159,197 research outputs found
A single E-box in the <i>Cel-lin-3</i> CRM is not sufficient for <i>lin-3</i> expression in the anchor cell of <i>C</i>. <i>elegans</i>.
No full text(A) New cis-regulatory lin-3 alleles with deleted E-boxL and NHR or NHR and E-boxR. (B) Quantification of vulval induction in these new mutants. Note the complete absence of any induction in the recovered lin-3 alleles (n>30). Scorings of lin-3(1417) animals are the same as those reported in Fig 5 and are used here to indicate that this mutation leads to vulval hypo-induction rather than no induction at all. (C-D) smFISH in lin-3(mf72) (C) and N2 (D) animals. Green spots correspond to lin-3 transcripts and red spots to lag-2 that is used as an anchor cell marker. Blue is DAPI staining of nuclei. Note the absence of lin-3 expression in the anchor cell in the lin-3(mf72) mutant animal. Absence of lin-3 signal in the anchor cell was also confirmed for the other lin-3 alleles.</p
On the sheaf-theoretic SL(2, C) Casson–Lin invariant
No full textWe prove that the (τ-weighted, sheaf-theoretic) SL(2, C) Casson–Lin invariant introduced by Manolescu and the first author is generically independent of the parameter τ and additive under connected sums of knots in integral homology 3-spheres. This addresses two questions asked by Manolescu and the first author. Our arguments involve a mix of topology and algebraic geometry, and rely crucially on the fact that the SL(2, C) Casson–Lin invariant admits an alternative interpretation via the theory of Behrend functions.</p
LIN-1 sumoylation is required for ventral toroid contraction.
No full text(A) Wild-type and K10A, K169A mutant LIN-1::GFP expression in L3 larvae at the Pn.px stage after VPC-specific degradation of AID::SMO-1 from the L2 stage onward. The 1° and 2° VPC descendants are underlined in white. The left panels show the corresponding DIC images overlaid with the LIN-1::GFP signal in green. (B) Quantification of LIN-1::GFP expression levels in 1° and 2° VPC descendants at the Pn.px stage in LIN-1::GFP wild-type and K10A, K169A double mutants under the indicated conditions. See S3 Fig for the corresponding measurements at the Pn.pxx stage. (C) Toroid morphogenesis defects in LIN-1 K10A and K169A single and double mutants at the L4 stage. Left panels show lateral views of z-projections. vulA and vulB1 toroids are outlined by the white rectangle in the top left panel and shown in top (xz) views in the right panels. (D) Quantification of vulA contraction, calculated as the ratio of the vulA and vulB1 toroid diameter. The box plots show the median values with the 25th and 75th percentiles and the whiskers indicate the maximum and minimum values. Where indicated, untreated controls are labelled with–IAA (blue) and animals treated with 1 mM auxin with +IAA (red). In each graph, the numbers of animals scored are indicated by the numbers in brackets. Statistical significance in (B) and (D) was calculated with unpaired two-tailed t-tests. p-values are indicated as * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. The scale bars are 10 μm.</p
Quantitative Insights into Developmental Signals and Phenotypes in C. elegans
Full text linkDesign of biomaterials and cellular scaffolds for tissue-engineering applications and regenerative medicine requires a precise understanding of the principles underlying multicellular patterning. Adhesion, migration, division, differentiation, and apoptosis are characteristic cellular behaviors, the engineering of which has the potential to allow creation of custom, multicellular structures. These cellular events occur naturally during embryonic and postembryonic development of multicellular organisms. Development thus offers the opportunity to learn about the design principles and molecular mechanisms that guide cellular patterning.
A key finding in developmental biology is that a limited set of conserved molecular signaling pathways act at multiple times and locations throughout the embryo to introduce cell-fate asymmetries in homogenous populations of cells. In turn, these asymmetries serve as starting points for the patterning of new organs. These signaling pathways interact quantitatively at multiple levels, including signaling cues, post-translational regulation, and gene-regulatory networks, to guide multicellular patterning.
How does the quantitative performance of these signaling networks ensure the intended phenotype pattern? How do changes in the quantitative performance of these networks, possibly over the course of evolution, give rise to new phenotypes? These are the central questions pursued in this thesis.
In order to answer such questions, we used vulva formation in the nematode Caenorhabditis elegans as a model system of cellular patterning. We formulated a mathematical model of the molecular network underlying cellular-fate specification in this system. Computational analysis of this molecular network reveals that cell–cell coupling through lateral LIN-12/Notch signaling amplifies the perception of the gradient in the epidermal-growth-factor-like soluble cue, LIN-3. Thus, the gradient in LIN-3 concentration produces an even steeper difference in LIN-3-mediated intracellular signals between adjoining cells. Such gradient amplification may be particularly important in converting a shallow, graded-specification signal into a spatial pattern of distinct fate choices.
Through quantitative perturbations of interaction strengths between components of the vulval patterning network, we further show that our modeling approach can correctly predict phenotype patterns observed in C. elegans mutation studies. This study generated a framework for quantitative analysis of molecular networks that links quantitative molecular perturbations to patterning outcomes. This framework will prove useful in the analysis of other systems involving cellular fate decisions and in tissue engineering applications where the generation of precise cell patterns is needed. We demonstrate the generality of our approach through an application to evolutionary developmental biology. Since molecular connectivity of the vulva patterning network of several closely related Caenorhabditis species is preserved, we correctly predict the quantitative diversification that must have occurred in this network during species evolution.</p
Somatic expression of <i>lin-35(+)</i> partially rescues <i>lin-35</i> mutant fertility defects.
No full text(A) Schematic of different somatic transgenes used in this study. Tissues expressing wild type lin-35(+) are shown in red/pink, tissues lacking lin-35 expression (lin-35(-)) are shown in blue (B) Mean brood size of fertile worms for each genotype across three different growth temperatures (continual growth at 20°C (blue), upshift from 20°C to 26°C at the L4 stage (yellow), and continual growth at 26°C (red)). Expression of lin-35(+) in lin-35 mutants from either somatic transgene partially rescued brood size at 20°C, while pan-somatic expression alone rescued brood size at 26°C. nd: no data, * significantly different than wild type at the same temperature, # significantly different than lin-35 mutants at the same temperature. P-value ≤ 0.05 using two-way ANOVA with Tukey correction. Error bars indicate ± SEM. (C) Percentage of hermaphrodites in the population that are fertile for each treatment. lin-35 mutants expressing intestinal lin-35(+) have significantly fewer fertile worms at 26°C than those expressing pan-somatic lin-35(+). * significantly different than wild type at the same temperature, # significantly different than lin-35 mutants at the same temperature, ** (red) significantly different than lin-35(n745); let-858p::lin-35. P-value ≤ 0.05 using a Fisher’s exact test. (D) The decrease in fertility with moderate temperature stress represented by the percentage of the mean brood size remaining with upshift or at 26°C compared to 20°C within the same strain. With upshift, lin-35 mutants show a smaller relative brood size than wild type even if expressing somatic lin-35(+). At 26°C, lin-35 mutants with pan-somatic, but not intestinal, expression of lin-35(+) retain a similar relative brood size as wild type. (E) Mean brood size of fertile worms for each treatment with expression of lin-35(+) in the somatic gonad. *significantly different than elt-2p::lin-35 at the same temperature. P-value ≤ 0.05 using two-way ANOVA with Tukey correction. Error bars indicated ± SEM.</p
Obtaining Thickness-Limited Electrospray Deposition for 3D coating
No full textThe electrospray process utilizes the balance of electrostatic forces and surface tension within a charged spray to produce charged microdroplets with a narrow dispersion in size. In electrospray deposition, each droplet carries a small quantity of suspended material to a target substrate. Past electrospray deposition results fall into two major categories: (1) continuous spray of films onto conducting substrates and (2) spray of isolated droplets onto insulating substrates. A cross-over regime, or a self-limited spray, has only been limitedly observed in the spray of insulating materials onto conductive substrates. In such sprays, a limiting thickness emerges where the accumulation of charge repels further spray. In this study, we examined the parametric spray of several glassy polymers to both categorize past electrospray deposition results and uncover the critical parameters for thickness-limited sprays. The key parameters for determining the limiting thickness were (1) field strength and (2) the spray temperature, related to (1) the necessary repulsive field and (2) the ability for the deposited materials to swell in the carrier solvent vapor and redistribute charge. These control mechanisms can be applied to the uniform or controllably varied microscale coating of complex 3D objects.Peer reviewe
LIN-2 and FRM-3 are required to maintain locomotory behaviour.
No full text(A) Genomic structure of lin-2 and frm-3 locus, mutant allele characterization, and protein architecture for LIN-2 and FRM-3. The whole promoter region of lin-2a and part of the kinase domain of LIN-2A is deleted in the e1309 mutants. The FERM domain in both frm-3a and frm-3b is deleted in the gk585 mutants. Syb1019 and syb1036 are stop codons in lin-2 and frm-3 that inactivate the expression of lin-2a and lin-2b, and frm-3a and frm-3b. (E, F) Locomotion speed is reduced by the loss of LIN-2 and FRM-3. Representative trajectories of locomotion in wild type (E) and mean locomotion speed in wild type, lin-2(e1309), frm-3(gk585), lin-2(syb1019), frm-3(syb1036), and lin-2(e1309);frm-3(gk585) mutants. To measure locomotion speed, young adult animals were washed with a drop of PBS and then transferred to fresh NGM plates with no bacterial lawn (30 worms per plate). Worm movement recordings (under room temperature 22°C) were started 10 min after the worms were transferred. A 2 min digital video of each plate was captured at 3.75 Hz frame rate by WormLab System (MBF Bioscience). Average speed and tracks were generated for each animal using WormLab software. To confirm the repeatability of the data, the locomotion speed was measured in two independent experiments in two days. For each mutant, around 10–40 animals were analyzed in one experiment. Significance was tested for each experiment. Data are mean ± SEM (**, p p < 0.001 when compared to wild type; n.s., non-significant; one-way ANOVA). The number of worms analyzed for each genotype is indicated in the bar.</p
Differential roles of the microRNA let-7 in C. elegans tissue development
Full text linkThe organs and tissues of the human body comprise of an astonishing variety of cells as different in morphology and function as muscle cells and neurons. Amazingly, despite their different protein contents, they largely contain the identical genomic information. In order to understand the processes that enable this differentiation, we need to determine the underlying regulatory mechanisms. A very recent discovery in this context was the posttranscriptional regulation of gene expression by microRNAs (miRNAs). miRNAs are small RNA molecules that mediate translational repression and degradation of mRNA transcripts through partial complementarity to their 3’ untranslated region (UTR) . Among the first miRNAs to be identified, let-7 stands out for its high conservation in sequence and developmental functions in development throughout the animal kingdom. During my PhD, I studied the role of let-7 in Caenorhabditis elegans in the context of two distinct processes of tissue development, namely differentiation of the epidermis (called hypodermis), and morphogenesis of the vulva. The functions of the let-7 miRNA in formation of the adult cuticle have been extensively studied and are well understood. let-7 controls differentiation of specific, mitotically active epidermal cells by inducing cell cycle exit, fusion, and switch to an adult specific transcriptional program upon repression of targets such as lin-41, daf-12, hbl-1 and let-60/ras. I set out to identify novel interactors of let-7 in a genome-wide RNAi screen for suppression of the lethal let-7 bursting phenotype. Candidates were then verified using fluorescence-based reporter systems for onset of hypodermis differentiation and intensity of repression of a known target. Thereby, I was able to validate a whole set of novel members of the let-7 network, comprising genes downstream in the pathway as well as potential regulators of let-7 activity. Notably, both groups of repressors contain factors required for cell cycle progression and mitosis, which indicates an active crosstalk between let-7 and the cell-cycle machinery. In a second project, I explored the molecular basis for the prominent let-7 vulval bursting phenotype. Despite the absence of overproliferation or any other obvious phenotype in vulval morphogenesis, I was able to show that let-7 activity is required in the vulva, and that its major function in this context is repression of a single target, namely lin-41. Disruption of let-7 binding to lin-41 through modification of the let-7 complementary sites by CRISPR/Cas9 mediated genome editing suffices to trigger the bursting phenotype, proving that repression of a single target is the key function of the miRNA in this context. In summary, my work shows that while both differentiation of hypodermis as well as vulval integrity are mediated through repression of lin-41, the downstream effect of this regulation seem to differ, suggesting that let-7 can be wired to control distinct processes depending on the cellular context. With respect to the latest findings both in C. elegans as well as in mammals, it will be interesting to determine if this depends on differential molecular functions of LIN-41 in the two tissues
Measurement of the ratio of prompt χ c to J / ψ production in pp collisions at √s = 7 TeV
Full text linkThe prompt production of charmonium χ c and J / ψ states is studied in proton-proton collisions at a centre-of-mass energy of √s = 7 TeV at the Large Hadron Collider. The χ c and J / ψ mesons are identified through their decays χ c → J / ψ γ and J / ψ → μ + μ - using 36 pb - 1 of data collected by the LHCb detector in 2010. The ratio of the prompt production cross-sections for χ c and J / ψ, σ (χ c → J / ψ γ) / σ (J / ψ), is determined as a function of the J / ψ transverse momentum in the range 2 < p T J / ψ < 15 GeV / c. The results are in excellent agreement with next-to-leading order non-relativistic expectations and show a significant discrepancy compared with the colour singlet model prediction at leading order, especially in the low p T J / ψ region
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