We recently introduced the Cre/Lox technology in our laboratory for both transient (mRNA injections) and stable/transgenic experiments. We experienced significant issues such as silencing, mosaicism, ...and partial recombination using both approaches. Reviewing the literature gave us the impression that these issues are common among the zebrafish community using the Cre/Lox system. While some researchers took advantage of these problems for specific applications, such as cell and lineage tracing using the Zebrabow construct, we tried here to improve the efficiency and reliability of this system by constituting and testing a new set of tools for zebrafish genetics. First, we implemented a codon-improved Cre version (iCre) designed for rodent studies to counteract some of the aforementioned problems. This eukaryotic-like iCre version was engineered to i) reduce silencing, ii) increase mRNA stability, iii) enhance translational efficiency, and iv) improve nuclear translocation. Second, we established a new set of tol2-kit compatible vectors to facilitate the generation of either iCre-mRNA or iCre-transgenes for transient and transgenic experiments, respectively. We then validated the use of this material and are providing tips for users. Interestingly, during the validation steps, we found that maternal iCRE-mRNA and/or protein deposition from female transgenics systematically led to complete/homogeneous conversion of all tested Lox-responder-transgenes, as opposed to some residual imperfect conversion when using males-drivers or mRNA injections. Considering that we did not find any evidence of Cre-protein soaking and injections in the literature as it is usually conducted with cells, we tested these approaches. While soaking of cell-permeant CRE-protein did not lead to any detectable Lox-conversion, 1ng–10 ng protein injections led to robust and homogeneous Lox-recombination, suggesting that the use of protein could be a robust option for exogenous delivery. This approach may be particularly useful to manipulate housekeeping genes involved in development, sex determination and reproduction which are difficult to investigate with traditional knockout approaches. All in all, we are providing here a new set of tools that should be useful in the field.
Abstract
The skeletal muscle T-tubule is a specialized membrane domain essential for coordinated muscle contraction. However, in the absence of genetically tractable systems the mechanisms involved ...in T-tubule formation are unknown. Here, we use the optically transparent and genetically tractable zebrafish system to probe T-tubule development in vivo. By combining live imaging of transgenic markers with three-dimensional electron microscopy, we derive a four-dimensional quantitative model for T-tubule formation. To elucidate the mechanisms involved in T-tubule formation in vivo, we develop a quantitative screen for proteins that associate with and modulate early T-tubule formation, including an overexpression screen of the entire zebrafish Rab protein family. We propose an endocytic capture model involving firstly, formation of dynamic endocytic tubules at transient nucleation sites on the sarcolemma, secondly, stabilization by myofibrils/sarcoplasmic reticulum and finally, delivery of membrane from the recycling endosome and Golgi complex.
When government imposes new taxes, rules, or regulations, it creates outcomes that often differ from the original intent. In some cases, these outcomes are so severe that they render the policy a ...failure. The law of unintended consequences has taken on an increasing importance during the era of ever-expanding government, and this book explores four important examples: cigarette taxes, alcohol prohibition, the minimum wage, and federal income tax.
The incidence of human papillomavirus (HPV)-related oropharynx cancer has steadily increased over the past two decades and now represents a majority of oropharyngeal cancer cases. Integration of the ...HPV genome into the host genome is a common event during carcinogenesis that has clinically relevant effects if the viral early genes are transcribed. Understanding the impact of HPV integration on clinical outcomes of head and neck squamous cell carcinoma (HNSCC) is critical for implementing deescalated treatment approaches for HPV
HNSCC patients. RNA sequencing (RNA-seq) data from HNSCC tumors (
= 84) were used to identify and characterize expressed integration events, which were overrepresented near known head and neck, lung, and urogenital cancer genes. Five genes were recurrent, including
A significant number of genes detected to have integration events were found to interact with Tp63, ETS, and/or FOX1A. Patients with no detected integration had better survival than integration-positive and HPV
patients. Furthermore, integration-negative tumors were characterized by strongly heightened signatures for immune cells, including CD4
, CD3
, regulatory, CD8
T cells, NK cells, and B cells, compared with integration-positive tumors. Finally, genes with elevated expression in integration-negative specimens were strongly enriched with immune-related gene ontology terms, while upregulated genes in integration-positive tumors were enriched for keratinization, RNA metabolism, and translation.
These findings demonstrate the clinical relevancy of expressed HPV integration, which is characterized by a change in immune response and/or aberrant expression of the integration-harboring cancer-related genes, and suggest strong natural selection for tumor cells with expressed integration events in key carcinogenic genes.
.
An experimentally well-studied model of RNA tertiary structures is a 58mer rRNA fragment, known as GTPase-associating center (GAC) RNA, in which a highly negative pocket walled by phosphate oxygen ...atoms is stabilized by a chelated cation. Although such deep pockets with more than one direct phosphate to ion chelation site normally include magnesium, as shown in one GAC crystal structure, another GAC crystal structure and solution experiments suggest potassium at this site. Both crystal structures also depict two magnesium ions directly bound to the phosphate groups comprising this controversial pocket. Here, we used classical molecular dynamics simulations as well as umbrella sampling to investigate the possibility of binding of potassium versus magnesium inside the pocket and to better characterize the chelation of one of the binding magnesium ions outside the pocket. The results support the preference of the pocket to accommodate potassium rather than magnesium and suggest that one of the closely binding magnesium ions can only bind at high magnesium concentrations, such as might be present during crystallization. This work illustrates the complementary utility of molecular modeling approaches with atomic-level detail in resolving discrepancies between conflicting experimental results.
The Varkud Satellite RNA contains a self-cleaving ribozyme that has been shown to function independently of its surroundings. This 160 nucleotide ribozyme adopts a catalytically active tertiary ...structure that includes a kissing hairpin complex formed by stem-loop I and stem-loop V (SLV). The five-nucleotide 5'-rUGACU loop of the isolated SLV has been shown to adopt a Mg(2+)-dependent U-turn structure by solution NMR. This U-turn hairpin is examined here by molecular dynamics simulations in the presence of monovalent and divalent ions. Simulations confirm on an all-atom level the hypotheses for the role of the Mg(2+) ions in stabilizing the loop, as well as the role of the solvent exposed U700 base. Additionally, these simulations suggest the Mg(2+)-free stem-loop adopts a wide range of structures, including energetically favorable structures similar to the Mg(2+)-bound loop structure. We propose this structure is a "gatekeeper" or precursor to Mg(2+) binding when those ions are present.
Biomarkers for early detection of breast cancer may complement population screening approaches to enable earlier and more precise treatment. The blood proteome is an important source for biomarker ...discovery but so far, few proteins have been identified with breast cancer risk. Here, we measure 2929 unique proteins in plasma from 598 women selected from the Karolinska Mammography Project to explore the association between protein levels, clinical characteristics, and gene variants, and to identify proteins with a causal role in breast cancer. We present 812 cis-acting protein quantitative trait loci for 737 proteins which are used as instruments in Mendelian randomisation analyses of breast cancer risk. Of those, we present five proteins (CD160, DNPH1, LAYN, LRRC37A2 and TLR1) that show a potential causal role in breast cancer risk with confirmatory results in independent cohorts. Our study suggests that these proteins should be further explored as biomarkers and potential drug targets in breast cancer.
Protein interaction networks are crucial for complex cellular processes. However, the elucidation of protein interactions occurring within highly specialised cells and tissues is challenging. Here, ...we describe the development, and application, of a new method for proximity-dependent biotin labelling in whole zebrafish. Using a conditionally stabilised GFP-binding nanobody to target a biotin ligase to GFP-labelled proteins of interest, we show tissue-specific proteomic profiling using existing GFP-tagged transgenic zebrafish lines. We demonstrate the applicability of this approach, termed BLITZ (Biotin Labelling In Tagged Zebrafish), in diverse cell types such as neurons and vascular endothelial cells. We applied this methodology to identify interactors of caveolar coat protein, cavins, in skeletal muscle. Using this system, we defined specific interaction networks within in vivo muscle cells for the closely related but functionally distinct Cavin4 and Cavin1 proteins.
By combining nanosphere lithography with template stripping, silicon wafers were patterned with hexagonal arrays of nanowells or pillars. These silicon masters were then replicated in gold by metal ...evaporation, resulting in wafer-scale hexagonal gratings for plasmonic applications. In the nanosphere lithography step, two-dimensional colloidal crystals of 510 nm diameter polystyrene spheres were assembled at the air–water interface and transferred to silicon wafers. The spheres were etched in oxygen plasma in order to define their size for masking of the silicon wafer. For fabrication of metallic nanopillar arrays, an alumina film was grown over the nanosphere layer and the spheres were then removed by bath sonication. The well pattern was defined in the silicon wafer by reactive ion etching in a chlorine plasma. For fabrication of metal nanowell arrays, the nanosphere monolayer was used directly as a mask and exposed areas of the silicon wafer were plasma-etched anisotropically in SF6/Ar. Both techniques could be used to produce subwavelength metal replica structures with controlled pillar or well diameter, depth, and profile, on the wafer scale, without the use of direct writing techniques to fabricate masks or masters.
PDF
