bluuebunny/crossref_metadata_2025 huggingface dataset:
Sustainable long-term conservation of rare cattle breeds using rotational AI sires
Authors: Colleau Jean-Jacques , Avon Laurent
Published: June 2009

The development of inbreeding in rotation breeding schemes, sequentially using artificial insemination (AI) sires over generations, was investigated for a full AI scheme. Asymptotic prediction formulae of inbreeding coefficients were established when the first rotation list of AI sires (possibly related) was in use. Simulated annealing provided the optimal rotation order of sires within this list, when the sires were related. These methods were also used for subsequent rotation lists, needed by the exhaustion of semen stores for the first bulls. Simulation was carried out starting with groups of independent sires, with different sizes. To generate a yearly inbreeding rate substantially lower than 0.05% (considered to be within reach by conventional conservation schemes using frequent replacements), the results obtained showed that the number of sires should be at least 10–15 and that the same sires should be used during at least 50 years. The ultimate objective was to examine the relevance of implementing rotation in breeding schemes on the actual rare French cattle breeds under conservation. The best candidate for such a test was the Villard-de-Lans breed (27 bulls and 73 000 doses for only 340 females) and it turned out to be the best performer with an inbreeding coefficient of only 7.4% after 500 years and five different sire lists. Due to the strong requirements on semen stores and on the stability of population size, actual implementation of this kind of conservation scheme was recommended only in special ('niche') cattle populations.
Conservation priorities for Ethiopian sheep breeds combining threat status, breed merits and contributions to genetic diversity
Authors: Gizaw Solomon , Komen Hans , Windig Jack J. , Hanotte Olivier , van Arendonk Johan A.M.
Published: June 2009

Prioritizing livestock breeds for conservation needs to incorporate both genetic and non-genetic aspects important for the survival of the breeds. Here, we apply a maximum-utility-strategy to prioritize 14 traditional Ethiopian sheep breeds based on their threat status, contributions to farmer livelihoods (current breed merits) and contributions to genetic diversity. Contributions of the breeds to genetic diversity were quantified using Eding's marker-estimated kinship approaches. Non-genetic aspects included threats (e.g. low population size, low preferences by farmers) and current merits (economic, ecological and cultural merits). Threat analysis identified eight of the 14 breeds as threatened. Analysis of current merits showed that sub-alpine and arid-lowland breeds contribute most to farmer livelihoods in comparison to other breeds. The highest contribution to the genetic diversity conserved was from the Simien breed. Simien showed high between-breed (low between-breed kinship = 0.04) as well as high within-breed diversity (low within-breed kinship = 0.09 and high H
E = 0.73 and allelic richness = 6.83). We combined the results on threat status, current breed merits and contributions to genetic diversity to produce a ranking of the 14 breeds for conservation purposes. Our results balance the trade-offs between conserving breeds as insurance against future uncertainties and current sustainable utilization. The ranking of breeds provides a basis for conservation strategies for Ethiopian sheep and contributes to a regional or global conservation plan.
Chromosomal mapping, differential origin and evolution of the S100 gene family
Authors: Shang Xuan , Cheng Hanhua , Zhou Rongjia
Published: June 2009

S100 proteins are calcium-binding proteins, which exist only in vertebrates and which constitute a large protein family. The origin and evolution of the S100 family in vertebrate lineages remain a challenge. Here, we examined the synteny conservation of mammalian S100A genes by analysing the sequence of available vertebrate S100 genes in databases. Five S100A gene members, unknown previously, were identified by chromosome mapping analysis. Mammalian S100A genes are duplicated and clustered on a single chromosome while two S100A gene clusters are found on separate chromosomes in teleost fish, suggesting that S100A genes existed in fish before the fish-specific genome duplication took place. During speciation, tandem gene duplication events within the cluster of S100A genes of a given chromosome have probably led to the multiple members of the S100A gene family. These duplicated genes have been retained in the genome either by neofunctionalisation and/or subfunctionalisation or have evolved into non-coding sequences. However in vertebrate genomes, other S100 genes are also present i.e. S100P, S100B, S100G and S100Z, which exist as single copy genes distributed on different chromosomes, suggesting that they could have evolved from an ancestor different to that of the S100A genes.
Genetic and morphological characterisation of the Ankole Longhorn cattle in the African Great Lakes region
Authors: Ndumu Deo B. , Baumung Roswitha , Hanotte Olivier , Wurzinger Maria , Okeyo Mwai A. , Jianlin Han , Kibogo Harrison , Sölkner Johann
Published: June 2009

The study investigated the population structure, diversity and differentiation of almost all of the ecotypes representing the African Ankole Longhorn cattle breed on the basis of morphometric (shape and size), genotypic and spatial distance data. Twentyone morphometric measurements were used to describe the morphology of 439 individuals from 11 sub-populations located in five countries around the Great Lakes region of central and eastern Africa. Additionally, 472 individuals were genotyped using 15 DNA microsatellites. Femoral length, horn length, horn circumference, rump height, body length and fore-limb circumference showed the largest differences between regions. An overall F
ST index indicated that 2.7% of the total genetic variation was present among sub-populations. The least differentiation was observed between the two sub-populations of Mbarara south and Luwero in Uganda, while the highest level of differentiation was observed between the Mugamba in Burundi and Malagarasi in Tanzania. An estimated membership of four for the inferred clusters from a model-based Bayesian approach was obtained. Both analyses on distance-based and model-based methods consistently isolated the Mugamba sub-population in Burundi from the others.
Inbreeding depression on beef cattle traits: Estimates, linearity of effects and heterogeneity among sire-families
Authors: Carolino Nuno , Gama Luis T.
Published: June 2009

Records from up to 19054 registered cows and 10297 calves in 155 herds of the Alentejana cattle breed were used to study the effects of individual (Fi) and maternal (Fm) inbreeding on reproductive, growth and carcass traits, as well as assessing the importance of non-linear associations between inbreeding and performance, and evaluating the differences among sire-families in the effect of Fi and Fm on calf weight at 7 months of age (W7M). Overall, regression coefficients of performance traits on inbreeding were small, indicating a minor but still detrimental effect of both Fi and Fm on most traits. The traits with the highest percentage impact of Fi were total number of calvings through life and calf weight at 3 months of age (W3M), followed by longevity and number of calves produced up to 7 years, while the highest effect of Fm was on W3M. Inbreeding depression on feed efficiency and carcass traits was extremely small and not significant. No evidence was found of a non-linear association between inbreeding and performance for the traits analyzed. Large differences were detected among sire-families in inbreeding depression on W7M, for both Fi and Fm, encouraging the possibility of incorporating sire effects on inbreeding depression into selection decisions.
Genotype by environment interaction for adult body weights of shrimp Penaeus vannamei when grown at low and high densitie
Authors: Ibarra Ana M , Famula Thomas R
Published: June 2009

Shrimp is one of few marine species cultured worldwide for which several selective breeding programs are being conducted. One environmental factor that can affect the response to selection in breeding programs is the density at which the shrimp are cultured (low-medium-high). Phenotypic plasticity in the growth response to different densities might be accompanied by a significant genotype by environment interaction, evidenced by a change in heritabilities between environments and by a genetic correlation less than one for a unique trait between environments. Our goal was to understand whether different growth densities affect estimates of those genetic parameters for adult body weight (BW) in the Pacific white shrimp (Penaeus vannamei). BW heritabilities were significantly different between environments, with the largest at high density. These differences resulted from both an increased additive genetic variance and a decreased environmental variance when grown at high density. The genetic correlation between BWs at the two environmental conditions was significantly less than one. Whereas these results might be suggestive for carrying out shrimp selective breeding for BW under high density conditions, further understanding of genetic correlations between growth and reproductive traits within a given environment is necessary, as there are indications of reduced reproductive fitness for shrimp grown at high densities.
Prion gene (PRNP) haplotype variation in United States goat breeds (Open Access publication)
Authors: White Stephen , Herrmann-Hoesing Lynn , O'rourke Katherine , Waldron Daniel , Rowe Joan , Alverson Janet
Published: June 2009

Scrapie eradication efforts cost 18 million dollars annually in the United States and rely heavily upon PRNP genotyping of sheep. Genetic resistance might reduce goat scrapie and limit the risk of goats serving as a scrapie reservoir, so PRNP coding sequences were examined from 446 goats of 10 breeds, 8 of which had not been previously examined at PRNP. The 10 observed alleles were all related to one of two central haplotypes by a single amino acid substitution. At least five of these alleles (M142, R143, S146, H154, and K222) have been associated with increased incubation time or decreased odds of scrapie. To the best of our knowledge, neither S146 nor K222 has been found in any goats with scrapie, though further evaluation will be required to demonstrate true resistance. S146 was more common, present in several breeds at widely varying frequencies, while K222 was observed only in two dairy breeds at low frequency. Overall, this study provides frequency data on PRNP alleles in US goats, shows the pattern of relationships between haplotypes, and demonstrates segregation of multiple scrapieassociated alleles in several breeds not examined before at PRNP.
Further insights of the variance component method for detecting QTL in livestock and aquacultural species: relaxing the assumption of additive effects
Authors: Martinez Victor
Published: June 2009

Complex traits may show some degree of dominance at the gene level that may influence the statistical power of simple models, i.e. assuming only additive effects to detect quantitative trait loci (QTL) using the variance component method. Little has been published on this topic even in species where relatively large family sizes can be obtained, such as poultry, pigs, and aquacultural species. This is important, when the idea is to select regions likely to be harbouring dominant QTL or in marker assisted selection. In this work, we investigated the empirical power and accuracy to both detect and localise dominant QTL with or without incorporating dominance effects explicitly in the model of analysis. For this purpose, populations with variable family sizes and constant population size and different values for dominance variance were simulated. The results show that when using only additive effects there was little loss in power to detect QTL and estimates of position, using or not using dominance, were empirically unbiased. Further, there was little gain in accuracy of positioning the QTL with most scenarios except when simulating an overdominant QTL.
Genetic variability in residual feed intake in rainbow trout clones and testing of indirect selection criteria (Open Access publication)
Authors: Grima Laure , Quillet Edwige , Boujard Thierry , Robert-Granié Christèle , Chatain Béatrice , Mambrini Muriel
Published: June 2009

Little is known about the genetic basis of residual feed intake (RFI) variation in fish, since this trait is highly sensitive to environmental influences, and feed intake of individuals is difficult to measure accurately. The purpose of this work was (i) to assess the genetic variability of RFI estimated by an X-ray technique and (ii) to develop predictive criteria for RFI. Two predictive criteria were tested: loss of body weight during feed deprivation and compensatory growth during re-feeding. Ten heterozygous rainbow trout clones were used. Individual intake and body weight were measured three times at threeweek intervals. Then, individual body weight was recorded after two cycles of a three-week feed deprivation followed by a three-week re-feeding. The ratio of the genetic variance to the phenotypic variance was found high to moderate for growth, feed intake, and RFI (VG/VP = 0.63 ± 0.11, 0.29 ± 0.11, 0.29 ± 0.09, respectively). The index that integrates performances achieved during deprivation and re-feeding periods explained 59% of RFI variations. These results provide a basis for further studies on the origin of RFI differences and show that indirect criteria are good candidates for future selective breeding programs.
Identification of a differentially expressed gene, ACL, between Meishan × Large White and Large White × Meishan F1 hybrids and their parents
Authors: Ren Zhu-Qing , Wang Yan , Xu Yong-Jie , Wang Lin-Jie , Lei Ming-Gang , Zuo Bo , Li Feng-E , Xu De-Quan , Zheng Rong , Deng Chang-Yan , Jiang Si-Wen , Xiong Yuan-Zhua
Published: June 2009

ATP-citrate lyase (ACL), one of the lipogenic enzymes, catalyses the formation of acetyl-coenzyme A (CoA) involved in the synthesis of fatty acid and cholesterol. In pig, very little is known about the ACL gene. In this work, the mRNA differential display technique was used to analyse the differences in gene expression between Meishan and Large White pigs and the F1 hybrids of both direct and reciprocal crosses. Our results show that among the differentially expressed genes ACL is up-regulated in the backfat of the F1 hybrids. After cloning and analysing the fulllength cDNA and the 870 bp 5'-flanking sequence of the porcine ACL gene, a C/T mutation at position -97 bp upstream of the transcription site was detected. Luciferase activity detection showed that this mutation changed the transcriptional activity. In F1 hybrids, the heterozygous genotype CT was more frequent than the homozygous genotypes CC and TT. Real-time PCR analysis showed that in Meishan pigs, ACL mRNA expression was more abundant in individuals with genotype CT than in those with genotype CC or TT or in Large White pigs. These results indicate that the C/T mutation affects ACL mRNA expression, probably via the activator protein 2.
Impact of strong selection for the PrP major gene on genetic variability of four French sheep breeds (Open Access publication)
Authors: Palhiere Isabelle , Brochard Mickaël , Moazami-Goudarzi Katayoun , Laloë Denis , Amigues Yves , Bed'hom Bertrand , Neuts Étienne , Leymarie Cyril , Pantano Thais , Cribiu Edmond Paul , Bibé Bernard , Verrier Étienne
Published: June 2009

Effective selection on the PrP gene has been implemented since October 2001 in all French sheep breeds. After four years, the ARR "resistant" allele frequency increased by about 35% in young males. The aim of this study was to evaluate the impact of this strong selection on genetic variability. It is focussed on four French sheep breeds and based on the comparison of two groups of 94 animals within each breed: the first group of animals was born before the selection began, and the second, 3–4 years later. Genetic variability was assessed using genealogical and molecular data (29 microsatellite markers). The expected loss of genetic variability on the PrP gene was confirmed. Moreover, among the five markers located in the PrP region, only the three closest ones were affected. The evolution of the number of alleles, heterozygote deficiency within population, expected heterozygosity and the Reynolds distances agreed with the criteria from pedigree and pointed out that neutral genetic variability was not much affected. This trend depended on breed, i.e. on their initial states (population size, PrP frequencies) and on the selection strategies for improving scrapie resistance while carrying out selection for production traits.
A genetic epidemiological model to describe resistance to an endemic bacterial disease in livestock: application to footrot in sheep
Authors: Nieuwhof Gert Jan , Conington Joanne , Bishop Stephen C
Published: June 2009

Selection for resistance to an infectious disease not only improves resistance of animals, but also has the potential to reduce the pathogen challenge to contemporaries, especially when the population under selection is the main reservoir of pathogens. A model was developed to describe the epidemiological cycle that animals in affected populations typically go through; viz. susceptible, latently infected, diseased and infectious, recovered and reverting back to susceptible through loss of immunity, and the rates at which animals move from one state to the next, along with effects on the pathogen population. The equilibrium prevalence was estimated as a function of these rates. The likely response to selection for increased resistance was predicted using a quantitative genetic threshold model and also by using epidemiological models with and without reduced pathogen burden. Models were standardised to achieve the same genetic response to one round of selection. The model was then applied to footrot in sheep. The only epidemiological parameters with major impacts for prediction of genetic progress were the rate at which animals recover from infection and the notional reproductive rate of the pathogen. There are few published estimates for these parameters, but plausible values for the rate of recovery would result in a response to selection, in terms of changes in the observed prevalence, double that predicted by purely genetic models in the medium term (e.g. 2–5 generations).
Genetic architecture of trout from Albania as revealed by mtDNA control region variation
Authors: Snoj Aleš , Marić Saša , Berrebi Patrick , Crivelli Alain J , Shumka Spase , Sušnik Simona
Published: June 2009

To determine the genetic architecture of trout in Albania, 87 individuals were collected from 19 riverine and lacustrine sites in Albania, FYROM and Greece. All individuals were analyzed for sequence variation in the mtDNA control region. Among fourteen haplotypes detected, four previously unpublished haplotypes, bearing a close relationship to haplotypes of the Adriatic andmarmoratuslineages ofSalmo trutta, were revealed. Ten previously described haplotypes, characteristic ofS. ohridanus,S. letnicaand the Adriatic and Mediterranean lineages ofS. trutta, were also detected. Haplotypes detected in this study were placed in a well supported branch ofS. ohridanus, and a cluster of Mediterranean – Adriatic –marmoratushaplotypes, which were further delimited into three subdivisions of Mediterranean,marmoratus, and a previously non-described formation of four Adriatic haplotypes (Balkan cluster). Haplotypes of the Balkan cluster and the other Adriatic haplotypes, do not represent a contiguous haplotype lineage and appear not to be closely related, indicating independent arrivals into the Adriatic drainage and suggesting successive colonization events. Despite the presence ofmarmoratushaplotypes in Albania, no marbled phenotype was found, confirming previously reported findings that there is no association between this phenotype andmarmoratushaplotypes.
Casein haplotypes and their association with milk production traits in Norwegian Red cattle
Authors: Nilsen Heidi , Olsen Hanne Gro , Hayes Ben , Sehested Erling , Svendsen Morten , Nome Torfinn , Meuwissen Theo , Lien Sigbjørn
Published: June 2009

A high resolution SNP map was constructed for the bovine casein region to identify haplotype structures and study associations with milk traits in Norwegian Red cattle. Our analyses suggest separation of the casein cluster into two haplotype blocks, one consisting of the CSN1S1, CSN2 and CSN1S2 genes and another one consisting of the CSN3 gene. Highly significant associations with both protein and milk yield were found for both single SNPs and haplotypes within the CSN1S1-CSN2-CSN1S2 haplotype block. In contrast, no significant association was found for single SNPs or haplotypes within the CSN3 block. Our results point towards CSN2 and CSN1S2 as the most likely loci harbouring the underlying causative DNA variation. In our study, the most significant results were found for the SNP CSN2_67 with the C allele consistently associated with both higher protein and milk yields. CSN2_67 calls a C to an A substitution at codon 67 in β-casein gene resulting in histidine replacing proline in the amino acid sequence. This polymorphism determines the protein variants A1/B (CSN2_67 A allele) versus A2/A3 (CSN2_67 C allele). Other studies have suggested that a high consumption of A1/B milk may affect human health by increasing the risk of diabetes and heart diseases. Altogether these results argue for an increase in the frequency of the CSN2_67 C allele or haplotypes containing this allele in the Norwegian Red cattle population by selective breeding.
Genomic scan for quantitative trait loci of chemical and physical body composition and deposition on pig chromosome X including the pseudoautosomal region of males
Authors: Duthie Carol-Anne , Simm Geoff , Pérez-Enciso Miguel , Doeschl-Wilson Andrea , Kalm Ernst , Knap Pieter W , Roehe Rainer
Published: June 2009

A QTL analysis of pig chromosome X (SSCX) was carried out using an approach that accurately takes into account the specific features of sex chromosomes i.e. their heterogeneity, the presence of a pseudoautosomal region and the dosage compensation phenomenon. A three-generation full-sib population of 386 animals was created by crossing Pietrain sires with a crossbred dam line. Phenotypic data on 72 traits were recorded for at least 292 and up to 315 F2 animals including chemical body composition measured on live animals at five target weights ranging from 30 to 140 kg, daily gain and feed intake measured throughout growth, and carcass characteristics obtained at slaughter weight (140 kg). Several significant and suggestive QTL were detected on pig chromosome X: (1) in the pseudoautosomal region of SSCX, a QTL for entire loin weight, which showed paternal imprinting, (2) closely linked to marker SW2456, a suggestive QTL for feed intake at which Pietrain alleles were found to be associated with higher feed intake, which is unexpected for a breed known for its low feed intake capacity, (3) at the telomeric end of the q arm of SSCX, QTL for jowl weight and lipid accretion and (4) suggestive QTL for chemical body composition at 30 kg. These results indicate that SSCX is important for physical and chemical body composition and accretion as well as feed intake regulation.
Eleven generations of selection for the duration of fertility in the intergeneric crossbreeding of ducks
Authors: Cheng Yu-Shin , Rouvier Roger , Liu Hsiao-Lung , Huang Shang-Chi , Huang Yu-Chia , Liao Chung-Wen , Liu Tai Jui-Jane , Tai Chein , Poivey Jean-Paul
Published: June 2009

A 12-generation selection experiment involving a selected line (S) and a control line (C) has been conducted since 1992 with the aim of increasing the number of fertile eggs laid by the Brown Tsaiya duck after a single artificial insemination (AI) with pooled Muscovy semen. On average, 28.9% of the females and 17.05% of the males were selected. The selection responses and the predicted responses showed similar trends. The average predicted genetic responses per generation in genetic standard deviation units were 0.40 for the number of fertile eggs, 0.45 for the maximum duration of fertility, and 0.32 for the number of hatched mule ducklings' traits. The fertility rates for days 2–8 after AI were 89.14% in the S line and 61.46% in the C line. Embryo viability was not impaired by this selection. The largest increase in fertility rate per day after a single AI was observed from d5 to d11. In G12, the fertility rate in the selected line was 91% at d2, 94% at d3, 92% at days 3 and 4 then decreased to 81% at d8, 75% at d9, 58% at d10 and 42% at d11. In contrast, the fertility rate in the control line showed an abrupt decrease from d4 (74%). The same tendencies were observed for the evolution of hatchability according to the egg set rates. It was concluded that selection for the number of fertile eggs after a single AI with pooled Muscovy semen could effectively increase the duration of the fertile period in ducks and that research should now be focused on ways to improve the viability of the hybrid mule duck embryo.
Insights into the role of Val45 and Gln182 of Escherichia coli MutY in DNA substrate binding and specificity
Authors: Chang Po-Wen , Madabushi Amrita , Lu A-Lien
Published: June 2009

Background

Escherichia coli MutY (EcMutY) reduces mutagenesis by removing adenines paired with guanines or 7,8-dihydro-8-oxo-guanines (8-oxoG). V45 and Q182 of EcMutY are considered to be the key determinants of adenine specificity. Both residues are spatially close to each other in the active site and are conserved in MutY family proteins but not in Methanobacterium thermoautotrophicum Mig.MthI T/G mismatch DNA glycosylase (A50 and L187 at the corresponding respective positions).

Results

Targeted mutagenesis study was performed to determine the substrate specificities of V45A, Q182L, and V45A/Q182L double mutant of EcMutY. All three mutants had significantly lower binding and glycosylase activities for A/G and A/8-oxoG mismatches than the wild-type enzyme. The double mutant exhibited an additive reduction in binding to both the A/G and A/GO in comparison to the single mutants. These mutants were also tested for binding and glycosylase activities with T/G- or T/8-oxoG-containing DNA. Both V45A and Q182L mutants had substantially increased affinities towards T/G, however, they did not exhibit any T/G or T/8-oxoG glycosylase activity. Surprisingly, the V45A/Q182L double mutant had similar binding affinities to T/G as the wild-type EcMutY. V45A, Q182L, and V45A/Q182L EcMutY mutants could not reduce the G:C to T:A mutation frequency of a mutY mutant. Expression of the V45A mutant protein caused a dominant negative phenotype with an increased G:C to A:T mutation frequency.

Conclusion

The substrate specificities are altered in V45A, Q182L, and V45A/Q182L EcMutY mutants. V45A and Q182L mutants had reduced binding and glycosylase activities for A/G and A/8-oxoG mismatches and increased affinities towards T/G mismatch. However, in contrast to a previous report that Mig.MthI thymine DNA glycosylase can be converted to a MutY-like adenine glycosylase by replacing two residues (A50V and L187Q), both V45A and Q182L EcMutY mutants did not exhibit any T/G or T/8-oxoG glycosylase activity. The dominant negative phenotype of V45A EcMutY mutant protein is probably caused by its increased binding affinity to T/G mismatch and thus inhibiting other repair pathways.
Improved results in proteomics by use of local and peptide-class specific false discovery rates
Authors: Sennels Lau , Bukowski-Wills Jimi-Carlo , Rappsilber Juri
Published: June 2009

Background

Proteomic protein identification results need to be compared across laboratories and platforms, and thus a reliable method is needed to estimate false discovery rates. The target-decoy strategy is a platform-independent and thus a prime candidate for standardized reporting of data. In its current usage based on global population parameters, the method does not utilize individual peptide scores optimally.

Results

Here we show that proteomic analyses largely benefit from using separate treatment of peptides matching to proteins alone or in groups based on locally estimated false discovery rates. Our implementation reduces the number of false positives and simultaneously increases the number of proteins identified. Importantly, single peptide identifications achieve defined confidence and the sequence coverage of proteins is optimized. As a result, we improve the number of proteins identified in a human serum analysis by 58% without compromising identification confidence.

Conclusion

We show that proteins can reliably be identified with a single peptide and the sequence coverage for multi-peptide proteins can be increased when using an improved estimation of false discovery rates.
The expression of the β-defensins hBD-2 and hBD-3 is differentially regulated by NF-κB and MAPK/AP-1 pathways in an in vitro model of Candida esophagitis
Authors: Steubesand Nadine , Kiehne Karlheinz , Brunke Gabriele , Pahl Rene , Reiss Karina , Herzig Karl-Heinz , Schubert Sabine , Schreiber Stefan , Fölsch Ulrich R , Rosenstiel Philip , Arlt Alexander
Published: June 2009

Background

Candida albicansresides on epithelial surfaces as part of the physiological microflora. However, under certain conditions it may cause life-threatening infections likeCandidasepsis. Human β-defensins (hBDs) are critical components of host defense at mucosal surfaces and we have recently shown that hBD-2 and hBD-3 are upregulated inCandidaesophagitis. We therefore studied the role ofCandidate signalling pathways in order to understand the mechanisms involved in regulation of hBD-expression byC. albicans. We used the esophageal cell line OE21 and analysed the role of paracrine signals from polymorphonuclear leukocytes (PMN) in anin vitromodel of esophageal candidiasis.

Results

Supernatants ofC. albicansor indirect coculture withC. albicansinduces upregulation of hBD-2 and hBD-3 expression. PMNs strongly amplifiesC. albicans-mediated induction of hBDs. By EMSA we demonstrate thatC. albicansactivates NF-κB and AP-1 in OE21 cells. Inhibition of these pathways revealed that hBD-2 expression is synergistically regulated by both NF-κB and AP-1. In contrast hBD-3 expression is independent of NF-κB and relies solely on an EGFR/MAPK/AP-1-dependent pathway.

Conclusion

Our analysis of signal transduction events demonstrate a functional interaction of epithelial cells with PMNs in response toCandidainfection involving divergent signalling events that differentially govern hBD-2 and hBD-3 expression.
Coordinated surface activities in Variovorax paradoxus EPS
Authors: Jamieson W David , Pehl Michael J , Gregory Glenn A , Orwin Paul M
Published: June 2009

Background

Variovorax paradoxus is an aerobic soil bacterium frequently associated with important biodegradative processes in nature. Our group has cultivated a mucoid strain of Variovorax paradoxus for study as a model of bacterial development and response to environmental conditions. Colonies of this organism vary widely in appearance depending on agar plate type.

Results

Surface motility was observed on minimal defined agar plates with 0.5% agarose, similar in nature to swarming motility identified in Pseudomonas aeruginosa PAO1. We examined this motility under several culture conditions, including inhibition of flagellar motility using Congo Red. We demonstrated that the presence of a wetting agent, mineral, and nutrient content of the media altered the swarming phenotype. We also demonstrated that the wetting agent reduces the surface tension of the agar. We were able to directly observe the presence of the wetting agent in the presence and absence of Congo Red, and found that incubation in a humidified chamber inhibited the production of wetting agent, and also slowed the progression of the swarming colony. We observed that swarming was related to both carbon and nitrogen sources, as well as mineral salts base. The phosphate concentration of the mineral base was critical for growth and swarming on glucose, but not succinate. Swarming on other carbon sources was generally only observed using M9 salts mineral base. Rapid swarming was observed on malic acid, d-sorbitol, casamino acids, and succinate. Swarming at a lower but still detectable rate was observed on glucose and sucrose, with weak swarming on maltose. Nitrogen source tests using succinate as carbon source demonstrated two distinct forms of swarming, with very different macroscopic swarm characteristics. Rapid swarming was observed when ammonium ion was provided as nitrogen source, as well as when histidine, tryptophan, or glycine was provided. Slower swarming was observed with methionine, arginine, or tyrosine. Large effects of mineral content on swarming were seen with tyrosine and methionine as nitrogen sources. Biofilms form readily under various culture circumstances, and show wide variance in structure under different conditions. The amount of biofilm as measured by crystal violet retention was dependent on carbon source, but not nitrogen source. Filamentous growth in the biofilm depends on shear stress, and is enhanced by continuous input of nutrients in chemostat culture.

Conclusion

Our studies have established that the beta-proteobacterium Variovorax paradoxus displays a number of distinct physiologies when grown on surfaces, indicative of a complex response to several growth parameters. We have identified a number of factors that drive sessile and motile surface phenotypes. This work forms a basis for future studies using this genetically tractable soil bacterium to study the regulation of microbial development on surfaces.
Lessons from the Pacific programme to eliminate lymphatic filariasis: a case study of 5 countries
Authors: Huppatz Clare , Capuano Corinne , Palmer Kevin , Kelly Paul M , Durrheim David N
Published: June 2009

Background

Lymphatic Filariasis (LF) is an important Neglected Tropical Disease, being a major cause of disability worldwide. The Global Programme to Eliminate Lymphatic Filariasis aims to eliminate LF as a public health problem by the year 2020, primarily through repeated Mass Drug Administration (MDA). The Pacific region programme commenced in 1999. By June 2007, five of the eleven countries classified as endemic had completed five MDA campaigns and post-MDA prevalence surveys to assess their progress. We review available programme data and discuss their implications for other LF elimination programs in developing countries.

Methods

Reported MDA coverage and results from initial surveys and post-MDA surveys of LF using the immunochromatographic test (ICT) from these five Pacific Island countries (Tonga, Niue, Vanuatu, Samoa and Cook Islands) were analysed to provide an understanding of their quality and programme progress towards LF elimination. Denominator data reported by each country programme for 2001 was compared to official sources to assess the accuracy of MDA coverage data.

Results

Initial survey results from these five countries revealed an ICT prevalence of between 2.7 and 8.6 percent in individuals tested prior to commencement of the programme. Country MDA coverage results varied depending on the source of denominator data. Of the five countries in this case study, three countries (Tonga, Niue and Vanuatu) reached the target prevalence of <1% antigenaemia following five rounds of MDA. However, endpoint data could not be reliably compared to baseline data as survey methodology varied.

Conclusion

Accurate and representative baseline and post-campaign prevalence data is crucial for determining program effectiveness and the factors contributing to effectiveness. This is emphasised by the findings of this case study. While three of the five Pacific countries reported achieving the target prevalence of <1% antigenaemia, limitations in the data preclude identification of key determinants of this achievement.
Homozygosity and risk of childhood death due to invasive bacterial disease
Authors: Lyons Emily J , Amos William , Berkley James A , Mwangi Isaiah , Shafi Mohammed , Williams Thomas N , Newton Charles R , Peshu Norbert , Marsh Kevin , Scott J Anthony G , Hill Adrian VS
Published: June 2009

Background

Genetic heterozygosity is increasingly being shown to be a key predictor of fitness in natural populations, both through inbreeding depression, inbred individuals having low heterozygosity, and also through chance linkage between a marker and a gene under balancing selection. One important component of fitness that is often highlighted is resistance to parasites and other pathogens. However, the significance of equivalent loci in human populations remains unclear. Consequently, we performed a case-control study of fatal invasive bacterial disease in Kenyan children using a genome-wide screen with microsatellite markers.

Methods

148 cases, comprising children aged <13 years who died of invasive bacterial disease, (variously, bacteraemia, bacterial meningitis or neonatal sepsis) and 137 age-matched, healthy children were sampled in a prospective study conducted at Kilifi District Hospital, Kenya. Samples were genotyped for 134 microsatellite markers using the ABI LD20 marker set and analysed for an association between homozygosity and mortality.

Results

At five markers homozygosity was strongly associated with mortality (odds ratio range 4.7 – 12.2) with evidence of interactions between some markers. Mortality was associated with different non-overlapping marker groups in Gram positive and Gram negative bacterial disease. Homozygosity at susceptibility markers was common (prevalence 19–49%) and, with the large effect sizes, this suggests that bacterial disease mortality may be strongly genetically determined.

Conclusion

Balanced polymorphisms appear to be more widespread in humans than previously appreciated and play a critical role in modulating susceptibility to infectious disease. The effect sizes we report, coupled with the stochasticity of exposure to pathogens suggests that infection and mortality are far from random due to a strong genetic basis.
Molecular analysis of the APC and MUTYH genes in Galician and Catalonian FAP families: a different spectrum of mutations?
Authors: Gómez-Fernández Nuria , Castellví-Bel Sergi , Fernández-Rozadilla Ceres , Balaguer Francesc , Muñoz Jenifer , Madrigal Irene , Milà Montserrat , Graña Begoña , Vega Ana , Castells Antoni , Carracedo Ángel , Ruiz-Ponte Clara
Published: June 2009

Background

Familial adenomatous polyposis (FAP) is an autosomal dominant-inherited colorectal cancer syndrome, caused by germline mutations in the APC gene. Recently, biallelic mutations in MUTYH have also been identified in patients with multiple colorectal adenomas and in APC-negative patients with FAP. The aim of this work is therefore to determine the frequency of APC and MUTYH mutations among FAP families from two Spanish populations.

Methods

Eighty-two unrelated patients with classical or attenuated FAP were screened for APC germline mutations. MUTYH analysis was then conducted in those APC-negative families and in 9 additional patients from a previous study. Direct sequencing, SSCP analysis and TaqMan genotyping were used to identify point and frameshift mutations, meanwhile large rearrangements in the APC gene were screened by multiplex ligation-dependent probe amplification (MLPA).

Results

APC germline mutations were found in 39% of the patients and, despite the great number of genetic variants described so far in this gene, seven new mutations were identified. The two hotspots at codons 1061 and 1309 of the APC gene accounted for 9,4% of the APC-positive families, although they were underrepresented in Galician samples. The deletion at codon 1061 was not found in 19 APC-positive Galician patients but represented 23% of the Catalonian positive families (p = 0,058). The same trend was observed at codon 1309, even though statistical analysis showed no significance between populations. Twenty-four percent of the APC-negative patients carried biallelic MUTYH germline mutations, and showed an attenuated polyposis phenotype generally without extracolonic manifestations. New genetic variants were found, as well as the two hotspots already reported (p.Tyr165Cys and p.Gly382Asp).

Conclusion

The results we present indicate that in Galician patients the frequency of the hotspot at codon 1061 in APC differs significantly from the Catalonian and also other Caucasian populations. Similar results had already been obtained in a previous study and could be due to the genetic isolation of the Galician population. MUTYH analysis is also recommended for all APC-negative families, even if a recessive inheritance is not confirmed.
Variations in the NBN/NBS1 gene and the risk of breast cancer in non-BRCA1/2French Canadian families with high risk of breast cancer
Authors: Desjardins Sylvie , Beauparlant Joly Charles , Labrie Yvan , Ouellette Geneviève ,   INHERIT BRCAs, Durocher Francine
Published: June 2009

Background

The Nijmegen Breakage Syndrome is a chromosomal instability disorder characterized by microcephaly, growth retardation, immunodeficiency, and increased frequency of cancers. Familial studies on relatives of these patients indicated that they also appear to be at increased risk of cancer.

Methods

In a candidate gene study aiming at identifying genetic determinants of breast cancer susceptibility, we undertook the full sequencing of theNBNgene in our cohort of 97 high-risk non-BRCA1and -BRCA2breast cancer families, along with 74 healthy unrelated controls, also from the French Canadian population.In silicoprograms (ESEfinder, NNSplice, Splice Site Finder and MatInspector) were used to assess the putative impact of the variants identified. The effect of the promoter variant was further studied by luciferase gene reporter assay in MCF-7, HEK293, HeLa and LNCaP cell lines.

Results

Twenty-four variants were identified in our case series and their frequency was further evaluated in healthy controls. The potentially deleterious p.Ile171Val variant was observed in one case only. The p.Arg215Trp variant, suggested to impair NBN binding to histone γ-H2AX, was observed in one breast cancer case and one healthy control. A promoter variant c.-242-110delAGTA displayed a significant variation in frequency between both sample sets. Luciferase reporter gene assay of the promoter construct bearing this variant did not suggest a variation of expression in the MCF-7 breast cancer cell line, but indicated a reduction of luciferase expression in both the HEK293 and LNCaP cell lines.

Conclusion

Our analysis ofNBNsequence variations indicated that potentialNBNalterations are present, albeit at a low frequency, in our cohort of high-risk breast cancer cases. Further analyses will be needed to fully ascertain the exact impact of those variants on breast cancer susceptibility, in particular for variants located inNBNpromoter region.
High-titer preparation of Bombyx mori nucleopolyhedrovirus (BmNPV) displaying recombinant protein in silkworm larvae by size exclusion chromatography and its characterization
Authors: Kato Tatsuya , Manoha Suganthi Lavender , Tanaka Shigeyasu , Park Enoch Y
Published: June 2009

Background

Budded baculoviruses are utilized for vaccine, the production of antibody and functional analysis of transmembrane proteins. In this study, we tried to produce and purify the recombinant Bombyx mori nucleopolyhedrovirus (rBmNPV-hPRR) that displayed human (pro)renin receptor (hPRR) connected with FLAG peptide sequence on its own surface. These particles were used for further binding analysis of hPRR to human prorenin. The rBmNPV-hPRR was produced in silkworm larvae and purified from its hemolymph using size exclusion chromatography (SEC).

Results

A rapid method of BmNPV titer determination in hemolymph was performed using quantitative real-time PCR (Q-PCR). A correlation coefficient of BmNPV determination between end-point dilution and Q-PCR methods was found to be 0.99. rBmNPV-hPRR bacmid-injected silkworm larvae produced recombinant baculovirus of 1.31 × 108 plaque forming unit (pfu) in hemolymph, which was 2.8 × 104 times higher than transfection solution in Bm5 cells. Its purification yield by Sephacryl S-1000 SF column chromatography was 264 fold from larval hemolymph at 4 days post-injection (p.i.), but 35 or 39 fold at 4.5 or 5 days p.i., respectively. Protein patterns of rBmNPV-hPRR purified at 4 and 5 days were the same and ratio of envelope proteins (76, 45 and 35 kDa) to VP39, one of nucleocapsid proteins, increased at 5 days p.i. hPRR was detected in only purified rBmNPV-hPRR at 5 days p.i..

Conclusion

The successful purification of rBmNPV-hPRR indicates that baculovirus production using silkworm larvae and its purification from hemolymph by Sephacryl S-1000 SF column chromatography can provide an economical approach in obtaining the purified BmNPV stocks with high titer for large-scale production of hPRR. Also, it can be utilized for further binding analysis and screening of inhibitors of hPRR.
Substantial reduction of inappropriate tablet splitting with computerised decision support: a prospective intervention study assessing potential benefit and harm
Authors: Quinzler Renate , Schmitt Simon PW , Pritsch Maria , Kaltschmidt Jens , Haefeli Walter E
Published: June 2009

Background

Currently ambulatory patients break one in four tablets before ingestion. Roughly 10% of them are not suitable for splitting because they lack score lines or because enteric or modified release coating is destroyed impairing safety and effectiveness of the medication. We assessed impact and safety of computerised decision support on the inappropriate prescription of split tablets.

Methods

We performed a prospective intervention study in a 1680-bed university hospital. Over a 15-week period we evaluated all electronically composed medication regimens and determined the fraction of tablets and capsules that demanded inappropriate splitting. In a subsequent intervention phase of 15 weeks duration for 10553 oral drugs divisibility characteristics were indicated in the system. In addition, an alert was generated and displayed during the prescription process whenever the entered dosage regimen demanded inappropriate splitting (splitting of capsules, unscored tablets, or scored tablets unsuitable for the intended fragmentation).

Results

During the baseline period 12.5% of all drugs required splitting and 2.7% of all drugs (257/9545) required inappropriate splitting. During the intervention period the frequency of inappropriate splitting was significantly reduced (1.4% of all drugs (146/10486); p = 0.0008). In response to half of the alerts (69/136) physicians adjusted the medication regimen. In the other half (67/136) no corrections were made although a switch to more suitable drugs (scored tablets, tablets with lower strength, liquid formulation) was possible in 82% (55/67).

Conclusion

This study revealed that computerised decision support can immediately reduce the frequency of inappropriate splitting without introducing new safety hazards.