Embryonic development of lake whitefish Coregonus clupeaformis: a staging series, analysis of growth and effects of fixation.

A reference staging series of 18 morphological stages of laboratory reared lake whitefish Coregonus clupeaformis is provided. The developmental processes of blastulation, gastrulation, neurulation as well as development of the eye, circulatory system, chromatophores and mouth are included and accompanied by detailed descriptions and live imaging. Quantitative measurements of embryo size and mass were taken at each developmental stage. Eggs were 3·19 ± 0·16 mm (mean ± s.d.) in diameter at fertilization and embryos reached a total length (LT ) of 14·25 ± 0·41 mm at hatch. Separated yolk and embryo dry mass were 0·25 ± 0·08 mg and 1·39 ± 0·17 mg, respectively, at hatch. The effects of two common preservatives (formalin and ethanol) were examined throughout development and post hatch. Embryo LT significantly decreased following fixation at all points in development. A correction factor to estimate live LT from corresponding fixed LT was determined as live LT = (fixed LT )(1·025) . Eye diameter and yolk area measurements significantly increased in fixed compared with live embryos up to 85-90% development for both measurements. The described developmental stages can be generalized to teleost species, and is particularly relevant for the study of coregonid development due to additionally shared developmental characteristics. The results of this study and staging series are therefore applicable across various research streams encompassing numerous species that require accurate staging of embryos and descriptions of morphological development.

Critical windows in embryonic development: Shifting incubation temperatures alter heart rate and oxygen consumption of Lake Whitefish (Coregonus clupeaformis) embryos and hatchlings.

Critical windows are periods of developmental susceptibility when the phenotype of an embryonic, juvenile or adult animal may be vulnerable to environmental fluctuations. Temperature has pervasive effects on poikilotherm physiology, and embryos are especially vulnerable to temperature shifts. To identify critical windows, we incubated whitefish embryos at control temperatures of 2°C, 5°C, or 8°C, and shifted treatments among temperatures at the end of gastrulation or organogenesis. Heart rate (fH) and oxygen consumption ( [Formula: see text] ) were measured across embryonic development, and [Formula: see text] was measured in 1-day old hatchlings. Thermal shifts, up or down, from initial incubation temperatures caused persistent changes in fH and [Formula: see text] compared to control embryos measured at the same temperature (2°C, 5°C, or 8°C). Most prominently, when embryos were measured at organogenesis, shifting incubation temperature after gastrulation significantly lowered [Formula: see text] or fH. Incubation at 2°C or 5°C through gastrulation significantly lowered [Formula: see text] (42% decrease) and fH (20% decrease) at 8°C, incubation at 2°C significantly lowered [Formula: see text] (40% decrease) and fH (30% decrease) at 5°C, and incubation at 5°C and 8°C significantly lowered [Formula: see text] at 2°C (27% decrease). Through the latter half of development, [Formula: see text] and fH in embryos were not different from control values for thermally shifted treatments. However, in hatchlings measured at 2°C, [Formula: see text] was higher in groups incubated at 5°C or 8°C through organogenesis, compared to 2°C controls (43 or 65% increase, respectively). Collectively, these data suggest that embryonic development through organogenesis represents a critical window of embryonic and hatchling phenotypic plasticity. This study presents an experimental design that identified thermally sensitive periods for fish embryos.

Retrospective study of outcomes, for patients admitted to a drug treatment centre board.

Retrospective study of urinary heroin outcomes of a cohort (123) of patients commenced on a methadone treatment program. Significantly poorer outcomes were associated with urines positive for cocaine (OR 0.69 CI 0.59-0.81) benzodiazepines (OR 0.7 CI 0.53-0.93) with prescribing of low dose methadone (OR 0.65 CI 0.48-0.87), with urines positive for heroin at time of admission (OR 0.74 CI 0.56-0.97) and with behavioural sanctions (OR 0.8, CI 0.65-0.98). Improved outcomes were associated with granting of take away methadone (OR 1.34 CI 1.1-1.62). with an indication of improved outcomes associated with alcohol positive urines (OR 1.34 CI 0.95-1.9) and increased duration of clinic attendance (OR 1.21 CI 0.99-1.47). On multiple regression analysis low dose methadone (0.07 CI 0.01-0.33) prescribing remained negatively associated with urine heroin outcomes.

Evaluation of headspace equilibration methods for quantifying greenhouse gases in groundwater.

The objective of the study was to evaluate the different headspace equilibration methods for the quantification of dissolved greenhouse gases in groundwater. Groundwater samples were collected from wells with contrasting hydrogeochemical properties and degassed using the headspace equilibration method. One hundred samples from each well were randomly selected, treatments were applied and headspace gases analysed by gas chromatography. Headspace equilibration treatments varied helium (He):water ratio, shaking time and standing time. Mean groundwater N(2)O, CO(2) and CH(4) concentrations were 0.024 mg N L(-1), 13.71 mg C L(-1) and 1.63 µg C L(-1), respectively. All treatments were found to significantly influence dissolved gas concentrations. Considerable differences in the optimal He:water ratio and standing time were observed between the three gases. For N(2)O, CO(2) and CH(4) the optimum operating points for He:water ratio was 4.4:1, 3:1 and 3.4:1; shaking time was 13, 12 and 13 min; and standing time was 63, 17 and 108 min, respectively. The headspace equilibration method needs to be harmonised to ensure comparability between studies. The experiment reveals that He:water ratio 3:1 and shaking time 13 min give better estimation of dissolved gases than any lower or higher ratios and shaking times. The standing time 63, 17 and 108 min should be applied for N(2)O, CO(2) and CH(4), respectively.

Adapting the buccal micronucleus cytome assay for use in wild birds: age and sex affect background frequency in pigeons.

Micronucleus (MN) formation has been used extensively as a biomarker of damage from genotoxic exposures. The Buccal MN Cytome (BMCyt) assay provides a noninvasive means of quantifying MN frequency in humans, but it has not been developed for use in wildlife. We adapted the BMCyt assay for use in wild birds, with a focus on feral pigeons (Columba livia) as a potential indicator species. Five of six urban bird species sampled using oral cavity swabs produced sufficient buccal cells for the BMCyt assay. The body size of species sampled ranged almost 100-fold (~60 to 5,000 g), but was a not major factor influencing the number of buccal cells collected. Pigeon cells were stained and scored following published BMCyt assay protocols for humans, but with a modified fixation approach using heat and methanol. Pigeons had the same common nuclear abnormalities reported in human studies, and a similar background MN formation frequency of 0.88 MN/1,000 cells. Adult pigeons had on average a threefold higher rate of MN formation than juveniles, and males had a 1.4- to 2.2-fold higher frequency than females. Domestic and feral pigeons did not differ in overall MN frequency. Our results indicate that the BMCyt assay can be used on wild birds, and could provide a means of assessing environmental genotoxicity in pigeons, a useful indicator species. However, bird age and sex are important factors affecting background MN frequency, and thereby the design of environmental studies.

Respiratory tract infection with Mycoplasma pneumoniae in interleukin-12 knockout mice results in improved bacterial clearance and reduced pulmonary inflammation.

Mycoplasma pneumoniae is a leading cause of pneumonia and is associated with asthma. Evidence links M. pneumoniae respiratory disease severity with interleukin-12 (IL-12) concentration in respiratory secretions. We evaluated the microbiologic, inflammatory, and pulmonary function indices of M. pneumoniae pneumonia in IL-12 (p35) knockout (KO) mice and wild-type (WT) mice to determine the role of IL-12 in M. pneumoniae respiratory disease. Eight-week-old wild-type BALB/c mice and 8-week-old IL-12 (p35) KO BALB/c mice were inoculated once intranasally with 10(7) CFU of M. pneumoniae. Mice were evaluated at days 2, 4, and 7 after inoculation. Outcome variables included quantitative bronchoalveolar lavage (BAL) M. pneumoniae culture, lung histopathologic scores (HPS), BAL cytokine concentrations determined by enzyme-linked immunosorbent assay (tumor necrosis factor alpha [TNF-alpha], gamma interferon [IFN-gamma], IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-10, and granulocyte-macrophage colony-stimulating factor) and plethysmography, before and after methacholine, to assess airway obstruction (AO) and airway hyperreactivity (AHR). IL-12 (p35) KO mice infected with M. pneumoniae were found to have significantly lower BAL M. pneumoniae concentrations compared with M. pneumoniae-infected WT mice. Lung HPS and the parenchymal pneumonia subscores (neutrophilic alveolar infiltrate), as well as AO, were significantly lower in infected KO mice. No difference was found for AHR. Infected KO mice had significantly lower BAL concentrations of IFN-gamma than WT mice; a trend toward lower BAL concentrations was observed for IL-10 (P = 0.065) and TNF-alpha (P = 0.078). No differences were found for IL-1beta, IL-2, IL-4, IL-5, or IL-6. The lack of IL-12 in experimental M. pneumoniae pneumonia was associated with less severe pulmonary disease and more rapid microbiologic and histologic resolution.

The adaptive response and protection against heritable mutations and fetal malformation.

There are a number of studies that show radiation can cause heritable mutations in the offspring of irradiated organisms. These "germ-line mutations" have been shown to occur in unique sequences of DNA called "minisatellite loci". The high frequencies of spontaneous and induced mutations at minisatellite loci allow mutation induction to be measured at low doses of exposure in a small population, making minisatellite mutation a powerful tool to investigate radiation-induced heritable mutations. However, the biological significance of these mutations is uncertain, and their relationship to health risk or population fitness is unknown. We have adopted this mutation assay to study the role of adaptive response in protecting mice against radiation-induced heritable defects. We have shown that male mice, adapted to radiation with a low dose priming exposure, do not pass on mutations to their offspring caused by a subsequent large radiation exposure to the adapted males. This presentation and paper provide a general overview of radiation-induced mutations in offspring and explain the effect of low dose exposures and the adaptive response on these mutations.It is also known that exposure of pregnant females to high doses of radiation can cause death or malformation (teratogenesis) in developing fetuses. Malformation can only occur during a specialized stage of organ formation known as organogenesis. Studies in rodents show that radiation-induced fetal death and malformation can be significantly reduced when a pregnant female is exposed to a prior low dose of ionizing radiation. The mechanism of this protective effect, through an adaptive response, depends on the stage of organogenesis when the low dose exposures are delivered. To better understand this process, we have investigated the role of an important gene known as p53. Therefore, this report will also discuss fetal effects of ionizing radiation and explain the critical stages of development when fetuses are at risk. Research will be explained that investigates the biological and genetic systems (p53) that protect the developing fetus and discuss the role of low dose radiation adaptive response in these processes.

Gamma radiation-induced heritable mutations at repetitive DNA loci in out-bred mice.

Recent studies have shown that expanded-simple-tandem-repeat (ESTR) DNA loci are efficient genetic markers for detecting radiation-induced germline mutations in mice. Dose responses following irradiation, however, have only been characterized in a small number of inbred mouse strains, and no studies have applied ESTRs to examine potential modifiers of radiation risk, such as adaptive response. We gamma-irradiated groups of male out-bred Swiss-Webster mice with single acute doses of 0.5 and 1.0 Gy, and compared germline mutation rates at ESTR loci to a sham-irradiated control. To test for evidence of adaptive response we treated a third group with a total dose of 1.1 Gy that was fractionated into a 0.1 Gy adapting dose, followed by a challenge dose of 1.0 Gy 24h later. Paternal mutation rates were significantly elevated above the control in the 0.5 Gy (2.8-fold) and 1.0 Gy (3.0-fold) groups, but were similar to each other despite the difference in radiation dose. The doubling dose for paternal mutation induction was 0.26 Gy (95% CI = 0.14-0.51 Gy). Males adapted with a 0.1 Gy dose prior to a 1.0 Gy challenge dose had mutation rates that were not significantly elevated above the control, and were 43% reduced compared to those receiving single doses. We conclude that pre-meiotic male germ cells in out-bred Swiss-Webster mice are sensitive to ESTR mutations induced by acute doses of ionizing radiation, but mutation induction may become saturated at a lower dose than in some strains of inbred mice. Reduced mutation rates in the adapted group provide intriguing evidence for suppression of ESTR mutations in the male germline through adaptive response. Repetitive DNA markers may be useful tools for exploration of biological factors affecting the probability of heritable mutations caused by low-dose ionizing radiation exposure. The biological significance of ESTR mutations in terms of radiation risk assessment, however, is still undetermined.

Haemophilus segnis: a rare cause of endocarditis.

This report presents a case of endocarditis due to Haemophilus segnis, which represents a speciation difficulty for the routine laboratory. In this study, a molecular approach provided speciation, which was confirmed phenotypically by a reference laboratory. The use of molecular genotypic analysis is an additional strategy in the investigation of endocarditis. It has applications not only in isolate identification but also in primary detection of infection, particularly in patients whose blood is culture negative by conventional methodologies.

Effectiveness of the "Baby Think It Over" teen pregnancy prevention program.

In an effort to reduce teen pregnancy, schools are purchasing a program called "Baby Think It Over," a computerized infant simulator intended to provide a realistic infant care experience. However, little empirical, especially experimental, program effectiveness data exist. This study determined if the program changed participants' attitudes toward parenting, as well as sexual and contraceptive behaviors linked to avoidance of teen pregnancy. Development of measurement tools was a part of the purpose. The study also asked teen-agers, through narrative questions, about their conscious perceptions of the baby's utility and impact. Participants included experimental (n = 151) and control (n = 62) groups of primarily White, middle class, suburban high school' students (mean age = 16.2). The quasi-experimental portion of the study failed to reveal a statistically significant effect. Narrative data revealed several positive and notable program effects.

Students' perceptions of parent-adolescent closeness and communication about sexuality: relations with sexual knowledge, attitudes, and behaviors.

The main goal of this study was to explore how parent-adolescent closeness and communication about sexuality were associated with three aspects of adolescent sexuality (sexual knowledge, attitudes and behaviors). Participants were 157 boys and girls in grades 9 to 12 from two suburban high schools in the Midwest. Canonical correlation analyses revealed two significant combinations of variables. First, younger age and less maternal and paternal communication were related to less sexual behavior and less sexual knowledge. Second, being younger and female and receiving less maternal communication was related to less sexual knowledge and more conservative attitudes. Contrary to expectation, higher levels of parental closeness in conjunction with parental communication did not have a significant influence on these adolescents' sexuality. Given the importance of both age and parental communication in predicting adolescent's sexuality in this study, implications concerning the timing of communication become evident.

Echocardiographic diagnosis of platypnoea-orthodeoxia syndrome after blunt chest trauma.

Platypnea-orthodeoxia syndrome is a rare entity most often related to an interatrial right-to-left shunt. We report the case of a patient who developed platypnoea-orthodeoxia shortly after a blunt chest wall trauma. Definite diagnosis was obtained with transoesophageal echocardiography using contrast in upright and recumbent position. A Medline search did not reveal any other case report of the syndrome after chest wall injury.

Ultrastructure of the proteoliaisin-ovoperoxidase complex and its spatial organization within the Strongylocentrotus purpuratus fertilization envelope.

Ovoperoxidase is a cortical granule-derived enzyme that hardens the sea urchin fertilization envelope by catalyzing the formation of dityrosine residues. Ovoperoxidase works in concert with a second protein, proteoliaisin, which anchors ovoperoxidase to the nascent fertilization envelope in a divalent cation-dependent manner. In this study, we examined the Ca(2+)-dependent interaction of proteoliaisin with ovoperoxidase in rotary-shadowed Pt replicas. Ovoperoxidase, a uniformly sized globular molecule, binds to a distal portion of rod-shaped proteoliaisin when low concentrations of Ca2+ are present. Higher Ca2+ concentrations lead to the formation of extended proteoliaisin strands that are decorated along their lengths with ovoperoxidase. Using immunogold labeling, we also examined the assimilation of these two proteins into the fertilization envelope in quick-frozen, deeply etched samples. Both proteins are abundant in the fertilization envelope as early as one minute after fertilization. Coincident with paracrystalline coating of the envelope, the labeling density is markedly reduced, suggesting that antigenic sites may be masked by the paracrystalline coat. This suggests that the ovoperoxidase-proteoliaisin complex resides within the central portion of the fertilization envelope, rather than in the paracrystalline coat.

Protein kinase C modulation of fibronectin matrix assembly.

Fibroblasts have cell surface sites that mediate the assembly of fibronectin (Fn) into the extracellular matrix. Treatment of fibroblasts with kinase inhibitors (ML-7, H7, HA1004, calphostin C, and staurosporine) resulted in the rapid decrease in the binding of 125I-labeled plasma Fn and iodinated amino-terminal fragments of Fn. The dose responses of the four inhibitors suggest that the target kinase is protein kinase C (PKC) rather than the cyclic AMP- or cyclic GMP-dependent kinases. Three different fibroblastic cells were similarly affected. The inhibition was rapid and reversible and could not be overcome by increasing concentrations of Fn. Treatment of fibroblasts with phorbol esters and other agents that activate PKC resulted in increased amounts of 125I-labeled Fn binding to the cell surface. These results imply that Fn matrix assembly is modulated by PKC-mediated phosphorylation.

Teaching health and safety: problems and possibilities for learner-centered training.

The University of Massachusetts at Lowell Work Environment Department, working jointly with four New England coalitions on occupational safety and health (COSH) groups, has developed and is delivering participatory and learner-centered health and safety training for hazardous waste site workers and emergency responders throughout New England. This consortium has created a technical curriculum for hazardous waste workers accessible to the nonspecialist and which, more importantly, draws upon workers' own experience and knowledge as a source of information, education, and experiential resources. In this article, we examine the goals of the consortium, the applicability of the training method, its successes and failures, and the wider implications such pedagogical techniques have for effective and empowering health and safety training. We conclude that such a teaching technique is a successful means of providing technical knowledge and skills in a positive and rewarding atmosphere. We also conclude, however, that the extent to which this form of education is empowering, as is claimed by many of its proponents, is less clear-cut.

Functional domains of proteoliaisin, the adhesive protein that orchestrates fertilization envelope assembly.

Ovoperoxidase, the enzyme that hardens the sea urchin fertilization envelope, is inserted into the assembling extracellular matrix through the action of an intermediary protein, proteoliaisin (PLN). The domain structure of PLN, a large, rod-shaped protein that binds to ovoperoxidase and the vitelline layer, was examined by limited proteolytic cleavage. Purified proteolytic fragments of PLN were tested for their ability to bind ovoperoxidase, inhibit the binding of 125I-PLN to the vitelline layer, or act as substrates for the hardening reaction. Based on these results, the vitelline layer-binding domain can be placed near the amino terminus, followed by the binding site for ovoperoxidase; the distal two-thirds of the protein contain sites for ovoperoxidase-catalyzed dityrosine formation. The pentapeptide GRGDS (but not RGD) inhibited PLN-vitelline layer binding half-maximally at 0.2 mM. Moreover, PLN promoted adhesion of bovine aortic endothelial cells to plastic dishes, a process inhibited by GRGDS. Thus PLN is a new member of the adhesive protein family, the function of which is to coordinate the morphogenesis of a specific, rapidly assembled extracellular matrix.