Selecting representative model micro-organisms

Background  

Micro-biological research relies on the use of model organisms that act as representatives of their species or subspecies, these are frequently well-characterized laboratory strains. However, it has often become apparent that the model strain initially chosen does not represent important features of the species. For micro-organisms, the diversity of their genomes is such that even the best possible choice of initial strain for sequencing may not assure that the genome obtained adequately represents the species. To acquire information about a species' genome as efficiently as possible, we require a method to choose strains for analysis on the basis of how well they represent the species.     

Modelling expected trout ranges under current and future water temperature regimes in the Eastern Cape,South Africa

Different values have resulted in conflicts between anglers and conservation lobbies in the management of trout in South Africa. Key to the conflict is the demarcation of boundaries to areas in which brown trout Salmo trutta and rainbow trout Oncorhynchus mykiss currently occur, or are likely to establish following stocking for angling. To provide a longer-term perspective on these areas, we developed models to link salmonid biological thermal thresholds to elevation. These, when applied spatially using a digital elevation model with a probability of occurrence model, provided the basis for estimating potentially available thermal habitat for these two cold water species. Here, we acknowledge that other variables (stocking history; river connectivity) also play a role in understanding trout distributions. Using a simple scenario of an increase in mean daily water temperatures of 2 °C, we demonstrated that both brown and rainbow trout are likely to exhibit considerable range reductions in the future. Because it is possible that these range restrictions will result in an increasing desire to introduce trout into areas above their current distribution limits for the maintenance of angling opportunities, conservation managers should prioritise these areas, with management interventions seeking to understand what will help to limit introductions.     

Myosin II Activity Softens Cells in Suspension

The cellular cytoskeleton is crucial for many cellular functions such as cell motility and wound healing, as well as other processes that require shape change or force generation. Actin is one cytoskeleton component that regulates cell mechanics. Important properties driving this regulation include the amount of actin, its level of cross-linking, and its coordination with the activity of specific molecular motors like myosin. While studies investigating the contribution of myosin activity to cell mechanics have been performed on cells attached to a substrate, we investigated mechanical properties of cells in suspension. To do this, we used multiple probes for cell mechanics including a microfluidic optical stretcher, a microfluidic microcirculation mimetic, and real-time deformability cytometry. We found that nonadherent blood cells, cells arrested in mitosis, and naturally adherent cells brought into suspension, stiffen and become more solidlike upon myosin inhibition across multiple timescales (milliseconds to minutes). Our results hold across several pharmacological and genetic perturbations targeting myosin. Our findings suggest that myosin II activity contributes to increased whole-cell compliance and fluidity. This finding is contrary to what has been reported for cells attached to a substrate, which stiffen via active myosin driven prestress. Our results establish the importance of myosin II as an active component in modulating suspended cell mechanics, with a functional role distinctly different from that for substrate-adhered cells.     

Bronchoabsorption; a novel bronchoscopic technique to improve biomarker sampling of the airway

Background

Current techniques used to obtain lung samples have significant limitations and do not provide reproducible biomarkers of inflammation. We have developed a novel technique that allows multiple sampling methods from the same area (or multiple areas) of the lung under direct bronchoscopic vision. It allows collection of mucosal lining fluid and bronchial brushing from the same site; biopsy samples may also be taken. The novel technique takes the same time as standard procedures and can be conducted safely.

Methods

Eight healthy smokers aged 40–65 years were included in this study. An absorptive filter paper was applied to the bronchial mucosa under direct vision using standard bronchoscopic techniques. Further samples were obtained from the same site using bronchial brushings. Bronchoalveolar lavage (BAL) was obtained using standard techniques. Chemokine (C-C Motif) Ligand 20 (CCL20), CCL4, CCL5, Chemokine (C-X-C Motif) Ligand 1 (CXCL1), CXCL8, CXCL9, CXCL10, CXCL11, Interleukin 1 beta (IL-1β), IL-6, Vascular endothelial growth factor (VEGF), Matrix metalloproteinase 8 (MMP-8) and MMP-9 were measured in exudate and BAL. mRNA was collected from the bronchial brushings for gene expression analysis.

Results

A greater than 10 fold concentration of all the biomarkers was detected in lung exudate in comparison to BAL. High yield of good quality RNA with RNA integrity numbers (RIN) between 7.6 and 9.3 were extracted from the bronchial brushings. The subset of genes measured were reproducible across the samples and corresponded to the inflammatory markers measured in exudate and BAL.

Conclusions

The bronchoabsorption technique as described offers the ability to sample lung fluid direct from the site of interest without the dilution effects caused by BAL. Using this method we were able to successfully measure the concentrations of biomarkers present in the lungs as well as collect high yield mRNA samples for gene expression analysis from the same site. This technique demonstrates superior sensitivity to standard BAL for the measurement of biomarkers of inflammation. It could replace BAL as the method of choice for these measurements. This method provides a systems biology approach to studying the inflammatory markers of respiratory disease progression.

Trial registration

NHS Health Research Authority (13/LO/0256).     

Mechanical difference between white and gray matter in the rat cerebellum measured by scanning force microscopy

The mechanical properties of tissues are increasingly recognized as important cues for cell physiology and pathology. Nevertheless, there is a sparsity of quantitative, high-resolution data on mechanical properties of specific tissues. This is especially true for the central nervous system (CNS), which poses particular difficulties in terms of preparation and measurement. We have prepared thin slices of brain tissue suited for indentation measurements on the micrometer scale in a near-native state. Using a scanning force microscope with a spherical indenter of radius ～20 μm we have mapped the effective elastic modulus of rat cerebellum with a spatial resolution of 100 μm. We found significant differences between white and gray matter, having effective elastic moduli of K=294±74 and 454±53 Pa, respectively, at 3 μm indentation depth (n_g=245, n_w=150 in four animals, p<0.05; errors are SD). In contrast to many other measurements on larger length scales, our results were constant for indentation depths of 2–4 μm indicating a regime of linear effective elastic modulus. These data, assessed with a direct mechanical measurement, provide reliable high-resolution information and serve as a quantitative basis for further neuromechanical investigations on the mechanical properties of developing, adult and damaged CNS tissue.     

Predicted warming and browning affect timing and magnitude of plankton phenological events in lakes: a mesocosm study

1. Aquatic ecosystems in Northern Europe are expected to face increases in temperature and water colour (TB) in future. While effects of these factors have been studied separately, it is unknown whether and how a combination of them might affect phenological events and trophic interactions. 2. In a mesocosm study, we combined both factors to create conditions expected to arise during the coming century. We focused on quantifying effects on timing and magnitude of plankton spring phenological events and identifying possible mismatches between resources (phytoplankton) and consumers (zooplankton). 3. We found that the increases in TB had important effects on timing and abundance of different plankton groups. While increased temperature led to an earlier peak in phytoplankton and zooplankton and a change in the relative timing of different zooplankton groups, increased water colour reduced chlorophyll‐a concentrations. 4. Increased TB together benefitted cladocerans and calanoid copepods and led to stronger top‐down control of algae by zooplankton. There was no sign of a mismatch between primary producers and grazers as reported from other studies. 5. Our results point towards an earlier onset of plankton spring growth in shallow lakes in future with a stronger top‐down control of phytoplankton by zooplankton grazers.     

Effects of brown and turbid water on piscivore–prey fish interactions along a visibility gradient

1. Environmental changes such as eutrophication and increasing inputs of humic matter (brownification) may have strong effects on predator–prey interactions in lakes through a reduction in the visual conditions affecting foraging behaviour of visually oriented predators. 2. In this experiment, we studied the effects of visual range (25–200 cm) in combination with optically deteriorating treatments (algae, clay or brown humic water) on predator–prey interactions between pike (Esox lucius) and roach (Rutilus rutilus). We measured effects on reaction distance and strike distance for pike and escape distance for roach, when pike individuals were exposed to free‐swimming roach as well as to roach held in a glass cylinder. 3. We found that reaction distance decreased with decreasing visual range caused by increasing levels of algae, clay or humic matter. The effect of reaction distance was stronger in turbid water (clay, algae) than in the brown water treatment. 4. Strike distance was neither affected by visual range nor by optical treatment, but we found shorter strike distances when pike attacked roach using visual cues only (roach held in a cylinder) compared to when pike could use multiple senses (free‐swimming roach). Escape distance for roach was longer in turbid than in brown water treatments. 5. Changes in environmental drivers, such as eutrophication and brownification, affecting the optical climate should thus have consequences for the strength of predator–prey interactions through changes in piscivore foraging efficiency and prey escape behaviour. This in turn may affect lake ecosystems through higher‐order interactions.     

Changes in ect2 localization couple actomyosin-dependent cell shape changes to mitotic progression

As they enter mitosis, animal cells undergo profound actin-dependent changes in shape to become round. Here we identify the Cdk1 substrate, Ect2, as a central regulator of mitotic rounding, thus uncovering a link between the cell-cycle machinery that drives mitotic entry and its accompanying actin remodeling. Ect2 is a RhoGEF that plays a well-established role in formation of the actomyosin contractile ring at mitotic exit, through the local activation of RhoA. We find that Ect2 first becomes active in prophase, when it is exported from the nucleus into the cytoplasm, activating RhoA to induce the formation of a mechanically stiff and rounded metaphase cortex. Then, at anaphase, binding to RacGAP1 at the spindle midzone repositions Ect2 to induce local actomyosin ring formation. Ect2 localization therefore defines the stage-specific changes in actin cortex organization critical for accurate cell division.     

The optical stretcher: a novel laser tool to micromanipulate cells.

When a dielectric object is placed between two opposed, nonfocused laser beams, the total force acting on the object is zero but the surface forces are additive, thus leading to a stretching of the object along the axis of the beams. Using this principle, we have constructed a device, called an optical stretcher, that can be used to measure the viscoelastic properties of dielectric materials, including biologic materials such as cells, with the sensitivity necessary to distinguish even between different individual cytoskeletal phenotypes. We have successfully used the optical stretcher to deform human erythrocytes and mouse fibroblasts. In the optical stretcher, no focusing is required, thus radiation damage is minimized and the surface forces are not limited by the light power. The magnitude of the deforming forces in the optical stretcher thus bridges the gap between optical tweezers and atomic force microscopy for the study of biologic materials.