The role of the NF-κB pathway in Her2-overexpressing breast cancer

Overexpression of the membrane-bound receptor tyrosine kinase Her2 (ErbB-2, EGFR2) occurs in approximately 30% of all breast cancers and typically correlates with poor prognosis. Overexpression of Her2 leads to activation of multiple downstream pathways including the MAPK, PI3K/Akt and NF-κB pathways. Her2 has been previously reported to activate the NF-κB pathway. However, the mechanism by which this occurs is poorly elucidated. In this report, we utilize an siRNA approach to investigate the role that the different Inhibitor of Kappa-B Kinase (IKK) subunits play in activation of NF-κB downstream of Her2. We show that IKKα plays a previously unreported role in NF-κB activation via the canonical pathway in Her2-overexpressing breast cancer cells. Furthermore, IKKα plays an important role in NF-κB regulated gene expression and induction of an invasive phenotype in these cells, independent of PI3K. This activation of NF-κB by Her2 also requires the NF-κB pathway kinase, TGF-β- activated kinase 1 (TAK1). Finally, we also show that inhibition of IKKα by siRNA leads to activation of SPARC, a member of the BM-40 family of genes which are often dysregulated in cancer. Activation of the NF-κB pathway can directly suppress expression of SPARC through a mechanism at the promoter of SPARC, proximal to the transcriptional start site. In summary, we present data elucidating the role and consequences of NF-κB activation in Her2-overexpressing breast cancer

IKKα and IKKβ Each Function to Regulate NF-κB Activation in the TNF-Induced/Canonical Pathway

Activation of the transcription factor NF-kappaB by cytokines is rapid, mediated through the activation of the IKK complex with subsequent phosphorylation and degradation of the inhibitory IkappaB proteins. The IKK complex is comprised of two catalytic subunits, IKKalpha and IKKbeta, and a regulatory protein known as NEMO. Using cells from mice that are genetically deficient in IKKbeta or IKKalpha, or using a kinase inactive mutant of IKKbeta, it has been proposed that IKKbeta is critical for TNF-induced IkappaB phosphorylation/degradation through the canonical pathway while IKKalpha has been shown to be involved in the non-canonical pathway for NF-kappaB activation. These conclusions have led to a focus on development of IKKbeta inhibitors for potential use in inflammatory disorders and cancer.Analysis of NF-kappaB activation in response to TNF in MEFs reveals that IKKbeta is essential for efficient phosphorylation and subsequent degradation of IkappaB alpha, yet IKKalpha contributes to the NF-kappaB activation response in these cells as measured via DNA binding assays. In HeLa cells, both IKKalpha and IKKbeta contribute to IkappaB alpha phosphorylation and NF-kappaB activation. A kinase inactive mutant of IKKbeta, which has been used as evidence for the critical importance of IKKbeta in TNF-induced signaling, blocks activation of NF-kappaB induced by IKKalpha, even in cells that are deficient in IKKbeta.These results demonstrate the importance of IKKalpha in canonical NF-kappaB activation, downstream of cytokine treatment of cells. The experiments suggest that IKKalpha will be a therapeutic target in inflammatory disorders

Genomic diversity of bacteriophages infecting Microbacterium spp

The bacteriophage population is vast, dynamic, old, and genetically diverse. The genomics of phages that infect bacterial hosts in the phylum Actinobacteria show them to not only be diverse but also pervasively mosaic, and replete with genes of unknown function. To further explore this broad group of bacteriophages, we describe here the isolation and genomic characterization of 116 phages that infect Microbacterium spp. Most of the phages are lytic, and can be grouped into twelve clusters according to their overall relatedness; seven of the phages are singletons with no close relatives. Genome sizes vary from 17.3 kbp to 97.7 kbp, and their G+C% content ranges from 51.4% to 71.4%, compared to ~67% for their Microbacterium hosts. The phages were isolated on five different Microbacterium species, but typically do not efficiently infect strains beyond the one on which they were isolated. These Microbacterium phages contain many novel features, including very large viral genes (13.5 kbp) and unusual fusions of structural proteins, including a fusion of VIP2 toxin and a MuF-like protein into a single gene. These phages and their genetic components such as integration systems, recombineering tools, and phage-mediated delivery systems, will be useful resources for advancing Microbacterium genetics

An inclusive Research and Education Community (iREC) model to facilitate undergraduate science education reform

Funding: This work was supported by Howard Hughes Medical Institute grants to DIH is GT12052 and MJG is GT15338.Over the last two decades, there have been numerous initiatives to improve undergraduate student outcomes in STEM. One model for scalable reform is the inclusive Research Education Community (iREC). In an iREC, STEM faculty from colleges and universities across the nation are supported to adopt and sustainably implement course-based research – a form of science pedagogy that enhances student learning and persistence in science. In this study, we used pathway modeling to develop a qualitative description that explicates the HHMI Science Education Alliance (SEA) iREC as a model for facilitating the successful adoption and continued advancement of new curricular content and pedagogy. In particular, outcomes that faculty realize through their participation in the SEA iREC were identified, organized by time, and functionally linked. The resulting pathway model was then revised and refined based on several rounds of feedback from over 100 faculty members in the SEA iREC who participated in the study. Our results show that in an iREC, STEM faculty organized as a long-standing community of practice leverage one another, outside expertise, and data to adopt, implement, and iteratively advance their pedagogy. The opportunity to collaborate in this manner and, additionally, to be recognized for pedagogical contributions sustainably engages STEM faculty in the advancement of their pedagogy. Here, we present a detailed pathway model of SEA that, together with underpinning features of an iREC identified in this study, offers a framework to facilitate transformations in undergraduate science education.Peer reviewe

Instructional Models for Course-Based Research Experience (CRE) Teaching

The course-based research experience (CRE) with its documented educational benefits is increasingly being implemented in science, technology, engineering, and mathematics education. This article reports on a study that was done over a period of 3 years to explicate the instructional processes involved in teaching an undergraduate CRE. One hundred and two instructors from the established and large multi-institutional SEA-PHAGES program were surveyed for their understanding of the aims and practices of CRE teaching. This was followed by large-scale feedback sessions with the cohort of instructors at the annual SEA Faculty Meeting and subsequently with a small focus group of expert CRE instructors. Using a qualitative content analysis approach, the survey data were analyzed for the aims of inquiry instruction and pedagogical practices used to achieve these goals. The results characterize CRE inquiry teaching as involving three instructional models: 1) being a scientist and generating data; 2) teaching procedural knowledge; and 3) fostering project ownership. Each of these models is explicated and visualized in terms of the specific pedagogical practices and their relationships. The models present a complex picture of the ways in which CRE instruction is conducted on a daily basis and can inform instructors and institutions new to CRE teaching

Models of classroom assessment for course-based research experiences

Course-based research pedagogy involves positioning students as contributors to authentic research projects as part of an engaging educational experience that promotes their learning and persistence in science. To develop a model for assessing and grading students engaged in this type of learning experience, the assessment aims and practices of a community of experienced course-based research instructors were collected and analyzed. This approach defines four aims of course-based research assessment—(1) Assessing Laboratory Work and Scientific Thinking; (2) Evaluating Mastery of Concepts, Quantitative Thinking and Skills; (3) Appraising Forms of Scientific Communication; and (4) Metacognition of Learning—along with a set of practices for each aim. These aims and practices of assessment were then integrated with previously developed models of course-based research instruction to reveal an assessment program in which instructors provide extensive feedback to support productive student engagement in research while grading those aspects of research that are necessary for the student to succeed. Assessment conducted in this way delicately balances the need to facilitate students’ ongoing research with the requirement of a final grade without undercutting the important aims of a CRE education

Dynamic histone acetylation is critical for cotranscriptional spliceosome assembly and spliceosomal rearrangements

Assembly of the spliceosome onto pre-mRNA is a dynamic process involving the ordered exchange of snRNPs to form the catalytically active spliceosome. These ordered rearrangements have recently been shown to occur cotranscriptionally, while the RNA polymerase is still actively engaged with the chromatin template. We previously demonstrated that the histone acetyltransferase Gcn5 is required for U2 snRNP association with the branchpoint. Here we provide evidence that histone acetylation and deacetylation facilitate proper cotranscriptional association of spliceosomal snRNPs. As with GCN5, mutation or deletion of Gcn5-targeted histone H3 residues leads to synthetic lethality when combined with deletion of the genes encoding the U2 snRNP components Lea1 or Msl1. Gcn5 associates throughout intron-containing genes and, in the absence of the histone deacetylases Hos3 and Hos2, enhanced histone H3 acetylation is observed throughout the body of genes. Deletion of histone deacetylaces also results in persistent association of the U2 snRNP and a severe defect in the association of downstream factors. These studies show that cotranscriptional spliceosome rearrangements are driven by dynamic changes in the acetylation state of histones and provide a model whereby yeast spliceosome assembly is tightly coupled to histone modification

IKKα and IKKβ each contribute to TNF-induced NF-κB activity in HeLa cells.

<p>HeLa cells, seeded in 24-well plates were transiently transfected with indicated siRNA constructs for 48 hr. Then media was replaced and cells were further transfected with NF-κB responsive 3x-κB luciferase and a control Renilla luciferase contructs. TNF was added (as indicated) and 24 hr later cells were lysed and dual lucifearse assay was performed. Luciferase readings in untreated and control vector transfected cells were normalized as 1.</p