Rodrigo Labouriau

Droplet Microfluidics Platform for Highly Sensitive and Quantitative Detection of Malaria-Causing Plasmodium Parasites Based on Enzyme Activity Measurement

We present an attractive new system for the specific and sensitive detection of the malaria-causing Plasmodium parasites. The system relies on isothermal conversion of single DNA cleavage-ligation events catalyzed specifically by the Plasmodium enzyme topoisomerase I to micrometer-sized products detectable at the single-molecule level. Combined with a droplet microfluidics lab-on-a-chip platform, this design allowed for sensitive, specific, and quantitative detection of all human-malaria-causing Plasmodium species in single drops of unprocessed blood with a detection limit of less than one parasite/μL. Moreover, the setup allowed for detection of Plasmodium parasites in noninvasive saliva samples from infected patients. During recent years malaria transmission has declined worldwide, and with this the number of patients with low-parasite density has increased. Consequently, the need for accurate detection of even a few parasites is becoming increasingly important for the continued combat against the disease. We believe that the presented droplet microfluidics platform, which has a high potential for adaptation to point-of-care setups suitable for low-resource settings, may contribute significantly to meet this demand. Moreover, potential future adaptation of the presented setup for the detection of other microorganisms may form the basis for the development of a more generic platform for diagnosis, fresh water or food quality control, or other purposes within applied or basic science.

Body metal concentrations and glycogen reserves in earthworms (Dendrobaena octaedra) from contaminated and uncontaminated forest soil.

Stress originating from toxicants such as heavy metals can induce compensatory changes in the energy metabolism of organisms due to increased energy expenses associated with detoxification and excretion processes. These energy expenses may be reflected in the available energy reserves such as glycogen. In a field study the earthworm, Dendrobaena octaedra, was collected from polluted areas, and from unpolluted reference areas. If present in the environment, cadmium, lead and copper accumulated to high concentrations in D. octaedra. In contrast, other toxic metals such as aluminium, nickel and zinc appeared to be regulated and kept at low internal concentrations compared to soil concentrations. Lead, cadmium and copper accumulation did not correlate with glycogen reserves of individual worms. In contrast, aluminium, nickel and zinc were negatively correlated with glycogen reserves. These results suggest that coping with different metals in earthworms is associated with differential energy demands depending on the associated detoxification strategy.

Selecting high-dimensional mixed graphical models using minimal AIC or BIC forests

BackgroundChow and Liu showed that the maximum likelihood tree for multivariate discrete distributions may be found using a maximum weight spanning tree algorithm, for example Kruskals algorithm. The efficiency of the algorithm makes it tractable for high-dimensional problems.ResultsWe extend Chow and Lius approach in two ways: first, to find the forest optimizing a penalized likelihood criterion, for example AIC or BIC, and second, to handle data with both discrete and Gaussian variables. We apply the approach to three datasets: two from gene expression studies and the third from a genetics of gene expression study. The minimal BIC forest supplements a conventional analysis of differential expression by providing a tentative network for the differentially expressed genes. In the genetics of gene expression context the method identifies a network approximating the joint distribution of the DNA markers and the gene expression levels.ConclusionsThe approach is generally useful as a preliminary step towards understanding the overall dependence structure of high-dimensional discrete and/or continuous data. Trees and forests are unrealistically simple models for biological systems, but can provide useful insights. Uses include the following: identification of distinct connected components, which can be analysed separately (dimension reduction); identification of neighbourhoods for more detailed analyses; as initial models for search algorithms with a larger search space, for example decomposable models or Bayesian networks; and identification of interesting features, such as hub nodes.

Cyclooxygenase-2, multidrug resistance 1, and breast cancer resistance protein gene polymorphisms and inflammatory bowel disease in the Danish population

Objective. Crohns disease (CD) and ulcerative colitis (UC) are characterized by an impaired mucosal defence to normal constituents of the intestinal flora and a dysregulated inflammatory response. The purpose of the study was to investigate whether single nucleotide polymorphisms (SNPs) in genes involved in these processes were associated with CD and UC. Material and methods. Allele frequencies of the cyclooxygenase 2 (COX-2/PTGS2/PGHS2) G-765C and breast cancer resistance protein (BCRP/ABCG2) C421A as well as allele and haplotype frequencies of multidrug resistance 1 (MDR1, ABCB1) SNPs G2677T/A, C3435T and G-rs3789243-A (intron 3) were assessed in a Danish case-control study comprising 373 CD and 541 UC patients and 796 healthy controls. Results. Carriers of the homozygous COX-2 and MDR1 intron 3 variant had a relatively high risk of CD, odds ratio (95% CI) (OR (95% CI))=2.86 ((1.34–5.88) p=0.006) and 1.39 ((0.99–1.92) p=0.054), respectively, and for UC of 2.63 ((1.33–5.26) p=0.005) and 1.28 ((0.96–1.51) p=0.093), respectively, assuming complete dominance. No association was found for BCRP or other MDR1 SNPs, or for selected MDR1 haplotypes. No effect-modification of smoking habit at the time of diagnosis was found. Conclusions. An effect of the COX-2 polymorphism on both CD and UC was shown which is compatible with the presence of a recessive allele in linkage equilibrium with the SNP marker in the COX-2 gene. The polymorphism located in intron 3 of the MDR1 gene showed a weak association with CD, and a marginally suggestive association with UC.

Probability of pregnancy as affected by oestrus number and days to first oestrus in dairy cows of three breeds and parities

An improved understanding of the animal factors that affect measures such as conception rate would contribute to solving the problem of impaired reproductive performance in modern dairy cows. A question of particular interest relates to the observed improvement in conception rates from first to second and third oestrus cycle: is the increase in conception rate related to cycle number per se or to increasing days from calving? A 5-year study using three breeds (Holstein, Jersey and Danish Red) allowed this issue to be examined. In 560 lactations, from calving until confirmed pregnancy or until 290 days after calving all cows were observed daily for signs of oestrus. Inseminations were carried out to all clear cases of oestrus after 35 days from calving until the cow was judged to be pregnant or until 290 days post-calving. Days from calving to the first oestrus was modeled using a frailty model. Danish Red cows had a significantly greater rate of occurrence of first oestrus over time. Generalized linear mixed models defined using a binomial distribution and logit link function were used to estimate probability of pregnancy as affected by: breed, parity, oestrus number and days from calving to first oestrus. In order to account for the paradox that the incidence of cows with inherently low fertility increases with oestrus number, different random components for cow were included. There was a significant increase in probability of pregnancy from first to second oestrus but no significant change in probability of pregnancy thereafter. There was a significant increase in probability of pregnancy with increasing days to first oestrus. This indicates that both oestrus number and days from calving play a role in determining the fertility of dairy cows. Correcting for inherent differences in fertility was shown to be important.

In vitro induction of the acrosome reaction in bull sperm and the relationship to field fertility using low-dose inseminations.

The acrosome reaction (AR) is a prerequisite for normal sperm fertilizing capability and can be studied in vitro after induction by various agents. The efficacy of a sperm population to undergo the AR in vivo is expected to influence male fertilizing potential. During the past two decades, a number of attempts have been made to relate the in vitro-induced AR to field fertility in several species. However, to our knowledge, no studies have combined in vitro induction of the AR with the simultaneous detection of sperm viability and acrosomal status using a high-precision flow cytometric technique. Furthermore, large-scale fertility trials using low-dose inseminations are pending. In the current study, the relationship between field fertility and the in vitro-induced AR was investigated using three ejaculates from each of 195 bulls, 156 Holstein and 39 Jersey bulls (Bos taurus), participating in a progeny test program including low-dose inseminations. A range of insemination doses, varying from 2.0 x 10(6) to 15 x 10(6) sperm/dose, was obtained by a controlled dilution process applied to each ejaculate. Different insemination doses were distributed at random among 75,610 experimental first inseminations in 4721 herds and 208 artificial insemination (AI) technicians. Simultaneous detection of sperm viability and acrosomal status was achieved using a triple color flow cytometric technique. Sperm samples from the bulls displayed a wide range of ability to acrosome react in response to calcium ionophore A23187. Both reproducibility of the AR response after induction and relationship between ability to acrosome react and field fertility was highly dependent on the definition of AR inducibility. Six basic and six combined AR indices were assessed. The AR index expressing the fraction of acrosome reacted sperm in the live sperm population after induction by ionophore had the highest repeatability, best described the biological variation in the studied population, and yielded the best significant predictive values on field fertility among the 12 indices considered. Moreover, the ability of sperm to acrosome react appeared to be a noncompensable trait that affects fertility regardless of the number of sperm per insemination dose. The current results therefore indicate that this sperm parameter is important in the field and also may play a role in the IVF laboratory.

Detection and estimation of conidial abundance of Penicillium verrucosum in soil by dilution plating on a selective and diagnostic agar medium (DYSG)

Penicillium verrucosum is one of the main producers of ochratoxin A (OA) in agricultural commodities. To forecast the risk of OA contamination, there is a need to improve our knowledge on the ecology of P. verrucosum in the field. Dilution plating on ‘dichloran yeast extract sucrose 18% glycerol agar’ (DYSG) offers a simple and very sensitive method of detecting P. verrucosum propagules in soil. The properties of DYSG are illustrated in a suspension mixture experiment and confirmed in a soil mixture experiment. In the latter, P. verrucosum could be detected in conidial concentrations below 200 colony forming units (cfu) g −1 soil even when it constituted no more than 0.3% of the cfu. Furthermore, the DYSG method can be used to estimate the abundance of P. verrucosum propagules in soil with good precision. In some of the analysed cases, however, it was necessary to use appropriate mathematical models to treat results with high numbers of cfu on the Petri dishes.

Relationship among seminal quality measures and field fertility of young dairy bulls using low-dose inseminations.

Optimal use of genetically superior bulls through artificial insemination (AI) is highly dependent on precise assessment of seminal quality which allows for reasonable estimations of field fertility with normal or low-dose inseminations. In the present study, seminal measures such as sperm motility and morphology, sperm viability, sperm DNA fragmentation, and the ability of the sperm to display an acrosome reaction were tested. The relationships between field fertility and the seminal measures were investigated using 3 ejaculates from each of 195 bulls (156 Holstein and 39 Jersey) participating in a progeny test program. A range of AI doses, varying from 2×10(6) to 15×10(6) sperm/straw, was obtained by a controlled dilution process applied to each ejaculate. The different AI doses were distributed at random among 75,610 experimental first inseminations in 4,721 herds and 208 AI technicians. Most of the seminal measures appeared to contain a predictive value for the nonreturn to estrus at 56 d post-AI (NRR56) regardless of the number of sperm per AI dose and can be regarded as noncompensable sperm traits. But, due to correlations between the individual measures, the best model for describing (and predicting) NRR56 was based on sperm concentration and viability in the neat (raw) semen, and post-thaw sperm viability. The statistical models for describing NRR56 included the following explanatory variables: strength of the estrus, number of sperm per AI dose, breed, parity, and random components representing herds and AI technicians. The present results show that the most precise estimation of a bulls NRR56 can be achieved through flow cytometric detection of sperm concentration and viability in neat semen as well as flow cytometric detection of post-thaw sperm viability.

Comment on "An Association Between the Kinship and Fertility of Human Couples"

Helgason et al. (Reports, 8 February 2008, p. 813) reported a positive association between kinship and fertility in the Icelandic population. We point out that the data further suggest that fertility initially increases with kinship and then decays. This is supported by another large study on the Danish population suggesting a superposition of effects of inbreeding and outbreeding depression on human fertility.

Distribution of ergosterol in organically and conventionally cultivated agricultural soils.

ABSTRACT Ergosterol is generally assumed to be one of the best indicators of fungal biomass and soil quality. The present case studies describe variations in ergosterol content in soil, sampled at three Danish farms. One is organically cultivated according to the biodynamical principles (ORG). The other two are conventionally cultivated, one with animal husbandry (CONV-H) and one with plant production (CONV-P). The analytical method combined extraction in a microwave oven, clean-up with a new SPE sorbent material (OasisTM), and quantification by HPLC. The average estimated concentrations of ergosterol were 1.66 μg g−1 in ORG, 2.35 μg g−1 in CONV-H and 1.13 μg g−1 in CONV-P. These results correlated well with some of the important variables in the management systems of the soils (frequency of grass and clover, input of animal manure, total-C, frequency of ploughing). However, the exact causal relationships cannot be revealed using the case-study approach. The statistical analysis of the spatial variation in ergosterol across the field demonstrated very clearly that in some soils, small-scale (few cm2) variations in ergosterol concentrations may be quite large in comparison with large scale (few hundred m2) variations. This should be taken into account when planning the sampling strategy.