Urinary 5-aminolevulinic acid in lead-exposed children

Lead intoxication can interfere with haem synthesis and alter the concentration of haem precursors, such as the neurotoxin 5-aminolevulinic acid, in plasma and urine. The relationship between blood lead concentration (PbB), a biomarker of lead exposure, and 5-aminolevulinic acid concentration in urine (ALAU), a biomarker of the early biological effect of lead, was examined in lead-exposed children. ALAU was assayed by chemical derivatization and high performance liquid chromatography with fluorescence detection. The study subjects were 79 children with moderate to high lead exposure recruited from a lead-poisoning prevention clinic. Their urine had been previously analysed for creatinine (CR) concentration and the benzene metabolite trans,trans- muconic acid, and their blood had been analysed for lead. We found that ALAU was not correlated with PbB (Spearman r=0.088, p=0.44), but the ratio ALAU/CR was correlated with PbB (Spearman r=0.22, p=0.054). Creatinine and ALAU concentrations were higher in urine samples collected in the afternoon than those collected in the morning, a finding that is consistent with known diurnal variation. However the ratio ALAU/CR was not different in morning and afternoon urines, supporting the use of creatinine adjustment of ALAU analysis of spot urine samples. In view of the neurotoxic properties of ALA, future validation studies of biomarkers of lead exposure and effect in children should include ALAU or ALAU/CR as potential markers of lead effect.     

Differentiation of Human Embryonic Stem Cells into Cells with Corneal Keratocyte Phenotype

Corneal transparency depends on a unique extracellular matrix secreted by stromal keratocytes, mesenchymal cells of neural crest lineage. Derivation of keratocytes from human embryonic stem (hES) cells could elucidate the keratocyte developmental pathway and open a potential for cell-based therapy for corneal blindness. This study seeks to identify conditions inducing differentiation of pluripotent hES cells to the keratocyte lineage. Neural differentiation of hES cell line WA01(H1) was induced by co-culture with mouse PA6 fibroblasts. After 6 days of co-culture, hES cells expressing cell-surface NGFR protein (CD271, p75NTR) were isolated by immunoaffinity adsorption, and cultured as a monolayer for one week. Keratocyte phenotype was induced by substratum-independent pellet culture in serum-free medium containing ascorbate. Gene expression, examined by quantitative RT-PCR, found hES cells co-cultured with PA6 cells for 6 days to upregulate expression of neural crest genes including NGFR, SNAI1, NTRK3, SOX9, and MSX1. Isolated NGFR-expressing cells were free of PA6 feeder cells. After expansion as a monolayer, mRNAs typifying adult stromal stem cells were detected, including BMI1, KIT, NES, NOTCH1, and SIX2. When these cells were cultured as substratum-free pellets keratocyte markers AQP1, B3GNT7, PTDGS, and ALDH3A1 were upregulated. mRNA for keratocan (KERA), a cornea-specific proteoglycan, was upregulated more than 10,000 fold. Culture medium from pellets contained high molecular weight keratocan modified with keratan sulfate, a unique molecular component of corneal stroma. These results show hES cells can be induced to differentiate into keratocytes in vitro. Pluripotent stem cells, therefore, may provide a renewable source of material for development of treatment of corneal stromal opacities.     

Hair snaring and molecular genetic identification for reconstructing the spatial structure of Eurasian lynx populations

Non-invasive genetic sampling (NGS) is being increasingly applied in wildlife monitoring and population genetic research. This study was designed to evaluate the use of NGS for reconstructing the spatial structure of populations of large felids. We developed a procedure for reliably genotyping individuals of Eurasian lynx (Lynx lynx) from samples obtained through a hair-trapping scheme based on a network of lynx scent-marking sites. The spatial locations of the identified genotypes were matched with the home ranges distribution of radio-tracked individuals, thus cross-checking the accuracy of the two methods. We analyzed DNA extracted from 170 hair samples and 11 blood samples from live-trapped lynx collected in 2004–2009 in the Bia?owie?a Primeval Forest, Poland. We obtained PCR products in 96 (67%) hair samples; 82 (85%) of them were reliably genotyped at 12 autosomal microsatellite loci following a multiple-tubes protocol and stringent quality-controls of the data set. The sample included 29 distinct genotypes; 18 were found only in hair samples, five were determined only in live-trapped animals, and six in both hair and blood samples. Based on linkage disequilibrium we estimated an effective population size N_e = 20.3 (90% CI = 15–28). The total population size estimated with Capwire was N_c = 32 (95% CI = 25–37) in close agreement with the observed number of genotypes. The genotypes obtained from hair samples were re-sampled on average 3.9 times and 50% of them were recorded for more than one year. The spatial distribution of six hair-genotypes was consistent with their home ranges obtained by radio-tracking in the same period. The distribution ranges of hair-trapped genotypes overlapped on average in 86.4% (mode 100%) with home ranges of the corresponding individuals. Hair-trapping and molecular identification is a reliable method for reconstructing the spatial organization of lynx population. It is likely to be also efficiently used in other rare and endangered species of felids in combination with data from other monitoring techniques, such as radio- and snow-tracking and photo-trapping.     

She gets many and she chooses the best: polygynandry in Salamandrina perspicillata (Amphibia: Salamandridae)

Polyandry is a widespread mating strategy, found in a broad number of taxa. Among amphibians, polyandry, and multiple paternity as its direct consequence, is quite common in salamanders, especially within Ambystomatidae and Plethodontidae. In the suborder Salamadroidea the existence of two different types of spermatheca allows several kinds of polyandry strategies to appear. We used multilocus microsatellite genotyping to investigate the presence of polyandry and its effects on the paternity in a previously unstudied species with a terrestrial habit, Salamandrina perspicillata. We collected gravid females in their natural habitat and analysed the paternity of the offspring by using the software COLONY and GERUD. We found that all the analysed clutches had been fertilized by 2–4 males and that in every clutch one male had sired most of the offspring. Our results confirmed that polyandry is an important component of the mating system of this species, suggesting that females are able to recognize the sperm of the male that will provide a genetic benefit for their offspring. We found evidence of female cryptic choice based on males' genetic dissimilarity: (1) males who sire most of the offspring of a given female tend to be genetically different from their sexual partner; (2) a same male, when mated with two females, sired a proportion of the offspring inversely correlated with his genetic similarity to the female; (3) genetic dissimilarity between mating partners is positively correlated with offspring heterozygosity. According to the genetic compatibility model, we hypothesized that in the observed non resource‐based mating system the indirect benefit for the offspring should reflect interactions between paternal and maternal genomes rather than the inheritance of the so‐called ‘good genes’. This study suggests a polygynandrous mating system for the study species and provides the first report in a salamandrid species in natural condition that reproductive success of males is correlated with genetic dissimilarity between mates. Moreover, we found evidence of an offspring benefit (higher heterozygosity) derived from the most genetically dissimilar father.     

Propagation of a segment of bacteriophage lamda-DNA in monkey cells after covalent linkage to a defective simian virus 40 genome.

A 520 base pair DNA segment was excised from the bacteriophage lamda-genome by cleavage with the bacterial restriction endonuclease, endo R. Hindll. This segment was covalently joined in vitro to an 880 base pair simian virus 40 (SV40) DNA segment which contains the initation site for SV40 DNA replication. The latter segment was derived from the genome of a defective reiteration mutant of SV40 also by endo R. Hindlll cleavage. When the recombinant molecule, together with wild-type SV40 DNA as helper, was introduced into monkey cells by DNA infection, replication of the lamda-DNA sequences was observed, and hybrid genomes were encapsidated into progeny SV40 virions. The structure of the lamda-DNA segment after serial passage in monkey cells was examined by use of restriction endonucleases and electron microscopic heteroduplex analysis.