CMS physics technical design report : Addendum on high density QCD with heavy ions

Peer reviewe

Synthesis and Evaluation of Cytosolic Phospholipase A₂ Activatable Fluorophores for Cancer Imaging

Activatable fluorophores selective to cytosolic phospholipase A₂ (cPLA₂) were synthesized and evaluated for their ability to image triple negative breast cancer cells. The activatable constructs were synthesized by esterification of a small molecule fluorophore with a fatty acid resulting in ablated fluorescence. Selectivity for cPLA₂ was generated through the choice of fluorophore and fatty acid. Esterification with arachidonic acid was sufficient to impart specificity to cPLA₂ when compared to esterification with palmitic acid. <i>In vitro</i> analysis of probes incorporated into phosphatidylcholine liposomes demonstrated that a nonselective phospholipase (sPLA₂ group IB) was able to hydrolyze both arachidonate and palmitate coupled fluorophores resulting in the generation of fluorescence. Of the four fluorophores tested, DDAO (7-hydroxy-9<i>H</i>-(1,3-dichloro-9,9-dimethylacridin-2-one)) was observed to perform optimally <i>in vitro</i> and was analyzed further in 4175-Luc+ cells, a metastatic triple negative human breast cancer cell line expressing high levels of cPLA₂. In contrast to the <i>in vitro</i> analysis, DDAO arachidonate was shown to activate selectively in 4175-Luc+ cells compared to the control DDAO palmitate as measured by fluorescence microscopy and quantitated with fluorescence spectroscopy. The addition of two agents known to activate cPLA₂ enhanced DDAO arachidonate fluorescence without inducing any change to DDAO palmitate. Inhibition of cPLA₂ resulted in reduced fluorescence of DDAO arachidonate but not DDAO palmitate. Together, we report the synthesis of a cPLA₂ selective activatable fluorophore capable of detecting cPLA₂ in triple negative breast cancer cells

Uncovering protein–protein interactions through a team-based undergraduate biochemistry course

How can we provide fertile ground for students to simultaneously explore a breadth of foundational knowledge, develop cross-disciplinary problem-solving skills, gain resiliency, and learn to work as a member of a team? One way is to integrate original research in the context of an undergraduate biochemistry course. In this Community Page, we discuss the development and execution of an interdisciplinary and cross-departmental undergraduate biochemistry laboratory course. We present a template for how a similar course can be replicated at other institutions and provide pedagogical and research results from a sample module in which we challenged our students to study the binding interface between 2 important biosynthetic proteins. Finally, we address the community and invite others to join us in making a larger impact on undergraduate education and the field of biochemistry by coordinating efforts to integrate research and teaching across campuses

In a semester of Biochemistry Superlab, students investigated the protein–protein interactions involved in the β-hydroxylation of the natural product skyllamycin.

<p>The skyllamycin peptide is constructed by <i>Streptomyces</i> bacteria via a NRPS involving 11 biosynthetic modules (“M”), composed of catalytic domains such as the A, PCP, and C domains. The <i>in trans</i> cytochrome P450 (P450_sky, orange) interacts with PCP-bound amino acids on modules 5, 7, and 11 to install β-hydroxyl groups (highlighted in orange on the structure of skyllamycin, right). As a class, we tackled the central question: What is the biochemical basis for the selectivity of the interaction of PCP from module 7 with P450_sky to install the hydroxyl group on the L-(OMe)-Tyr (incorporated at the boxed position of skyllamycin)? A, adenylation; C, condensation; NRPS, non-ribosomal peptide synthetase; PCP, peptidyl carrier protein.</p

SV-AUC data collected and analyzed by students to obtain dissociation constants for P450_sky and mutants of P450_sky interacting with inhibitor-bound PCP7_sky (L-imidazoyl-PCP7_sky).

<p>Comparisons of the <i>c(s)</i> distributions are shown for 10 μM P450_sky wild type alone and in complex with 60 μM L-imidazoyl-PCP7_sky L62A, L-imidazoyl-PCP7_sky F66A, and L-imidazoyl-PCP7_sky wild type. A) 280 nm (protein), B) 418 nm (heme). In general, shifts to the right suggest that the reaction boundary favors tighter binding. SV-AUC, sedimentation velocity experiments with an analytical ultracentrifuge.</p

General workflow for students investigating the noncovalent interactions involved in P450_sky-catalyzed β-hydroxylation of L-(OMe)-Tyr.

<p>This involves computational analysis (Step 1), molecular biology or synthetic chemistry (Step 2), protein purification (Step 3), chemoenzymatic assays (Step 4), and biochemical and biophysical experiments (Step 5). This workflow is a template for realizing an integrated science curriculum, as described and assessed by the Interdisciplinary Learning Consortium [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003145#pbio.2003145.ref009" target="_blank">9</a>]. PCP, peptidyl carrier protein.</p