular-weight molecules, including proteins and peptides, which then acquire its longer half-life, thereby protecting the bound species from kidney clearance. We developed an experimental method to isolate albumin in its native state and to then identify [mass spectrometry (MS) sequencing] the corresponding bound low–molecular-weight molecules. We used this method to analyze pooled sera from a human disease study set (high-risk persons without cancer, n= 40; stage I ovarian cancer, n = 30; stage III ovarian cancer, n = 40) to demonstrate the feasibility of this approach as a discovery method. Methods Albumin was isolated by solid-phase affinity capture under native binding and washing conditions. Captured albumin-associated proteins and peptides were separated by gel electrophoresis and subjected to iterative MS sequencing by microcapillary reversed-phase tandem MS. Selected albumin-bound protein fragments were confirmed in human sera by Western blotting and immunocompetition. Results In total, 1208 individual protein sequences were predicted from all 3 pools. The predicted sequences were largely fragments derived from proteins with diverse biological functions. More than one third of these fragments were identified by multiple peptide sequences, and more than one half of the identified species were in vivo cleavage products of parent proteins. An estimated 700 serum peptides or proteins were predicted that had not been reported in previous serum databases. Several proteolytic fragments of larger molecules that may be cancer-related were confirmed immunologically in blood by Western blotting and peptide immunocompetition. BRCA2, a 390-kDa low-abundance nuclear protein linked to cancer susceptibility, was represented in sera as a series of specific fragments bound to albumin. Conclusion Carrier-protein harvesting provides a rich source of candidate peptides and proteins with potential diverse tissue and cellular origins that may reflect important disease-related information.

Comparison of block and event-related experimental designs in diffusion-weighted functional MRI

PURPOSE To compare diffusion-weighted functional magnetic resonance imaging (DfMRI), a novel alternative to the blood oxygenation level-dependent (BOLD) contrast, in a functional MRI experiment. MATERIALS AND METHODS Nine participants viewed contrast reversing (7.5 Hz) black-and-white checkerboard stimuli using block and event-related paradigms. DfMRI (b = 1800 mm/s2 ) and BOLD sequences were acquired. Four parameters describing the observed signal were assessed: percent signal change, spatial extent of the activation, the Euclidean distance between peak voxel locations, and the time-to-peak of the best fitting impulse response for different paradigms and sequences. RESULTS The BOLD conditions showed a higher percent signal change relative to DfMRI; however, event-related DfMRI showed the strongest group activation (t = 21.23, P < 0.0005). Activation was more diffuse and spatially closer to the BOLD response for DfMRI when the block design was used. DfMRIevent showed the shortest TTP (4.4 +/- 0.88 sec). CONCLUSION The hemodynamic contribution to DfMRI may increase with the use of block designs.

A biomechanical analysis of growing rods used in the management of early onset scoliosis

INTRODUCTION Managing spinal deformities in young children is challenging, particularly early-onset scoliosis (EOS). Any progressive spinal deformity particularly in early life presents significant health risks for the child and a challenge for the treating surgeon. Surgical intervention is often required if EOS has been unresponsive to conservative treatment particularly with rapidly progressive curves. An emerging treatment option particularly for EOS is fusionless scoliosis surgery. Similar to bracing this surgical option potentially harnesses growth, motion and function of the spine along with correcting spinal deformity. Dual growing rods is one such fusionless treatment, which aims to modulate growth of the vertebrae. The aim of this study was to ascertain the extent to which semi-constrained growing rods (Medtronic, Memphis, TN) with a telescopic sleeve component, reduce rotational constraint on the spine compared with standard rigid rods and hence potentially provide a more physiological mechanical environment for the growing spine. METHODS Six 40-60kg English Large White porcine spines served as a model for the paediatric human spine. Each spine was dissected into 7 level thoracolumbar multi-segment unit (MSU) spines, removing all non-ligamentous soft tissues. Appropriately sized semi-constrained growing rods and rigid rods were secured by multi-axial screws (Medtronic) prior to testing in alternating sequences for each spine. Pure nondestructive moments of +/4Nm at a constant rotation rate of 8deg/s was applied to the mounted MSU spines. Displacement of each level was captured using an Optotrak (Northern Digital Inc, Waterloo, ON). The range of motion (ROM), neutral zone (NZ) size and stiffness (Nm/deg) were calculated from the Instron load-displacement data and intervertebral ROM was calculated through a MATLAB algorithm from Optotrak data. RESULTS Irrespective of sequence order rigid rods significantly reduced the total ROM (deg) than compared to semi-constrained rods (p<0.05) and resulted in a significantly stiffer (Nm/deg) spine for both left and right axial rotation testing (p<0.05). Analysing the intervertebral motion within the instrumented levels, rigid rods showed reduced ROM (Deg) than compared to semi-constrained growing rods and the un-instrumented (UN-IN) test sequences. CONCLUSION The semi-constrained growing rods maintained rotation similar to UN-IN spines while the rigid rods showed significantly reduced axial rotation across all instrumented levels. Clinically the effect of semi-constrained growing rods evaluated in this study is that they will allow growth via the telescopic rod components while maintaining the axial rotation ability of the spine, which may also reduce the occurrence of the crankshaft phenomenon.

Serum 25-hydroxy vitamin D concentrations are more deficient/insufficient in peritoneal dialysis than haemodialysis patients in a sunny climate

Background Research has identified associations between serum 25(OH)D and a range of clinical outcomes in chronic kidney disease and wider populations. The present study aimed to investigate vitamin D deficiency/insufficiency in dialysis patients and the relationship with vitamin D intake and sun exposure. Methods A cross-sectional study was used. Participants included 30 peritoneal dialysis (PD) (43.3% male; 56.87 ± 16.16 years) and 26 haemodialysis (HD) (80.8% male; 63.58 ± 15.09 years) patients attending a department of renal medicine. Explanatory variables were usual vitamin D intake from diet/supplements (IU day−1) and sun exposure (min day−1). Vitamin D intake, sun exposure and ethnic background were assessed by questionnaire. Weight, malnutrition status and routine biochemistry were also assessed. Data were collected during usual department visits. The main outcome measure was serum 25(OH)D (nm). Results Prevalence of inadequate/insufficient vitamin D intake differed between dialysis modality, with 31% and 43% found to be insufficient (<50 nm) and 4% and 33% found to be deficient (<25 nm) in HD and PD patients, respectively (P < 0.001). In HD patients, there was a correlation between diet and supplemental vitamin D intake and 25(OH)D (ρ = 0.84, P < 0.001) and average sun exposure and 25(OH)D (ρ = 0.50, P < 0.02). There were no associations in PD patients. The results remained significant for vitamin D intake after multiple regression, adjusting for age, gender and sun exposure. Conclusions The results highlight a strong association between vitamin D intake and 25(OH)D in HD but not PD patients, with implications for replacement recommendations. The findings indicate that, even in a sunny climate, many dialysis patients are vitamin D deficient, highlighting the need for exploration of determinants and consequences.

Optimizing I/O cost and managing memory for composition vector method based on correlation matrix calculation in bioinformatics

The generation of a correlation matrix for set of genomic sequences is a common requirement in many bioinformatics problems such as phylogenetic analysis. Each sequence may be millions of bases long and there may be thousands of such sequences which we wish to compare, so not all sequences may fit into main memory at the same time. Each sequence needs to be compared with every other sequence, so we will generally need to page some sequences in and out more than once. In order to minimize execution time we need to minimize this I/O. This paper develops an approach for faster and scalable computing of large-size correlation matrices through the maximal exploitation of available memory and reducing the number of I/O operations. The approach is scalable in the sense that the same algorithms can be executed on different computing platforms with different amounts of memory and can be applied to different bioinformatics problems with different correlation matrix sizes. The significant performance improvement of the approach over previous work is demonstrated through benchmark examples.