Medium-Throughput Processing of Whole Mount In Situ Hybridisation Experiments into Gene Expression Domains

Understanding the function and evolution of developmental regulatory networks requires the characterisation and quantification of spatio-temporal gene expression patterns across a range of systems and species. However, most high-throughput methods to measure the dynamics of gene expression do not preserve the detailed spatial information needed in this context. For this reason, quantification methods based on image bioinformatics have become increasingly important over the past few years. Most available approaches in this field either focus on the detailed and accurate quantification of a small set of gene expression patterns, or attempt high-throughput analysis of spatial expression through binary pattern extraction and large-scale analysis of the resulting datasets. Here we present a robust, “medium-throughput” pipeline to process in situ hybridisation patterns from embryos of different species of flies. It bridges the gap between high-resolution, and high-throughput image processing methods, enabling us to quantify graded expression patterns along the antero-posterior axis of the embryo in an efficient and straightforward manner. Our method is based on a robust enzymatic (colorimetric) in situ hybridisation protocol and rapid data acquisition through wide-field microscopy. Data processing consists of image segmentation, profile extraction, and determination of expression domain boundary positions using a spline approximation. It results in sets of measured boundaries sorted by gene and developmental time point, which are analysed in terms of expression variability or spatio-temporal dynamics. Our method yields integrated time series of spatial gene expression, which can be used to reverse-engineer developmental gene regulatory networks across species. It is easily adaptable to other processes and species, enabling the in silico reconstitution of gene regulatory networks in a wide range of developmental contexts.     

Prevalence of a Nosema sp. (Microsporida: Nosematidae) in Natural Populations of Chorthippus brunneus (Orthoptera: Gomphocerinae) in Southern Spain

A microsporidium Nosema sp. was isolated from the grasshopper Chorthippus brunneus (Thunberg) collected in Spain. Infected individuals were found throughout most of the season when nymphs and adults of C. brunneus were present (March-June). Other acridoids collected in the same area were not found to be infected, indicating that this microsporidium is probably host specific. Attempts to artificially infect Dociostarus maroccanus (Thunberg) nymphs failed.     

Proficiency testing in cytology laboratories in Ontario, Canada: a decade of experience. III. A precision study of consistency and reproducibility in cytology reporting

The Ontario Laboratory Proficiency Testing Program's testing model and the results of early surveys of cytology laboratories have been previously described. To test consistency and accuracy, the same slides from one survey were recirculated to the same laboratory in the next survey. There was no statistical difference in either the accuracy or consistency in reporting by the various categories of personnel. Analysis of the data by category of diagnosis showed the reporting of "no abnormal cells" and "benign atypia" to be slightly more accurate and "moderate dysplasia" and "severe dysplasia" to be slightly less accurate than the reporting in other categories. Consistency in reporting appeared to be slightly less accurate in severe dysplasia than in the other categories. The analysis, which includes the results from all licensed cytology laboratories in Ontario, provides evidence that, in the reporting of cervical cytologic material, it is feasible to differentiate among the various categories within the spectrum of squamous epithelial abnormalities with a general level of accuracy and consistency that, although satisfactory, is not yet ideal.     

Physiological characteristics of serotonin transporters on rat platelets

Physiological characteristics of serotonin (5-hydroxytryptamine, 5HT) transport through the platelet membrane was investigated in Wistar rats with our recently developed method permitting repetitive measurements of transporter kinetics in individual animals. Full kinetic analysis in the population of 91 animals revealed Michaelis constant (K_m) of 0.158±0.025 μM and maximal velocity (V_max) of 5HT uptake of 225±32 pmol per 10⁸platelets min⁻¹ (mean±S.D.). Both kinetic parameters demonstrated normal distribution curves, which for V_max were slightly skewed toward higher than average values. No gender effect was shown in frequency distributions, mean values and variability of kinetic parameters. A significant intraindividual correlation between kinetic parameters was found suggesting compensation at the level of the plasma membrane. Kinetic parameters were not influenced by age (until the middle age) or annual cycle (under laboratory conditions) and were shown to be fairly stable in time, supporting the view that platelet 5HT transport kinetics could be a useful biological trait marker.     

A newly recorded entomopoxvirus B in Anacridium aegyptium (Orthoptera: Acrididae)

An entomopoxvirus infecting fat body cells of Anacridium aegyptium is reported. Spheroid virus occlusion bodies, ranging in size from 3 to 8 μm, contain virions 336–356 nm long and 210–217 nm wide. The virion ultrastructure indicates that the newly discovered virus belongs to the genus Entomopoxvirus B known to infect Lepidoptera and Orthoptera.     

Alcohol dependence and polymorphisms of serotonin-related genes: association studies

Variations in 5HT-related genes contribute to the alterations of serotonergic neurotransmission, which is implicated in the etiopathology of alcoholism. In this preliminary study we have tested polymorphisms of genes involved in 5HT transport and turnover for their association with alcohol dependence. A case group of males with type 2 alcoholism (N=59) and a control group of healthy males (N=282), both of Croatian origin, were analyzed for the frequency distribution of polymorphisms in 5HT transporter (5HTT-VNTR2, 5HTT-LPR), monoamine oxidase A (MAOA-uVNTR) and B (MAOB-A/G) and tryptophan hydroxylase 1 (TPH1 A218C) and 2 (TPH2 G-703T) genes. An increase in the frequencies of 10-repeat allele (p = 0.010; OR = 1.73; 95% CI = 1.14-2.60) and 10/10 genotype (p = 0.006; OR = 2.57; 95% CI = 1.32-5.00) of the 5HTT-VNTR2 polymorphism was found in alcoholic patients. No differences between case and control groups were observed for the other tested polymorphisms. Present results support earlier studies implicating the role of 5HTT gene in alcoholism. The increase of sample size (in progress) is expected to enable search of more subtle differences, as well as re-evaluation of these preliminary findings.     

Pharmacological characterization of vasomotor activity of human musculocutaneous perforator artery and vein.

Vasospasm is one of the main causes of skin ischemic necrosis in cutaneous and musculocutaneous flap surgery, but the pathogenic mechanism is unclear. We planned to test the hypothesis derived from clinical impression that veins are more susceptible to vasospasm than arteries in flap surgery and, once established, that venous vasospasm is difficult to resolve and more detrimental than arterial vasospasm. To this end, we investigated the differences in sensitivity to vasoconstrictors and vasodilators between the human musculocutaneous perforator (MCP) artery and vein by measuring the isometric tension of arterial and venous rings suspended in organ chambers. Vascular contraction was expressed as a percentage of the tension induced by 50 mM KCl. Relaxation was expressed as a percentage of contraction induced by a submaximal concentration (3 x 10(-9) M) of endothelin-1 (ET-1). We observed that the vasoconstrictor potency of norepinephrine was significantly higher in the MCP vein than in the MCP artery. The vasoconstrictor potency of ET-1 and the thromboxane A(2) mimetic U-46619 were similar in the MCP vein and artery, but the maximal contraction induced by ET-1 and U-46619 was significantly higher in the MCP vein than in the MCP artery. On the other hand, the MCP vein was less sensitive than the MCP artery to the relaxation effect of nitroglycerin, nifedipine, and lidocaine. These differences between the human MCP artery and vein in response to vasoactive agents lend support to the clinical impression in flap surgery that veins appear to be more susceptible to vasospasm than arteries and venous vasospasm seems to be more difficult to resolve than arterial vasospasm in cutaneous and musculocutaneous flap surgery.     

Serotonin transporter kinetics in rats selected for extreme values of platelet serotonin level

By selective breeding of Wistar rats for the extreme values of platelet serotonin (5HT) level (PSL), we have developed earlier two sublines of animals differing markedly in this parameter. Further studies, performed on the protein and mRNA levels, revealed platelet serotonin transporter (5HTt) as parameter underlying mentioned differences in PSL between sublines. In this work, we have performed full-kinetic analysis of platelet serotonin uptake (PSU) in animals from the genetically selected sublines. The results demonstrated marked differences in maximal velocity (V(max)) of the 5HT transporter, as contrasted to the lack of any difference in the Michaelis constant (K(m)). High correlation between PSL and V(max) of PSU was demonstrated, revealing that the number of membrane 5HT transporter sites is under genetic control and responsible for marked differences in PSL between high- and low-5HT sublines. These results enabled further selective breeding of animals for the extremes of V(max) of platelet 5HT transporter, and so the development of more specific model "Wistar-Zagreb 5HT rats".