National Centre for Biomedical Engineering Science (Scholarly Articles)

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Differential release of high mannose structural isoforms by fungal and bacterial endo-β-N-acetylglucosaminidases
(Royal Society of Chemistry, 2012-02-28) Gerlach, Jared Q.; Kilcoyne, Michelle; Farrell, Mark P.; Kane, Marian; Joshi, Lokesh
Endo-beta-N-acetylglucosaminidases (ENGases) are widely used to remove N-linked oligosaccharides from glycoproteins for glycomic and proteomic studies and biopharmaceutical processes. Although several ENGases are widely available and their main oligosaccharide structural preferences are generally known (i.e. high mannose, hybrid or complex), the preferences of ENGases from different kingdoms for individual structural isoforms within the major classes of N-linked oligosaccharides have previously not been compared. In this work, a fungal ENGase (Endo Tv) was purified for the first time from a commercial Trichoderma viride chitinase mixture by sequential anion exchange and size exclusion chromatography, a commonly used strategy for purification of chitinases and endo enzymes. Oligosaccharides released from substrate glycoproteins by Endo Tv were identified and quantified by high pH anion exchange chromatography with pulsed amperometric detection and verified by mass spectrometric analysis. Unlike the widely-used bacterial ENGases, Endo H and Endo F1, Endo Tv released exclusively high mannose N-linked oligosaccharides from RNase B, ovalbumin, and yeast invertase. Endo Tv did not hydrolyze fucosylated, hybrid, complex type or bisecting N-acetylglucosamine-containing structures from bovine fetuin, ovalbumin and IgG. When compared to the bacterial ENGase, Endo H, the relative ratio of high-mannose oligosaccharide structural isoforms released from RNase B by Endo Tv was found to differ, with Endo Tv releasing more Man(5)GlcNAc and Man(7)GlcNAc isoform I and less Man(9)GlcNAc from RNase B. Based on these data, it is suggested that use of ENGases from multiple sources may serve to balance an introduced bias in quantitative analysis of released structural isoforms and may further prove valuable in biochemical structure-function studies.
Campylobacter jejuni strain discrimination and temperature-dependent glycome expression profiling by lectin microarray
(Elsevier, 2014-02-12) Kilcoyne, Michelle; Twomey, Marcus E.; Gerlach, Jared Q.; Kane, Marian; Moran, Anthony P.; Joshi, Lokesh; Science Foundation Ireland; European Regional Development Fund; Seventh Framework Programme
Gram-negative Campylobacter jejuni is the leading cause of bacterial gastroenteritis in humans worldwide and the most frequently identified infectious trigger in patients developing Guillain-BarrÃ© syndrome (GBS). While C. jejuni is pathogenic in humans, it is a commensal in avian hosts. Bacterial cell surface carbohydrates are important virulence factors and play roles in adherence, colonisation and infection. The mechanisms leading to infection or persistent colonisation of C. jejuni are not well understood but host temperature may provide an important stimulus for specific adaptation. Thus, examination of the modulation of the total surface glycome of C. jejuni in response to temperature may help shed light on commensal and pathogenic mechanisms for this species. C. jejuni strains 81116 and 81-176 were cultured at 37 and 42Â°C to simulate human and avian host conditions, respectively, and whole cells were profiled on lectin microarrays constructed to include a wide range of binding specificities. C. jejuni 81116 profiles indicated that the previously characterised lipopolysaccharide (LPS)-like molecule and N-linked glycans were the predominantly recognised cell surface structures while capsular polysaccharide (CPS), lipooligosaccharides (LOS) and N-linked glycosylation were best recognised for strain 81-176 at 37Â°C. The profiles of both strains varied and were distinguishable at both temperatures. At the higher temperature, reduced dominance of the LPS-like structure was associated with strain 81116 and a change in the relative distribution of CPS and LOS structures was indicated for strain 81-176. This change in LOS molecular mass species distribution between temperatures was confirmed by SDS-PAGE analysis. Additionally, opposite behaviour of certain lectins was noted between the plate agglutination assay and the microarray platform. Insights into the important glycosylation involved in C. jejuni host cell tropism at different growth temperatures were gained using the lectin microarray platform.
Complex and dynamic transcriptional changes allow the helminth Fasciola gigantica to adjust to its intermediate snail and definitive mammalian hosts
(BMC (part of Springer Nature), 2019-10-12) Zhang, Xiao-Xuan; Cwiklinski, Krystyna; Hu, Rui-Si; Zheng, Wen-Bin; Sheng, Zhao-An; Zhang, Fu-Kai; Elsheikha, Hany M.; Dalton, John P.; Zhu, Xing-Quan; Science Foundation Ireland; National Basic Research Program of China (973 Program); Elite Program of Chinese Academy of Agricultural Sciences
The tropical liver fluke, Fasciola gigantica causes fasciolosis, an important disease of humans and livestock. We characterized dynamic transcriptional changes associated with the development of the parasite in its two hosts, the snail intermediate host and the mammalian definitive host. Differential gene transcription analysis revealed 7445 unigenes transcribed by all F. gigantica lifecycle stages, while the majority (n¿=¿50,977) exhibited stage-specific expression. Miracidia that hatch from eggs are highly transcriptionally active, expressing a myriad of genes involved in pheromone activity and metallopeptidase activity, consistent with snail host finding and invasion. Clonal expansion of rediae within the snail correlates with increased expression of genes associated with transcription, translation and repair. All intra-snail stages (miracidia, rediae and cercariae) require abundant cathepsin L peptidases for migration and feeding and, as indicated by their annotation, express genes putatively involved in the manipulation of snail innate immune responses. Cercariae emerge from the snail, settle on vegetation and become encysted metacercariae that are infectious to mammals; these remain metabolically active, transcribing genes involved in regulation of metabolism, synthesis of nucleotides, pH and endopeptidase activity to assure their longevity and survival on pasture. Dramatic growth and development following infection of the mammalian host are associated with high gene transcription of cell motility pathways, and transport and catabolism pathways. The intra-mammalian stages temporally regulate key families of genes including the cathepsin L and B proteases and their trans-activating peptidases, the legumains, during intense feeding and migration through the intestine, liver and bile ducts. While 70% of the F. gigantica transcripts share homology with genes expressed by the temperate liver fluke Fasciola hepatica, gene expression profiles of the most abundantly expressed transcripts within the comparable lifecycle stages implies significant species-specific gene regulation. Transcriptional profiling of the F. gigantica lifecycle identified key metabolic, growth and developmental processes the parasite undergoes as it encounters vastly different environments within two very different hosts. Comparative analysis with F. hepatica provides insight into the similarities and differences of these parasites that diverged >¿20 million years ago, crucial for the future development of novel control strategies against both species.
Effects of combined progesterone and 17 beta-estradiol treatment on the transcriptome of cultured human myometrial smooth muscle cells
(American Physiological Society, 2016-01-01) Chandran, Sreenath; Cairns, Michael T.; O'Brien, Margaret; O'Connell, Enda; Mashayekhi, Kaveh; Smith, Terry J.; |~|
A transcriptomic analysis of cultured human uterine smooth muscle cells (hUtSMCs) was performed to examine gene expression profiles in smooth muscle in an environment containing the two major steroid hormones that regulate the human myometrium in physiological states associated with estrous, pregnancy, labor, and pathophysiological states such as leiomyoma and endometrial cancer. hUtSMCs were treated with progesterone (P4) and 17 beta-estradiol (E2) individually and in combination, in the presence and absence of RU486 (mifepristone). Transcription of many genes was modulated in the presence of P4 or E2 alone, but almost six times more genes were transcriptionally modulated in the presence of the P4/E2 hormone combination. In total 796 annotated genes were significantly differentially expressed in the presence of both P4 and E2 relative to their expression in untreated cells. Functional withdrawal of P4 by addition of RU486 effectively reversed almost all transcriptional changes caused by P4/E2 treatment. Gene ontology analysis of differentially expressed genes revealed a strong association between P4/E2 treatment and downregulated expression of genes involved in cell communication, signal transduction, channel activity, inflammatory response, and differentiation. Upregulated processes included cell survival, gene transcription, steroid hormone biosynthesis, muscle development, insulin receptor signaling, and cell growth.
Development of a rapid recombinase polymerase amplification assay for the detection of Streptococcus pneumoniae in whole blood
(BioMed Central, 2015-10-29) Clancy, Eoin; Higgins, Owen; Boo, Teck Wee; Cormican, Martin; Barry, Thomas; Smith, Terry J.; |~|
Background: Streptococcus pneumoniae is an important cause of microbial disease in humans. The introduction of multivalent vaccines has coincided with a dramatic decrease in the number of pneumococcal-related deaths. In spite of this, at a global level, pneumococcal infection remains an important cause of death among children under 5 years of age and in adults 65 years of age or older. In order to properly manage patients and control the spread of infection, a rapid and highly sensitive diagnostic method is needed for routine implementation, especially in resource-limited regions where pneumococcal disease is most prevalent.Methods: Using the gene encoding leader peptidase A as a molecular diagnostics target, a real-time RPA assay was designed and optimised for the detection of S. pneumoniae in whole blood. The performance of the assay was compared to real-time PCR in terms of its analytical limit of detection and specificity. The inhibitory effect of human genomic DNA on amplification was investigated. The potential clinical utility of the assay was investigated using a small number of clinical samples.Results: The RPA assay has a limit of detection equivalent to PCR (4.0 and 5.1 genome equivalents per reaction, respectively) and was capable of detecting the equivalent of
Alignment-free clustering of transcription factor binding motifs using a genetic-k-medoids approach
(BioMed Central, 2015-01-28) Ó Broin, Pilib; Smith, Terry J.; Golden, Aaron; |~|
Background: Familial binding profiles (FBPs) represent the average binding specificity for a group of structurally related DNA-binding proteins. The construction of such profiles allows the classification of novel motifs based on similarity to known families, can help to reduce redundancy in motif databases and de novo prediction algorithms, and can provide valuable insights into the evolution of binding sites. Many current approaches to automated motif clustering rely on progressive tree-based techniques, and can suffer from so-called frozen sub-alignments, where motifs which are clustered early on in the process remain 'locked' in place despite the potential for better placement at a later stage. In order to avoid this scenario, we have developed a genetic-k-medoids approach which allows motifs to move freely between clusters at any point in the clustering process.Results: We demonstrate the performance of our algorithm, GMACS, on multiple benchmark motif datasets, comparing results obtained with current leading approaches. The first dataset includes 355 position weight matrices from the TRANSFAC database and indicates that the k-mer frequency vector approach used in GMACS outperforms other motif comparison techniques. We then cluster a set of 79 motifs from the JASPAR database previously used in several motif clustering studies and demonstrate that GMACS can produce a higher number of structurally homogeneous clusters than other methods without the need for a large number of singletons. Finally, we show the robustness of our algorithm to noise on multiple synthetic datasets consisting of known motifs convolved with varying degrees of noise.Conclusions: Our proposed algorithm is generally applicable to any DNA or protein motifs, can produce highly stable and biologically meaningful clusters, and, by avoiding the problem of frozen sub-alignments, can provide improved results when compared with existing techniques on benchmark datasets.
Analysis of stress-induced hepatic gene expression in rainbow trout (Oncorhynchus mykiss) selected for high- and low-responsiveness to stress
(Elsevier, 2011-12) Pemmasani, Jhansi K.; Pottinger, Tom G.; Cairns, Michael T.; |~|
The production and welfare of intensively reared fish would be improved by reducing stress responsiveness. One approach to achieving this goal is selective breeding utilising stress-responsive genes as direct genetic markers of the desirable trait. As a first step in this process, microarray analysis has been carried out on liver tissues of rainbow trout selectively bred for high (HR) or low (LR) responsiveness to a stressor. Microarray hybridizations provided gene expression profiles for pooled samples of fish confined for 6 h, 24 h and 168 h and for individual fish (168 h only). 161 genes were shown to be differentially regulated in HR and LR fish during confinement exposure and eight of these gene expression profiles were validated by quantitative PCR. Genes of particular interest included intelectin-2 precursor which showed greater than 100-fold higher expression in HR fish compared to LR fish irrespective of whether the fish were confined or not; interferon inducible transmembrane protein 3 which was differentially stress-induced between the two lines; and hepatic pro-opiomelanocortin B (POMC B) which was upregulated during stress in HR fish but downregulated in LR fish. All these offer potential as direct markers of low stress responsiveness in a marker-assisted selection scheme. (C) 2011 Elsevier Inc. All rights reserved.
A note on the evaluation of a beta-casein variant in bovine breeds by allele-specific PCR and relevance to beta-casomorphin
(Teagasc; De Gruyter Open, 2008) Keating, Aileen F.; Smith, Terry J.; Cairns, Michael T.; |~|
Two genetic variants of the bovine beta-casein gene (A(1) and B) encode a histidine residue at codon 67, resulting in potential liberation of a bioactive peptide, beta-casomorphin, upon digestion. An allele-specific PCR (AS-PCR) was evaluated to distinguish between the beta-casomorphin-releasing variants (A(1) and B) and the non-releasing variants. AS-PCR successfully distinguished P-casein variants in 41 of 42 animals as confirmed by sequence analysis. Overall, while the incidence of the homozygous A, and B animals (i.e., homozygous for the histidine residue; 21.4%) was lower than that for animals without the histidine residue (30.9% respectively), 69% of animals carried at least one allele for the histidine residue at codon 67.
A single nucleotide polymorphism in the bovine beta-casein promoter region across different bovine breeds
(Cambridge Journals, 2006-02-14) Keating, Aileen F.; Smith, Terry J.; Cairns, Michael T.; |~|
The bovine β-casein (CSN2) gene has been shown to span a region of 8·5 kb, containing nine exons and eight intervening introns (Bonsing et al. 1988; Martin et al. 2002). The exons range in size from 24 to 498 bp; the introns, however, are much larger and account for 85% of the gene. Twelve genetic variants in the coding sequence of the β-casein gene have been reported (Farrell et al. 2004). The A2 allele of the β-casein gene has been associated with a higher milk production (Lin et al. 1986; Bech & Kristiansen, 1990) while the B variant has been associated with an increase in protein content and better cheesemaking properties (Marziali & Ng-Hang-Kwai, 1986). The β-casein gene codes for a protein of 209 amino acids with varying regions at codons 67, 106 and 122. The A1 variant differs from A2 at position 67, where a histidine replaces a proline (Lien et al. 1992). The β-casein A2 variant has histidine and the A3 variant has glycine at position 106 (Lien et al. 1992); the β-casein A2 variant has serine at position 122 and the β-casein B variant has arginine at this codon (Stewart et al. 1987; Damiani et al. 1992).
Isolation and characterization of the bovine Stearoyl-CoAdesaturase promoter and analysis of polymorphisms in the promoter region in dairy cows
(Springer, 2005-03) Keating, Aileen F.; Murphy, John J.; Smith, Terry J.; Cairns, Michael T.; |~|
Conjugated linoleic acid (CLA) in milk arises through microbial biohydrogenation of dietary polyunsaturated fatty acids (PUFA) in the rumen, and by the action of mammary Stearoyl-CoA desaturase (Scd). A large variation (up to ten-fold) in the concentration of this fatty acid in milk have been observed, even in cows receiving the same diet. The reasons for this variation are not well understood. In this study, the bovine core promoter region was isolated by a genome walking strategy from genomic DNA GenomeWalker libraries and then cloned and characterised. This core promoter sequence extended approximately 600bp upstream of the translation start site. The presence of putative transcription factor binding sites conserved in bovine, human, and mouse promoters were observed. Evidence that this promoter fragment was functional in vivo was obtained from expression studies in a mammary cell line. The promoter sequence of the scd gene was compared between cows selected for the ability to produce high fatty acid methyl esters (FAME) (2.22-2.72) in their milk, with the same promoter region of low FAME producing cows (0.81-1.12). However, such comparisons of the sequences of the scd promoter region of cows producing high milk CLA compared with low CLA revealed no polymorphisms in this promoter segment. Furthermore, no sequence polymorphisms were observed between the scd promoter region of Holstein Friesian, Montbeliarde, Normande, Norwegian Red, Charlois, Limousin and Kerry breeds.
Bovine kappa-casein gene promoter haplotypes with potential implications for milk protein expression
(Elsevier ScienceDirect, 2007-05-25) Keating, A. F.; Davoren, P.; Smith, T. J.; Cairns, Michael T.; |~|
Genetic analysis of the kappa-casein gene (CSN3) promoter regions of 42 cattle representing 9 different breeds revealed that 2 distinct haplotypes (A and B) exist at this locus, differing from each other by single base changes at positions -514 (T/G), -426 (T/C), and -384 ( T/C), where haplotype A has bases T, T, and T and haplotype B has bases G, C, and C. The AA and AB haplotypes were found to occur at a higher frequency in the animals tested, with 69.0 and 21.4% being homozygous and heterozygous, respectively. The sequences that include these polymorphisms are potentially important in transcriptional regulation of the kappa-casein gene, because they contain putative sites for binding of many transcription factors. Linkage disequilibrium between the kappa-casein promoter haplotype and either one of the 2 major kappa-casein coding sequence haplotypes was not evident. The A allele is dominant in all groups (dairy, beef, and dual purpose) with an allele frequency of 80% and is higher among high-yielding dairy animals (88.9%) than among beef animals (75%). The AB haplotype is comparatively rare in the dairy cattle (11.1%) compared with both beef and dual-purpose animals. The BB haplotype, though rare overall (9.5%), is much higher in dual-purpose animals (18.8%) than dairy (5.6%) animals. In contrast, the B allele is much more representative of the kappa-casein promoters from other ruminants.
Cloning and characterization of two genes encoding rainbow trout homologues of the IFITM protein family
(Elsevier, 2006-04-15) Johnson, Mark C.; Sangrador-Vegas, Amaia; Smith, Terry; Cairns, Michael T.; |~|
To identify genes connected with leucocyte function in rainbow trout a cDNA library derived front head kidney leucocytes exposed to phytohemagglutinin (PHA) was screened. A clone with homology to the previously described human interferon-inducible transmembrane proteins (IFITM) was isolated. This clone was used to screen a trout genomic library identifying two trout IFITM genes. Both genes are composed of two exons, in common with human IFITM genes. The promoter regions of both genes were examined and found to contain interferon, immune and stress-related transcription factor binding sites. Transcripts for both genes were found in most tissues examined. Trout IFITM1 mRNA levels were highest in head kidney, but present in all tissues studied. IFITM2 mRNA levels were highest in gill, heart and liver, and absent in head kidney and blood. In response to conditions mimicking viral infection, transcription of the trout IFITM genes was elevated in a trout spleen macrophage cell line (RTS11) over the examined timecourse (72 h). This study suggests that the organization and transcription of the IFITM genes is well conserved between human and rainbow trout. (c) 2006 Elsevier B.V. All rights reserved.
A cDNA microarray assessment of gene expression in the liver of rainbow trout (Oncorhynchus mykiss) in response to a handling and confinement stressor
(Elsevier, 2008-03) Cairns, Michael T.; Johnson, M. C.; Talbot, A. T.; Pernmasani, J. K.; McNeill, R. E.; Houeix, Benoit; Sangrador-Vegas, A.; Pottinger, T. G.; |~|
A purpose-designed microarray platform (Stressgenes, Phase 1) was utilised to investigate the changes in gene expression within the liver of rainbow trout during exposure to a prolonged period of confinement. Tissue and blood samples were collected from trout at intervals up to 648 h after transfer to a standardised confinement stressor, together with matched samples from undisturbed control fish. Plasma ACTH, cortisol, glucose and lactate were analysed to confirm that the neuroendocrine response to confinement was consistent with previous findings and to provide a phenotypic context to assist interpretation of gene expression data. Liver samples for suppression subtractive hybridisation (SSH) library construction were selected from within the experimental groups comprising “early” stress (2–48 h) and “late” stress (96–504 h). In order to reduce redundancy within the four SSH libraries and yield a higher number of unique clones an additional subtraction was carried out. After printing of the arrays a series of 55 hybridisations were executed to cover 6 time points. At 2 h, 6 h, 24 h, 168 h and 504 h 5 individual confined fish and 5 individual control fish were used with control fish only at 0 h. A preliminary list of 314 clones considered differentially regulated over the complete time course was generated by a combination of data analysis approaches and the most significant gene expression changes were found to occur during the 24 h to 168 h time period with a general approach to control levels by 504 h. Few changes in expression were apparent over the first 6 h. The list of genes whose expression was significantly altered comprised predominantly genes belonging to the biological process category (response to stimulus) and one cellular component category (extracellular region) and were dominated by so-called acute phase proteins. Analysis of the gene expression profile in liver tissue during confinement revealed a number of significant clusters. The major patterns comprised genes that were up-regulated at 24 h and beyond, the primary examples being haptoglobin, β-fibrinogen and EST10729. Two representative genes from each of the six k-means clusters were validated by qPCR. Correlations between microarray and qPCR expression patterns were significant for most of the genes tested. qPCR analysis revealed that haptoglobin expression was up-regulated approximately 8-fold at 24 h and over 13-fold by 168 h.
Use of microarray technology to assess the time course of liver stress response after confinement exposure in gilthead sea bream (Sparus aurata L.)
(BioMed Central, 2010-03-22) Davey, Grace; Houeix, Benoit; Talbot, Anita; Cairns, Michael T.; |~|
Background: Selection programs for growth and stress traits in cultured fish are fundamental to the improvement of aquaculture production. The gilthead sea bream ( Sparus aurata) is the main aquacultured species in the Mediterranean area and there is considerable interest in the genetic improvement of this species. With the aim of increasing the genomic resources in gilthead sea bream and identifying genes and mechanisms underlying the physiology of the stress response, we developed a cDNA microarray for gilthead sea bream that is enriched by suppression substractive hybridization with stress and immunorelevant genes. This microarray is used to analyze the dynamics of gilthead sea bream liver expression profile after confinement exposure.Results: Groups of confined and control juvenile fish were sampled at 6, 24, 72 and 120 h post exposure. GeneSpring analyses identified 202 annotated genes that appeared differentially expressed at least at one sampling time (P
Effects of combined progesterone and beta-estradiol treatment on the transcriptome of cultured human myometrial smooth muscle cells
(American Physiological Society, 2015) Chandran, Sreenath; Cairns, Michael T.; O Brien, Margaret; O Connell, Enda; Mashayekhi, Kaveh; Smith, Terry; |~|1267880|~|
A transcriptomic analysis of cultured human uterine smooth muscle cells (hUtSMCs) was performed in order to examine gene expression profiles in smooth muscle in an environment containing the two major steroid hormones that regulate the human myometrium in physiological states associated with estrous, pregnancy, labor, and pathophysiological states such as leiomyoma and endometrial cancer. hUtSMCs were treated with progesterone (P4) and 17β-estradiol (E2) individually and in combination, in the presence and absence of RU486 (mifepristone). Transcription of many genes was modulated in the presence of P4 or E2 alone but almost six times more genes were transcriptionally modulated in the presence of the P4/E2 hormone combination. In total 796 annotated genes were significantly differentially expressed in the presence of both P4 and E2 relative to their expression in untreated cells. Functional withdrawal of progesterone by addition of RU486 effectively reversed almost all transcriptional changes caused by P4/E2 treatment. Gene ontology analysis of differentially expressed genes revealed a strong association between P4/E2 treatment and down-regulated expression of genes involved in cell communication, signal transduction, channel activity, inflammatory response and differentiation. Up-regulated processes included cell survival, gene transcription, steroid hormone biosynthesis, muscle development, insulin receptor signalling and cell growth.
Dietary vegetable oils do not alter the intestine transcriptome of gilthead sea bream (Sparus aurata), but modulate the transcriptomic response to infection with Enteromyxum leei
(BioMed Central, 2012) Cairns, Michael T.; Davey, Grace C.; |~|
Background: Studies conducted with gilthead sea bream (Sparus aurata L.) have determined the maximum dietary replacement of fish meal and oil without compromising growth or product quality. The present study aimed to analyze the effect of the nutritional background on fish health and fish fed plant protein-based diets with fish oil (FO diet) or a blend of vegetable oils (66VO diet) were exposed for 102 days to the intestinal myxosporean parasite Enteromyxum leei, and the intestine transcriptome was analyzed with a customized oligo-microarray of 7,500 annotated genes.Results: Infection prevalence was high and similar in the two diet groups, but the outcome of the disease was more pronounced in fish fed the 66VO diet. No differences were found in the transcriptome of both diet control groups, whereas the number of differentially expressed genes in infected groups was considerable. K-means clustering of these differentially expressed genes identified four expression patterns that reflected the progression of the disease with the magnitude of the fold-change being higher in infected 66VO fish. A positive correlation was found between the time of infection and the magnitude of the transcriptional change within the 66VO group, being higher in early infected animals. Within this diet group, a strong up-regulation of many components of the immune specific response was evidenced, whereas other genes related to complement response and xenobiotic metabolism were down-regulated.Conclusions: The high replacement of fish oil by vegetable oils in practical fish feeds did not modify the intestine transcriptome of gilthead sea bream, but important changes were apparent when fish were exposed to the myxosporean E. leei. The detected changes were mostly a consequence rather than a cause of the different disease progression in the two diet groups. Hence, the developed microarray constitutes an excellent diagnostic tool to address changes associated with the action of intestinal pathogens, but lacks a prognostic value to predict in advance the different susceptibility of growing fish to the current pathogen.
Predicting the elastic properties of selective laser sintered PCL/b-TCP bone scaffold materials using computational modelling.
(Springer, 2013) Doyle, Heather; Lohfeld, Stefan; McHugh, Peter E.; |~|6201984|~|
Abstract This study assesses the ability of finite element (FE) models to capture the mechanical behaviour of sintered orthopaedic scaffold materials. Individual scaffold struts were fabricated from a 50:50 wt% poly-e-caprolactone (PCL)/b-tricalcium phosphate (b-TCP) blend, using selectivelaser sintering. The tensile elastic modulus of single struts was determined experimentally. High resolution FE models of single struts were generated from micro-CT scans (28.8 lmresolution) and an effective strut elastic modulus was calculated from tensile loading simulations. Three material assignment methods were employed: (1) homogeneous PCL elastic constants, (2) composite PCL/b-TCP elastic constants based on rule of mixtures, and (3) heterogeneous distribution of micromechanically-determined elastic constants. In comparisonwith experimental results, the use of homogeneous PCL properties gave a good estimate of strut modulus; however it is not sufficiently representative of the real material as it neglects the b-TCP phase. The rule of mixtures method significantly overestimated strut modulus, while there was no significant difference between strut modulus evaluated using the micromechanically-determined elastic constants and experimentally evaluated strut modulus. These results indicate that the multi-scale approach of linking micromechanical modelling of the sintered scaffold material with macroscale modelling gives an accurate prediction of the mechanical behaviour of the sintered structure.
Laser sintering for the fabrication of tissue engineering scaffolds
(Springer Science+Business Media, 2012) Lohfeld, Stefan; McHugh, Peter E.; |~|
Laser sintering (LS) utilises a laser to sinter powder particles. A volumetric model is sliced and processedcross section by cross section to create a physical part. In theory, all powdered materials are suitable forsintering; however, only few have been tested successfully. For tissue engineering (TE) applications of thisrapid prototyping technology it is an advantage that no toxic solvents or binders are necessary. This chapterreviews the direct and indirect use of LS to fabricate scaffolds for TE from single and multiphase materials.
Interaction of cell culture with composition effects on the mechanical properties of polycaprolactone-hydroxyapatite scaffolds fabricated via selective laser sintering (SLS)
(Elsevier, 2012) Eosoly, Szilvia; Vrana, Nihal Engin; Lohfeld, Stefan; Hindie, Mathilde; Looney, Lisa; |~|; Marie Curie Early Stage Research Training Fellowship of the European Community's Sixth Framework Programme under contract number MEST-CT-2005-020621
In the current study PCL/HA composites were fabricated using SLS as two- and three-dimensional lattice structures and exposed to a cellular component (MC 313 osteoblast-like cells). The main aims were to determine the mechanical differences due to powder composition and to observe the physical and mechanical changes pertaining to cell presence. These structures were characterized by compressive mechanical testing, and the effects of cell culturing and degradation on mechanical properties of the scaffolds with different PCL/HA compositions were determined. Moreover, changes in the scaffold morphology due to the cell culture conditions were determined by mu-CF analysis.Cells steadily grew on the scaffolds for 21 days with preferential distribution around the macropores and initially PCL/HA(15%) composites had higher cell numbers. Removal of loosely sintered parts was observable during the culturing period. Cell culture conditions did not change the compressive moduli significantly but had a distinct effect on compressive strength. For PCL/HA(15%) composites, an initial loss in strength caused by cell culture was reversed by longer cell exposure, with compressive strength of the structures restored to the initial properties (p <= 0.05). mu-CT measurements showed widespread morphological changes in the scaffolds, such as a decrease in the roughness of the struts. In general, in the initial period composites with lower HA content (15 wt.%) showed better metabolic activity compared to the higher HA content, however by day 14 the performance of the two compositions was equal. These results suggest that changes in sintering due to the differences in powder composition can have profound effects on the short and long term mechanical properties of the scaffold particularly:under cell culture conditions, and this should be closely considered for SLS processing of scaffolds. (C) 2012 Elsevier B.V. All rights reserved.
Finite element predictions compared to experimental results for the effective modulus of bone tissue engineering scaffolds fabricated by selective laser sintering
(Springer Verlag, 2009) Lohfeld, Stefan; McHugh, Peter E.; Cahill, Senan; |~|; Programme for Research in Third Level Institutions (PRTLI); EU Framework Programme 6 Project (STEPS, Contract NMP3-CT- 2005-500465)
A current challenge in bone tissue engineering is to create scaffolds with suitable mechanical properties, high porosity, full interconnectivity and suitable pore size. In this paper, polyamide and polycaprolactone scaffolds were fabricated using a solid free form technique known as selective laser sintering. These scaffolds had fully interconnected pores, minimized strut thickness, and a porosity of approximately 55%. Tensile and compression tests as well as finite element analysis were carried out on these scaffolds. It was found that the values predicted for the effective modulus by the FE model were much higher than the actual values obtained from experimental results. One possible explanation for this discrepancy, viz. the surface roughness of the scaffold and the presence of micropores in the scaffold struts, was investigated with a view to making recommendations on improving FE model configurations for accurate effective property predictions.

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