Assessment of treatment-specific tethering survival bias for the juvenile blue crab Callinectes sapidus in a simulated salt marsh.

The blue crab (Callinectes sapidus) is ecologically and economically important in Chesapeake Bay. Nursery habitats, such as seagrass beds, disproportionately contribute individuals to the adult segment of populations. Salt marshes dominated by smooth cordgrass Spartina alterniflora are intertidal nursery habitats which may serve as a refuge from predation for juvenile blue crabs. However, the effects of various characteristics of salt marshes on nursery metrics, such as survival, have not been quantified. Comparisons of juvenile survival between salt marshes and other habitats often employ tethering to assess survival. Although experimental bias when tethering juvenile prey is well recognized, the potential for habitat-specific bias in salt marshes has not been experimentally tested. Using short-term mesocosm predation experiments, we tested if tethering in simulated salt marsh habitats produces a habitat-specific bias. Juvenile crabs were tethered or un-tethered and randomly allocated to mesocosms at varying simulated shoot densities and unstructured sand. Tethering reduced survival, and its effect was not habitat specific, irrespective of shoot density, as evidenced by a non-significant interaction effect between tethering treatment and habitat. Thus, tethering juvenile blue crabs in salt marsh habitat did not produce treatment-specific bias relative to unvegetated habitat across a range of shoot densities; survival of tethered and un-tethered crabs was positively related to shoot density. These findings indicate that tethering is a useful method for assessing survival in salt marshes, as with other nursery habitats including seagrass beds, algae and unstructured sand.

Phase separation provides a mechanism to reduce noise in cells.

Expression of proteins inside cells is noisy, causing variability in protein concentration among identical cells. A central problem in cellular control is how cells cope with this inherent noise. Compartmentalization of proteins through phase separation has been suggested as a potential mechanism to reduce noise, but systematic studies to support this idea have been missing. In this study, we used a physical model that links noise in protein concentration to theory of phase separation to show that liquid droplets can effectively reduce noise. We provide experimental support for noise reduction by phase separation using engineered proteins that form liquid-like compartments in mammalian cells. Thus, phase separation can play an important role in biological signal processing and control.

Long-term persistence of structured habitats: seagrass meadows as enduring hotspots of biodiversity and faunal stability.

Ecological studies indicate that structurally complex habitats support elevated biodiversity, stability and resilience. The long-term persistence of structured habitats and their importance in maintaining biodiverse hotspots remain underexplored. We combined geohistorical data (dead mollusc assemblages, 'DA') and contemporary surveys (live mollusc assemblages, 'LA') to assess the persistence of local seagrass habitats over multi-centennial timescales and to evaluate whether they acted as long-term drivers of biodiversity, stability and resilience of associated fauna. We sampled structured seagrass meadows and open sandy bottoms along Florida's Gulf Coast. Results indicated that: (i) LA composition differed significantly between the two habitat types, (ii) LA from seagrass sites were characterized by significantly elevated local biodiversity and significantly higher spatial stability, (iii) DA composition differed significantly between the two habitat types, and (iv) fidelity between LA and DA was significantly greater for seagrass habitats. Contemporary results support the hypotheses that local biodiversity and spatial stability of marine benthos are both elevated in structured seagrass habitats. Geohistorical results suggest that structured habitats persist as local hotspots of elevated biodiversity and faunal stability over centennial-to-millennial timescales; indicating that habitat degradation and concomitant loss within structurally complex marine systems is a key driver of declining biodiversity and resilience.

Piezo1 channels are mechanosensors in human fetoplacental endothelial cells.

STUDY QUESTION: Does the shear stress sensing ion channel subunit Piezo1 have an important mechanotransduction role in human fetoplacental endothelium? SUMMARY ANSWER: Piezo1 is present and functionally active in human fetoplacental endothelial cells, and disruption of Piezo1 prevents the normal response to shear stress. WHAT IS KNOWN ALREADY: Shear stress is an important stimulus for maturation and function of placental vasculature but the molecular mechanisms by which the force is detected and transduced are unclear. Piezo1 channels are Ca2+-permeable non-selective cationic channels which are critical for shear stress sensing and maturation of murine embryonic vasculature. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: We investigated the relevance of Piezo1 to placental vasculature by studying human fetoplacental endothelial cells (FpECs) from healthy pregnancies. Endothelial cells were isolated from placental cotyledons and cultured, for the study of tube formation and cell alignment to shear stress. In addition, human placental arterial endothelial cells were isolated and studied immediately by patch-clamp electrophysiology. MAIN RESULTS AND THE ROLE OF CHANCE: The synthetic Piezo1 channel agonist Yoda1 caused strong elevation of the intracellular Ca2+ concentration with a 50% effect occurring at about 5.4 µM. Knockdown of Piezo1 by RNA interference suppressed the Yoda1 response, consistent with it being mediated by Piezo1 channels. Alignment of cells to the direction of shear stress was also suppressed by Piezo1 knockdown without loss of cell viability. Patch-clamp recordings from freshly isolated endothelium showed shear stress-activated single channels which were characteristic of Piezo1. LIMITATIONS, REASONS FOR CAUTION: The in vitro nature of fetoplacental endothelial cell isolation and subsequent culture may affect FpEC characteristics and PIEZO1 expression. In addition to Piezo1, alternative shear stress sensing mechanisms have been suggested in other systems and might also contribute in the placenta. WIDER IMPLICATIONS OF THE FINDINGS: These data suggest that Piezo1 is an important molecular determinant of blood flow sensitivity in the placenta. Establishing and manipulating the molecular mechanisms regulating shear stress sensing could lead to novel therapeutic strategies to improve blood flow in the placenta. LARGE-SCALE DATA: Not applicable. STUDY FUNDING/COMPETING INTEREST(S): LCM was funded by a Clinical Research Training Fellowship from the Medical Research Council and by the Royal College of Obstetricians and Gynaecologists, and has received support from a Wellcome Trust Institutional Strategic Support Fund. JS was supported by the Wellcome Trust and a BHF Intermediate Research Fellowship. HJG, CW, AJH and PJW were supported by PhD Studentships from BHF, BBSRC and the Leeds Teaching Hospitals Charitable Foundation respectively. All authors declare no conflict of interest.

Piezo1 Channels in Vascular Development and the Sensing of Shear Stress.

A critical point in mammalian development occurs before mid-embryogenesis when the heart starts to beat, pushing blood into the nascent endothelial lattice. This pushing force is a signal, detected by endothelial cells as a frictional force (shear stress) to trigger cellular changes that underlie the essential processes of vascular remodeling and expansion required for embryonic growth. The processes are complex and multifactorial and Piezo1 became a recognized player only 2years ago, 4years after Piezo1's initial discovery as a functional membrane protein. Piezo1 is now known to be critical in murine embryonic development just at the time when the pushing force is first detected by endothelial cells. Murine Piezo1 gene disruption in endothelial cells is embryonic lethal and mutations in human PIEZO1 associate with severe disease phenotype due to abnormal lymphatic vascular development. Piezo1 proteins coassemble to form calcium-permeable nonselective cationic channels, most likely as trimers. They are large proteins with little if any resemblance to other proteins or ion channel subunits. The channels appear to sense mechanical force directly, including the force imposed on endothelial cells by physiological shear stress. Here, we review current knowledge of Piezo1 in the vascular setting and discuss hypotheses about how it might serve its vascular functions and integrate with other mechanisms. Piezo1 is a new important player for investigators in this field and promises much as a basis for better understanding of vascular physiology and pathophysiology and perhaps also discovery of new therapies.

Temporal models for mitotic phase labelling.

With the widespread use of time-lapse data to understand cellular function, there is a need for tools which facilitate high-throughput analysis of data. Fluorescence microscopy of genetically engineered cell lines in culture can be used to visualise the progression of these cells through the cell cycle, including distinctly identifiable sequential stages of cell division (mitotic phases). We present a system for automated segmentation and mitotic phase labelling using temporal models. This work takes the novel approach of using temporal features evaluated over the whole of the mitotic phases rather than over single frames, thereby capturing the distinctive behaviour over the phases. We compare and contrast three different temporal models: Dynamic Time Warping, Hidden Markov Models, and Semi Markov Models. A new loss function is proposed for the Semi Markov model to make it more robust to inconsistencies in data annotation near transition boundaries. The models are tested under two different experimental conditions to explore robustness to changes in biological conditions.

Multiplex cytological profiling assay to measure diverse cellular states.

Computational methods for image-based profiling are under active development, but their success hinges on assays that can capture a wide range of phenotypes. We have developed a multiplex cytological profiling assay that "paints the cell" with as many fluorescent markers as possible without compromising our ability to extract rich, quantitative profiles in high throughput. The assay detects seven major cellular components. In a pilot screen of bioactive compounds, the assay detected a range of cellular phenotypes and it clustered compounds with similar annotated protein targets or chemical structure based on cytological profiles. The results demonstrate that the assay captures subtle patterns in the combination of morphological labels, thereby detecting the effects of chemical compounds even though their targets are not stained directly. This image-based assay provides an unbiased approach to characterize compound- and disease-associated cell states to support future probe discovery.

Quantifying structure and performance diversity for sets of small molecules comprising small-molecule screening collections.

Using a diverse collection of small molecules we recently found that compound sets from different sources (commercial; academic; natural) have different protein-binding behaviors, and these behaviors correlate with trends in stereochemical complexity for these compound sets. These results lend insight into structural features that synthetic chemists might target when synthesizing screening collections for biological discovery. We report extensive characterization of structural properties and diversity of biological performance for these compounds and expand comparative analyses to include physicochemical properties and three-dimensional shapes of predicted conformers. The results highlight additional similarities and differences between the sets, but also the dependence of such comparisons on the choice of molecular descriptors. Using a protein-binding dataset, we introduce an information-theoretic measure to assess diversity of performance with a constraint on specificity. Rather than relying on finding individual active compounds, this measure allows rational judgment of compound subsets as groups. We also apply this measure to publicly available data from ChemBank for the same compound sets across a diverse group of functional assays. We find that performance diversity of compound sets is relatively stable across a range of property values as judged by this measure, both in protein-binding studies and functional assays. Because building screening collections with improved performance depends on efficient use of synthetic organic chemistry resources, these studies illustrate an important quantitative framework to help prioritize choices made in building such collections.

Small molecules of different origins have distinct distributions of structural complexity that correlate with protein-binding profiles.

Using a diverse collection of small molecules generated from a variety of sources, we measured protein-binding activities of each individual compound against each of 100 diverse (sequence-unrelated) proteins using small-molecule microarrays. We also analyzed structural features, including complexity, of the small molecules. We found that compounds from different sources (commercial, academic, natural) have different protein-binding behaviors and that these behaviors correlate with general trends in stereochemical and shape descriptors for these compound collections. Increasing the content of sp(3)-hybridized and stereogenic atoms relative to compounds from commercial sources, which comprise the majority of current screening collections, improved binding selectivity and frequency. The results suggest structural features that synthetic chemists can target when synthesizing screening collections for biological discovery. Because binding proteins selectively can be a key feature of high-value probes and drugs, synthesizing compounds having features identified in this study may result in improved performance of screening collections.

Small-Molecule Suppressors of Cytokine-Induced beta-Cell Apoptosis.

Pancreatic beta-cell apoptosis is a critical event during the development of type-1 diabetes. The identification of small molecules capable of preventing cytokine-induced apoptosis could lead to avenues for therapeutic intervention. We developed a set of phenotypic cell-based assays designed to identify such small-molecule suppressors. Rat INS-1E cells were simultaneously treated with a cocktail of inflammatory cytokines and a collection of 2,240 diverse small molecules and screened using an assay for cellular ATP levels. Forty-nine top-scoring compounds included glucocorticoids, several pyrazole derivatives, and known inhibitors of glycogen synthase kinase-3beta. Two compounds were able to increase cellular ATP levels, reduce caspase-3 activity and nitrite production, and increase glucose-stimulated insulin secretion in the presence of cytokines. These results indicate that small molecules identified by this screening approach may protect beta cells from autoimmune attack and may be good candidates for therapeutic intervention in early stages of type-1 diabetes.

Small-molecule fluorophores to detect cell-state switching in the context of high-throughput screening.

A small molecule capable of distinguishing the distinct states resulting from cellular differentiation would be of enormous value, for example, in efforts aimed at regenerative medicine. We screened a collection of fluorescent small molecules for the ability to distinguish the differentiated state of a mouse skeletal muscle cell line. High-throughput fluorescence-based screening of C2C12 myoblasts and myotubes resulted in the identification of six compounds with the desired selectivity, which was confirmed by high-content screening in the same cell states. The compound that resulted in the greatest fluorescence intensity difference between the cell states was used as the screening agent in a pilot screen of 84 kinase inhibitors, each present in four doses, for inhibition of myogenesis. Of the kinase inhibitors, 17 resulted in reduction of fluorescence at one or more concentrations; among the "hits" included known inhibitors of myogenesis, confirming that this compound is capable of detecting the differentiated myotube state. We suggest that the strategy of screening for screening agents reported here may be extended more broadly in the future.

ChemBank: a small-molecule screening and cheminformatics resource database.

ChemBank (http://chembank.broad.harvard.edu/) is a public, web-based informatics environment developed through a collaboration between the Chemical Biology Program and Platform at the Broad Institute of Harvard and MIT. This knowledge environment includes freely available data derived from small molecules and small-molecule screens and resources for studying these data. ChemBank is unique among small-molecule databases in its dedication to the storage of raw screening data, its rigorous definition of screening experiments in terms of statistical hypothesis testing, and its metadata-based organization of screening experiments into projects involving collections of related assays. ChemBank stores an increasingly varied set of measurements derived from cells and other biological assay systems treated with small molecules. Analysis tools are available and are continuously being developed that allow the relationships between small molecules, cell measurements, and cell states to be studied. Currently, ChemBank stores information on hundreds of thousands of small molecules and hundreds of biomedically relevant assays that have been performed at the Broad Institute by collaborators from the worldwide research community. The goal of ChemBank is to provide life scientists unfettered access to biomedically relevant data and tools heretofore available primarily in the private sector.

Straight GDP-tubulin protofilaments form in the presence of taxol.

Microtubules exist in dynamic equilibrium, growing and shrinking by the addition or loss of tubulin dimers from the ends of protofilaments. The hydrolysis of GTP in beta-tubulin destabilizes the microtubule lattice by increasing the curvature of protofilaments in the microtubule and putting strain on the lattice. The observation that protofilament curvature depends on GTP hydrolysis suggests that microtubule destabilizers and stabilizers work by modulating the curvature of the microtubule lattice itself. Indeed, the microtubule destabilizer MCAK has been shown to increase the curvature of protofilaments during depolymerization. Here, we show that the atomic force microscopy (AFM) of individual tubulin protofilaments provides sufficient resolution to allow the imaging of single protofilaments in their native environment. By using this assay, we confirm previous results for the effects of GTP hydrolysis and MCAK on the conformation of protofilaments. We go on to show that taxol stabilizes microtubules by straightening the GDP protofilament and slowing down the transition of protofilaments from straight to a curved configuration.

A recombineering pipeline for functional genomics applied to Caenorhabditis elegans.

We present a new concept in DNA engineering based on a pipeline of serial recombineering steps in liquid culture. This approach is fast, straightforward and facilitates simultaneous processing of multiple samples in parallel. We validated the approach by generating green fluorescent protein (GFP)-tagged transgenes from Caenorhabditis briggsae genomic clones in a multistep pipeline that takes only 4 d. The transgenes were engineered with minimal disturbance to the natural genomic context so that the correct level and pattern of expression will be secured after transgenesis. An example transgene for the C. briggsae ortholog of lin-59 was used for ballistic transformation in Caenorhabditis elegans. We show that the cross-species transgene is correctly expressed and rescues RNA interference (RNAi)-mediated knockdown of the endogenous C. elegans gene. The strategy that we describe adapts the power of recombineering in Escherichia coli for fluent DNA engineering to a format that can be directly scaled up for genomic projects.

Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans.

A key challenge of functional genomics today is to generate well-annotated data sets that can be interpreted across different platforms and technologies. Large-scale functional genomics data often fail to connect to standard experimental approaches of gene characterization in individual laboratories. Furthermore, a lack of universal annotation standards for phenotypic data sets makes it difficult to compare different screening approaches. Here we address this problem in a screen designed to identify all genes required for the first two rounds of cell division in the Caenorhabditis elegans embryo. We used RNA-mediated interference to target 98% of all genes predicted in the C. elegans genome in combination with differential interference contrast time-lapse microscopy. Through systematic annotation of the resulting movies, we developed a phenotypic profiling system, which shows high correlation with cellular processes and biochemical pathways, thus enabling us to predict new functions for previously uncharacterized genes.

Antiproliferative effects of calcitonin gene-related peptide in aortic and pulmonary artery smooth muscle cells.

Pulmonary hypertension is characterized by vascular remodeling involving smooth muscle cell proliferation and migration. Calcitonin gene-related peptide (CGRP) and nitric oxide (NO) are potent vasodilators, and the inhibition of aortic smooth muscle cell (ASMC) proliferation by NO has been documented, but less is known about the effects of CGRP. The mechanism by which overexpression of CGRP inhibits proliferation in pulmonary artery smooth muscle cells (PASMC) and ASMC following in vitro transfection by the gene coding for prepro-CGRP was investigated. Increased expression of p53 is known to stimulate p21, which inhibits G(1) cyclin/cdk complexes, thereby inhibiting cell proliferation. We hypothesize that p53 and p21 are involved in the growth inhibitory effect of CGRP. In this study, CGRP was shown to inhibit ASMC and PASMC proliferation. In PASMC transfected with CGRP and exposed to a PKA inhibitor (PKAi), cell proliferation was restored. p53 and p21 expression increased in CGRP-treated cells but decreased in cells treated with CGRP and PKAi. PASMC treated with CGRP and a PKG inhibitor (PKGi) recovered from inhibition of proliferation induced by CGRP. ASMC treated with CGRP and then PKAi or PKGi recovered only when exposed to the PKAi and not PKGi. Although CGRP is thought to act through a cAMP-dependent pathway, cGMP involvement in the response to CGRP has been reported. It is concluded that p53 plays a role in CGRP-induced inhibition of cell proliferation and cAMP/PKA appears to mediate this effect in ASMC and PASMC, whereas cGMP appears to be involved in PASMC proliferation.

Current Canadian approaches to dialysis for acute renal failure in the ICU.

BACKGROUND: Although there is a very high mortality rate (>50%) with acute renal failure (ARF) in the intensive care unit (ICU), there is no general consensus on the best dialysis treatment for this condition. METHODS: We surveyed by mail questionnaire, all adult academic and community registered Canadian nephrology centers that offer treatment for ARF. RESULTS: The overall response rate was 59% (53/90). Comparing current dialysis methods with those utilized 5 years ago, the largest increase was in continuous renal replacement therapies (CRRT) (26 vs. 9%). Both intermittent hemodialysis (IHD) and peritoneal dialysis decreased in utilization. The predominant current CRRT methods utilized venovenous access (80%), as compared to 5 years ago when arteriovenous was the most common (52%). Despite data from chronic dialysis (and preliminary data in ARF) suggesting reduced mortality and morbidity with increasing dialysis dose, there was no formal method of dialysis prescription monitoring in over 75% of the centers. CONCLUSION: Notwithstanding a lack of definitive evidence of superior outcomes with CRRT compared to older methods, the utilization of CRRT is dramatically increasing for the treatment of ARF in Canada. Whether this shift towards CRRT, and whether more attention to dialysis dose in ARF, might be expected to lead to better outcomes, requires further evaluation.

Aurora-A kinase is required for centrosome maturation in Caenorhabditis elegans.

Centrosomes mature as cells enter mitosis, accumulating gamma-tubulin and other pericentriolar material (PCM) components. This occurs concomitant with an increase in the number of centrosomally organized microtubules (MTs). Here, we use RNA-mediated interference (RNAi) to examine the role of the aurora-A kinase, AIR-1, during centrosome maturation in Caenorhabditis elegans. In air-1(RNAi) embryos, centrosomes separate normally, an event that occurs before maturation in C. elegans. After nuclear envelope breakdown, the separated centrosomes collapse together, and spindle assembly fails. In mitotic air-1(RNAi) embryos, centrosomal alpha-tubulin fluorescence intensity accumulates to only 40% of wild-type levels, suggesting a defect in the maturation process. Consistent with this hypothesis, we find that AIR-1 is required for the increase in centrosomal gamma-tubulin and two other PCM components, ZYG-9 and CeGrip, as embryos enter mitosis. Furthermore, the AIR-1-dependent increase in centrosomal gamma-tubulin does not require MTs. These results suggest that aurora-A kinases are required to execute a MT-independent pathway for the recruitment of PCM during centrosome maturation.