Detection and correction of blinking bias in image correlation transport measurements of quantum dot tagged macromolecules

Semiconductor nanocrystals or quantum dots (QDs) are becoming widely used as fluorescent labels for biological applications. Here we demonstrate that fluorescence fluctuation analysis of their diffusional mobility using temporal image correlation spectroscopy is highly susceptible to systematic errors caused by fluorescence blinking of the nanoparticles. Temporal correlation analysis of fluorescence microscopy image time series of streptavidin-functionalized (CdSe)ZnS QDs freely diffusing in two dimensions shows that the correlation functions are fit well to a commonly used diffusion decay model, but the transport coefficients can have significant systematic errors in the measurements due to blinking. Image correlation measurements of the diffusing QD samples measured at different laser excitation powers and analysis of computer simulated image time series verified that the effect we observe is caused by fluorescence intermittency. We show that reciprocal space image correlation analysis can be used for mobility measurements in the presence of blinking emission because it separates the contributions of fluctuations due to photophysics from those due to transport. We also demonstrate application of the image correlation methods for measurement of the diffusion coefficient of glycosyl phosphatidylinositol-anchored proteins tagged with QDs as imaged on living fibroblasts.     

Anthrax in the Gambia: an epidemiological study

Epidemiological data on 448 cases of human cutaneous anthrax from the Gambia showed that this particular strain of anthrax bacillus causes widespread morbidity and some mortality with, at the same time, subclinical infection. Analysis also showed that anthrax is not an occupationally related disease in the Gambia.The possibility of human-to-human spread, affecting all age groups and both sexes, by means of a communal toilet article was also shown. The fact that the strain is a good toxin producer but contains a weak antigen may have accounted for the repeated clinical infection and the fact that antibody titres were generally transient. Subclinical infection in animals was also found, particularly in sheep and goats, and also, with an unusually low mortality, in cows. Insect vectors were not excluded, but were unlikely. Vultures may spread the disease from village to village. Some possible public health and immunization procedures are discussed, with a view to containing this difficult problem in this part of west Africa.     

Pseudogene: lessons from PCR bias,identification and resurrection

Pseudogenes are fragments of non-functional genomic DNA with high sequences similarity to normal functional genes. They are a kind of non-coding DNA produced by gene duplications or retrotranspositions. Pseudogenes exist in human genome at a large quantity which is nearly as much as that of normal functional genes. They could cause PCR bias in molecular biology experiments and confuse related analysis. On the other hand, pesudogenes are important elements in genomics study for getting an integral picture of genome annotation. They give diverse information of evolutionary history and are regarded as genome fossils. Worldwide research project “encyclopedia of DNA elements”(ENCODE) founded in recent years have enhanced our understanding of pseudogenes. Approaches established to identify pseudogenes include PseudoPipe, HAVANA method, PseudoFinder, RetroFinder, GIS-PET method and consensus method. This paper discuss pseudogenes with respect to the formation mechanisms, distribution, and problems for PCR, importance and identification of pseudogenes. Furthermore, potential resurrection of pseudogenes and their potential function are discussed.     

Metabolic footprint of diabetes: a multiplatform metabolomics study in an epidemiological setting

Background

Metabolomics is the rapidly evolving field of the comprehensive measurement of ideally all endogenous metabolites in a biological fluid. However, no single analytic technique covers the entire spectrum of the human metabolome. Here we present results from a multiplatform study, in which we investigate what kind of results can presently be obtained in the field of diabetes research when combining metabolomics data collected on a complementary set of analytical platforms in the framework of an epidemiological study.

Methodology/Principal Findings

40 individuals with self-reported diabetes and 60 controls (male, over 54 years) were randomly selected from the participants of the population-based KORA (Cooperative Health Research in the Region of Augsburg) study, representing an extensively phenotyped sample of the general German population. Concentrations of over 420 unique small molecules were determined in overnight-fasting blood using three different techniques, covering nuclear magnetic resonance and tandem mass spectrometry. Known biomarkers of diabetes could be replicated by this multiple metabolomic platform approach, including sugar metabolites (1,5-anhydroglucoitol), ketone bodies (3-hydroxybutyrate), and branched chain amino acids. In some cases, diabetes-related medication can be detected (pioglitazone, salicylic acid).

Conclusions/Significance

Our study depicts the promising potential of metabolomics in diabetes research by identification of a series of known and also novel, deregulated metabolites that associate with diabetes. Key observations include perturbations of metabolic pathways linked to kidney dysfunction (3-indoxyl sulfate), lipid metabolism (glycerophospholipids, free fatty acids), and interaction with the gut microflora (bile acids). Our study suggests that metabolic markers hold the potential to detect diabetes-related complications already under sub-clinical conditions in the general population.     

Estimating local interaction from spatiotemporal forest data, and Monte Carlo bias correction

We point out a general problem in fitting continuous time spatially explicit models to a temporal sequence of spatial data observed at discrete times. To illustrate the problem, we examined the continuous time Markov model for forest gap dynamics. A forest is assumed to be apportioned into discrete cells (or sites) arranged in a regular square lattice. Each site is characterized as either a gap or a non-gap site according to the vegetation height of trees. The model incorporates the influence of neighboring sites on transition rate: transition rate from a non-gap to a gap site increases linearly with the number of neighbors that are currently in the gap state, and vice versa. We fitted the model to the spatiotemporal data of canopy height observed at the permanent plot in Barro Colorado Island (BCI). When we used the approximate maximum likelihood method to estimate the parameters of the model, the estimated transition rates included a large bias-in particular, the strength of interaction between nearby sites was underestimated. This bias originated from the assumption that each transition between two observation times is independent. The interaction between sites at local scale creates a long chain of transitions within a single census interval, which violates the independence of each transition. We show that a computer-intensive method, called Monte Carlo bias correction (MCBC), is very effective in removing the bias included in the estimate. The global and local gap densities measuring spatial aggregation of gap sites were computed from simulated and real gap dynamics to assess the model. When the approximate likelihood estimates were applied to the model, the predicted local gap density was clearly lower than the observed one. The use of MCBC estimates, suggesting a strong interaction between sites, improved this discrepancy.     

The transmission of campylobacter in piggeries; an epidemiological study

The campylobacter infection of 10 sows and their piglets was monitored. These pigs werekept on two multiplier farms. Rectal faeces samples were taken from the sows shortly beforelittering and at different intervals after littering. Swab samples of rectal content were taken fromsix piglets per sow at different intervals after birth. Nine sows were shown to be infected withcampylobacter before litter and all sows after litter, with an average colony count of 4·1in log N g^–1 of faeces. Half of the piglets became infected withcampylobacter during the first week of life and 85%, after four weeks. Two genetic subtypingmethods (ERIC-PCR and RFLP) were used to study the relationships between campylobacterisolates from sows and piglets. A large diversity of campylobacter subtypes was found.Nevertheless, piglets and their mothers often harboured campylobacter isolates with identicalgenetic subtyping profiles, suggesting that piglets become infected via their mothers. However,observed similarities in genetic subtyping profiles between campylobacters isolated on differentfarms made this difficult to prove.     

Systematic bias in high-throughput sequencing data and its correction by BEADS

Genomic sequences obtained through high-throughput sequencing are not uniformly distributed across the genome. For example, sequencing data of total genomic DNA show significant, yet unexpected enrichments on promoters and exons. This systematic bias is a particular problem for techniques such as chromatin immunoprecipitation, where the signal for a target factor is plotted across genomic features. We have focused on data obtained from Illumina's Genome Analyser platform, where at least three factors contribute to sequence bias: GC content, mappability of sequencing reads, and regional biases that might be generated by local structure. We show that relying on input control as a normalizer is not generally appropriate due to sample to sample variation in bias. To correct sequence bias, we present BEADS (bias elimination algorithm for deep sequencing), a simple three-step normalization scheme that successfully unmasks real binding patterns in ChIP-seq data. We suggest that this procedure be done routinely prior to data interpretation and downstream analyses.     

Perspectives for an epidemiological study of hairy cell leukemia

A strong male predominance (4/1) has been noticed in all series of hairy cell leukemia (HCL) and we wonder whether there could be a link between male predominance and occupation. From a series of 161 patients observed by two different groups, the repartition of profession suggest an aetiological link between HCL and occupational exposure, particularly radiation, benzene and other solvents, since it appears that the proportion of medical workers (6%), mechanic divers (22%), printers and painters (10%) and farmers (11%) represent a high proportion of patients. Aware of the relative rarity of the disease we suggested to set up a national registry of the cases of HCL, the precise incidence of which remains unknown, and to start a classical case-referent study.     

Postpartum anestrus in French beef cattle: an epidemiological study

Postpartum anestrus of lactating beef cows was studied by means of an epidemiological study carried out on 878 lactating beef cows in 60 French herds. The cows calved between October 1992 and March 1993 and were housed 2 mo after calving, when the anestrus status was determined by progesterone radioimmunoassays. Data analysis was performed using a multiple logistic model in order to adjust for confounding and interaction. Fifty-one percent of the primiparous and 23% of the multiparous cows were found to be in anestrus. Factors significantly related to anestrus were parity (primiparous); breed (Charolais); housing type (tie housing); suckling (compared to weaning at birth); and, among those that were under the control of the farmer, calving conditions (manual exploration of the birth canal); body condition score at calving (3 or less, on a 5-point scale); and loss in body condition score after calving (1 point or more within 2 mo). Previous reproductive performance for multiparous cows such as a long calving interval and induced estrus in the previous year also appeared to be related to anestrus.     

Pulsed-field gel electrophoresis as an epidemiological tool for clonal identification of Aeromonas hydrophila

Pulsedfield gel electrophoresis (PFGE) was used to characterize Aeromonas hydrophila strains isolated from a cluster of hospital-acquired infections that occurred over approximately 1 month in a French hospital. Five isolates from patients and 10 isolates from the water supply were characterized by biotyping and antibiotic susceptibility patterns and compared with 10 epidemiologically unrelated strains isolated from patients and rivers, by PFGE of digests of chromosomal DNA. Five environmental and four clinical isolates belonged to the same biotype and antibiotic susceptibility pattern type. The endonucleases XbaI, SpeI and SwaI gave satisfactory profiles whereas DraI did not. The profiles were stable, reproducible and discriminatory. The 10 epidemiologically unrelated strains exhibited 10 different patterns after digestion with XbaI , the least expensive, suitable endonuclease. PFGE is a rapid and discriminatory technique for the typing of Aeromonas hydrophila where a common origin of infection is suspected.     

Some comments on anderson and pospahala's correction of bias in line transect sampling

ANDERSON and POSPAHALA (1970) investigated the estimation of wildlife population size using the belt or line transect sampling method and devised a correction for bias, thus leading to an estimator with interesting characteristics. This work was given a uniform mathematical framework in BURNHAM and ANDERSON (1976). In this paper we show that the ANDERSON-POSPAHALA estimator is optimal in the sense of being the (unique) best linear unbiased estimator within the class of estimators which are linear combinations of cell frequencies, provided certain assumptions are met.     

The identification, typing and fingerprinting of Salmonella: laboratory aspects and epidemiological applications

Since the 1930s, traditional methods of strain identification based on serotyping and phage typing have been the foundation of salmonella epidemiology. Although the incidence of diseases such as typhoid and paratyphoid has decreased in recent years, food-poisoning caused by non-typhoidal salmonella strains has now reached epidemic proportions in many countries, despite improvements in sanitation and hygiene. Precise strain identification is an essential prerequisite for epidemiological investigations aimed at combating the spread of these strains and eradicating the sources of infection. Modern methods of genotypic typing, particularly those based on physical characterization of the plasmid content of the organism have already proved invaluable for the identification and differentiation of strains in many outbreaks. These plasmid typing methods are now increasingly used with serotyping and phage typing for many epidemiological investigations. Other methods of genotypic typing, particularly those based on recognition of small differences in chromosome structure, are not yet practical for the examination of large numbers of strains. Nevertheless, improvements in small-scale methods for chromosomal DNA extraction coupled with the increasing use of non-isotopic labels for identification of restriction fragment length polymorphisms may provide a new dimension to Salmonella epidemiology.     

Onychomycosis: Multicentre epidemiological, clinical and mycological study

BackgroundOnychomycosis accounts for up to 50% of all nail disorders. They can be caused by: yeasts, dermatophytes and non-dermatophyte moulds.Objectives and methodsA multicentre study designed to determine the prevalence, mycological test results, aetiological agents, and clinical presentation of onychomycosis was carried out. All fingernail and toenail samples taken during a one year period at 9 diagnostic centres were included.ResultsA total of 5,961 samples were analysed, of which 82.3% were from toenails and 17.7% from fingernails. The mean age of the patients was 49.7 years, and 66% were females. Direct microscopic examination was positive in 61% of the samples. In adults, 61.2% of toenails were positive using potassium hydroxide (KOH), and 43.7% were positive in cultures. The prevailing aetiological agents belong to the dermatophyte group (82.8%), and distal subungual was the most common clinical form. In fingernails, direct examination showed 59.8% positive samples, and cultures were positive in 52.9%. The prevailing agents were yeasts belonging to Candida species, and onycholysis was the most common lesion.ConclusionsDirect mycological examinations were positive in 61%, a higher value than that found in other series. Dermatophytes were prevalent in toenails of both sexes, and in finger nails yeast were prevalent in females, and dermatophytes in males. Non-dermatophyte moulds corresponded to 4.8% of toenail and 2.05% of fingernails isolates.