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.     

Computer simulation of antibody binding specificity

A Monte Carlo algorithm that searches for the optimal docking configuration of hen egg white lysozyme to an antibody is developed. Both the lysozyme and the antibody are kept rigid. Unlike the work of other authors, our algorithm does not attempt to explicitly maximize surface contact, but minimizes the energy computed using coarse-grained pair potentials. The final refinement of our best solutions using all-atom OPLS potentials (Jorgensen and Tirado-Rives⁸) consistently yields the native conformation as the preferred solution for three different antibodies. We find that the use of an exponential distance-dependent dielectric function is an improvement over the more commonly used linear form. © 1993 Wiley-Liss, Inc.     

Bromocriptine in management of large pituitary tumours.

Bromocriptine has an accepted place in the management of small pituitary tumours that secrete either prolactin or growth hormone. The treatment of large tumours with extrasellar extensions is more difficult, however: though surgery is the standard treatment, it is often unsuccessful in returning excessive hormone secretion to normal and may cause hypopituitarism. A prospective trial was undertaken to assess the frequency with which changes in pituitary function and size of large tumours occurs. Nineteen patients were studied before and during treatment with bromocriptine (7.5 to 60 ml/day) for three to 22 months, using contrast radiology and a detailed assessment of pituitary function. Eighteen patients had hyperprolactinaemia and two of these also had raised concentrations of growth hormones; one patient had an apparently non-functioning tumour. In 12 patients (63%) tumour size decreased with bromocriptine and no tumour enlarged. Nine patients had visual-field defects, which improved in seven, becoming normal in five. Pituitary function improved in nine patients (47%) becoming entirely normal in three. Bromocriptine should be the treatment of choice in patients with large pituitary tumours with extrasellar extensions, provided close supervision is maintained.     

Unexplained hepatitis following halothane.

Full clinical and laboratory details of 203 patients with postoperative jaundice were submitted to a panel of hepatologists. All patients whose jaundice may have had an identifiable cause were excluded, which left 76 patients with unexplained hepatitis following halothane anaesthesia (UHFH). Hepatitis in 95% of these cases followed multiple exposure to halothane, with repeated exposure within four weeks in 55% of cases. Twenty-nine patients were obese, 52 were aged 41-70, and 53 were women. Thirteen patients died in acute hepatic failure. Rapid onset of jaundice after anaesthesia, male sex, and obesity in either sex were poor prognostic signs. Of the clinical stigmata of hypersensitivity, only eosinophilia was impressive. The UHFH group had a much greater incidence of liver kidney microsomal (LKM) and thyroid antibodies and autoimmune complement fixation than those patients whose jaundice related to identifiable factors. Thirteen of the 19 patients with LKM antibodies also had thyroid antibodies. In six patients retested two to three years later LKM antibodies had disappeared, although thyroid antibodies persisted. Rapidly repeated exposure to halothane may cause hepatitis, but such a complication is probably rare. Possibly obese women with a tendency to organ-specific autoimmunity may be more at risk. Nevertheless, the comparative risks of rapidly repeated halothane or non-halothane anaesthesia cannot be determined from the present data. If alternative satisfactory agents are available halothane should be avoided in patients with unexplained hepatitis after previous exposure, although in three to five patients with UHFH who were re-exposed to halothane jaundice did not recur.     

Effects of diet,habitat, and phylogeny on the fecal microbiome of wild African savanna (Loxodonta africana) and forest elephants (L. cyclotis)

The gut microbiome, or the community of microorganisms inhabiting the digestive tract, is often unique to its symbiont and, in many animal taxa, is highly influenced by host phylogeny and diet. In this study, we characterized the gut microbiome of the African savanna elephant (Loxodonta africana) and the African forest elephant (Loxodonta cyclotis), sister taxa separated by 2.6–5.6 million years of independent evolution. We examined the effect of host phylogeny on microbiome composition. Additionally, we examined the influence of habitat types (forest versus savanna) and diet types (crop‐raiding versus noncrop‐raiding) on the microbiome within L. africana. We found 58 bacterial orders, representing 16 phyla, across all African elephant samples. The most common phyla were Firmicutes, Proteobacteria, and Bacteroidetes. The microbiome of L. africana was dominated by Firmicutes, similar to other hindgut fermenters, while the microbiome of L. cyclotis was dominated by Proteobacteria, similar to more frugivorous species. Alpha diversity did not differ across species, habitat type, or diet, but beta diversity indicated that microbial communities differed significantly among species, diet types, and habitat types. Based on predicted KEGG metabolic pathways, we also found significant differences between species, but not habitat or diet, in amino acid metabolism, energy metabolism, and metabolism of terpenoids and polyketides. Understanding the digestive capabilities of these elephant species could aid in their captive management and ultimately their conservation.     

Validating the use of stereo‐video cameras to conduct remote measurements of sea turtles

Point 1: Stereo‐video camera systems (SVCSs) are a promising tool to remotely measure body size of wild animals without the need for animal handling. Here, we assessed the accuracy of SVCSs for measuring straight carapace length (SCL) of sea turtles.Point 2: To achieve this, we hand captured and measured 63 juvenile, subadult, and adult sea turtles across three species: greens, Chelonia mydas (n = 52); loggerheads, Caretta caretta (n = 8); and Kemp''s ridley, Lepidochelys kempii (n = 3) in the waters off Eleuthera, The Bahamas and Crystal River, Florida, USA, between May and November 2019. Upon release, we filmed these individuals with the SVCS. We performed photogrammetric analysis to extract stereo SCL measurements (eSCL), which were then compared to the (manual) capture measurements (mSCL).Point 3: mSCL ranged from 25.9 to 89.2 cm, while eSCL ranged from 24.7 to 91.4 cm. Mean percent bias of eSCL ranged from −0.61% (±0.11 SE) to −4.46% (±0.31 SE) across all species and locations. We statistically analyzed potential drivers of measurement error, including distance of the turtle to the SVCS, turtle angle, image quality, turtle size, capture location, and species.Point 4: Using a linear mixed effects model, we found that the distance between the turtle and the SVCS was the primary factor influencing measurement error. Our research suggests that stereo‐video technology enables high‐quality measurements of sea turtle body size collected in situ without the need for hand‐capturing individuals. This study contributes to the growing knowledge base that SVCS are accurate for body size measurements independent of taxonomic clade.     

Current approaches using genetic distances produce poor estimates of landscape resistance to interindividual dispersal

Landscape resistance reflects how difficult it is for genes to move across an area with particular attributes (e.g. land cover, slope). An increasingly popular approach to estimate resistance uses Mantel and partial Mantel tests or causal modelling to relate observed genetic distances to effective distances under alternative sets of resistance parameters. Relatively few alternative sets of resistance parameters are tested, leading to relatively poor coverage of the parameter space. Although this approach does not explicitly model key stochastic processes of gene flow, including mating, dispersal, drift and inheritance, bias and precision of the resulting resistance parameters have not been assessed. We formally describe the most commonly used model as a set of equations and provide a formal approach for estimating resistance parameters. Our optimization finds the maximum Mantel r when an optimum exists and identifies the same resistance values as current approaches when the alternatives evaluated are near the optimum. Unfortunately, even where an optimum existed, estimates from the most commonly used model were imprecise and were typically much smaller than the simulated true resistance to dispersal. Causal modelling using Mantel significance tests also typically failed to support the true resistance to dispersal values. For a large range of scenarios, current approaches using a simple correlational model between genetic and effective distances do not yield accurate estimates of resistance to dispersal. We suggest that analysts consider the processes important to gene flow for their study species, model those processes explicitly and evaluate the quality of estimates resulting from their model.