Genetic interplay between transcription factor Pou4f1/Brn3a and neurotrophin receptor Ret in retinal ganglion cell type specification.

BACKGROUND: While the transcriptional code governing retinal ganglion cell (RGC) type specification begins to be understood, its interplay with neurotrophic signaling is largely unexplored. In mice, the transcription factor Brn3a/Pou4f1 is expressed in most RGCs, and is required for the specification of RGCs with small dendritic arbors. The Glial Derived Neurotrophic Factor (GDNF) receptor Ret is expressed in a subset of RGCs, including some expressing Brn3a, but its role in RGC development is not defined. METHODS: Here we use combinatorial genetic experiments using conditional knock-in reporter alleles at the Brn3a and Ret loci, in combination with retina- or Ret specific Cre drivers, to generate complete or mosaic genetic ablations of either Brn3a or Ret in RGCs. We then use sparse labelling to investigate Brn3a and Ret gene dosage effects on RGC dendritic arbor morphology. In addition, we use immunostaining and/or gene expression profiling by RNASeq to identify transcriptional targets relevant for the potential Brn3a-Ret interaction in RGC development. RESULTS: We find that mosaic gene dosage manipulation of the transcription factor Brn3a/Pou4f1 in neurotrophic receptor Ret heterozygote RGCs results in altered cell fate decisions and/or morphological dendritic defects. Specific RGC types are lost if Brn3a is ablated during embryogenesis and only mildly affected by postnatal Brn3a ablation. Sparse but not complete Brn3a heterozygosity combined with complete Ret heterozygosity has striking effects on RGC type distribution. Brn3a only mildly modulates Ret transcription, while Ret knockouts exhibit slightly skewed Brn3a and Brn3b expression during development that is corrected by adult age. Brn3a loss of function modestly but significantly affects distribution of Ret co-receptors GFRα1-3, and neurotrophin receptors TrkA and TrkC in RGCs. CONCLUSIONS: Based on these observations, we propose that Brn3a and Ret converge onto developmental pathways that control RGC type specification, potentially through a competitive mechanism requiring signaling from the surrounding tissue.

Natural and Artificial Mechanisms of Mitochondrial Genome Elimination.

The generally accepted theory of the genetic drift of mitochondrial alleles during mammalian ontogenesis is based on the presence of a selective bottleneck in the female germline. However, there is a variety of different theories on the pathways of genetic regulation of mitochondrial DNA (mtDNA) dynamics in oogenesis and adult somatic cells. The current review summarizes present knowledge on the natural mechanisms of mitochondrial genome elimination during mammalian development. We also discuss the variety of existing and developing methodologies for artificial manipulation of the mtDNA heteroplasmy level. Understanding of the basics of mtDNA dynamics will shed the light on the pathogenesis and potential therapies of human diseases associated with mitochondrial dysfunction.

Two Separate Cases: Complex Chromosomal Abnormality Involving Three Chromosomes and Small Supernumerary Marker Chromosome in Patients with Impaired Reproductive Function.

For medical genetic counseling, estimating the chance of a child being born with chromosome abnormality is crucially important. Cytogenetic diagnostics of parents with a balanced karyotype are a special case. Such chromosome rearrangements cannot be detected with comprehensive chromosome screening. In the current paper, we consider chromosome diagnostics in two cases of chromosome rearrangement in patients with balanced karyotype and provide the results of a detailed analysis of complex chromosomal rearrangement (CCR) involving three chromosomes and a small supernumerary marker chromosome (sSMC) in a patient with impaired reproductive function. The application of fluorescent in situ hybridization, microdissection, and multicolor banding allows for describing analyzed karyotypes in detail. In the case of a CCR, such as the one described here, the probability of gamete formation with a karyotype, showing a balance of chromosome regions, is extremely low. Recommendation for the family in genetic counseling should take into account the obtained result. In the case of an sSMC, it is critically important to identify the original chromosome from which the sSMC has been derived, even if the euchromatin material is absent. Finally, we present our view on the optimal strategy of identifying and describing sSMCs, namely the production of a microdissectional DNA probe from the sSMC combined with a consequent reverse painting.

Postnatal developmental dynamics of cell type specification genes in Brn3a/Pou4f1 Retinal Ganglion Cells.

BACKGROUND: About 20-30 distinct Retinal Ganglion Cell (RGC) types transmit visual information from the retina to the brain. The developmental mechanisms by which RGCs are specified are still largely unknown. Brn3a is a member of the Brn3/Pou4f transcription factor family, which contains key regulators of RGC postmitotic specification. In particular, Brn3a ablation results in the loss of RGCs with small, thick and dense dendritic arbors ('midget-like' RGCs), and morphological changes in other RGC subpopulations. To identify downstream molecular mechanisms underlying Brn3a effects on RGC numbers and morphology, our group recently performed a RNA deep sequencing screen for Brn3a transcriptional targets in mouse RGCs and identified 180 candidate transcripts. METHODS: We now focus on a subset of 28 candidate genes encoding potential cell type determinant proteins. We validate and further define their retinal expression profile at five postnatal developmental time points between birth and adult stage, using in situ hybridization (ISH), RT-PCR and fluorescent immunodetection (IIF). RESULTS: We find that a majority of candidate genes are enriched in the ganglion cell layer during early stages of postnatal development, but dynamically change their expression profile. We also document transcript-specific expression differences for two example candidates, using RT-PCR and ISH. Brn3a dependency could be confirmed by ISH and IIF only for a fraction of our candidates. CONCLUSIONS: Amongst our candidate Brn3a target genes, a majority demonstrated ganglion cell layer specificity, however only around two thirds showed Brn3a dependency. Some were previously implicated in RGC type specification, while others have known physiological functions in RGCs. Only three genes were found to be consistently regulated by Brn3a throughout postnatal retina development - Mapk10, Tusc5 and Cdh4.

Avian Influenza Virus with Hemagglutinin-Neuraminidase Combination H8N8, Isolated in Russia.

We report the genome sequence of an avian influenza virus (AIV) subtype H8N8, isolated in Russia. The genome analysis shows that all genes belong to AIV Eurasian lineages. The PB2 gene was similar to a Mongolian low-pathogenic (LP) AIV H7N1 and a Chinese high-pathogenic (HP) AIV H5N2.

Assessment of potential hazards during the process of house building in Estonia.

The aim of this study was to evaluate new working conditions in the construction industry in Estonia. Three construction sites were investigated: a dwelling house at the stage of renovation, a warehouse at the stage of excavation and preparation work, and a hotel, where we examined the working conditions of the finishers. Microclimate: air temperature, relative humidity and air velocity were determined at every workplace. Full-shift personal exposure measurements of dust and gas exposure were performed among 97 construction workers in breathing zone air. The concentration of lead in the air was analysed by AAS. Vapours of benzene, styrene and toluene concentrations were determined by gas chromatography. The noise level was measured with the help of the sound-level meter at every workplace. All microclimatic indices were sometimes below or above exposure standards for indoor work. High levels of airborne dust and increased concentrations of lead were observed during repair work. Low concentrations of organic solvents in the air were determined when using paints and some synthetic substances. The noise level exceeded the permitted limit when an excavator and powerful electric appliances were used. A questionnaire determined the prevalence of subjectively experienced musculoskeletal strain in 30% of workers. The working conditions in the construction industry in Estonia are approaching European level. However, it is necessary to improve working conditions permanently by the use of new harmless materials, personal protective equipment, and through influencing worker health by permanent education and medical watch. We consider that questionnaires are very important in the control of the work environment, in subjective health assessments, and as a source of useful proposals to improve working conditions.

Benzene exposure assessed by metabolite excretion in Estonian oil shale mineworkers: influence of glutathione s-transferase polymorphisms.

Measurement of urinary excretion of the benzene metabolites S-phenylmercapturic acid (S-PMA) and trans,trans-muconic acid (t,t-MA) has been proposed for assessing benzene exposure, in workplaces with relatively high benzene concentrations. Excretion of S-PMA and t,t-MA in underground workers at an oil shale mine were compared with the excretion in workers engaged in various production assignments above ground. In addition, possible modifying effects of genetic polymorphisms in glutathione S-transferases T1 (GSTT1), M1 (GSTM1), and P1 (GSTP1) on the excretion of S-PMA and t,t-MA were investigated. Fifty underground workers and 50 surface workers participated. Blood samples and three urine samples were collected from each worker: (a) a preshift sample collected the morning after a weekend, (b) a postshift sample 1 collected after the first shift, and (c) a postshift sample 2 collected after the last shift of the week. Personal benzene exposure was 114 +/- 35 mug/m(3) in surface workers (n = 15) and 190 +/- 50 mug/m(3) in underground workers (n = 15) in measurements made prior to the study. We found t,t-MA excretion to be significantly higher in underground workers after the end of shifts 1 and 2 compared with the corresponding surface workers. The same picture, although not significant, was seen for S-PMA excretion. Excretion of S-PMA and t,t-MA was found to increase significantly during the working week in underground workers but not in those employed on the surface. Both t,t-MA and S-PMA excretion were significantly higher in smokers compared with nonsmokers. Subjects carrying the GSTT1 wild-type excreted higher concentrations of S-PMA than subjects carrying the null genotype, suggesting that it is a key enzyme in the glutathione conjugation that leads to S-PMA. The results support the use of benzene metabolites as biomarkers for assessment of exposure at modest levels and warrant for further investigations of health risks of occupational benzene exposure in shale oil mines.

Effects of occupational exposure to diesel exhaust on porphyrin metabolism in lymphocytes of workers employed at black coal and oil-shale mines.

BACKGROUND: This pilot study was conducted to investigate biochemical effects of exposure to diesel engine exhaust at two mines. For this purpose, heme biosynthesis, and PP association with DNA (PP/DNA) in lymphocytes of miners exposed to diesel exhaust were determined. METHODS: The pilot study was carried out at a black coal mine in Czech Republic, and at an oil-shale mine in Estonia. The subjects were ten drivers from each mine, who were exposed to diesel exhaust (underground workers). Control groups consisted of ten maintenance workers from each mine (surface workers). The content of 1-nitropyrene (NP) in respirable dust was measured using GC-MS. The levels of PP, PP/DNA, heme, and activity of ferrochelatase (FC) were determined by spectrophotometry. RESULTS: The exposure to diesel exhaust evaluated as the level of NP associated to particulate matter in air. NP level was significantly higher in oil-shale mine compared with coal mine. The values of PP and PP/DNA in lymphocytes appeared to be significantly increased only in miners in the oil-shale mine. There was no difference in the levels of PP and PP/DNA and FC activity between surface workers and miners at the coal mine. The level of heme in lymphocytes of coal mine miners was significantly higher than in miners of the oil-shale mine. The activity of FC was significantly lower in underground workers compared to surface workers at this mine. High level of NP was accompanied by an increase of alterations in cells porphyrin metabolism in lymphocytes of miners. CONCLUSIONS: Alterations of porphyrin and heme metabolism in peripheral lymphocytes may serve as biomarker in assessment of exposure to diesel exhaust effects.

Biomarkers of carcinogenic contaminants in Baltic flounder (Platichthys flesus): temporal changes in urban and non-urban sites in Tallinn bay.

In order to study effects of environmental contamination, a suite of biomarkers were measured over the period 1996 to 1999 in livers of flounder (Platichthys flesus) from two urban embayments and one non-urban reference site of the Gulf of Finland in the vicinity of Tallinn, Estonia. Total cytochrome P450 (CYP) level, aryl hydrocarbon hydroxylase (AHH), 5-aminolevulinic acid synthetase (ALA-S), and heme synthetase (HEM-S) activities were quantified by means of spectrophotometry. These data were compared to results obtained in 1994 for the same biomarkers at one of the urban embayments and the non-urban site, as measured by the same protocols. For the flounder collected from the non-urban site, changes occurred in AHH activity and the total CYP level, which were significantly lower in 1996 and 1999 compared with 1994 (p < 0.05). Activity of ALA-S decreased slightly over this same period. The activity of HEM-S increased between 1996 and 1999. In the urban site first investigated in 1994, the activities of AHH and ALA-S, as well as the total level of CYP in flounder liver were significantly higher compared with 1999 (p < 0.05). HEM-S activities did not show any significant changes over this time period. AHH activities of flounder collected in another urban site decreased slightly between 1996 and 1999, in contrast to data on the total CYP level which diminished drastically over these years (p < 0.05). Activities of HEM-S increased significantly (p < 0.05) during the period investigated, while activities of ALA-S remained unchanged. These findings suggest that contamination of the marine environments by PAHs has gone down everywhere in the Tallinn area during the last 3 to 5 years. However, the results indicate that the area is still contaminated, as indicated by elevated heme synthesis enzymes and the total CYP content, and that monitoring of contaminants and their effects should be continued in this region.