Combining Motion Analysis and Microfluidics – A Novel Approach for Detecting Whole-Animal Responses to Test Substances

Small, early life stages, such as zebrafish embryos are increasingly used to assess the biological effects of chemical compounds in vivo. However, behavioural screens of such organisms are challenging in terms of both data collection (culture techniques, drug delivery and imaging) and data evaluation (very large data sets), restricting the use of high throughput systems compared to in vitro assays. Here, we combine the use of a microfluidic flow-through culture system, or BioWell plate, with a novel motion analysis technique, (sparse optic flow - SOF) followed by spectral analysis (discrete Fourier transformation - DFT), as a first step towards automating data extraction and analysis for such screenings. Replicate zebrafish embryos housed in a BioWell plate within a custom-built imaging system were subject to a chemical exposure (1.5% ethanol). Embryo movement was videoed before (30 min), during (60 min) and after (60 min) exposure and SOF was then used to extract data on movement (angles of rotation and angular changes to the centre of mass of embryos). DFT was subsequently used to quantify the movement patterns exhibited during these periods and Multidimensional Scaling and ANOSIM were used to test for differences. Motion analysis revealed that zebrafish had significantly altered movements during both the second half of the alcohol exposure period and also the second half of the recovery period compared to their pre-treatment movements. Manual quantification of tail flicking revealed the same differences between exposure-periods as detected using the automated approach. However, the automated approach also incorporates other movements visible in the organism such as blood flow and heart beat, and has greater power to discern environmentally-driven changes in the behaviour and physiology of organisms. We suggest that combining these technologies could provide a highly efficient, high throughput assay, for assessing whole embryo responses to various drugs and chemicals.     

Long-term effects of dietary isoflavones on uterine gene expression profiles

Isoflavones (ISOs) are bioactive food ingredients of the traditional East Asian diet and currently discussed as alternatives to classical hormone replacement therapies and for reducing the prevalence of hormone-dependent cancers. Although there are many studies on ISOs, not much is known about their long-term effects.Therefore, we performed an animal experiment analyzing the effects of three different diets: a phytoestrogen-free diet, a diet supplemented with genistein (700 μg/g diet) and an ISO-high diet (232 μg daidzein and 240 μg genistein/g) at two distinct time points, juvenile (21 days) and adult (97 days). Exposure started prior to mating of the parents and throughout the life of the offspring.We observed a stronger increase of uterine wet weights in juvenile offspring with genistein exposure (1018 ± 350 mg/kg BW) than with ISO-high diet (497 ± 133 mg/kg BW). Whereas the expression of proliferation related genes (PCNA; Ki67; IGF-1; IGF-1R), analyzed by real-time-qPCR and Western blot, were significantly down-regulated in juvenile animals exposed to genistein. Additionally, genistein exposure led to estrogenic responses, observed upon increase of complement C3 and decrease of estrogen receptors gene expressions, while the exposure to ISO-high diet did not show these effects.In conclusion, both the time point on which phytoestrogen exposure starts together with the composition of the ingested phytoestrogen containing diet are of great importance for the biological response of the offspring.     

Chemical composition and ultrastructure of the epicuticular wax in four mutants of Pisum sativum (L)

The action of mutations affecting the epicuticular wax of Pisum sativum has been investigated at the chemical and ultrastructural level. Upper and lower surfaces of the leaves were found to differ markedly in both ultrastructure and chemistry. Mutations affected primarily either the lower (wa, wb and wsp) or the upper surface (wlo), but some effects of all 4 genes could be seen on both surfaces. Specific biochemical lesions could be implied for wsp and wa but the chemical effects of wb and wlo were more diffuse. Generally a close relation between chemical composition and crystallite form of the wax was evident throughout the work.     

Territorial behaviour in southern impala rams (Aepyceros melampus Lichtenstein)

The role of territoriality was investigated by studying 25 impala rams at a reserve in the Waterberg region of South Africa (23°45′S, 28°23′E). Mean territorial tenure was 67.25 days (range 23–99), with a mean territory size of 21.0 ± 11.27 ha, compared with home ranges of 34.1 ± 9.03 ha for territorial rams and 58.8 ± 33.35 ha for bachelor rams, using the fixed kernel method. Territory boundaries remained constant, whilst the area surrounding important features such as water holes, appears to be neutral in terms of territoriality. The rut, as evidenced from peaks in chasing and roaring, lasted for 2 months from 10 April to 10 June 2001, with intensified behaviour including matings observed from 16 May to 4 June 2001. Territorial rams chase and roar more than bachelors. Flehmen and display behaviours are performed by all rams, whilst fights and other reproductive behaviours are generally rare. Bachelors browse more than territorial rams. Only bachelors spar and allogroom, and orally groom themselves more than territorial rams.     

New selective and differential medium for Vibrio cholerae and Vibrio vulnificus

Thiosulfate-citrate-bile salts-sucrose agar has been routinely used for the isolation of pathogenic vibrios, although its selectivity for Vibrio cholerae and Vibrio vulnificus is inadequate. Therefore, a new plating medium, cellobiose-polymyxin B-colistin agar, was developed for the isolation of these two species. Cellobiose-polymyxin B-colistin agar demonstrated a significant advantage over other media designed for the isolation or differentiation of vibrios: of both the 136 strains representing 19 Vibrio species and the marine isolates of the genera Pseudomonas, Flavobacterium, and Photobacterium, only V. vulnificus and V. cholerae were able to grow. Furthermore, the fermentation of cellobiose by V. vulnificus allowed for the easy differentiation of these two species. This medium offers significant potential as a selective and differential medium for these two pathogenic vibrios.     

Amino acid-dependent transport of sugars by Fusobacterium nucleatum ATCC 10953.

Resting cells of Fusobacterium nucleatum 10953 (grown previously in a medium containing glucose) failed to accumulate glucose under aerobic or anaerobic conditions. However, the addition of glutamic acid, lysine, or histidine to anaerobic suspensions of cells caused the immediate and rapid accumulation of glucose. Except for the amino acid-dependent transport of galactose and fructose (the latter being transported at approximately one-third the rate of glucose), no other sugars tested were accumulated by the resting cells. Amino acid-dependent uptake of sugar(s) by F. nucleatum was abolished by exposure of cells to air, and under aerobic conditions the rates of fermentation of glutamic acid and lysine were less than 15% of the rates determined anaerobically. The energy necessary for active transport of the sugars (acetyl phosphate and ATP) is derived from the anaerobic fermentation of glutamic acid, lysine, or histidine. Competition studies revealed that glucose and galactose were mutual and exclusive inhibitors of transport, and it is suggested that the two sugars (Km = 14 microM) are translocated via a common carrier. The products of amino acid-dependent sugar transport were recovered from resting cells as ethanol-precipitable, high-molecular-weight polymers. Polymer formation by F. nucleatum, during growth in medium containing glucose or galactose, was confirmed by electron microscopy.     

Age-related changes in oxidized proteins

We have previously described the oxidative inactivation of several key metabolic enzymes by a variety of mixed function oxidation systems. Because many of the enzymes which are inactivated have been shown by others to accumulate as inactive or less active forms during cellular aging, we have examined the levels of oxidatively modified proteins in two model systems used for studies on aging. The results show that levels of oxidatively modified proteins increase with age in circulating erythrocytes, and this change is correlated with the loss of marker enzyme activity. Our studies also show that in cultured fibroblasts from normal donors the levels of oxidatively modified proteins increase only after the age of 60. However, the levels of oxidatively modified proteins in fibroblasts from individuals with progeria or Werner's syndrome are significantly higher than age-matched controls. Moreover, treatment of glucose-6-phosphate dehydrogenase with a mixed function oxidation system leads to oxidative modification and increased heat lability of the enzyme. Taken together these results suggest that loss of functional enzyme activity and increased heat lability of enzymes during aging may be due in part to oxidative modification by mixed function oxidation systems.     

Sterol Composition and Ecdysteroid Content of Eggs of the Root-knot Nematodes Meloidogyne incognita and M. arenaria

Free and esterified sterols of eggs of the root-knot nematodes Meloidogyne incognita races 2 and 3 and M. arenaria race 1 were isolated and identified by gas-liquid chromatography-mass spectrometry. The major sterols of eggs of each race were 24-ethylcholesterol (33.4-38.8% of total sterol), 24-ethylcholestanol (18.3-25.3%), 24-methylcholesterol (8.6-11.7%), 24-methylcholestanol (7.7-12.5%), and cholesterol (4.6-11.6%). Consequently, the major metabolic transformation performed by Meloidogyne females or eggs upon host sterols appeared to be saturation of the sterol nucleus. The free and esterified sterols of the same race did not differ appreciably, except for a slight enrichment of the steryl esters in cholesterol. Although the sterol composition of Meloidogyne eggs differed from that of other life stages of other genera of plant-parasitic nematodes, the three Meloidogyne races could not be distinguished from each other by their egg sterols. Ecdysteroids, compounds with hormonal function in insects, were not detected by radioimmunoassay in the Meloidogyne eggs either as free ecdysteroids or as polar conjugates.     

Effects of microclimate on daily flight activity ofSimulium arcticum Malloch (Diptera: Simuliidae)

Microclimate was found to affect the daily flight activity ofSimulium arcticum in a pasture situation in central Alberta, Canada. Vapour pressure, air temperature, light intensity, and wind gust velocity had the greatest correlation to flight activity. These variables were correlated to support the concept that they influence the energy and water balances of a black fly. Flight activity is a response of the black fly to alterations of its energy and water balances.