Identification Of Target Sites Research Articles

COTiR: Molecular Communication Model for Synthetic Exosome-Based Tissue Regeneration.

Mesenchymal stem cell (MSC)-derived exosomes are recognized as an unparalleled therapy for tissue damage rendered by COVID-19 infection and subsequent hyper-inflammatory immune response. However, the natural targeting mechanism of exosomes is challenging to detect the damaged tissue over long diffusion distances efficiently. The coordinated movement of exosomes is desired for successful identification of target sites. In this work, we propose a molecular communication model, CoTiR, with a bio-inspired directional migration strategy (DMS) for guided propagation of exosomes to target the damaged tissues. The model includes directional propagation, reception, and regeneration of tissue. The proposed model has the potential to be used in designing efficient communication systems in the nanodomain. We compare the proposed model to the basic random propagation model and show the efficacy of our model regarding the detection of multiple targets and the detection time required. Simulation results indicate that the proposed model requires a shorter period of time for a similar number of exosomes to detect the targets compared to the basic random propagation model. Furthermore, the results reveal a 99.96% decrease in the collagen concentration in the absence of inflammatory cytokine molecules compared to the collagen concentration in the presence of inflammatory cytokine molecules.

3D Structural Analyses of Bifunctional Protein MdtA for Target Site Identification

3D Structural Analyses of Bifunctional Protein MdtA for Target Site Identification

Inferring RNA-binding protein target preferences using adversarial domain adaptation.

Precise identification of target sites of RNA-binding proteins (RBP) is important to understand their biochemical and cellular functions. A large amount of experimental data is generated by in vivo and in vitro approaches. The binding preferences determined from these platforms share similar patterns but there are discernable differences between these datasets. Computational methods trained on one dataset do not always work well on another dataset. To address this problem which resembles the classic “domain shift” in deep learning, we adopted the adversarial domain adaptation (ADDA) technique and developed a framework (RBP-ADDA) that can extract RBP binding preferences from an integration of in vivo and vitro datasets. Compared with conventional methods, ADDA has the advantage of working with two input datasets, as it trains the initial neural network for each dataset individually, projects the two datasets onto a feature space, and uses an adversarial framework to derive an optimal network that achieves an optimal discriminative predictive power. In the first step, for each RBP, we include only the in vitro data to pre-train a source network and a task predictor. Next, for the same RBP, we initiate the target network by using the source network and use adversarial domain adaptation to update the target network using both in vitro and in vivo data. These two steps help leverage the in vitro data to improve the prediction on in vivo data, which is typically challenging with a lower signal-to-noise ratio. Finally, to further take the advantage of the fused source and target data, we fine-tune the task predictor using both data. We showed that RBP-ADDA achieved better performance in modeling in vivo RBP binding data than other existing methods as judged by Pearson correlations. It also improved predictive performance on in vitro datasets. We further applied augmentation operations on RBPs with less in vivo data to expand the input data and showed that it can improve prediction performances. Lastly, we explored the predictive interpretability of RBP-ADDA, where we quantified the contribution of the input features by Integrated Gradients and identified nucleotide positions that are important for RBP recognition.

Palindromic target site identification in SARS-CoV-2, MERS-CoV and SARS-CoV-1 by adopting CRISPR-Cas technique.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated Cas protein (CRISPR-Cas) has turned out to be a very important tool for the rapid detection of viruses. This can be used for the identification of the target site in a virus by identifying a 3–6 nt length Protospacer Adjacent Motif (PAM) adjacent to the potential target site, thus motivating us to adopt CRISPR-Cas technique to identify SARS-CoV-2 as well as other members of Coronaviridae family. In this regard, we have developed a fast and effective method using k-mer technique in order to identify the PAM by scanning the whole genome of the respective virus. Subsequently, palindromic sequences adjacent to the PAM locations are identified as the potential target sites. Palindromes are considered in this work as they are known to identify viruses. Once all the palindrome-PAM combinations are identified, PAMs specific for the RNA-guided DNA Cas9/Cas12 endonuclease are identified to bind and cut the target sites. In this regard, PAMs such as 5’-TGG-3’ and 5’-TTTA-3’ in NSP3 and Exon for SARS-CoV-2, 5’-GGG-3’ and 5’-TGG-3’ in Exon and NSP2 for MERS-CoV and 5’-AGG-3’ and 5’-TTTG-3’ in Helicase and NSP3 respectively for SARS-CoV-1 are identified corresponding to SpCas9 and FnCas12a endonucleases. Finally, to recognise the target sites of Coronaviridae family as cleaved by SpCas9 and FnCas12a, complements of the palindromic target regions are designed as primers or guide RNA (gRNA). Therefore, such complementary gRNAs along with respective Cas proteins can be considered in assays for the identification of SARS-CoV-2, MERS-CoV and SARS-CoV-1.

Separation and Characterization of Protein-DNA Complexes by EMSA and In-Gel Footprinting.

In-gel footprinting enables the precise identification of protein binding sites on the DNA after separation of free and protein-bound DNA molecules by gel electrophoresis in native conditions and subsequent digestion by the nuclease activity of the 1,10-phenanthroline-copper ion [(OP)2-Cu+] within the gel matrix. Hence, the technique combines the resolving power of protein-DNA complexes in the electrophoretic mobility shift assay (EMSA) with the precision of target site identification by chemical footprinting. This approach is particularly well suited to characterize distinct molecular assemblies in a mixture of protein-DNA complexes and to identify individual binding sites within composite operators, when the concentration-dependent occupation of binding sites, with a different affinity, results in the generation of complexes with a distinct stoichiometry and migration velocity in gel electrophoresis.

Direct Target Site Identification of a Sulfonyl-Triazole Covalent Kinase Probe by LC-MS Chemical Proteomics.

Chemical proteomics is widely used for the global investigation of protein activity and binding of small molecule ligands. Covalent probe binding and inhibition are assessed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to gain molecular information on targeted proteins and probe-modified sites. The identification of amino acid sites modified by large complex probes, however, is particularly challenging because of the increased size, hydrophobicity, and charge state of peptides derived from modified proteins. These studies are important for direct evaluation of proteome-wide selectivity of inhibitor scaffolds used to develop targeted covalent inhibitors. Here, we disclose reverse-phase chromatography and MS dissociation conditions tailored for binding site identification using a clickable covalent kinase inhibitor containing a sulfonyl-triazole reactive group (KY-26). We applied this LC-MS/MS strategy to identify tyrosine and lysine sites modified by KY-26 in functional sites of kinases and other ATP-/NAD-binding proteins (>65 in total) in live cells. Our studies revealed key bioanalytical conditions to guide future chemical proteomic workflows for direct target site identification of complex irreversible probes and inhibitors.

Say no to drugs: Bioactive macromolecular therapeutics without conventional drugs

The vast majority of nanomedicines (NM) investigated today consists of a macromolecular carrier and a drug payload (conjugated or encapsulated), with a purpose of preferential delivery of the drug to the desired site of action, either through passive accumulation, or by active targeting via ligand-receptor interaction. Several drug delivery systems (DDS) have already been approved for clinical use. However, recent reports are corroborating the notion that NM do not necessarily need to include a drug payload, but can exert biological effects through specific binding/blocking of important target proteins at the site of action. The seminal work of Kopeček et al. on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers containing biorecognition motifs (peptides or oligonucleotides) for crosslinking cell surface non-internalizing receptors of malignant cells and inducing their apoptosis, without containing any low molecular weight drug, led to the definition of a special group of NM, termed Drug-Free Macromolecular Therapeutics (DFMT). Systems utilizing this approach are typically designed to employ pendant targeting-ligands on the same macromolecule to facilitate multivalent interactions with receptors. The lack of conventional small molecule drugs reduces toxicity and adverse effects at off-target sites.In this review, we describe different types of DFMT that possess biological activity without attached low molecular weight drugs. We classified the relevant research into several groups by their mechanisms of action, and compare the advantages and disadvantages of these different approaches. We show that identification of target sites, specificity of attached targeting ligands, binding affinity and the synthesis of carriers of defined size and ligand spacing are crucial aspects of DFMT development. We further discuss how knowledge in the field of NM accumulated in the past few decades can help in the design of a successful DFMT to speed up the translation into clinical practice.

Abstract 2757: Discovery and characterization of covalent Pin1 inhibitors targeted to an active site cysteine

Abstract Proline-directed phosphorylation at serine or threonine residues (pSer/Thr-Pro) regulates numerous cellular processes, including the cell cycle, transcription, and differentiation. Deregulation of such signaling networks is a hallmark of transformation and oncogenesis. Pin1, a peptidyl-prolyl isomerase, regulates the function and stability of phosphoproteins by catalyzing the cis/trans isomerization of pSer/Thr-Pro motifs. Pin1 is frequently overexpressed in human cancers, including pancreatic ductal adenocarcinoma (PDAC), and Pin1 is required for activated Ras to induce tumorigenesis. While mutations in KRAS are observed in 90-95% of human PDAC cases, it has historically proven very challenging to develop small molecules that inhibit mutant Ras function. Consequently, drug discovery efforts have turned to targets required for Ras-mediated transformation, such as Pin1. However, existing Pin1 inhibitors lack the potency, selectivity, and/or cell permeability to serve as informative cellular probes. We report a highly potent, cell-permeable Pin1 inhibitor that covalently targets Cys113, a conserved cysteine residue in the Pin1 active site. Through iterative rounds of synthesis and characterization, we developed inhibitor 1b. With a Ki of 15 nM as measured in biochemical binding and isomerase inhibition assays, 1bis currently the most potent Pin1 inhibitor available. Furthermore, in a chemoproteomic study using Covalent Inhibitor Target Site Identification (CITe-Id) to quantify the dose-dependent covalent labeling of 1b to individual cysteines across the proteome, Pin1 Cys113 was the only identified target, highlighting the pronounced selectivity of 1b for Pin1. We show that treatment with 1b diminishes viability of human PDAC cell lines, which can be fully rescued in corresponding Pin1 knockout cells generated using CRISPR/Cas9, showing that this phenotype is on-target. In parallel to inhibitor development, we used CRISPR/Cas9 GFP-dropout screens to further validate the dependence of these cell lines on Pin1. Genetic disruption of Pin1 led to antiproliferative effects, confirming the results of 1b treatment. We also employed the degradation tag (dTAG) approach to assess the effects of rapid and selective targeted Pin1 degradation through generation of FKBP12F36V-Pin1, Pin1-/-human PDAC cell lines. Treatment with a small molecule FKBP12F36V-degrader led to rapid ubiquitination and degradation of FKBP12F36V-Pin1, enabling comparisons of targeted inhibition and Pin1 degradation. Through the development of a selective Pin1 inhibitor coupled with genetic approaches and the chemical-genetic dTAG strategy, we demonstrate that Pin1 inhibition represents a tractable strategy in PDAC. Citation Format: Benika Pinch, Zainab Doctor, Christopher M. Browne, Hyuk-Soo Seo, Behnam Nabet, Shingo Kozono, Xiaolan Lian, Daniel Zaidman, Dina Daitchman, Nir London, Lu Gong, Theresa Manz, Yujin Chun, Li Tan, Jarrod Marto, Stephen Buratowski, Sirano Dhe-Paganon, Xiao Zhou, Kun Ping Lu, Nathanael S. Gray. Discovery and characterization of covalent Pin1 inhibitors targeted to an active site cysteine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2757.

Development of a neurotoxicity assay that is tuned to detect mitochondrial toxicants

Many neurotoxicants affect energy metabolism in man, but currently available test methods may still fail to predict mito- and neurotoxicity. We addressed this issue using LUHMES cells, i.e., human neuronal precursors that easily differentiate into mature neurons. Within the NeuriTox assay, they have been used to screen for neurotoxicants. Our new approach is based on culturing the cells in either glucose or galactose (Glc–Gal–NeuriTox) as the main carbohydrate source during toxicity testing. Using this Glc–Gal–NeuriTox assay, 52 mitochondrial and non-mitochondrial toxicants were tested. The panel of chemicals comprised 11 inhibitors of mitochondrial respiratory chain complex I (cI), 4 inhibitors of cII, 8 of cIII, and 2 of cIV; 8 toxicants were included as they are assumed to be mitochondrial uncouplers. In galactose, cells became more dependent on mitochondrial function, which made them 2–3 orders of magnitude more sensitive to various mitotoxicants. Moreover, galactose enhanced the specific neurotoxicity (destruction of neurites) compared to a general cytotoxicity (plasma membrane lysis) of the toxicants. The Glc–Gal–NeuriTox assay worked particularly well for inhibitors of cI and cIII, while the toxicity of uncouplers and non-mitochondrial toxicants did not differ significantly upon glucose ↔ galactose exchange. As a secondary assay, we developed a method to quantify the inhibition of all mitochondrial respiratory chain functions/complexes in LUHMES cells. The combination of the Glc–Gal–NeuriTox neurotoxicity screening assay with the mechanistic follow up of target site identification allowed both, a more sensitive detection of neurotoxicants and a sharper definition of the mode of action of mitochondrial toxicants.

Magnetic resonance imaging guidance for the optimization of ventricular tachycardia ablation.

Catheter ablation has an important role in the management of patients with ventricular tachycardia (VT) but is limited by modest long-term success rates. Magnetic resonance imaging (MRI) can provide valuable anatomic and functional information as well as potentially improve identification of target sites for ablation. A major limitation of current MRI protocols is the spatial resolution required to identify the areas of tissue responsible for VT but recent developments have led to new strategies which may improve substrate assessment. Potential ways in which detailed information gained from MRI may be utilized during electrophysiology procedures include image integration or performing a procedure under real-time MRI guidance. Image integration allows pre-procedural magnetic resonance (MR) images to be registered with electroanatomical maps to help guide VT ablation and has shown promise in preliminary studies. However, multiple errors can arise during this process due to the registration technique used, changes in ventricular geometry between the time of MRI and the ablation procedure, respiratory and cardiac motion. As isthmus sites may only be a few millimetres wide, reducing these errors may be critical to improve outcomes in VT ablation. Real-time MR-guided intervention has emerged as an alternative solution to address the limitations of pre-acquired imaging to guide ablation. There is now a growing body of literature describing the feasibility, techniques, and potential applications of real-time MR-guided electrophysiology. We review whether real-time MR-guided intervention could be applied in the setting of VT ablation and the potential challenges that need to be overcome.

Research directions in plant protection chemistry

This Opinion paper briefly summarizes the views of the authors on the directions of research in the area of plant protection chemistry. We believe these directions need to focus on (1) the discovery of new pesticide active ingredients, and (2) the protection of human health and the environment. Research revenues are discussed thematically in topics of target site identification, pesticide discovery, environmental aspects, as well as keeping track with the international trends. The most fundamental approach, target site identification, covers both computer-aided molecular design and research on biochemical mechanisms. The discovery of various classes of pesticides is reviewed including classes that hold promise to date, as well as up-to-date methods of innovation, e.g. utilization of plant metabolomics in identification of novel target sites of biological activity. Environmental and ecological aspects represent a component of increasing importance in pesticide development by emphasizing the need to improve methods of environmental analysis and assess ecotoxicological side-effects, but also set new directions for future research. Last, but not least, pesticide chemistry and biochemistry constitute an integral part in the assessment of related fields of plant protection, e.g. agricultural biotechnology, therefore, issues of pesticide chemistry related to the development and cultivation of genetically modified crops are also discussed.

A Guide to Computational Tools and Design Strategies for Genome Editing Experiments in Zebrafish Using CRISPR/Cas9.

The development of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology for mainstream biotechnological use based on its discovery as an adaptive immune mechanism in bacteria has dramatically improved the ability of molecular biologists to modify genomes of model organisms. The zebrafish is highly amenable to applications of CRISPR/Cas9 for mutation generation and a variety of DNA insertions. Cas9 protein in complex with a guide RNA molecule recognizes where to cut the homologous DNA based on a short stretch of DNA termed the protospacer-adjacent motif (PAM). Rapid and efficient identification of target sites immediately preceding PAM sites, quantification of genomic occurrences of similar (off target) sites and predictions of cutting efficiency are some of the features where computational tools play critical roles in CRISPR/Cas9 applications. Given the rapid advent and development of this technology, it can be a challenge for researchers to remain up to date with all of the important technological developments in this field. We have contributed to the armamentarium of CRISPR/Cas9 bioinformatics tools and trained other researchers in the use of appropriate computational programs to develop suitable experimental strategies. Here we provide an in-depth guide on how to use CRISPR/Cas9 and other relevant computational tools at each step of a host of genome editing experimental strategies. We also provide detailed conceptual outlines of the steps involved in the design and execution of CRISPR/Cas9-based experimental strategies, such as generation of frameshift mutations, larger chromosomal deletions and inversions, homology-independent insertion of gene cassettes and homology-based knock-in of defined point mutations and larger gene constructs.

CRISPRstrand: predicting repeat orientations to determine the crRNA-encoding strand at CRISPR loci.

Motivation: The discovery of CRISPR-Cas systems almost 20 years ago rapidly changed our perception of the bacterial and archaeal immune systems. CRISPR loci consist of several repetitive DNA sequences called repeats, inter-spaced by stretches of variable length sequences called spacers. This CRISPR array is transcribed and processed into multiple mature RNA species (crRNAs). A single crRNA is integrated into an interference complex, together with CRISPR-associated (Cas) proteins, to bind and degrade invading nucleic acids. Although existing bioinformatics tools can recognize CRISPR loci by their characteristic repeat-spacer architecture, they generally output CRISPR arrays of ambiguous orientation and thus do not determine the strand from which crRNAs are processed. Knowledge of the correct orientation is crucial for many tasks, including the classification of CRISPR conservation, the detection of leader regions, the identification of target sites (protospacers) on invading genetic elements and the characterization of protospacer-adjacent motifs.Results: We present a fast and accurate tool to determine the crRNA-encoding strand at CRISPR loci by predicting the correct orientation of repeats based on an advanced machine learning approach. Both the repeat sequence and mutation information were encoded and processed by an efficient graph kernel to learn higher-order correlations. The model was trained and tested on curated data comprising >4500 CRISPRs and yielded a remarkable performance of 0.95 AUC ROC (area under the curve of the receiver operator characteristic). In addition, we show that accurate orientation information greatly improved detection of conserved repeat sequence families and structure motifs. We integrated CRISPRstrand predictions into our CRISPRmap web server of CRISPR conservation and updated the latter to version 2.0.Availability: CRISPRmap and CRISPRstrand are available at http://rna.informatik.uni-freiburg.de/CRISPRmap.Contact: backofen@informatik.uni-freiburg.deSupplementary information: Supplementary data are available at Bioinformatics online.

MicroRNA target site identification by integrating sequence and binding information.

High-throughput sequencing has opened numerous possibilities for the identification of regulatory RNA-binding events. Cross-linking and immunoprecipitation of Argonaute protein members can pinpoint microRNA target sites within tens of bases, but leaves the identity of the microRNA unresolved. A flexible computational framework that integrates sequence with cross-linking features reliably identifies the microRNA family involved in each binding event, considerably outperforms sequence-only approaches, and quantifies the prevalence of noncanonical binding modes.

Individually tailored left ventricular lead placement: lessons from multimodality integration between three-dimensional echocardiography and coronary sinus angiogram

Non-responder rates for cardiac resynchronization therapy (CRT) vary from 11% to 46%. Retrospective data imply a better outcome with stimulation of the latest contracting left ventricular (LV) region. Our study analysed the feasibility, safety and clinical outcome of prospectively planned LV lead placement at the site of latest mechanical activation. Thirty-eight heart failure patients with CRT indication were assessed by three-dimensional (3D) transoesophageal echocardiography and rotation angiography of the coronary sinus (CS). Both images were merged into a single 3D-model to identify CS target veins close to the site of latest mechanical activation. Subsequently, LV lead deployment was attempted at the desired target position. Patients were clinically and echocardiographically evaluated at baseline, after 3 and 6 months. The area of latest mechanical activation covered 6 ± 2 segments (38 ± 13% of LV surface) and was found lateral in 24 of 37 (65%), anterior in 11 of 37 (30%), inferior in 2 of 37 (5%), and septal in 1 of 37 (3%) patients. In 36 of 37 (97%) patients an appropriate target vein was identified and successful implantation could be performed in 34 of 37 (92%) patients. Among those patients clinical and echocardiographic response was observed in 91% and 81%, respectively. Individualized lead placement at the latest contracting LV site can be performed safely and successfully in the majority of patients. Initial clinical outcome data are encouraging. Identification of target sites requires multimodality integration between LV wall motion data and CS anatomy. Future developments need to improve those technologies and require randomized data on clinical outcome parameters.

Effect of Helical Conformation and Side Chain Structure on γ-Secretase Inhibition by β-Peptide Foldamers: Insight into Substrate Recognition

Substrate-selective inhibition or modulation of the activity of γ-secretase, which is responsible for the generation of amyloid-β peptides, might be an effective strategy for prevention and treatment of Alzheimer's disease. We have shown that helical β-peptide foldamers are potent and specific inhibitors of γ-secretase. Here we report identification of target site of the foldamers by using a photoaffinity probe. The photoprobe directly and specifically labeled the N-terminal fragment of presenilin 1, in which the initial substrate docking site is predicted to be located. We also optimized the foldamer structure by preparing a variety of derivatives and obtained two highly potent foldamers by incorporation of a hydrophilic and neutral functional group into the parent structure. The class of side chain functional group and the position of incorporation were both important for γ-secretase-inhibitory activity. The substrate selectivity of the inhibitory activity was also quite sensitive to the class of side chain group incorporated.

Quantifying transient binding of ISWI chromatin remodelers in living cells by pixel-wise photobleaching profile evolution analysis

Interactions between nuclear proteins and chromatin frequently occur on the time scale of seconds and below. These transient binding events are important for the fast identification of target sites as concluded from our previous analysis of the human chromatin remodelers Snf2H and Snf2L from the imitation switch (ISWI) family. Both ATP-driven molecular motor proteins are able to translocate nucleosomes along the DNA and appear to exert this activity only on a small number of nucleosomes to which they bind more tightly. For mechanistic studies, one needs to distinguish such translocation reactions or other long-lived interactions associated with conformational changes and/or ATP hydrolysis from nonproductive chromatin sampling during target search. These processes can be separated by measuring the duration of nucleosome binding with subsecond time resolution. To reach this goal, we have developed a fluorescence bleaching technique termed pixel-wise photobleaching profile evolution analysis (3PEA). It exploits the inherent time structure of confocal microscopy images and yields millisecond resolution. 3PEA represents a generally applicable approach to quantitate transient chromatin interactions in the 2- to 500-ms time regime within only ∼1 s needed for a measurement. The green autofluorescent protein (GFP)-tagged Snf2H and Snf2L and the inactive Snf2L+13 splice variant were studied by 3PEA in comparison to the isolated GFP or red autofluorescent protein and a GFP pentamer. Our results reveal that the residence time for transient chromatin binding of Snf2H and Snf2L is <2 ms, and strongly support the view that ISWI-type remodelers are only rarely active in unperturbed cells during G1 phase.

Computational Identification and Modeling of Crosstalk between Phosphorylation, O-β-glycosylation and Methylation of FoxO3 and Implications for Cancer Therapeutics

FoxO3 is a member of the forkhead class of transcription factors and plays a major role in the regulation of diverse cellular processes, including cell cycle arrest, DNA repair, and protection from stress stimuli by detoxification of reactive oxygen species. In addition, FoxO3 is a tumor suppressor and has been considered as a novel target for cancer therapeutics. Phosphorylation of FoxO3 via the AKT, IKK, and ERK pathways leads to deregulation, cytoplasmic retention, degradation of FoxO3 and favors tumor progression. Identification of the amino acid residues that are the target of different posttranslational modifications (PTMs) provides a foundation for understanding the molecular mechanisms of FoxO3 modifications and associated outcomes. In addition to phosphorylation, serine and threonine residues of several proteins are regulated by a unique type of PTM known as O-β-glycosylation, which serves as a functional switch. We sought to investigate the crosstalk of different PTMs on the FoxO3 which leads to the onset/progression of various cancers and that could also potentially be targeted as a therapeutic point of intervention. A computational workflow and set of selection parameters have been defined for the identification of target sites and crosstalk between different PTMs. We identified phosphorylation, O-β-GlcNAc modification, and Yin Yang sites on Ser/Thr residues, and propose a potential novel mechanism of crosstalk between these PTMs. Furthermore, methylation potential of human FoxO3 at arginine and lysine residues and crosstalk between methylation and phosphorylation have also been described. Our findings may facilitate the study of therapeutic strategies targeting posttranslational events.

A functional assay for microRNA target identification and validation

MicroRNAs (miRNA) are a class of small RNA molecules that regulate numerous critical cellular processes and bind to partially complementary sequences resulting in down-regulation of their target genes. Due to the incomplete homology of the miRNA to its target site identification of miRNA target genes is difficult and currently based on computational algorithms predicting large numbers of potential targets for a given miRNA. To enable the identification of biologically relevant miRNA targets, we describe a novel functional assay based on a 3′-UTR-enriched library and a positive/negative selection strategy. As proof of principle we have used mir-130a and its validated target MAFB to test this strategy. Identification of MAFB and five additional targets and their subsequent confirmation as mir-130a targets by western blot analysis and knockdown experiments validates this strategy for the functional identification of miRNA targets.

Endovascular Thoracic Aortic Aneurysm Repair without Angiography

: Intravascular ultrasound (IVUS) generates high definition circumferential cross-sectional images and provides real-time readout of vascular dimensions, including visualization of vessel branches. We have used it as an alternative to angiography in the endovascular thoracic aneurysm repair work-up. : Out of consecutive 203 patients with descending thoracic aortic aneurysm, 89 (43.8%) received endovascular treatment [mean age, 68 ± 8 years; range, 29-82; male, 79 (88.7%); female, 10 (11.3%)] without using angiography during the endovascular procedure. IVUS (6 F, 12.5 MHz probe or 10 F 9 MHz) coupled with fluoroscopy for the placement of radiopaque markers was used for target site identification, landing zone measurement, device positioning, and assessment of endovascular repair. : Hospital mortality was 4/89 (4.5%). Number of devices implanted in each patient was 1.2 (range, 1-3). X-ray exposure time was 12 ± 8 minutes. Median procedure time was 63 ± 20 minutes. Conversion to open surgery was necessary in one patient (1.1%) because of aortic dissection. In nine patients (10.1%) left subclavian artery was covered because of a short neck. Two patients (2.2%) had vascular access lesions and required surgical repair. One patient developed paraplegia (1.1%). Early endoleak was observed in eight patients (8.9%) and 4 (4.5%) required additional procedures (proximal or distal extensions). Late conversion was necessary in one patient (1.1%). : IVUS provides all information necessary for device selection, target site identification as well as safe and correct deployment of thoracic endoprostheses and makes periprocedural angiography unnecessary, thus avoiding the risk of renal failure because of contrast medium.