A highly selective, label-free, homogenous luminescent switch-on probe for the detection of nanomolar transcription factor NF-kappaB

Transcription factors are involved in a number of important cellular processes. The transcription factor NF-κB has been linked with a number of cancers, autoimmune and inflammatory diseases. As a result, monitoring transcription factors potentially represents a means for the early detection and prevention of diseases. Most methods for transcription factor detection tend to be tedious and laborious and involve complicated sample preparation, and are not practical for routine detection. We describe herein the first label-free luminescence switch-on detection method for transcription factor activity using Exonuclease III and a luminescent ruthenium complex, [Ru(phen)2(dppz)]2+. As a proof of concept for this novel assay, we have designed a double-stranded DNA sequence bearing two NF-κB binding sites. The results show that the luminescence response was proportional to the concentration of the NF-κB subunit p50 present in the sample within a wide concentration range, with a nanomolar detection limit. In the presence of a known NF-κB inhibitor, oridonin, a reduction in the luminescence response of the ruthenium complex was observed. The reduced luminescence response of the ruthenium complex in the presence of small molecule inhibitors allows the assay to be applied to the high-throughput screening of chemical libraries to identify new antagonists of transcription factor DNA binding activity. This will allow the rapid and low cost identification and development of novel scaffolds for the treatment of diseases caused by the deregulation of transcription factor activity

Spectroscopy of kaonic atoms at DAFNE and J-PARC

The interaction of antikaons (K^{-}) with nucleons and nuclei in the low-energy regime represents a very active research field in hadron physics. A unique and rather direct experimental access to the antikaon-nucleon scattering lengths is provided by precision X-ray spectroscopy of transitions in low-lying states in the lightest kaonic atoms (i.e. kaonic hydrogen and deuterium). In the SIDDHARTA experiment at the electron-positron collider DAFNE of LNFINFN we measured the most precise values of the strong interaction observables in conic hydrogen. The strong interaction on the 1s ground state of the electromagnetically bound K-p atom causes an energy shift and broadening of the 1s state. SIDDHARTA will extend the spectroscopy to kaonic deuterium to get access to the antikaon-neutron interaction and thus the isospin dependent scattering lengths. At J-PARC a kaon beam is used in a complementary experiment with a different setup for spectroscopy of kaonic deuterium atoms. The talk will give an overview of the of the upcoming experiments SIDDHARTA and the complementary experiment at J-PARC.Furthermore, the implications of the experiments for the theory of low-energy strong interaction with strangeness will be discussed

Characterization of the SIDDHARTA-2 luminosity monitor

A luminosity monitor, based on plastic scintillator detectors, has been developed for the SIDDHARTA-2 experiment aiming to perform high precision measurements of kaonic atoms and was installed in 2020 on the DAFNE $e^+e^-$ collider at LNF (Laboratori Nazionali di Frascati, INFN). The main goal of this system is to provide the~instantaneous and integrated luminosity of the DAFNE facility by measuring the rate of $K^+K^-$ correlated pairs emitted by the phi meson decay. This task requires an accurate timing of the DAQ signals, as well as timing resolution below 1ns, in order to disentangle the $K^\pm$ signals from the background minimum ionizing particles (MIPs) produced during the $e^+e^-$ collisions at DAFNE. In this paper the luminosity monitor concept as well as its laboratory characterization and the first results inside DAFNE are presented.Comment: Published in JINS

Nuclear receptor corepressors

The ability of NR LBDs to transfer repression function to a heterologous DNA binding domain, and the cross-squelching of repression by untethered LBDs, has suggested that repression is mediated by interactions with putative cellular corepressor proteins. The yeast-two hybrid screen for protein interactors has proven to be the key to the isolation and characterization of corepressors. This short review will focus on N-CoR and SMRT

Analysis of Thyroid Response Element Activity during Retinal Development

Thyroid hormone (TH) signaling components are expressed during retinal development in dynamic spatial and temporal patterns. To probe the competence of retinal cells to mount a transcriptional response to TH, reporters that included thyroid response elements (TREs) were introduced into developing retinal tissue. The TREs were placed upstream of a minimal TATA-box and two reporter genes, green fluorescent protein (GFP) and human placental alkaline phosphatase (PLAP). Six of the seven tested TREs were first tested in vitro where they were shown to drive TH-dependent expression. However, when introduced into the developing retina, the TREs reported in different cell types in both a TH-dependent and TH-independent manner, as well as revealed specific spatial patterns in their expression. The role of the known thyroid receptors (TR), TRα and TRβ, was probed using shRNAs, which were co-electroporated into the retina with the TREs. Some TREs were positively activated by TR+TH in the developing outer nuclear layer (ONL), where photoreceptors reside, as well as in the outer neuroblastic layer (ONBL) where cycling progenitor cells are located. Other TREs were actively repressed by TR+TH in cells of the ONBL. These data demonstrate that non-TRs can activate some TREs in a spatially regulated manner, whereas other TREs respond only to the known TRs, which also read out activity in a spatially regulated manner. The transcriptional response to even simple TREs provides a starting point for understanding the regulation of genes by TH, and highlights the complexity of transcriptional regulation within developing tissue

kaonic atoms experiment at the daφne collider by siddharta siddharta 2

The excellent quality kaon beam provided by the DA\PhiΦNE collider of LNF-INFN (Italy) together with SIDDHARTA/SIDDHARTA-2 new experimental techniques, as very precise and fast-response X-ray detectors, allow to perform unprecedented measurements on light kaonic atoms crucial for a deeper understanding of the low-energy quantum chromodynamics (QCD) in the strangeness sector. In this paper an overview of the main results obtained by the SIDDHARTA collaboration, as well as the future plans related to the SIDDHARTA-2 experiment, are discussed

Autophagy Induction as a Therapeutic Strategy for Neurodegenerative Diseases.

Autophagy is a major, conserved cellular pathway by which cells deliver cytoplasmic contents to lysosomes for degradation. Genetic studies have revealed extensive links between autophagy and neurodegenerative disease, and disruptions to autophagy may contribute to pathology in some cases. Autophagy degrades many of the toxic, aggregate-prone proteins responsible for such diseases, including mutant huntingtin (mHTT), alpha-synuclein (α-syn), tau, and others, raising the possibility that autophagy upregulation may help to reduce levels of toxic protein species, and thereby alleviate disease. This review examines autophagy induction as a potential therapy in several neurodegenerative diseases-Alzheimer's disease, Parkinson's disease, polyglutamine diseases, and amyotrophic lateral sclerosis (ALS). Evidence in cells and in vivo demonstrates promising results in many disease models, in which autophagy upregulation is able to reduce the levels of toxic proteins, ameliorate signs of disease, and delay disease progression. However, the effective therapeutic use of autophagy induction requires detailed knowledge of how the disease affects the autophagy-lysosome pathway, as activating autophagy when the pathway cannot go to completion (e.g., when lysosomal degradation is impaired) may instead exacerbate disease in some cases. Investigating the interactions between autophagy and disease pathogenesis is thus a critical area for further research

Multiple domains of the glucocorticoid receptor involved in synergism with the CACCC box factor(s).

Steroid induction of responsive genes functions through the synergistic activity of steroid receptor-binding sequences with adjacent transcription factor-binding sites. To analyze the mechanism of synergy we tested different human glucocorticoid receptor mutants for synergistic function with another transcription factor in comparison with intrinsic trans-activation obtained with a single receptor binding site (glucocorticoid response element). Multiple domains were found to be involved in synergistic activity of the glucocorticoid receptor with the CACCC box factor. Deletions within the N-terminal receptor half affected simultaneously intrinsic trans-activation and synergism. However, deletion of the hormone-binding domain mainly impaired synergism rather than intrinsic trans-activation, clearly showing that this domain synergizes by a mechanism independent of intrinsic activation. A chimeric protein where the DNA-binding domain of the glucocorticoid receptor was replaced by that of the yeast GAL4 protein also showed functional synergism. These data suggest that some of the receptor domains outside the DNA-binding domain synergize by their intrinsic trans-activating property, but the hormone-binding domain contributes to synergism by a different mechanism

Enhancement of human estrogen receptor activity by SPT6: a potential coactivator

The conserved nature of the transcriptional machinery between yeast and higher eukaryotes makes the yeast system suitable to genetically dissect the signal transduction pathway of steroid hormone receptors. This report describes the yeast protein, SPT6, which modulates the transcriptional activity of the human estrogen receptor (hER) by affecting the C-terminal activation domain. It is demonstrated that SPT6 is able to potentiate hER activity in yeast and also in mammalian cells in vivo. SPT6 interacts specifically with the hormone-binding domain of hER in vivo. The in vivo studies are substantiated by specific protein-protein interactions between SPT6 and the hormone-binding domain of hER in vitro. Therefore, the data suggest that the SPT6 protein may be involved in signal transmission of ER by acting as a coactivator