Automatic detection and analysis of cell motility in phase-contrast time-lapse images using a combination of maximally stable extremal regions and Kalman filter approaches.

Phase-contrast illumination is simple and most commonly used microscopic method to observe nonstained living cells. Automatic cell segmentation and motion analysis provide tools to analyze single cell motility in large cell populations. However, the challenge is to find a sophisticated method that is sufficiently accurate to generate reliable results, robust to function under the wide range of illumination conditions encountered in phase-contrast microscopy, and also computationally light for efficient analysis of large number of cells and image frames. To develop better automatic tools for analysis of low magnification phase-contrast images in time-lapse cell migration movies, we investigated the performance of cell segmentation method that is based on the intrinsic properties of maximally stable extremal regions (MSER). MSER was found to be reliable and effective in a wide range of experimental conditions. When compared to the commonly used segmentation approaches, MSER required negligible preoptimization steps thus dramatically reducing the computation time. To analyze cell migration characteristics in time-lapse movies, the MSER-based automatic cell detection was accompanied by a Kalman filter multiobject tracker that efficiently tracked individual cells even in confluent cell populations. This allowed quantitative cell motion analysis resulting in accurate measurements of the migration magnitude and direction of individual cells, as well as characteristics of collective migration of cell groups. Our results demonstrate that MSER accompanied by temporal data association is a powerful tool for accurate and reliable analysis of the dynamic behaviour of cells in phase-contrast image sequences. These techniques tolerate varying and nonoptimal imaging conditions and due to their relatively light computational requirements they should help to resolve problems in computationally demanding and often time-consuming large-scale dynamical analysis of cultured cells.

Osteogenic medium is superior to growth factors in differentiation of human adipose stem cells towards bone-forming cells in 3D culture.

Human adipose stem cells (hASCs) have been recently used to treat bone defects in clinical practice. Yet there is a need for more optimal scaffolds and cost-effective approaches to induce osteogenic differentiation of hASCs. Therefore, we compared the efficiency of bone morphogenetic proteins (BMP-2 and BMP-7), vascular endothelial growth factor (VEGF), and osteogenic medium (OM) for the osteo-induction of hASCs in 3D culture. In addition, growth factors were tested in combination with OM. Commercially available bioactive glass scaffolds (BioRestore) and biphasic calcium phosphate granules (BoneCeramic) were evaluated as prospective carriers for hASCs. Both biomaterials supported hASC-viability, but BioRestore resulted in higher cell number than BoneCeramic, whereas BoneCeramic supported more significant collagen production. The most efficient osteo-induction was achieved with plain OM, promoting higher alkaline phosphatase activity and collagen production than growth factors. In fact, treatment with BMP-2 or VEGF did not increase osteogenic differentiation or cell number significantly more than maintenance medium with either biomaterial. Moreover, BMP-7 treatment consistently inhibited proliferation and osteogenic differentiation of hASCs. Interestingly, there was no benefit from growth factors added to OM. This is the first study to demonstrate that OM enhances hASC-differentiation towards bone-forming cells significantly more than growth factors in 3D culture.

Perinatal outcome of children born after frozen and fresh embryo transfer: the Finnish cohort study 1995-2006.

BACKGROUND: The number of children born after frozen embryo transfer (FET) is steadily rising. However, studies on obstetric and perinatal outcomes are limited. Our primary aim was to compare the perinatal health of children born after FET and fresh embryo transfer, and to use data from children born after spontaneous conception as a reference. METHODS: In a register-based cohort study we evaluated the obstetric and perinatal outcomes of children born after FET (n = 2293), fresh embryo transfer (n = 4151) and those born after spontaneous pregnancy (reference group; n = 31 946). Data were collected from the registers of two infertility outpatient clinics, two university hospitals and the Finnish Medical Birth Register (1995-2006). RESULTS: After adjusting for confounding factors the FET group showed decreased risks of preterm birth [adjusted odd ratio (AOR) 0.83, 95% confidence interval (CI) 0.71-0.97], low birthweight (AOR 0.74; 0.62-0.88) and being small for gestational age (AOR 0.63; 0.49-0.83) compared with the fresh embryo transfer group. Mean birthweight was 134 g higher in the FET singletons versus the fresh embryo transfer singletons (P< 0.0001). When FET singletons were compared with the reference group, increased risks of preterm birth (AOR 1.45; 1.25-1.68) and low birthweight (AOR 1.22; 1.03-1.45) and a decreased risk of being small for gestational age (AOR 0.71; 0.54-0.92) were found. No excess of perinatal and infant mortality occurred between the groups. CONCLUSIONS: Embryo freezing does not adversely affect perinatal outcome in terms of prematurity, low birthweight and being small for gestational age versus the fresh embryo transfer and the outcome is similar or even better, particularly regarding fetal growth. Our study, which is one of the largest on FET pregnancies, provides further evidence on the safety of FET.

Inter- and intra-specific responses to elevated ozone and chamber climate in northern birches.

We studied the responses of micropropagated, northern provenances of downy, mountain and silver birches to elevated ozone (O(3)) and changing climate using open-top chambers (OTCs). Contrary to our hypothesis, northern birches were sensitive to O(3), i.e. O(3) levels of 31-36 ppb reduced the leaf and root biomasses by -10%, whereas wood biomass was affected to a lesser extent. The warmer and drier OTC climate enhanced growth in general, though there were differences among the species and clones, e.g. in bud burst and biomass production. Inter- and intra-specific responses to O(3) and changing climate relate to traits such as allocation patterns between the above- and belowground parts (i.e. root/shoot ratio), which further relate to nutrient and water economy. Our experiments may have mimicked future conditions quite well, but only long-term field studies can yield the information needed to forecast responses at both tree and ecosystem levels.

Anaerobic digestion of grass silage in batch leach bed processes for methane production.

Anaerobic digestion of grass silage in batch leach bed reactors, with and without a second stage upflow anaerobic sludge blanket (UASB) reactor, was evaluated. Sixty six percent of the methane potential in grass was obtained within the 55 days solids retention time in the leach bed-UASB process without pH adjustment, whereas in the one-stage leach bed process 20% of the methane potential in grass was extracted. In two-stage operation, adjustment of the pH of influent to the leach bed reactor to 6 with HCl led to inhibition of both hydrolysis/acidogenesis and methanogenesis. In the leach bed-UASB process 39% of the carbohydrates and 58% of the acid soluble lignin were solubilised within the 49 days of operation, whereas Klason lignin was most recalcitrant. The methane potential of the digestates varied from 0.141 to 0.204 m3 CH4 kg(-1) added volatile solids.

Changes in the concentrations of phenolics and photosynthates in Scots pine (Pinus sylvestris L.) seedlings exposed to nickel and copper.

Studies were done on the effects of elevated soil concentrations of copper (Cu) and (Ni) on foliar carbohydrates and phenolics in Scots pine (Pinus sylvestris L.). Four year-old seedlings were planted in pots filled with metal-treated mineral forest soil in early June. The experimental design included all combinations of four levels of Cu (0, 25, 40 and 50 mg kg(-1) soil dw) and Ni (0, 5, 15 and 25 mg kg(-1) soil dw). Current year needles were sampled for soluble sugar, starch and phenolics at the end of September. Ni increased sucrose concentration in the needles, indicating disturbances in carbohydrate metabolism. Trees exposed to Ni had higher concentrations of condensed tannins compared with controls. In contrast, concentrations of several other phenolic compounds decreased when seedlings were exposed to high levels of Cu or to a combination of Ni and Cu. The results suggest that concentrations of phenolics in Scots pine needles vary in their responses to Ni and Cu in the forest soil.

UV-absorbing compounds in subarctic herbarium bryophytes.

The UV-B-absorbing compounds of herbarium specimens of 10 subarctic bryophyte species collected during the years 1926-1996 and available at the Botanical Museum, University of Oulu, were studied. We studied whether herbarium specimens reflect changes in the past radiation climate through their methanol-extractable compounds. The order of gametophytes based on the average amount of total compounds (sum of A280-320 nm) per mass from the lowest to the highest was Polytrichum commune, Pleurozium schreberi, Hylocomium splendens, Sphagnum angustifolium, Dicranum scoparium, Funaria hygrometrica, Sphagnum fuscum, Sphagnum warnstorfii, Sphagnum capillifolium and Polytrichastrum alpinum, and the amount of UV-B-absorbing compounds per specific surface area correlated with the summertime daily global radiation and latitude. P. alpinum, F. hygrometrica and three Sphagnum species seem to be good indicators for further studies. The amount of UV-B-absorbing compounds revealed no significant trends from the 1920s till the 1990s, with the exception of S. capillifolium, which showed a significant decreasing trend.

Environmental specimen bank samples of Pleurozium schreberi and Hylocomium splendens as indicators of the radiation environment at the surface.

Pleurozium schreberi (Brid.) Mitt. and Hylocomium splendens (Hedw.) Schimp. samples from the Finnish Environmental Specimen Bank were used to study the UV-B-absorbing compounds as potential screens of the past radiation environment. The first series from southern and central Finland consisted of samples collected from 18 P. schreberi or H. splendens dominated sites in 1985 and 1995. The second series from four H. splendens dominated sites (two in southern and two in northern Finland) and two P. schreberi dominated sites (one southern and one northern) were from the years 1985, 1990, 1995 and 2000. In the first series, the moss surface area of the analyzed specimens (5 +/- 0.2 mg DW) was smaller in 1995 (40% of both species collected in June) than in 1985 (40% of P. schreberi and 55% of H. splendens collected in September). The methanol-extractable UV-B-absorbing compounds of P. schreberi in 1985 and 1995 measured as absorbance at 10 nm intervals at 280-360 nm per mg DW revealed a negative correlation with reconstructed UV-radiation data. In the second series, the UV-B-absorbing compounds per specific surface area (surface area mm2 per DW) showed significant differences between the years. The highest values in both species were obtained in 1990. In H. splendens, a difference between the southern and northern samples was observed. The preliminary study encouraged the use of environmental specimen bank samples to indicate changes in surface radiation conditions.

A multilocus microsatellite phylogeny of the Drosophila virilis group.

We used a set of 48 polymorphic microsatellites derived from Drosophila virilis to infer phylogenetic relationships in the D. virilis clade. Consistent with previous studies, D. virilis and D. lummei were the most basal species of the group. Within the D. montana phylad, the phylogenetic relationship could not be resolved. Special attention was given to the differentiation between D. americana texana, D. americana americana and D. novamexicana. Significant differences between these three groups were detected by F(ST) analyses. Similarly, a model-based clustering method for multilocus genotype data also provided strong support for the presence of three differentiated groups. This genome-wide differentiation between D. americana texana and D. americana americana contrasts with previous analyses based on DNA sequence data.

QTL analysis of variation in male courtship song characters in Drosophila virilis.

We have used a quantitative trait locus (QTL) mapping approach to study the genetic basis of differences between two Drosophila virilis strains representing extreme phenotypes in two song characters, the number of pulses in a pulse train (PN) and the length of a pulse train (PTL). Variation in these characters among 520 F2 males was studied by single-marker analysis and composite interval mapping (CIM) using a recombination linkage map constructed for 26 microsatellite markers. In single-marker analysis, two adjacent microsatellite markers on the third chromosome, msat19 and vir84 explained 13.8 and 12.4% of the variation in PN and 9.9 and 6.5% of the variation in PTL, respectively. CIM analysis revealed significant QTLs affecting PN, located on the X and the second, third and fourth chromosome of D. virilis, while variation in PTL was attributable to QTLs located only on the third chromosome.

Nucleotide variation at the no-on-transient A gene in Drosophila littoralis.

The no-on-transient A (nonA) gene encodes a putative RNA-binding protein, and mutations in this gene are known to affect vision, male courtship song and viability in Drosophila melanogaster. Here we have sequenced the coding region of the nonA gene of Drosophila littoralis and compared it with those of Drosophila virilis and D. melanogaster. All portions of nonA appeared to be conserved between D. littoralis and D. virilis, while the 5' region of the gene of these two species showed high divergence from that of a more distantly-related species, D. melanogaster. The same was true for the glycine repeat regions. No significant deviation from neutrality was observed in the analysis of intraspecific nucleotide variation in 5' or 3' region of the nonA gene in D. littoralis population. Also, comparison of D. littoralis sequences with homologous sequence of D. virilis suggests that the gene is evolving neutrally in D. virilis group. Divergence of the 5' regions between D. virilis group species and D. melanogaster could be a result of positive selection, but this finding is obscured by the long divergence time of the species groups.

Identification of X chromosomal restriction fragment length polymorphism markers and their use in a gene localization study in Drosophila virilis and D. littoralis.

We have identified six restriction fragment length polymorphism (RFLP) markers based on unique gene sequences on the X chromosome of Drosophila virilis and D. littoralis. The markers were localized by in situ hybridization on larval polytene chromosomes, and the conjugation of the X chromosomes of the two species was studied in salivary glands of interspecific hybrid female larvae. The gene arrangement of D. virilis and D. littoralis appeared to be very different at the proximal end of the X chromosome preventing recombination between RFLP markers located in this area. Simple quantitative trait loci (QTL) analysis showed that five of our marker genes (including nonA and Dmca1A, previously found to affect male courtship song in D. melanogaster) are linked with a gene(s) having a major effect on species differences in the male courtship song between D. virilis and D. littoralis. This shows that the song gene(s) may be located inside a large X-chromosomal inversion in D. littoralis (as previously suggested), but that it may also be located on an area between this inversion and the centromere, close to nonA and Dmca1A. Localization of this gene or gene complex will be continued with the aid of our newly identified RFLP markers by making interspecific crosses between D. virilis group species with more similar X chromosomes.

UV-absorbing compounds and waxes of Scots pine needles during a third growing season of supplemental UV-B.

Methanol-extractable UV-absorbing compounds, wax tube distribution and the chloroform-soluble waxes of the needles of mature Scots pines were studied in a UV-B field experiment in Oulu (65 degrees N). Throughout the experiment, UV-B lamp banks were placed over the same selected branch and each year needle samples were taken from the same branch. In the third exposure year, needle samples were taken twice a month from 3-day-old needles (18 June) to fully developed needles (13 August). On 28 September, the previous year's needles (c + 1, c + 2) were also collected. There was a significant negative correlation between the amount of waxes and UV-absorbing compounds. A high amount of UV-absorbing compounds was observed early and late in the season when the amount of waxes was low and epicuticular waxes were undeveloped (youngest needles) or already eroded (c + 2 needles). The amount of UV-absorbing compounds (A310/cm2 and A320/cm2) was significantly (30-day-old needles) or slightly (all the other needle ages) higher in the ambient needles compared to the needles under supplemental UV-B. This possibly indicated the already inhibited pigment synthesis in the UV-B-treated needles during the third year of supplemental UV-B. This observation could mean that the protective mechanisms may not be effective under accumulated UV-B dose.

The glutathione status of mature Scots pines during the third season of UV-B radiation exposure.

Impacts of UV-B radiation on the glutathione level were studied in mature Scots pine needles (Pinus sylvestris L.) during the third season of a UV-B field experiment. Studies were made on 4-week-old (July) to 14-week-old (September) current-year needles and 3-year-old needles which had their third UV-B-exposure season in progress. Depending on the season and the year (1996-98), the supplemental UV-B dose varied from 0.92 to 5.09 kJ m-2 day-1 UV-BBE compared to 0.47-2.44 kJ m-2 day-1 UV-BBE under the ambient treatment. Fully grown UV-B-treated current-year needles showed lower total glutathione concentrations after the vegetation period in September, whereas in UV-B-treated 3-year-old needles the total glutathione content was significantly lower and the proportion of oxidized glutathione (GSSG%) 56% higher in July. The significant differences in total glutathione in current-year needles in September and in active 3-year-old needles in July seem to indicate that the effect of enhanced UV-B radiation on glutathione status could be cumulative.

Ovulation side and cycle fecundity: a retrospective analysis of frozen/thawed embryo transfer cycles.

The aim of the study was to evaluate a possible lateral difference in ovarian activity and its effect on cycle fecundity. A database was analysed retrospectively which covered 477 cycles in which frozen/thawed embryo transfer had been carried out. The cycles were spontaneous, with no hormonal treatment. Women with ovulation problems as a reason for infertility treatment were excluded. Factors investigated were the side of ovulation, endometrial thickness on cycle days 10-12 and on the day of embryo transfer, and pregnancy rate per embryo transfer. Ovulation was right-sided in 273 of the 477 cycles (57.2%) and left-sided in 204 of the cycles (42. 8%) (95% CI 38.3-47.2, P = 0.002). In the age category of 30-37 years, covering 288 cycles, the incidence of left-sided ovulation was 126 (43.7%, 95% CI 38.0-49.5, P = 0.034). In this category, the endometrial thickness (+/-SD) was significantly greater on the day of embryo transfer (i.e. at time of implantation) when there had been left-sided ovulation compared with right-sided [9.6 mm (2.0) versus 9.1 mm (1.8), P = 0.037]. In addition, the pregnancy rate per embryo transfer was higher when ovulation had been on the left side [32/126 (25.2%) versus right side 24/162 (14.8%), P = 0.035, 95% CI 0.0122-0.199]. In conclusion, right-sided ovulation was more frequent than left-sided in the whole group. This is the first study to report that the side of ovulation has a clinical impact. These data support the hypothesis that the side of ovulation is significant in terms of embryo implantation.

Stress indications in copper- and nickel-exposed Scots pine seedlings.

Scots pine nursery seedlings were planted in pots, five seedlings per treatment, and placed in an experimental field at the University of Oulu in northern Finland at the beginning of June 1997. Copper and nickel sulphates were mixed with forest mineral soil before seedling planting. The metal levels ranged from 0 to 25 mg Ni kg(-1) dry soil and 0 to 50 mg Cu kg(-1) in dry soil and in combinations of both metals. Current year's needles for element analyses, EDS microanalyses, microscopy and glutathione and peroxidase activity analyses were collected from 1-5 seedlings per treatment in September. Seedling biomass in controls, Cu25 and Cu50 differed significantly from the Ni25Cu50 treatment. The root/shoot ratio was highest in the Ni5 treatment, indicating good root growth, though the roots were visibly healthier in the Cu25 treatment than in the Ni5 treatment. At higher Ni levels, the condition of roots deteriorated. The proportion of plasmolysed mesophyll cells was highest in the Ni25 treatment. Copper-treated seedlings did not suffer from Cu stress, because no severe injuries were seen in either the roots or the needles in Cu-exposed seedlings. The needle concentrations of Cu increased only slightly due to treatments. Ni accumulation in needles increased with increasing concentrations in soil. Needles of Cu-treated seedlings had less oxidized glutathione than those of Ni-treated seedlings, but the roots had higher, not significantly, peroxidase activity levels. Light-colored, swollen thylakoids were occasionally observed in the Ni25Cu50 treatment, indicating some interaction between Ni and Cu. Ni seemed to cause more oxidative stress to the seedlings than copper, which was manifested as a decreased GSH level and an increased proportion of GSSG in the Ni treatments. Copper together with nickel strongly decreased root growth, the root/shoot ratio being lowest in the Ni25Cu50 treatment.

Response of needle sulphur and nitrogen concentrations of Scots pine versus Norway spruce to SO2 and NO2.

The results of two field studies and an open-top chamber fumigation experiment showed that the response of mature Scots pine to SO(2) and NO(2) differed from that of mature Norway spruce. Moreover, the response of pine seedlings to SO(2) and NO(2) differed from that of mature trees. The greater increase in the needle total S concentrations of pine suggested more abundant stomatal uptake of SO(2) compared to spruce. Both pine seedlings and mature trees also seemed to absorb more N from atmospheric deposition. Mature pine was able to assimilate SO(4)(2-) derived from SO(2) into organic S more effectively than mature spruce at the high S and N deposition sites, whereas both pine and spruce seedlings accumulated SO(4)-S under NO(2)+SO(2) exposure. Spruce, in turn, accumulated SO(4)-S even when well supplied with N. Net assimilation of SO(4)(2-) in conifer seedlings was enhanced markedly by elevated temperature. To protect the northern coniferous forests against the harmful effects of S and N deposition, it is recommended that the critical level for SO(2) as a growing season mean be set at 5-10 microg m(-3) and NO(2) at 10-15 microg m(-3), depending on the 'effective temperature sum' and/or whether SO(2) and NO(2) occur alone or in combination.

Obstetric and perinatal outcome of pregnancies after intrauterine insemination.

The main aim of this study was to evaluate the obstetric and perinatal outcome of pregnancies after intrauterine insemination (IUI) with the partner's spermatozoa combined with ovarian stimulation. Information concerning the antenatal care and obstetric and perinatal outcome of IUI pregnancies (n = 111), spontaneous (n = 333) and in-vitro fertilization (IVF) (n = 333) was obtained from the Finnish Medical Birth Register (MBR). The multiple birth rate in the IUI group was 17% (19/111). Significantly less antenatal care was required by the IUI group than the IVF group. The frequency of Caesarean section was 25% for IUI singletons and 58% for IUI multiples, similar to the other groups. The mean (SD) gestational age for IUI singletons at birth was 39.5 (1.8) weeks, with a mean birth weight of 3285 (575) g, compared with 3448 (600) g in non-assisted singletons (P < 0.05). For IUI multiples the mean gestational age at birth was 36.0 (2.8) weeks and the mean birth weight was 2449 (678) g. The incidence of preterm birth, low birth weight or low Apgar scores and the need for neonatal care were similar in all groups. One case of major malformation and two perinatal deaths were recorded in the IUI group. In conclusion, IUI treatment did not appear to increase obstetric or perinatal risks compared with matched spontaneous or IVF pregnancies. Most problems were associated with patient characteristics and multiple pregnancy. Reduction of the high incidence of multiple pregnancies after assisted reproductive technology is essential to improve its outcome.

Intrauterine insemination treatment in subfertility: an analysis of factors affecting outcome.

A total of 811 intrauterine insemination (IUI) cycles in which clomiphene citrate/human menopausal gonadotrophin (HMG) was used for ovarian stimulation were analysed retrospectively to identify prognostic factors regarding treatment outcome. The overall pregnancy rate was 12.6% per cycle, the multiple pregnancy rate 13.7%, and the miscarriage rate 23.5%. Logistic regression analysis revealed five predictive variables as regards pregnancy: number of the treatment cycle (P = 0.009), duration of infertility (P = 0.017), age (P = 0.028), number of follicles (P = 0.031) and infertility aetiology (P = 0.045). The odds ratios for age < 40 years, unexplained infertility aetiology (versus endometriosis) and duration of infertility < or = 6 years were 3.24, 2.79 and 2.33, respectively. A multifollicular ovarian response to clomiphene citrate/HMG resulted in better treatment success than a monofollicular response, and 97% of the pregnancies were obtained in the first four treatment cycles. The results indicate that clomiphene citrate/HMG/IUI is a useful and cost-effective treatment option in women < 40 years of age with infertility duration < or = 6 years, who do not suffer from endometriosis.

Effects of the ultraviolet-B radiation (UV-B) on conifers: a review.

The current knowledge on conifer responses to enhanced ultraviolet-B (UV-B) radiation is mainly based on greenhouse or growth chamber experiments of one growing season in duration. However, the biomass losses observed in greenhouses do not occur in field-grown trees in their natural habitats. Moreover, the majority of the 20 conifer species studied have been 1-year-old seedlings, and no studies have been undertaken on mature trees. Fully grown needles, with their glaucous waxy surfaces and thick epidermal cells with both soluble and wall-bound UV-B screening metabolites, are well protected against UV-B radiation. However, it is not known whether these are sufficient protectants in young emerging needles or during the early spring period of high UV-B levels reflected from snow. In order to understand all the mechanisms that result in the protection of conifer needles against UV-B radiation, future research should focus on the epidermal layer, separating the waxes, cuticle and epidermal and hypodermal cells. Parallel studies should consist of wall-bound and soluble secondary metabolite analysis, antioxidant measurements and microscopic observations.