Green Fluorescent Protein in the sea urchin: new experimental approaches to transcriptional regulatory analysis in embryos and larvae

The use of Green Fluorescent Protein (GFP) as a reporter for expression transgenes opens the way to several new experimental strategies for the study of gene regulation in sea urchin development. A GFP coding sequence was associated with three different previously studied cis-regulatory systems, viz those of the SM50 gene, expressed in skeletogenic mesenchyme, the CyIIa gene, expressed in archenteron, skeletogenic and secondary mesenchyme, and the Endo16 gene, expressed in vegetal plate, archenteron and midgut. We demonstrate that the sensitivity with which expression can be detected is equal to or greater than that of whole-mount in situ hybridization applied to detection of CAT mRNA synthesized under the control of the same cis-regulatory systems. However, in addition to the important feature that it can be visualized nondestructively in living embryos, GFP has other advantages. First, it freely diffuses even within fine cytoplasmic cables, and thus reveals connections between cells, which in sea urchin embryos is particularly useful for observations on regulatory systems that operate in the syncytial skeletogenic mesenchyme. Second, GFP expression can be dramatically visualized in postembryonic larval tissues. This brings postembryonic larval developmental processes for the first time within the easy range of gene transfer analyses. Third, GFP permits identification and segregation of embryos in which the clonal incorporation of injected DNA has occurred in any particular desired region of the embryo. Thus, we show explicitly that, as expected, GFP transgenes are incorporated in the same nuclei together with other transgenes with which they are co-injected

Compensation effects in GaN:Mg probed by Raman spectroscopy and photoluminescence measurements

This article may be downloaded for personal use only. Any other use requires prior permission of the author and AIP Publishing. This article appeared in J. Appl. Phys. 113, 103504 (2013) and may be found at https://doi.org/10.1063/1.4794094.Compensation effects in metal organic chemical vapour deposition grown GaN doped with magnesium are investigated with Raman spectroscopy and photoluminescence measurements. Examining the strain sensitive E2(high) mode, an increasing compressive strain is revealed for samples with Mg-concentrations lower than 7 × 1018 cm−3. For higher Mg-concentrations, this strain is monotonically reduced. This relaxation is accompanied by a sudden decrease in crystal quality. Luminescence measurements reveal a well defined near band edge luminescence with free, donor bound, and acceptor bound excitons as well as a characteristic donor acceptor pair (DAP) luminescence. Following recent results, three acceptor bound excitons and donor acceptor pairs are identified. Along with the change of the strain, a strong modification in the luminescence of the dominating acceptor bound exciton and DAP luminescence is observed. The results from Raman spectroscopy and luminescence measurements are interpreted as fingerprints of compensation effects in GaN:Mg leading to the conclusion that compensation due to defect incorporation triggered by Mg-doping already affects the crystal properties at doping levels of around 7 × 1018 cm−3. Thereby, the generation of nitrogen vacancies is introduced as the driving force for the change of the strain state and the near band edge luminescence.DFG, 43659573, SFB 787: Halbleiter - Nanophotonik: Materialien, Modelle, Bauelement

Nicotine' actions on energy balance: friend or foe?

Teixit adipós; Receptors nicotínics; ObesitatTejido adiposo; Receptores nicotínicos; ObesidadAdipose tissue; Nicotinic receptors; ObesityObesity has reached pandemic proportions and is associated with severe comorbidities, such as type 2 diabetes mellitus, hepatic and cardiovascular diseases, and certain cancer types. However, the therapeutic options to treat obesity are limited. Extensive epidemiological studies have shown a strong relationship between smoking and body weight, with non-smokers weighing more than smokers at any age. Increased body weight after smoking cessation is a major factor that interferes with their attempts to quit smoking. Numerous controlled studies in both humans and rodents have reported that nicotine, the main bioactive component of tobacco, exerts a marked anorectic action. Furthermore, nicotine is also known to modulate energy expenditure, by regulating the thermogenic activity of brown adipose tissue (BAT) and the browning of white adipose tissue (WAT), as well as glucose homeostasis. Many of these actions occur at central level, by controlling the activity of hypothalamic neuropeptide systems such as proopiomelanocortin (POMC), or energy sensors such as AMP-activated protein kinase (AMPK). However, direct impact of nicotine on metabolic tissues, such as BAT, WAT, liver and pancreas has also been described. Here, we review the actions of nicotine on energy balance. The relevance of this interaction is interesting, because considering the restricted efficiency of obesity treatments, a possible complementary approach may focus on compounds with known pharmacokinetic profile and pharmacological actions, such as nicotine or nicotinic acetylcholine receptors signaling.Xunta de Galicia (RN: 2016-PG057; ML: 2016-PG068); Ministerio de Economía y Competitividad (MINECO) co-funded by the FEDER Program of EU (RN: RTI2018-099413-B-I00; CD: BFU2017- 87721-P; ML: RTI2018-101840-B-I00; JMF-R and ML: BFU2017- 90578-REDT/Adipoplast); Instituto de Salud Carlos III (JMF-R: PI15–01934); Atresmedia Corporación (RN and ML); Fundación BBVA (RN); “la Caixa” Foundation (ID 100010434), under the agreement LCF/PR/HR19/52160022 (ML); European Foundation for the Study of Diabetes (RN); ERC Synergy Grant-2019-WATCH- 810331 (RN); US Na- tional Institutes of Health (KR: HL084207); the US Department of Vet- erans Affairs (KR: I01BX004249); The University of Iowa Fraternal Order of Eagles Diabetes Research Center (KR). PS-C is recipient of a fel- lowship from Xunta de Galicia (ED481B 2018/050). The CiMUS is sup- ported by the Xunta de Galicia (2016-2019, ED431G/05). CIBER de Fisiopatología de la Obesidad y Nutrición is an initiative of ISCIII

Polarity Control in Group-III Nitrides beyond Pragmatism

Controlling the polarity of polar semiconductors on nonpolar substrates offers a wealth of device concepts in the form of heteropolar junctions. A key to realize such structures is an appropriate buffer-layer design that, in the past, has been developed by empiricism. GaN or ZnO on sapphire are prominent examples for that. Understanding the basic processes that mediate polarity, however, is still an unsolved problem. In this work, we study the structure of buffer layers for group-III nitrides on sapphire by transmission electron microscopy as an example. We show that it is the conversion of the sapphire surface into a rhombohedral aluminum-oxynitride layer that converts the initial N-polar surface to Al polarity. With the various AlxOyNz phases of the pseudobinary Al2O3-AlN system and their tolerance against intrinsic defects, typical for oxides, a smooth transition between the octahedrally coordinated Al in the sapphire and the tetrahedrally coordinated Al in AlN becomes feasible. Based on these results, we discuss the consequences for achieving either polarity and shed light on widely applied concepts in the field of group-III nitrides like nitridation and low-temperature buffer layers

Scintillation proximity assay for measurement of RNA methylation

Methylation of RNA by methyltransferases is a phylogenetically ubiquitous post-transcriptional modification that occurs most extensively in transfer RNA (tRNA) and ribosomal RNA (rRNA). Biochemical characterization of RNA methyltransferase enzymes and their methylated product RNA or RNA–protein complexes is usually done by measuring the incorporation of radiolabeled methyl groups into the product over time. This has traditionally required the separation of radiolabeled product from radiolabeled methyl donor through a filter binding assay. We have adapted and optimized a scintillation proximity assay (SPA) to replace the more costly, wasteful and cumbersome filter binding assay and demonstrate its utility in studies of three distinct methyltransferases, RmtA, KsgA and ErmC’. In vitro, RmtA and KsgA methylate different bases in 16S rRNA in 30S ribosomal particles, while ErmC’ most efficiently methylates protein-depleted or protein-free 23S rRNA. This assay does not utilize engineered affinity tags that are often required in SPA, and is capable of detecting either radiolabeled RNA or RNA–protein complex. We show that this method is suitable for quantitating extent of RNA methylation or active RNA methyltransferase, and for testing RNA-methyltransferase inhibitors. This assay can be carried out with techniques routinely used in a typical biochemistry laboratory or could be easily adapted for a high throughput screening format

Modelling with non-stratified chain event graphs

© 2019, Springer Nature Switzerland AG. Chain Event Graphs (CEGs) are recent probabilistic graphical modelling tools that have proved successful in modelling scenarios with context-specific independencies. Although the theory underlying CEGs supports appropriate representation of structural zeroes, the literature so far does not provide an adaptation of the vanilla CEG methods for a real-world application presenting structural zeroes also known as the non-stratified CEG class. To illustrate these methods, we present a non-stratified CEG representing a public health intervention designed to reduce the risk and rate of falling in the elderly. We then compare the CEG model to the more conventional Bayesian Network model when applied to this setting

Deciphering interplay between Salmonella invasion effectors

Bacterial pathogens have evolved a specialized type III secretion system (T3SS) to translocate virulence effector proteins directly into eukaryotic target cells. Salmonellae deploy effectors that trigger localized actin reorganization to force their own entry into non-phagocytic host cells. Six effectors (SipC, SipA, SopE/2, SopB, SptP) can individually manipulate actin dynamics at the plasma membrane, which acts as a ‘signaling hub’ during Salmonella invasion. The extent of crosstalk between these spatially coincident effectors remains unknown. Here we describe trans and cis binary entry effector interplay (BENEFIT) screens that systematically examine functional associations between effectors following their delivery into the host cell. The results reveal extensive ordered synergistic and antagonistic relationships and their relative potency, and illuminate an unexpectedly sophisticated signaling network evolved through longstanding pathogen–host interaction

Robust methods for purification of histones from cultured mammalian cells with the preservation of their native modifications

Post-translational modifications (PTMs) of histones play a role in modifying chromatin structure for DNA-templated processes in the eukaryotic nucleus, such as transcription, replication, recombination and repair; thus, histone PTMs are considered major players in the epigenetic control of these processes. Linking specific histone PTMs to gene expression is an arduous task requiring large amounts of highly purified and natively modified histones to be analyzed by various techniques. We have developed robust and complementary procedures, which use strong protein denaturing conditions and yield highly purified core and linker histones from unsynchronized proliferating, M-phase arrested and butyrate-treated cells, fully preserving their native PTMs without using enzyme inhibitors. Cell hypotonic swelling and lysis, nuclei isolation/washing and chromatin solubilization under mild conditions are bypassed to avoid compromising the integrity of histone native PTMs. As controls for our procedures, we tested the most widely used conventional methodologies and demonstrated that they indeed lead to drastic histone dephosphorylation. Additionally, we have developed methods for preserving acid-labile histone modifications by performing non-acid extractions to obtain highly purified H3 and H4. Importantly, isolation of histones H3, H4 and H2A/H2B is achieved without the use of HPLC. Functional supercoiling assays reveal that both hyper- and hypo-phosphorylated histones can be efficiently assembled into polynucleosomes. Notably, the preservation of fully phosphorylated mitotic histones and their assembly into polynucleosomes should open new avenues to investigate an important but overlooked question: the impact of mitotic phosphorylation in chromatin structure and function