Dynamic Characterization of the IKK:κBα:NFκB Negative Feedback Loop Using Real-Time Bioluminescence Imaging

The transcription factor NF-κB is a pivotal regulator of mammalian cell function, modulating genes implicated in cellular stress responses, proliferation, differentiation, cell survival and apoptosis, as well as immune and inflammatory responses. Improper regulation of NF-κB signaling has been implicated in a myriad of human pathological disorders, including cardiovascular and neurodegenerative diseases, chronic inflammation, and various cancers. A key regulatory node within canonical NF-κB signaling is the IKK:NF-κB: IκBα negative feedback loop that plays a major role in regulating the strength and duration of NF-κB transcriptional activity. We have developed and characterized an unique bioluminescent reporter (κB5àIκBα-FLuc) that recapitulates this transcriptionally coupled negative feedback loop, and have extensively utilized this reporter to interrogate how diverse stimuli (i.e., ligand type, duration, concentration, sequential stimulation, etc.) impact the IKK:NF-κB: IκBα negative feedback loop in cellulo and in vivo. We found that the negative feedback loop exhibits differential and reproducible dynamic patterns in response to modulation of TNFα; concentration or pulse duration, and that responses to TNFα exhibited a remarkable degree of synchronicity at the level of single cells, cell populations, and in vivo. Furthermore, we discovered a TNFα-induced transient refractory period (lasting up to 120 min) during which cells were unable to fully degrade IκBα following a second TNFα challenge, and identified nuclear export of NF-κB: IκBα complexes as a rate-limiting step that may impact this refractory period. A high-throughput RNAi screen to identify new phosphatase and kinase regulators of TNFα-induced IKK:NF-κB: IκBα negative feedback loop dynamics revealed a vast array of different IκBα-FLuc dynamic profiles, highlighting the large number and diverse activities of kinases and phosphatases regulating the NF-κB pathway. Two of these hits, PTPRJ and DAPK3, have been validated and are the subjects of current investigations to understand the physiological and/or pathophysiological relevance in NF-κB, especially in the context of TNFα signaling during cancer and inflammation in the liver. In conclusion, our studies using dynamic, real-time bioluminescence imaging have demonstrated the utility of employing bioluminescent reporters alongside traditional biochemical assays, in silico modeling, and cell/molecular biology techniques to rigorously interrogate how diverse stimuli impact the IKK:NF-κB: IκBα negative feedback loop in single cells, cell populations, and at the organ- and tissue-level in vivo

Original Article

本研究の目的は,健康な小児ががんや白血病といった病名からどのようなイメージを抱くのか,自分が大きな病気になったとしたら,病名や治療などについて教えて欲しいと思っているのかを明らかにすることである。千葉県内の小中高校に通っている小学校5年生から高校3年生までの児童生徒1964名から得られたアンケートの回答を,統計ソフトSPSSにて分析を行い以下の結果を得た。1.健康な小児が抱くがんのイメージは,「死ぬ・治らない」「重い・危険・苦しい」といった悲観的なのものが多く,全体の約65%を占めていた。「聞いたことがない・わからない」と答えたものもおり全体の約15%であり,どの学年でも同様の傾向であった。2.健康な小児が抱く白血病のイメージは「病態や特徴について」「原因や治療について」といったものが,全体の約38%を占めていた。「聞いたことがない・わからない」と答えたものは全体の約33%を占め,小学生では半数以上が「聞いたことがない・わからない」と答えていた。3.自分が病気になったときにされる説明については小学生の80%,中学生の85%,高校生の91%が真実を伝えられることを求めていた。その理由として自分の知る権利,治療に前向きに取り組めるといった姿勢の向上,知らないことが不安になる,残された命を有意義に悔いのないように過ごしたいといったものがあげられていた。The purposes of this study were to identify the images of healthy children on cancer and leukemia, and the way of thinking of truth telling with disease. The number of subjects were 1964. They belonged to between the fifth grade of primary school and the third year in high school. They answered someitem-questionnaire, and the answers were analyzed using SPSS. The results were as follows: 1. Sixty-five percent of the images of cancer were pessimistic, like death or incurable and serious or painful . About fifteen percent were having no images. 2. Thirty-eight percent of the images of leukemia were the feature and the cause or the treatment . About thirty-three percent were having no images. 3. Truth telling was desired by eighty percent of students of primary schools, eighty-five percent of junior high schools and ninety-one percent of a high school. The reasons were the rights to know , to be patient with treatments , to become more anxious without truth telling , and to live the remaining days without regrets

Veni, Vidi, Vici: In Vivo Molecular Imaging of Immune Response

“I came, I saw, I conquered,” Julius Caesar proclaimed, highlighting the importance of direct visualization as a winning strategy. Continuing the “From the Field” series (see Editorial [2007] 26, 131), Gross et al. summarize how modern molecular imaging techniques can successfully dissect the complexities of immune response in vivo

Evaluation to Improve a High School Summer Science Outreach Program

The goal of the Young Scientist Program (YSP) at Washington University School of Medicine in St. Louis (WUSM) is to broaden science literacy and recruit talent for the scientific future. In particular, YSP seeks to expose underrepresented minority high school students from St. Louis public schools (SLPS) to a wide variety of careers in the sciences. The centerpiece of YSP, the Summer Focus Program (SFP), is a nine-week, intensive research experience for competitively chosen rising high school seniors (Scholars). Scholars are paired with volunteer graduate student, medical student, or postdoctoral fellow mentors who are active members of the practicing scientific community and serve as guides and exemplars of scientific careers. The SFP seeks to increase the number of underrepresented minority students pursuing STEM undergraduate degrees by making the Scholars more comfortable with science and science literacy. The data presented here provide results of the objective, quick, and simple methods developed by YSP to assess the efficacy of the SFP from 2006 to 2013. We demonstrate that the SFP successfully used formative evaluation to continuously improve the various activities within the SFP over the course of several years and in turn enhance student experiences within the SFP. Additionally we show that the SFP effectively broadened confidence in science literacy among participating high school students and successfully graduated a high percentage of students who went on to pursue science, technology, engineering, and mathematics (STEM) majors at the undergraduate level

Rate Motifs Tune Auxin/Indole-3-Acetic Acid Degradation Dynamics

Ubiquitin-mediated protein degradation is a common feature in diverse plant cell signaling pathways; however, the factors that control the dynamics of regulated protein turnover are largely unknown. One of the best-characterized families of E3 ubiquitin ligases facilitates ubiquitination of auxin (aux)/indole-3-acetic acid (IAA) repressor proteins in the presence of auxin. Rates of auxin-induced degradation vary widely within the Aux/IAA family, and sequences outside of the characterized degron (the minimum region required for auxin-induced degradation) can accelerate or decelerate degradation. We have used synthetic auxin degradation assays in yeast (Saccharomyces cerevisiae) and in plants to characterize motifs flanking the degron that contribute to tuning the dynamics of Aux/IAA degradation. The presence of these rate motifs is conserved in phylogenetically distant members of the Arabidopsis (Arabidopsis thaliana) Aux/IAA family, as well as in their putative Brassica rapa orthologs. We found that rate motifs can act by enhancing interaction between repressors and the E3, but that this is not the only mechanism of action. Phenotypes of transgenic plants expressing a deletion in a rate motif in IAA28 resembled plants expressing degron mutations, underscoring the functional relevance of Aux/IAA degradation dynamics in regulating auxin responses

Evaluation of oligomerization potential of SCF^TIR1 subunits.

<p><b>(a-d)</b> BiFC-based evaluation of TIR1, CUL1, ASK1 oligomerization and ASK1-TIR1 interaction, respectively, following transient expression in Nicotiana leaves. <b>(a'-d')</b> Propidium iodide staining of the nucleus. <b>(e)</b> Assessment of TIR1-TIR1 protein interaction in a Y2H assay. Images of single colonies expressing the designated constructs and grown on histidine plates (top panel) and test plates containing the indicated concentrations of 3-AT in the absence of histidine (bottom panels). <b>(f)</b> Co-IP-based assessment of TIR1 oligomerization following transient expression in Nicotiana leaves. HA:TIR1 and Myc:TIR1 were co-injected in leaves. Protein extracts were subjected to immunoprecipitation using anti-Myc antibody. Immunoprecipitates were examined by western-blotting using anti-Myc and anti-HA antibodies. (<b>g</b>) Co-IP-based assessment of TIR1 oligomerization in double <i>pTIR1</i>:<i>TIR1–GUS</i> and <i>pTIR1</i>:<i>gTIR1–VENUS</i> transgenic Arabidopsis plants. Protein extracts were subjected to immunoprecipitation using anti-GUS antibody. Immunoprecipitates were examined by western-blotting using anti-GUS and anti-GFP antibodies.</p

A set of spatially-clustered amino acids are critical for TIR1 oligomerization.

<p><b>(a, c, e, g, i</b> and <b>k)</b> BiFC-based assessment of TIR1 oligomerization using wild-type and G142A, F143A, I151A, V117A and G147A mutants, respectively, following transient expression in Nicotiana leaves. <b>(b, d, f, h, j</b> and <b>l)</b> Assessment of TIR1-ASK1 interaction using wild-type and G142A, F143A, I151A, V117A and G147A mutants respectively, following transient expression in Nicotiana leaves. <b>(m</b> and <b>n)</b> Assessment of TIR1 interaction with D170A and D170E mutants using BiFC. <b>(o)</b> Co-IP experiments in Nicotiana leaves. HA-tagged wild-type and mutant Flag tagged TIR1 constructs were co-injected. Protein extracts were subjected to immuno-precipitation using anti-HA conjugated beads. The immune-precipitates were examined by western-blotting using anti-Flag and anti-HA antibodies. <b>(p)</b> Side view of TIR1-IAA7 peptide structure (PDB 2P1Q); residues critical for TIR1 oligomerization are depicted in orange.</p

TIR1 oligomerization is essential for auxin signaling.

<p><b>(a)</b> Semi <i>in vitro</i> pull-down assays using GST-IAA3 and Nicotiana cell extracts expressing wild-type and mutant HA:TIR1 proteins. Values represent fold-change compared to TIR1:HA samples (all values were corrected for equal loading among samples). <b>(b)</b> Assessment of TIR1-IAA7 protein interaction in Y2H assay in the presence of auxin (25 μM). Images of yeast cells expressing the designated constructs and grown on histidine plates and test plates containing 10 mM of 3-AT. <b>(c)</b> Degradation of YFP-IAA fusion proteins in yeast in the presence of wild-type and mutant TIR1 proteins after the addition of auxin. Yeast cells were imaged using time-lapse flow cytometry. Degradation curves were normalized to the starting fluorescence absorbance unit (a.u.). <b>(d)</b> Five day old seedlings were transferred to 0.5x MS mediums containing the indicated concentrations of 2,4-D. After 5 days of growth, the length of primary root was measured, and expressed relative to growth on control plates. <b>(e)</b> Schematic model of SCF^TIR1 dimerization.</p