Free Brick1 Is a Trimeric Precursor in the Assembly of a Functional Wave Complex

Background: The Wave complex activates the Arp2/3 complex, inducing actin polymerization in lamellipodia and membrane ruffles. The Wave complex is composed of five subunits, the smallest of which, Brick1/Hspc300 (Brk1), is the least characterized. We previously reported that, unlike the other subunits, Brk1 also exists as a free form. Principal Findings: Here we report that this free form of Brk1 is composed of homotrimers. Using a novel assay in which purified free Brk1 is electroporated into HeLa cells, we were able to follow its biochemical fate in cells and to show that free Brk1 becomes incorporated into the Wave complex. Importantly, incorporation of free Brk1 into the Wave complex was blocked upon inhibition of protein synthesis and incorporated Brk1 was found to associate preferentially with neosynthesized subunits. Brk1 depleted HeLa cells were found to bleb, as were Nap1, Wave2 or ARPC2 depleted cells, suggesting that this blebbing phenotype of Brk1 depleted cells is due to an impairment of the Wave complex function rather than a specific function of free Brk1. Blebs of Brk1 depleted cells were emitted at sites where lamellipodia and membrane ruffles were normally emitted. In Brk1 depleted cells, the electroporation of free Brk1 was sufficient to restore Wave complex assembly and to rescue the blebbing phenotype. Conclusion: Together these results establish that the free form of Brk1 is an essential precursor in the assembly of

Coral microbiome composition along the northern Red Sea suggests high plasticity of bacterial and specificity of endosymbiotic dinoflagellate communities

Background The capacity of reef-building corals to tolerate (or adapt to) heat stress is a key factor determining their resilience to future climate change. Changes in coral microbiome composition (particularly for microalgal endosymbionts and bacteria) is a potential mechanism that may assist corals to thrive in warm waters. The northern Red Sea experiences extreme temperatures anomalies, yet corals in this area rarely bleach suggesting possible refugia to climate change. However, the coral microbiome composition, and how it relates to the capacity to thrive in warm waters in this region, is entirely unknown. Results We investigated microbiomes for six coral species (Porites nodifera, Favia favus, Pocillopora damicornis, Seriatopora hystrix, Xenia umbellata, and Sarcophyton trocheliophorum) from five sites in the northern Red Sea spanning 4° of latitude and summer mean temperature ranges from 26.6 °C to 29.3 °C. A total of 19 distinct dinoflagellate endosymbionts were identified as belonging to three genera in the family Symbiodiniaceae (Symbiodinium, Cladocopium, and Durusdinium). Of these, 86% belonged to the genus Cladocopium, with notably five novel types (19%). The endosymbiont community showed a high degree of host-specificity despite the latitudinal gradient. In contrast, the diversity and composition of bacterial communities of the surface mucus layer (SML)—a compartment particularly sensitive to environmental change—varied significantly between sites, however for any given coral was species-specific. Conclusion The conserved endosymbiotic community suggests high physiological plasticity to support holobiont productivity across the different latitudinal regimes. Further, the presence of five novel algal endosymbionts suggests selection of certain genotypes (or genetic adaptation) within the semi-isolated Red Sea. In contrast, the dynamic composition of bacteria associated with the SML across sites may contribute to holobiont function and broaden the ecological niche. In doing so, SML bacterial communities may aid holobiont local acclimatization (or adaptation) by readily responding to changes in the host environment. Our study provides novel insight about the selective and endemic nature of coral microbiomes along the northern Red Sea refugia

Benthic and Hyporheic Macroinvertebrate Distribution Within the Heads and Tails of Riffles During Baseflow Conditions

The distribution of lotic fauna is widely acknowledged to be patchy reflecting the interaction between biotic and abiotic factors. In an in-situ field study, the distribution of benthic and hyporheic invertebrates in the heads (downwelling) and tails (upwelling) of riffles were examined during stable baseflow conditions. Riffle heads were found to contain a greater proportion of interstitial fine sediment than riffle tails. Significant differences in the composition of benthic communities were associated with the amount of fine sediment. Riffle tail habitats supported a greater abundance and diversity of invertebrates sensitive to fine sediment such as EPT taxa. Shredder feeding taxa were more abundant in riffle heads suggesting greater availability of organic matter. In contrast, no significant differences in the hyporheic community were recorded between riffle heads and tails. We hypothesise that clogging of hyporheic interstices with fine sediments may have resulted in the homogenization of the invertebrate community by limiting faunal movement into the hyporheic zone at both the riffle head and tail. The results suggest that vertical hydrological exchange significantly influences the distribution of fine sediment and macroinvertebrate communities at the riffle scale

The contribution of refractoriness to arrhythmic substrate in hypokalemic Langendorff-perfused murine hearts

The clinical effects of hypokalemia including action potential prolongation and arrhythmogenicity suppressible by lidocaine were reproduced in hypokalemic (3.0 mM K(+)) Langendorff-perfused murine hearts before and after exposure to lidocaine (10 μM). Novel limiting criteria for local and transmural, epicardial, and endocardial re-excitation involving action potential duration (at 90% repolarization, APD(90)), ventricular effective refractory period (VERP), and transmural conduction time (Δlatency), where appropriate, were applied to normokalemic (5.2 mM K(+)) and hypokalemic hearts. Hypokalemia increased epicardial APD(90) from 46.6 ± 1.2 to 53.1 ± 0.7 ms yet decreased epicardial VERP from 41 ± 4 to 29 ± 1 ms, left endocardial APD(90) unchanged (58.2 ± 3.7 to 56.9 ± 4.0 ms) yet decreased endocardial VERP from 48 ± 4 to 29 ± 2 ms, and left Δlatency unchanged (1.6 ± 1.4 to 1.1 ± 1.1 ms; eight normokalemic and five hypokalemic hearts). These findings precisely matched computational predictions based on previous reports of altered ion channel gating and membrane hyperpolarization. Hypokalemia thus shifted all re-excitation criteria in the positive direction. In contrast, hypokalemia spared epicardial APD(90) (54.8 ± 2.7 to 60.6 ± 2.7 ms), epicardial VERP (84 ± 5 to 81 ± 7 ms), endocardial APD(90) (56.6 ± 4.2 to 63.7 ± 6.4 ms), endocardial VERP (80 ± 2 to 84 ± 4 ms), and Δlatency (12.5 ± 6.2 to 7.6 ± 3.4 ms; five hearts in each case) in lidocaine-treated hearts. Exposure to lidocaine thus consistently shifted all re-excitation criteria in the negative direction, again precisely agreeing with the arrhythmogenic findings. In contrast, established analyses invoking transmural dispersion of repolarization failed to account for any of these findings. We thus establish novel, more general, criteria predictive of arrhythmogenicity that may be particularly useful where APD(90) might diverge sharply from VERP

Defoliation and Soil Compaction Jointly Drive Large-Herbivore Grazing Effects on Plants and Soil Arthropods on Clay Soil

In addition to the well-studied impacts of defecation and defoliation, large herbivores also affect plant and arthropod communities through trampling, and the associated soil compaction. Soil compaction can be expected to be particularly important on wet, fine-textured soils. Therefore, we established a full factorial experiment of defoliation (monthly mowing) and soil compaction (using a rammer, annually) on a clay-rich salt marsh at the Dutch coast, aiming to disentangle the importance of these two factors. Additionally, we compared the effects on soil physical properties, plants, and arthropods to those at a nearby cattle-grazed marsh under dry and under waterlogged conditions. Soil physical conditions of the compacted plots were similar to the conditions at cattle-grazed plots, showing decreased soil aeration and increased waterlogging. Soil salinity was doubled by defoliation and quadrupled by combined defoliation and compaction. Cover of the dominant tall grass Elytrigia atherica was decreased by 80% in the defoliated plots, but cover of halophytes only increased under combined defoliation and compaction. Effects on soil micro-arthropods were most severe under waterlogging, showing a fourfold decrease in abundance and a smaller mean body size under compaction. Although the combined treatment of defoliation and trampling indeed proved most similar to the grazed marsh, large discrepancies remained for both plant and soil fauna communities, presumably because of colonization time lags. We conclude that soil compaction and defoliation differently affect plant and arthropod communities in grazed ecosystems, and that the magnitude of their effects depends on herbivore density, productivity, and soil physical properties

Line-Scanning Particle Image Velocimetry: An Optical Approach for Quantifying a Wide Range of Blood Flow Speeds in Live Animals

The ability to measure blood velocities is critical for studying vascular development, physiology, and pathology. A key challenge is to quantify a wide range of blood velocities in vessels deep within living specimens with concurrent diffraction-limited resolution imaging of vascular cells. Two-photon laser scanning microscopy (TPLSM) has shown tremendous promise in analyzing blood velocities hundreds of micrometers deep in animals with cellular resolution. However, current analysis of TPLSM-based data is limited to the lower range of blood velocities and is not adequate to study faster velocities in many normal or disease conditions.We developed line-scanning particle image velocimetry (LS-PIV), which used TPLSM data to quantify peak blood velocities up to 84 mm/s in live mice harboring brain arteriovenous malformation, a disease characterized by high flow. With this method, we were able to accurately detect the elevated blood velocities and exaggerated pulsatility along the abnormal vascular network in these animals. LS-PIV robustly analyzed noisy data from vessels as deep as 850 µm below the brain surface. In addition to analyzing in vivo data, we validated the accuracy of LS-PIV up to 800 mm/s using simulations with known velocity and noise parameters.To our knowledge, these blood velocity measurements are the fastest recorded with TPLSM. Partnered with transgenic mice carrying cell-specific fluorescent reporters, LS-PIV will also enable the direct in vivo correlation of cellular, biochemical, and hemodynamic parameters in high flow vascular development and diseases such as atherogenesis, arteriogenesis, and vascular anomalies

Marine probiotics: increasing coral resistance to bleaching through microbiome manipulation

Although the early coral reef-bleaching warning system (NOAA/USA) is established, there is no feasible treatment that can minimize temperature bleaching and/or disease impacts on corals in the field. Here, we present the first attempts to extrapolate the widespread and well-established use of bacterial consortia to protect or improve health in other organisms (e.g., humans and plants) to corals. Manipulation of the coral-associated microbiome was facilitated through addition of a consortium of native (isolated from Pocillopora damicornis and surrounding seawater) putatively beneficial microorganisms for corals (pBMCs), including five Pseudoalteromonas sp., a Halomonas taeanensis and a Cobetia marina-related species strains. The results from a controlled aquarium experiment in two temperature regimes (26 °C and 30 °C) and four treatments (pBMC; pBMC with pathogen challenge – Vibrio coralliilyticus, VC; pathogen challenge, VC; and control) revealed the ability of the pBMC consortium to partially mitigate coral bleaching. Significantly reduced coral-bleaching metrics were observed in pBMC-inoculated corals, in contrast to controls without pBMC addition, especially challenged corals, which displayed strong bleaching signs as indicated by significantly lower photopigment contents and Fv/Fm ratios. The structure of the coral microbiome community also differed between treatments and specific bioindicators were correlated with corals inoculated with pBMC (e.g., Cobetia sp.) or VC (e.g., Ruegeria sp.). Our results indicate that the microbiome in corals can be manipulated to lessen the effect of bleaching, thus helping to alleviate pathogen and temperature stresses, with the addition of BMCs representing a promising novel approach for minimizing coral mortality in the face of increasing environmental impacts