Dynamic Analysis of Vascular Morphogenesis Using Transgenic Quail Embryos

Background: One of the least understood and most central questions confronting biologists is how initially simple clusters or sheet-like cell collectives can assemble into highly complex three-dimensional functional tissues and organs. Due to the limits of oxygen diffusion, blood vessels are an essential and ubiquitous presence in all amniote tissues and organs. Vasculogenesis, the de novo self-assembly of endothelial cell (EC) precursors into endothelial tubes, is the first step in blood vessel formation [1]. Static imaging and in vitro models are wholly inadequate to capture many aspects of vascular pattern formation in vivo, because vasculogenesis involves dynamic changes of the endothelial cells and of the forming blood vessels, in an embryo that is changing size and shape. Methodology/Principal Findings: We have generated Tie1 transgenic quail lines Tg(tie1:H2B-eYFP) that express H2B-eYFP in all of their endothelial cells which permit investigations into early embryonic vascular morphogenesis with unprecedented clarity and insight. By combining the power of molecular genetics with the elegance of dynamic imaging, we follow the precise patterning of endothelial cells in space and time. We show that during vasculogenesis within the vascular plexus, ECs move independently to form the rudiments of blood vessels, all while collectively moving with gastrulating tissues that flow toward the embryo midline. The aortae are a composite of somatic derived ECs forming its dorsal regions and the splanchnic derived ECs forming its ventral region. The ECs in the dorsal regions of the forming aortae exhibit variable mediolateral motions as they move rostrally; those in more ventral regions show significant lateral-to-medial movement as they course rostrally. Conclusions/Significance: The present results offer a powerful approach to the major challenge of studying the relative role(s) of the mechanical, molecular, and cellular mechanisms of vascular development. In past studies, the advantages of the molecular genetic tools available in mouse were counterbalanced by the limited experimental accessibility needed for imaging and perturbation studies. Avian embryos provide the needed accessibility, but few genetic resources. The creation of transgenic quail with labeled endothelia builds upon the important roles that avian embryos have played in previous studies of vascular development

Using Workflows to Explore and Optimise Named Entity Recognition for Chemistry

Chemistry text mining tools should be interoperable and adaptable regardless of system-level implementation, installation or even programming issues. We aim to abstract the functionality of these tools from the underlying implementation via reconfigurable workflows for automatically identifying chemical names. To achieve this, we refactored an established named entity recogniser (in the chemistry domain), OSCAR and studied the impact of each component on the net performance. We developed two reconfigurable workflows from OSCAR using an interoperable text mining framework, U-Compare. These workflows can be altered using the drag-&-drop mechanism of the graphical user interface of U-Compare. These workflows also provide a platform to study the relationship between text mining components such as tokenisation and named entity recognition (using maximum entropy Markov model (MEMM) and pattern recognition based classifiers). Results indicate that, for chemistry in particular, eliminating noise generated by tokenisation techniques lead to a slightly better performance than others, in terms of named entity recognition (NER) accuracy. Poor tokenisation translates into poorer input to the classifier components which in turn leads to an increase in Type I or Type II errors, thus, lowering the overall performance. On the Sciborg corpus, the workflow based system, which uses a new tokeniser whilst retaining the same MEMM component, increases the F-score from 82.35% to 84.44%. On the PubMed corpus, it recorded an F-score of 84.84% as against 84.23% by OSCAR

Multiple TORC1-Associated Proteins Regulate Nitrogen Starvation-Dependent Cellular Differentiation in Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae undergoes differentiation into filamentous-like forms and invades the growth medium as a foraging response to nutrient and environmental stresses. These developmental responses are under the downstream control of effectors regulated by the cAMP/PKA and MAPK pathways. However, the upstream sensors and signals that induce filamentous growth through these signaling pathways are not fully understood. Herein, through a biochemical purification of the yeast TORC1 (Target of Rapamycin Complex 1), we identify several proteins implicated in yeast filamentous growth that directly associate with the TORC1 and investigate their roles in nitrogen starvation-dependent or independent differentiation in yeast.We isolated the endogenous TORC1 by purifying tagged, endogenous Kog1p, and identified associated proteins by mass spectrometry. We established invasive and pseudohyphal growth conditions in two S. cerevisiae genetic backgrounds (Σ1278b and CEN.PK). Using wild type and mutant strains from these genetic backgrounds, we investigated the roles of TORC1 and associated proteins in nitrogen starvation-dependent diploid pseudohyphal growth as well as nitrogen starvation-independent haploid invasive growth.We show that several proteins identified as associated with the TORC1 are important for nitrogen starvation-dependent diploid pseudohyphal growth. In contrast, invasive growth due to other nutritional stresses was generally not affected in mutant strains of these TORC1-associated proteins. Our studies suggest a role for TORC1 in yeast differentiation upon nitrogen starvation. Our studies also suggest the CEN.PK strain background of S. cerevisiae may be particularly useful for investigations of nitrogen starvation-induced diploid pseudohyphal growth

RNA Methylation by the MIS Complex Regulates a Cell Fate Decision in Yeast

For the yeast Saccharomyces cerevisiae, nutrient limitation is a key developmental signal causing diploid cells to switch from yeast-form budding to either foraging pseudohyphal (PH) growth or meiosis and sporulation. Prolonged starvation leads to lineage restriction, such that cells exiting meiotic prophase are committed to complete sporulation even if nutrients are restored. Here, we have identified an earlier commitment point in the starvation program. After this point, cells, returned to nutrient-rich medium, entered a form of synchronous PH development that was morphologically and genetically indistinguishable from starvation-induced PH growth. We show that lineage restriction during this time was, in part, dependent on the mRNA methyltransferase activity of Ime4, which played separable roles in meiotic induction and suppression of the PH program. Normal levels of meiotic mRNA methylation required the catalytic domain of Ime4, as well as two meiotic proteins, Mum2 and Slz1, which interacted and co-immunoprecipitated with Ime4. This MIS complex (Mum2, Ime4, and Slz1) functioned in both starvation pathways. Together, our results support the notion that the yeast starvation response is an extended process that progressively restricts cell fate and reveal a broad role of post-transcriptional RNA methylation in these decisions

Integration of Global Signaling Pathways, cAMP-PKA, MAPK and TOR in the Regulation of FLO11

The budding yeast, Saccharomyces cerevisiae, responds to various environmental cues by invoking specific adaptive mechanisms for their survival. Under nitrogen limitation, S. cerevisiae undergoes a dimorphic filamentous transition called pseudohyphae, which helps the cell to forage for nutrients and reach an environment conducive for growth. This transition is governed by a complex network of signaling pathways, namely cAMP-PKA, MAPK and TOR, which controls the transcriptional activation of FLO11, a flocculin gene that encodes a cell wall protein. However, little is known about how these pathways co-ordinate to govern the conversion of nutritional availability into gene expression. Here, we have analyzed an integrative network comprised of cAMP-PKA, MAPK and TOR pathways with respect to the availability of nitrogen source using experimental and steady state modeling approach. Our experiments demonstrate that the steady state expression of FLO11 was bistable over a range of inducing ammonium sulphate concentration based on the preculturing condition. We also show that yeast switched from FLO11 expression to accumulation of trehalose, a STRE response controlled by a transcriptional activator Msn2/4, with decrease in the inducing concentration to complete starvation. Steady state analysis of the integrative network revealed the relationship between the environment, signaling cascades and the expression of FLO11. We demonstrate that the double negative feedback loop in TOR pathway can elicit a bistable response, to differentiate between vegetative growth, filamentous growth and STRE response. Negative feedback on TOR pathway function to restrict the expression of FLO11 under nitrogen starved condition and also with re-addition of nitrogen to starved cells. In general, we show that these global signaling pathways respond with specific sensitivity to regulate the expression of FLO11 under nitrogen limitation. The holistic steady state modeling approach of the integrative network revealed how the global signaling pathways could differentiate between multiple phenotypes

Unanesthetized Auditory Cortex Exhibits Multiple Codes for Gaps in Cochlear Implant Pulse Trains

Cochlear implant listeners receive auditory stimulation through amplitude-modulated electric pulse trains. Auditory nerve studies in animals demonstrate qualitatively different patterns of firing elicited by low versus high pulse rates, suggesting that stimulus pulse rate might influence the transmission of temporal information through the auditory pathway. We tested in awake guinea pigs the temporal acuity of auditory cortical neurons for gaps in cochlear implant pulse trains. Consistent with results using anesthetized conditions, temporal acuity improved with increasing pulse rates. Unlike the anesthetized condition, however, cortical neurons responded in the awake state to multiple distinct features of the gap-containing pulse trains, with the dominant features varying with stimulus pulse rate. Responses to the onset of the trailing pulse train (Trail-ON) provided the most sensitive gap detection at 1,017 and 4,069 pulse-per-second (pps) rates, particularly for short (25 ms) leading pulse trains. In contrast, under conditions of 254 pps rate and long (200 ms) leading pulse trains, a sizeable fraction of units demonstrated greater temporal acuity in the form of robust responses to the offsets of the leading pulse train (Lead-OFF). Finally, TONIC responses exhibited decrements in firing rate during gaps, but were rarely the most sensitive feature. Unlike results from anesthetized conditions, temporal acuity of the most sensitive units was nearly as sharp for brief as for long leading bursts. The differences in stimulus coding across pulse rates likely originate from pulse rate-dependent variations in adaptation in the auditory nerve. Two marked differences from responses to acoustic stimulation were: first, Trail-ON responses to 4,069 pps trains encoded substantially shorter gaps than have been observed with acoustic stimuli; and second, the Lead-OFF gap coding seen for <15 ms gaps in 254 pps stimuli is not seen in responses to sounds. The current results may help to explain why moderate pulse rates around 1,000 pps are favored by many cochlear implant listeners

α7-Nicotinic Acetylcholine Receptor: Role in Early Odor Learning Preference in Mice

Recently, we have shown that mice with decreased expression of α7-nicotinic acetylcholine receptors (α7) in the olfactory bulb were associated with a deficit in odor discrimination compared to wild-type mice. However, it is unknown if mice with decreased α7-receptor expression also show a deficit in early odor learning preference (ELP), an enhanced behavioral response to odors with attractive value observed in rats. In this study, we modified ELP methods performed in rats and implemented similar conditions in mice. From post-natal days 5–18, wild-type mice were stroked simultaneously with an odor presentation (conditioned odor) for 90 s daily. Control mice were only stroked, exposed to odor, or neither. On the day of testing (P21), mice that were stroked in concert with a conditioned odor significantly investigated the conditioned odor compared to a novel odor, as observed similarly in rats. However, mice with a decrease in α7-receptor expression that were stroked during a conditioned odor did not show a behavioral response to that odorant. These results suggest that decreased α7-receptor expression has a role in associative learning, olfactory preference, and/or sensory processing deficits

Staphylococcus aureus forms spreading dendrites that have characteristics of active motility

Staphylococcus aureus is historically regarded as a non-motile organism. More recently it has been shown that S. aureus can passively move across agar surfaces in a process called spreading. We re-analysed spreading motility using a modified assay and fo- cused on observing the formation of dendrites: branching structures that emerge from the central colony. We discovered that S. aureus can spread across the surface of media in struc- tures that we term ‘comets’, which advance outwards and precede the formation of dendrites. We observed comets in a diverse selection of S. aureus isolates and they exhibit the following behaviours: (1) They consist of phenotypically distinct cores of cells that move forward and seed other S. aureus cells behind them forming a comet ‘tail’; (2) they move when other cells in the comet tail have stopped moving; (3) the comet core is held together by a matrix of slime; and (4) the comets etch trails in the agar as they move forwards. Comets are not con- sistent with spreading motility or other forms of passive motility. Comet behaviour does share many similarities with a form of active motility known as gliding. Our observations therefore suggest that S. aureus is actively motile under certain conditions