Unraveling the Allosteric Mechanism of Serine Protease Inhibition by an Antibody

SummaryRecent structural studies have outlined the mechanism of protease inhibition by active site-directed antibodies. However, the molecular basis of allosteric inhibition by antibodies has been elusive. Here we report the 2.35 Å resolution structure of the trypsin-like serine protease hepatocyte growth factor activator (HGFA) in complex with the allosteric antibody Ab40, a potent inhibitor of HGFA catalytic activity. The antibody binds at the periphery of the substrate binding cleft and imposes a conformational change on the entire 99-loop (chymotrypsinogen numbering). The altered conformation of the 99-loop is incompatible with substrate binding due to the partial collapse of subsite S2 and the reorganization of subsite S4. Remarkably, a single residue deletion of Ab40 abolished inhibition of HGFA activity, commensurate with the reversal of the 99-loop conformation to its “competent” state. The results define an “allosteric switch” mechanism as the basis of protease inhibition by an allosteric antibody

MicroRNA-203 predicts human survival after resection of colorectal liver metastasis.

BackgroundResection of colorectal liver metastasis (CRLM) can be curative. Predicting which patients may benefit from resection, however, remains challenging. Some microRNAs (miRNAs) become deregulated in cancers and contribute to cancer progression. We hypothesized that miRNA expression can serve as a prognostic marker of survival after CRLM resection.ResultsMiR-203 was significantly overexpressed in tumors of short-term survivors compared to long-term survivors. R1/R2 margin status and high clinical risk score (CRS) were also significantly associated with short-term survival (both p = 0.001). After adjusting for these variables, higher miR-203 expression remained an independent predictor of shorter survival (p = 0.010). In the serum cohort, high CRS and KRAS mutation were significantly associated with short-term survival (p = 0.005 and p = 0.026, respectively). After adjusting for CRS and KRAS status, short-term survivors were found to have significantly higher miR-203 levels (p = 0.016 and p = 0.033, respectively).Materials and methodsWe employed next-generation sequencing of small-RNAs to profile miRNAs in solid tumors obtained from 38 patients who underwent hepatectomy for CRLM. To validate, quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed on 91 tumor samples and 46 preoperative serum samples.ConclusionsAfter CRLM resection, short-term survivors exhibited significantly higher miR-203 levels relative to long-term survivors. MiR-203 may serve as a prognostic biomarker and its prognostic capacity warrants further investigation

El Niño–Southern Oscillation in Tropical and Midlatitude Column Ozone

The impacts of El Niño–Southern Oscillation (ENSO) on the tropical total column ozone, the tropical tropopause pressure, and the 3.5-yr ozone signal in the midlatitude total column ozone were examined using the Goddard Earth Observing System Chemistry–Climate Model (GEOS CCM). Observed monthly mean sea surface temperature and sea ice between 1951 and 2004 were used as boundary conditions for the model. Since the model includes no solar cycle, quasi-biennial oscillation, or volcanic forcing, the ENSO signal was found to dominate the tropical total column ozone variability. Principal component analysis was applied to the detrended, deseasonalized, and low-pass filtered model outputs. The first mode of model total column ozone captured 63.8% of the total variance. The spatial pattern of this mode was similar to that in Total Ozone Mapping Spectrometer (TOMS) observations. There was also a clear ENSO signal in the tropical tropopause pressure in the GEOS CCM, which is related to the ENSO signal in the total column ozone. The regression coefficient between the model total column ozone and the model tropopause pressure was 0.71 Dobson units (DU) hPa^(−1). The GEOS CCM was also used to investigate a possible mechanism for the 3.5-yr signal observed in the midlatitude total column ozone. The 3.5-yr signal in the GEOS CCM column ozone is similar to that in the observations, which suggests that a model with realistic ENSO can reproduce the 3.5-yr signal. Hence, it is likely that the 3.5-yr signal was caused by ENSO

PCSK9 variation and association with blood pressure in African Americans: Preliminary findings from the HyperGEN and REGARDS studies

Proprotein convertase subtilisin/kexin type 9 (encoded by PCSK9) plays a well-known role in the regulation of low-density lipoprotein (LDL) receptors, and an inhibitor of this enzyme is a promising new therapeutic for hyperlipidemia. Recently, animal and human studies also implicate PCSK9 genetic variation in the regulation of blood pressure. The goal of this study was to examine if common and rare polymorphisms in PCSK9 are associated with blood pressure in an African-American population at high risk for cardiovascular disease. Using genomic data assayed on the Affymetrix 6.0 array (n = 1199) and the Illumina HumanExome Beadchip (n = 1966) from the Hypertension Genetic Epidemiology Network (HyperGEN), we tested the association of PCSK9 polymorphisms with blood pressure. We used linear mixed models and the sequence kernel association test (SKAT) to assess the association of 31 common and 19 rare variants with blood pressure. The models were adjusted for age, sex, center, smoking status, principal components for ancestry and diabetes as fixed effects and family as a random effect. The results showed a marginally significant effect of two genome-wide association study (GWAS) single-nucleotide polymorphisms (SNPs) (rs12048828: β = 1.8, P = 0.05 and rs9730100: β = 1.0, P = 0.05) with diastolic blood pressure (DBP); however these results were not significant after correction for multiple testing. Rare variants were cumulatively associated with DBP (P = 0.04), an effect that was strengthened by restriction to nonsynonymous or stop-gain SNPs (P = 0.02). While gene-based results for DBP did not replicate (P = 0.36), we found an association with SBP (P = 0.04) in the Reasons for Geographic And Racial Differences in Stroke study (REGARDS). The findings here suggest rare variants in PCSK9 may influence blood pressure among African Americans, laying the ground work for further validation studies

In vitro modification of human centromere protein CENP-C fragments by small ubiquitin-like modifier (SUMO) protein: Definitive identification of the modification sites by tandem mass spectrometry analysis of the isopeptides

Protein sumoylation by small ubiquitin-like modifier (SUMO) proteins is an important post-translational regulatory modification. A role in the control of chromosome dynamics was first suggested when SUMO was identified as high-copy suppressor of the centromere protein CENP-C mutants. CENP-C itself contains a consensus sumoylation sequence motif that partially overlaps with its DNA binding and centromere localization domain. To ascertain whether CENP-C can be sumoylated, tandem mass spectrometry (MS) based strategy was developed for high sensitivity identification and sequencing of sumoylated isopeptides present among in-gel-digested tryptic peptides of SDS-PAGE fractionated target proteins. Without a predisposition to searching for the expected isopeptides based on calculated molecular mass and relying instead on the characteristic MS/MS fragmentation pattern to identify sumolylation, we demonstrate that several other lysine residues located not within the perfect consensus sumoylation motif {psi}KXE/D, where {psi} represents a large hydrophobic amino acid, and X represnts any amino acid, can be sumolylated with a reconstituted in vitro system containing only the SUMO proteins, E1-activating enzyme and E2-conjugating enzyme (Ubc9). In all cases, target sites that can be sumoylated by SUMO-2 were shown to be equally susceptible to SUMO-1 attachments which include specific sites on SUMO-2 itself, Ubc9, and the recombinant CENP-C fragments. Two non-consensus sites on one of the CENP-C fragments were found to be sumoylated in addition to the predicted site on the other fragment. The developed methodologies should facilitate future studies in delineating the dynamics and substrate specificities of SUMO-1/2/3 modifications and the respective roles of E3 ligases in the process

Phase II randomized preoperative window-of-opportunity study of the PI3K inhibitor pictilisib plus anastrozole compared with anastrozole alone in patients with estrogen receptor-positive breast cancer

Purpose: Preclinical data support a key role for the PI3K pathway in estrogen receptor-positive breast cancer and suggest that combining PI3K inhibitors with endocrine therapy may overcome resistance. This preoperative window study assessed whether adding the PI3K inhibitor pictilisib (GDC-0941) can increase the antitumor effects of anastrozole in primary breast cancer and aimed to identify the most appropriate patient population for combination therapy. Patients and Methods: In this randomized, open-label phase II trial, postmenopausal women with newly diagnosed operable estrogen receptor-positive, human epidermal growth factor receptor 2 (HER2)-negative breast cancers were recruited. Participants were randomly allocated (2:1, favoring the combination) to 2 weeks of preoperative treatment with anastrozole 1 mg once per day (n = 26) or the combination of anastrozole 1 mg with pictilisib 260 mg once per day (n = 49). The primary end point was inhibition of tumor cell proliferation as measured by change in Ki-67 protein expression between tumor samples taken before and at the end of treatment. Results: There was significantly greater geometric mean Ki-67 suppression of 83.8% (one-sided 95% CI, ≥ 79.0%) for the combination and 66.0% (95% CI, ≤ 75.4%) for anastrozole (geometric mean ratio [combination: anastrozole], 0.48; 95% CI, ≤ 0.72; P = .004). PIK3CA mutations were not predictive of response to pictilisib, but there was significant interaction between response to treatment and molecular subtype (P =.03);for patients with luminal B tumors, the combination:anastrozole geometric mean ratio of Ki-67 suppression was 0.37 (95% CI, ≤ 0.67; P = .008), whereas no significant Ki-67 response was observed for pictilisib in luminal A tumors (1.01; P = .98). Multivariable analysis confirmed Ki-67 response to the combination treatment of patients with luminal B tumors irrespective of progesterone receptor status or baseline Ki-67 expression. Conclusion: Adding pictilisib to anastrozole significantly increases suppression of tumor cell proliferation in luminal B primary breast cancer

dOCRL maintains immune cell quiescence in Drosophila by regulating endosomal traffic

Lowe Syndrome is a developmental disorder characterized by eye, kidney, and neurological pathologies, and is caused by mutations in the phosphatidylinositol-5-phosphatase OCRL. OCRL plays diverse roles in endocytic and endolysosomal trafficking, cytokinesis, and ciliogenesis, but it is unclear which of these cellular functions underlie specific patient symptoms. Here, we show that mutation of Drosophila OCRL causes cell-autonomous activation of hemocytes, which are macrophage-like cells of the innate immune system. Among many cell biological defects that we identified in docrl mutant hemocytes, we pinpointed the cause of innate immune cell activation to reduced Rab11-dependent recycling traffic and concomitantly increased Rab7-dependent late endosome traffic. Loss of docrl amplifies multiple immune-relevant signals, including Toll, Jun kinase, and STAT, and leads to Rab11-sensitive mis-sorting and excessive secretion of the Toll ligand Spåtzle. Thus, docrl regulation of endosomal traffic maintains hemocytes in a poised, but quiescent state, suggesting mechanisms by which endosomal misregulation of signaling may contribute to symptoms of Lowe syndrome

Results of a Survey Assessing the Impact of Astronomy Village: Investigating the Universe

Abstract This paper discusses the results of a follow-up questionnaire administered to over 300 registered owners of Astronomy Village ® : Investigating the Universe™. The results indicate that teachers who used the software were very pleased with it. They felt that students were learning a variety of science related skills. It was also determined that the greatest barriers teachers face in adopting Astronomy Village are lack of proper equipment and lack of time