Development of a selection assay for small guide RNAs that drive efficient site-directed RNA editing.

A major challenge confronting the clinical application of site-directed RNA editing (SDRE) is the design of small guide RNAs (gRNAs) that can drive efficient editing. Although many gRNA designs have effectively recruited endogenous Adenosine Deaminases that Act on RNA (ADARs), most of them exceed the size of currently FDA-approved antisense oligos. We developed an unbiased in vitro selection assay to identify short gRNAs that promote superior RNA editing of a premature termination codon. The selection assay relies on hairpin substrates in which the target sequence is linked to partially randomized gRNAs in the same molecule, so that gRNA sequences that promote editing can be identified by sequencing. These RNA substrates were incubated in vitro with ADAR2 and the edited products were selected using amplification refractory mutation system PCR and used to regenerate the substrates for a new round of selection. After nine repetitions, hairpins which drove superior editing were identified. When gRNAs of these hairpins were delivered in trans, eight of the top ten short gRNAs drove superior editing both in vitro and in cellula. These results show that efficient small gRNAs can be selected using our approach, an important advancement for the clinical application of SDRE.

Structure-based validation can drastically underestimate error rate in proteome-wide cross-linking mass spectrometry studies.

Thorough quality assessment of novel interactions identified by proteome-wide cross-linking mass spectrometry (XL-MS) studies is critical. Almost all current XL-MS studies have validated cross-links against known three-dimensional structures of representative protein complexes. Here, we provide theoretical and experimental evidence demonstrating that this approach can drastically underestimate error rates for proteome-wide XL-MS datasets, and propose a comprehensive set of four data-quality metrics to address this issue.

A massively parallel barcoded sequencing pipeline enables generation of the first ORFeome and interactome map for rice.

Systematic mappings of protein interactome networks have provided invaluable functional information for numerous model organisms. Here we develop PCR-mediated Linkage of barcoded Adapters To nucleic acid Elements for sequencing (PLATE-seq) that serves as a general tool to rapidly sequence thousands of DNA elements. We validate its utility by generating the ORFeome for Oryza sativa covering 2,300 genes and constructing a high-quality protein-protein interactome map consisting of 322 interactions between 289 proteins, expanding the known interactions in rice by roughly 50%. Our work paves the way for high-throughput profiling of protein-protein interactions in a wide range of organisms.

MaXLinker: Proteome-wide Cross-link Identifications with High Specificity and Sensitivity.

Protein-protein interactions play a vital role in nearly all cellular functions. Hence, understanding their interaction patterns and three-dimensional structural conformations can provide crucial insights about various biological processes and underlying molecular mechanisms for many disease phenotypes. Cross-linking mass spectrometry (XL-MS) has the unique capability to detect protein-protein interactions at a large scale along with spatial constraints between interaction partners. The inception of MS-cleavable cross-linkers enabled the MS2-MS3 XL-MS acquisition strategy that provides cross-link information from both MS2 and MS3 level. However, the current cross-link search algorithm available for MS2-MS3 strategy follows a "MS2-centric" approach and suffers from a high rate of mis-identified cross-links. We demonstrate the problem using two new quality assessment metrics ["fraction of mis-identifications" (FMI) and "fraction of interprotein cross-links from known interactions" (FKI)]. We then address this problem, by designing a novel "MS3-centric" approach for cross-link identification and implementing it as a search engine named MaXLinker. MaXLinker outperforms the currently popular search engine with a lower mis-identification rate, and higher sensitivity and specificity. Moreover, we performed human proteome-wide cross-linking mass spectrometry using K562 cells. Employing MaXLinker, we identified a comprehensive set of 9319 unique cross-links at 1% false discovery rate, comprising 8051 intraprotein and 1268 interprotein cross-links. Finally, we experimentally validated the quality of a large number of novel interactions identified in our study, providing a conclusive evidence for MaXLinker's robust performance.

Carbonic anhydrase XII in valve interstitial cells promotes the regression of calcific aortic valve stenosis.

AIMS: Calcific aortic valve stenosis (CAVS) is the most common heart valve disease. In the present work we sought to determine the reversibility of mineralization in the aortic valve. METHODS AND RESULTS: By using in vitro analyses we found that valve interstitial cells (VICs) have the ability to resorb minerals. We documented that agonist of P2Y2 receptor (P2Y2R) promoted the expression of carbonic anhydrase XII (CAXII) at the cell membrane of VICs, whereby minerals are resorbed. P2Y2R-mediated mineral resorption was corroborated by using mouse VICs isolated from wild type and P2Y2R(-/-) mice. Measurements of extracellular pH (pHe) by using core-shell nanosensors revealed that P2Y2R-mediated CAXII export to the cell membrane led to an acidification of extracellular space, whereby minerals are resorbed. In vivo, we next treated LDLR(-/-)/ApoB(100/100)/IGF2 mice, which had developed CAVS under a high-fat/high-sucrose diet for 8 months, with 2-thioUTP (a P2Y2R agonist) or saline for the next 2 months. The administration of 2-thioUTP (2mg/kg/day i.p.) reduced the mineral volume in the aortic valve measured with serial microCT analyses, which improved hemodynamics and reduced left ventricular hypertrophy (LVH). Examination of leaflets at necropsy confirmed a lower level of mineralization and fibrosis along with higher levels of CAXII in mice under 2-thioUTP. In another series of experiment, the administration of acetazolamide (a CA inhibitor) prevented the acidification of leaflets and the regression of CAVS induced by 2-thioUTP in LDLR(-/-)/ApoB(100/100)/IGF2 mice. CONCLUSION: P2Y2R-mediated expression of CAXII by VICs acidifies the extracellular space and promotes the regression of CAVS.

Chronic TNF exposure induces glucocorticoid-like immunosuppression in the alveolar macrophages of aged mice that enhances their susceptibility to pneumonia.

Chronic low-grade inflammation, particularly elevated tumor necrosis factor (TNF) levels, occurs due to advanced age and is associated with greater susceptibility to infection. One reason for this is age-dependent macrophage dysfunction (ADMD). Herein, we use the adoptive transfer of alveolar macrophages (AM) from aged mice into the airway of young mice to show that inherent age-related defects in AM were sufficient to increase the susceptibility to Streptococcus pneumoniae, a Gram-positive bacterium and the leading cause of community-acquired pneumonia. MAPK phosphorylation arrays using AM lysates from young and aged wild-type (WT) and TNF knockout (KO) mice revealed multilevel TNF-mediated suppression of kinase activity in aged mice. RNAseq analyses of AM validated the suppression of MAPK signaling as a consequence of TNF during aging. Two regulatory phosphatases that suppress MAPK signaling, Dusp1 and Ptprs, were confirmed to be upregulated with age and as a result of TNF exposure both ex vivo and in vitro. Dusp1 is known to be responsible for glucocorticoid-mediated immune suppression, and dexamethasone treatment increased Dusp1 and Ptprs expression in cells and recapitulated the ADMD phenotype. In young mice, treatment with dexamethasone increased the levels of Dusp1 and Ptprs and their susceptibility to infection. TNF-neutralizing antibody reduced Dusp1 and Ptprs levels in AM from aged mice and reduced pneumonia severity following bacterial challenge. We conclude that chronic exposure to TNF increases the expression of the glucocorticoid-associated MAPK signaling suppressors, Dusp1 and Ptprs, which inhibits AM activation and increases susceptibility to bacterial pneumonia in older adults.

Synthesis and biological evaluation of novel quinazoline-4-piperidinesulfamide derivatives as inhibitors of NPP1.

The ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) was recently shown to promote mineralization of the aortic valve, hence, its inhibition represents a significant target. A quinazoline-4-piperidine sulfamide compound (QPS1) has been described as a specific and non-competitive inhibitor of NPP1. We report herein the synthesis and in vitro inhibition studies of novel quinazoline-4-piperidine sulfamide analogues using QPS1 as the lead compound. Of the 26 derivatives prepared, four compounds were found to have Ki < 105 nM against human NPP1.

Quinazoline-4-piperidine sulfamides are specific inhibitors of human NPP1 and prevent pathological mineralization of valve interstitial cells.

BACKGROUND AND PURPOSE: Ectonucleotide pyrophosphatase/PDE1 (NPP1) is an ectoenzyme, which plays a role in several disorders including calcific aortic valve disease (CAVD). So far, compounds that have been developed as inhibitors of NPP1 lack potency and specificity. Quinazoline-4-piperidine sulfamides (QPS) have been described as potent inhibitors of NPP1. However, their mode of inhibition as well as their selectivity and capacity to modify biological processes have not been investigated. EXPERIMENTAL APPROACH: In the present series of experiments, we have evaluated the efficacy of two derivatives, QPS1-2, in inhibiting human NPP1, and we have evaluated the effect of the most potent derivative (QPS1) on other ectonucleotidases as well as on the ability of this compound to prevent phosphate-induced mineralization of human primary aortic valve interstitial cells (VICs). KEY RESULTS: The QPS1 derivative is a potent (Ki 59.3 ± 5.4 nM) and selective non-competitive inhibitor of human NPP1. Moreover, QPS1 also significantly inhibited the K121Q NPP1 gene variant (Ki 59.2 ± 14.5 nM), which is prevalent in the general population. QPS1 did not significantly alter the activity of other nucleotide metabolizing ectoenzymes expressed at the cell surface, namely NPP3, NTPDases (1-3), ecto-5'-nucleotidase and ALP. Importantly, QPS1 in the low micromolar range (≤10 µM) prevented phosphate-induced mineralization of VICs and lowered the rise of osteogenic genes as expected for NPP1 inhibition. CONCLUSIONS AND IMPLICATIONS: We have provided evidence that QPS1 is a potent and selective non-competitive inhibitor of NPP1 and that it prevented pathological mineralization in a cellular model.

Polymorphisms of the ADAM33 gene and chronic obstructive pulmonary disease risk: a meta-analysis.

BACKGROUND: The T1 (rs2280091), S1 (rs3918396) and S2 (rs528557) polymorphisms in a disintegrin and metalloprotease (ADAM33) gene has been implicated in susceptibility of chronic obstructive pulmonary disease (COPD). But, a number of studies have reported inconclusive results. The aim of this study is to investigate the relationship between T1 (rs2280091), S1 (rs3918396) and S2 (rs528557) polymorphisms in ADAM33 gene and COPD risk by meta-analysis. METHODS: We searched PubMed database, Embase database, Chinese National Knowledge Infrastructure database and Wanfang database, covering all studies till September 5, 2012. Statistical analysis was performed using software METAGEN (STATA 12.0) and Revman5.0. RESULTS: A total of 2139 COPD cases and 3765 controls in 10 case-control studies were included in this study. The results showed that S2 (rs528557) and T1 (rs2280091) polymorphisms did not result in an increased or a decreased risk of COPD. The analysis described in this report demonstrated that S1 (rs3918396) polymorphism (GG + AG vs AA) was significantly associated with the total and Asian. Odds ratio (OR)total = 1.27 [95% confidence interval (CI) 1.03-1.56, P = 0.03], ORAsian = 1.44 (95% CI 1.13-1.83, P = 0.003) but not with Caucasians. CONCLUSIONS: This meta-analysis suggested that S1 (rs3918396) polymorphism of ADAM33 is associated with increased risk of COPD in Asian (China) but not in Caucasians. Future studies are needed to validate our conclusions.