CRISPR/Cas9-based editing of a sensitive transcriptional regulatory element to achieve cell type-specific knockdown of the NEMO scaffold protein

The use of alternative promoters for the cell type-specific expression of a given mRNA/protein is a common cell strategy. NEMO is a scaffold protein required for canonical NF-κB signaling. Transcription of the NEMO gene is primarily controlled by two promoters: one (promoter B) drives NEMO transcription in most cell types and the second (promoter D) is largely responsible for NEMO transcription in liver cells. Herein, we have used a CRISPR/Cas9-based approach to disrupt a core sequence element of promoter B, and this genetic editing essentially eliminates expression of NEMO mRNA and protein in 293T human kidney cells. By cell subcloning, we have isolated targeted 293T cell lines that express no detectable NEMO protein, have defined genomic alterations at promoter B, and do not support activation of canonical NF-κB signaling in response to treatment with tumor necrosis factor. Nevertheless, noncanonical NF-κB signaling is intact in these NEMO-deficient cells. Expression of ectopic wildtype NEMO, but not certain human NEMO disease mutants, in the edited cells restores downstream NF-κB signaling in response to tumor necrosis factor. Targeting of the promoter B element does not substantially reduce NEMO expression (from promoter D) in the human SNU423 liver cancer cell line. Thus, we have created a strategy for selectively eliminating cell typespecific expression from an alternative promoter and have generated 293T cell lines with a functional knockout of NEMO. The implications of these findings for further studies and for therapeutic approaches to target canonical NF-κB signaling are discussed.Published versio

CRISPR/Cas9-based editing of a sensitive transcriptional regulatory element to achieve cell type-specific knockdown of the NEMO scaffold protein

The use of alternative promoters for the cell type-specific expression of a given mRNA/protein is a common cell strategy. NEMO is a scaffold protein required for canonical NF-κB signaling. Transcription of the NEMO gene is primarily controlled by two promoters: one (promoter B) drives NEMO transcription in most cell types and the second (promoter A) is largely responsible for NEMO transcription in liver cells. Herein, we have used a CRISPR/Cas9-based approach to disrupt a core sequence element of promoter B, and this genetic editing essentially eliminates expression of NEMO mRNA and protein in 293T human kidney cells. By cell subcloning, we have isolated targeted 293T cell lines that express no detectable NEMO protein, have defined genomic alterations at promoter B, and do not support canonical NF-κB signaling in response to treatment with tumor necrosis factor (TNF). Nevertheless, non-canonical NF-κB signaling is intact in these NEMO-deficient cells. Expression of ectopic NEMO in the edited cells restores downstream NF-κB signaling in response to TNF. Targeting of the promoter B element does not substantially reduce NEMO expression (from promoter A) in the human SNU-423 liver cancer cell line. We have also used homology directed repair (HDR) to fix the promoter B element in a 293T cell clone. Overall, we have created a strategy for selectively eliminating cell type-specific expression from an alternative promoter and have generated 293T cell lines with a functional knockout of NEMO. The implications of these findings for further studies and for therapeutic approaches to target canonical NF-κB signaling are discussed.GM117350 - National Institutes of Health; CA077474 - National Institutes of HealthPublished versio

Design and Experimental Evaluation of a Route Optimisation Solution for NEMO

An important requirement for Internet protocol (IP) networks to achieve the aim of ubiquitous connectivity is network mobility (NEMO). With NEMO support we can provide Internet access from mobile platforms, such as public transportation vehicles, to normal nodes that do not need to implement any special mobility protocol. The NEMO basic support protocol has been proposed in the IETF as a first solution to this problem, but this solution has severe performance limitations. This paper presents MIRON: Mobile IPv6 route optimization for NEMO, an approach to the problem of NEMO support that overcomes the limitations of the basic solution by combining two different modes of operation: a Proxy-MR and an address delegation with built-in routing mechanisms. This paper describes the design and rationale of the solution, with an experimental validation and performance evaluation based on an implementation.Publicad

The DNA damage response promotes Polyomavirus JC infection by nucleus to cytoplasm NF-Kappa B activation.

Background: Infection of glial cells by human neurotropic polyomavirus JC (JCV), the causative agent of the CNS demyelinating disease progressive multifocal leukoencephalopathy (PML), rapidly inflicts damage to cellular DNA. This activates DNA damage response (DDR) signaling including induction of expression of DNA repair factor Rad51. We previously reported that Rad51 co-operates with the transcription factor NF-κB p65 to activate JCV early transcription. Thus Rad51 induction by JCV infection may provide positive feedback for viral activation early in JCV infection. DDR is also known to stimulate NF-κB activity, a phenomenon known as nucleus to cytoplasm or “insideout” NF-κB signaling, which is initiated by Ataxia telangiectasia mutated (ATM) protein, a serine/threonine kinase recruited and activated by DNA double-strand breaks. Downstream of ATM, there occurs a series of posttranslational modifications of NF-κB essential modulator (NEMO), the γ regulatory subunit of inhibitor of NF-κB (IκB) kinase (IKK), resulting in NF-κB activation. Methods: We analyzed the effects of downstream pathways in the DDR by phosphospecific Western blots and analysis of the subcellular distribution of NEMO by cell fractionation and immunocytochemistry. The role of DDR in JCV infection was analyzed using a small molecule inhibitor of ATM (KU-55933). NEMO sumoylation was investigated by Western and association of ATM and NEMO by immunoprecipitation/Western blots. Results: We show that JCV infection caused phosphorylation and activation of ATM while KU-55933 inhibited JCV replication. JCV infection caused a redistribution of NEMO from cytoplasm to nucleus. Co-expression of JCV large Tantigen and FLAG-tagged NEMO showed the occurrence of sumoylation of NEMO, while co-expression of ATM and FLAG-NEMO demonstrated physical association between ATM and NEMO. Conclusions: We propose a model where JCV infection induces both overexpression of Rad51 protein and activation of the nucleus to cytoplasm NF-κB signaling pathway, which then act together to enhance JCV gene expression

Limits on different majoron decay modes of 100Mo and 82Se for neutrinoless double beta decays in the NEMO-3 experiment

The NEMO-3 tracking detector is located in the Fréjus Underground Laboratory. It was designed to study double beta decay in a number of different isotopes. Presented here are the experimental half-life limits on the double beta decay process for the isotopes 100Mo and 82Se for different majoron emission modes and limits on the effective neutrino–majoron coupling constants. In particular, new limits on “ordinary” majoron (spectral index 1) decay of 100Mo (T1/2>2.7×1022 yr) and 82Se (T1/2>1.5×1022 yr) have been obtained. Corresponding bounds on the majoron–neutrino coupling constant are gee<(0.4–1.8)×10−4 and <(0.66–1.9)×10−4

Antimicrobial activity of biogenically produced spherical Se-nanomaterials embedded in organic material against Pseudomonas aeruginosa and Staphylococcus aureus strains on hydroxyapatite-coated surfaces

In an effort to prevent the formation of pathogenic biofilms on hydroxyapatite (HA)-based clinical devices and surfaces, we present a study evaluating the antimicrobial efficacy of Spherical biogenic Se-Nanostructures Embedded in Organic material (Bio Se-NEMO-S) produced by Bacillus mycoides SelTE01 in comparison with two different chemical selenium nanoparticle (SeNP) classes. These nanomaterials have been studied as potential antimicrobials for eradication of established HA-grown biofilms, for preventing biofilm formation on HA-coated surfaces and for inhibition of planktonic cell growth of Pseudomonas aeruginosa NCTC 12934 and Staphylococcus aureus ATCC 25923. Bio Se-NEMO resulted more efficacious than those chemically produced in all tested scenarios. Bio Se-NEMO produced by B.&nbsp;mycoides SelTE01 after 6 or 24&nbsp;h of Na 2 SeO 3 exposure show the same effective antibiofilm activity towards both P.&nbsp;aeruginosa and S.&nbsp;aureus strains at 0.078&nbsp;mg&nbsp;ml −1 (Bio Se-NEMO 6 ) and 0.3125&nbsp;mg&nbsp;ml −1 (Bio Se-NEMO 24 ). Meanwhile, chemically synthesized SeNPs at the highest tested concentration (2.5&nbsp;mg&nbsp;ml −1 ) have moderate antimicrobial activity. The confocal laser scanning micrographs demonstrate that the majority of the P.&nbsp;aeruginosa and S.&nbsp;aureus cells exposed to biogenic SeNPs within the biofilm are killed or eradicated. Bio Se-NEMO therefore displayed good antimicrobial activity towards HA-grown biofilms and planktonic cells, becoming possible candidates as new antimicrobials

What have they been up to in Lübeck recently

This talk will give an overview over three related research prototypes for ambient interactive systems. We start by introducing NEMO, the Network Environment for Multimedia Objects. NEMO is a smart media environment for semantically rich, personalised, and device-specific access to and interaction with multimedia objects. Next, a shared electronic whiteboard called ShareBoard is decribed. The goal of ShareBoard is to deliver a natural user interface to working with electronic whiteboards. Integrated within ShareBoard are input devices to recognise the movement of users in the surrounding space and for sensing 3D-gesture. ShareBoard can make use of media objects in NEMO. Last, we introduce the Modular Awareness Construction Kit. MACK is a framework for developing context aware, ambient intelligent systems that blend seamlessly with the users’ everyday route, enabling unobtrusive in-situ interaction and facilitating enhanced cooperation and communication. In the future, MACK is to deliver contextual information to both NEMO and ShareBoard

Recent Developments of NEMO: Detection of Solar Eruptions Characteristics

The recent developments in space instrumentation for solar observations and telemetry have caused the necessity of advanced pattern recognition tools for the different classes of solar events. The Extreme ultraviolet Imaging Telescope (EIT) of solar corona on-board SOHO spacecraft has uncovered a new class of eruptive events which are often identified as signatures of Coronal Mass Ejection (CME) initiations on solar disk. It is evident that a crucial task is the development of an automatic detection tool of CMEs precursors. The Novel EIT wave Machine Observing (NEMO) (http://sidc.be/nemo) code is an operational tool that detects automatically solar eruptions using EIT image sequences. NEMO applies techniques based on the general statistical properties of the underlying physical mechanisms of eruptive events on the solar disc. In this work, the most recent updates of NEMO code - that have resulted to the increase of the recognition efficiency of solar eruptions linked to CMEs - are presented. These updates provide calculations of the surface of the dimming region, implement novel clustering technique for the dimmings and set new criteria to flag the eruptive dimmings based on their complex characteristics. The efficiency of NEMO has been increased significantly resulting to the extraction of dimmings observed near the solar limb and to the detection of small-scale events as well. As a consequence, the detection efficiency of CMEs precursors and the forecasts of CMEs have been drastically improved. Furthermore, the catalogues of solar eruptive events that can be constructed by NEMO may include larger number of physical parameters associated to the dimming regions.Comment: 12 Pages, 5 figures, submitted to Solar Physic

Effects of architectural issues on a km3 scale detector

Simulation results showing the comparison between the performance of different km3 detector geometries are reported. Effective neutrino areas and angular resolutions are reported for three different geometries based on NEMO-towers and strings. The results show that the NEMO-tower based detector has the best performance concerning both the effective area and the angular resolution isotropyComment: to be published on VVVNT2 proceedings (Catania, Italy, November 8-11, 2005