Addressing the unmet clinical need for low-volume assays in early diagnosis of pancreatic cancer

The incidental detection of pancreatic cysts, an opportunity for the early detection of pancreatic cancer, is increasing, owing to an aging population and improvements in imaging technology. The classification of pancreatic cystic precursors currently relies on imaging and cyst fluid evaluations, including cytology and protein and genomic analyses. However, there are persistent limitations that obstruct the accuracy and quality of information for clinicians, including the limited volume of the complex, often acellular, and proteinaceous milieu that comprises pancreatic cyst fluid. The constraints of currently available clinical assays lead clinicians to the subjective and inconsistent application of diagnostic tools, which can contribute to unnecessary surgery and missed pancreatic cancers. Herein, we describe the pathway toward pancreatic cyst classification and diagnosis, the volume requirements for several clinically available diagnostic tools, and some analytical and diagnostic limitations for each assay. We then discuss current and future work on novel markers and methods, and how to expand the utility of clinical pancreatic cyst fluid samples. Results of ongoing studies applying SERS as a detection mode suggest that 50 µL of pancreatic cyst fluid is more than sufficient to accurately rule out non-mucinous pancreatic cysts with no malignant potential from further evaluation. This process is expected to leave sufficient fluid to analyze a follow-up, rule-in panel of markers currently in development that can stratify grades of dysplasia in mucinous pancreatic cysts and improve clinical decision-making

A synergistic antiproliferation effect of curcumin and docosahexaenoic acid in SK-BR-3 breast cancer cells: unique signaling not explained by the effects of either compound alone

<p>Abstract</p> <p>Background</p> <p>Breast cancer is a collection of diseases in which molecular phenotypes can act as both indicators and mediators of therapeutic strategy. Therefore, candidate therapeutics must be assessed in the context of multiple cell lines with known molecular phenotypes. Docosahexaenoic acid (DHA) and curcumin (CCM) are dietary compounds known to antagonize breast cancer cell proliferation. We report that these compounds in combination exert a variable antiproliferative effect across multiple breast cell lines, which is synergistic in SK-BR-3 cells and triggers cell signaling events not predicted by the activity of either compound alone.</p> <p>Methods</p> <p>Dose response curves for CCM and DHA were generated for five breast cell lines. Effects of the DHA+ CCM combination on cell proliferation were evaluated using varying concentrations, at a fixed ratio, of CCM and DHA based on their individual ED₅₀. Detection of synergy was performed using nonlinear regression of a sigmoid dose response model and Combination Index approaches. Cell molecular network responses were investigated through whole genome microarray analysis of transcript level changes. Gene expression results were validated by RT-PCR, and western blot analysis was performed for potential signaling mediators. Cellular curcumin uptake, with and without DHA, was analyzed via flow cytometry and HPLC.</p> <p>Results</p> <p>CCM+DHA had an antiproliferative effect in SK-BR-3, MDA-MB-231, MDA-MB-361, MCF7 and MCF10AT cells. The effect was synergistic for SK-BR-3 (ER^-PR^-Her2⁺) relative to the two compounds individually. A whole genome microarray approach was used to investigate changes in gene expression for the synergistic effects of CCM+DHA in SK-BR-3 cells lines. CCM+DHA triggered transcript-level responses, in disease-relevant functional categories, that were largely non-overlapping with changes caused by CCM or DHA individually. Genes involved in cell cycle arrest, apoptosis, inhibition of metastasis, and cell adhesion were upregulated, whereas genes involved in cancer development and progression, metastasis, and cell cycle progression were downregulated. Cellular pools of PPARγ and phospho-p53 were increased by CCM+DHA relative to either compound alone. DHA enhanced cellular uptake of CCM in SK-BR-3 cells without significantly enhancing CCM uptake in other cell lines.</p> <p>Conclusions</p> <p>The combination of DHA and CCM is potentially a dietary supplemental treatment for some breast cancers, likely dependent upon molecular phenotype. DHA enhancement of cellular curcumin uptake is one potential mechanism for observed synergy in SK-BR-3 cells; however, transcriptomic data show that the antiproliferation synergy accompanies many signaling events unique to the combined presence of the two compounds.</p

Crystallization of human thymidylate synthase

Human thymidylate synthase has been crystallized in the absence of ligands and diffracts beyond 3.0 A. The protein was cloned and expressed in Escherichia coli and then crystallized from ammonium sulfate in the presence of beta-mercaptoethanol at a variety of pH values. The crystals are trigonal in the space-group P3(1)21; the unit cell dimensions are a = b = 96.7 A, c = 84.1 A

Flexibility of PCNA-Protein Interface Accommodates Differential Binding Partners

<div><p>The expanding roles of PCNA in functional assembly of DNA replication and repair complexes motivated investigation of the structural and dynamic properties guiding specificity of PCNA-protein interactions. A series of biochemical and computational analyses were combined to evaluate the PIP Box recognition features impacting complex formation. The results indicate subtle differences in topological and molecular descriptors distinguishing both affinity and stoichiometry of binding among PCNA-peptide complexes through cooperative effects. These features were validated using peptide mimics of p85α and Akt, two previously unreported PCNA binding partners. This study characterizes for the first time a reverse PIP Box interaction with PCNA. Small molecule ligand binding at the PIP Box interaction site confirmed the adaptive nature of the protein in dictating overall shape and implicates allosterism in transmitting biological effects.</p></div

Crystal structure of human thymidylate synthase: a structural mechanism for guiding substrates into the active site

The crystal structure of human thymidylate synthase, a target for anti-cancer drugs, is determined to 3.0 A resolution and refined to a crystallographic residual of 17.8%. The structure implicates the enzyme in a mechanism for facilitating the docking of substrates into the active site. This mechanism involves a twist of approximately 180 degrees of the active site loop, pivoted around the neighboring residues 184 and 204, and implicates ordering of external, eukaryote specific loops along with the well-characterized closure of the active site upon substrate binding. The highly conserved, but eukaryote-specific insertion of twelve residues 90-101 (h117-128), and of eight residues between 156 and 157 (h146-h153) are known to be alpha-helical in other eukaryotes, and lie close together on the outside of the protein in regions of disordered electron density in this crystal form. Two cysteines [cys 202 (h199) and 213 (h210)] are close enough to form a disulfide bond within each subunit, and a third cysteine [cys 183 (h180)] is positioned to form a disulfide bond with the active site cysteine [cys 198 (h195)] in its unliganded conformation. The amino terminal 27 residues, unique to human TS, contains 8 proline residues, is also in a region of disordered electron density, and is likely to be flexible prior to substrate binding. The drug resistance mutation, Y6H, confers a 4-fold reduction in FdUMP affinity and 8-fold reduction in kcat for the dUMP reaction. Though indirectly connected to the active site, the structure suggests a mechanism of resistance that possibly involves a change in structure. This structure offers a unique opportunity for structure-based drug design aimed at the unliganded form of the human enzyme

ANCHOR Results of Short PIP Box Peptides Binding to PCNA.

<p>Changes in the SASA of the short PIP Box peptide mimic upon ligand binding, as determined by the average trajectory model exported from the molecular dynamic simulations, were calculated using ANCHOR. Values within the heat map indicate ΔSASA between the bound and unbound forms.</p

PIP Box Peptide Competition of PCNA-PL Interactions by Fluorescence Polarization.

<p>Competition of 50-PL and 1 µM recombinant PCNA protein (monomer concentration) with increasing amounts of unlabeled PL (squares) and p21 (circles) peptides. Anisotropy values (<i>N</i> = 4) were converted to fractional occupancy, <i>f_b</i>, values using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102481#pone.0102481.e005" target="_blank">Eq. (3)</a> and represented as mean ± standard error of mean (SEM). Error bars associated with specific data points may be within the data points themselves.</p