Quantification of Intact and Truncated Stromal Cell-Derived Factor-1α in Circulation by Immunoaffinity Enrichment and Tandem Mass Spectrometry

Stromal cell-derived factor 1α (SDF-1α) or CXCL12 is a small pro-inflammatory chemoattractant cytokine and a substrate of dipeptidyl peptidase IV (DPP-IV). Proteolytic cleavage by DPP-IV inactivates SDF-1α and attenuates its interaction with CXCR4, its cell surface receptor. To enable investigation of suppression of such inactivation with pharmacologic inhibition of DPP-IV, we developed quantitative mass spectrometric methods that differentiate intact SDF-1α from its inactive form. Using top-down strategy in quantification, we demonstrated the unique advantage of keeping SDF-1α’s two disulfide bridges intact in the analysis. To achieve the optimal sensitivity required for quantification of intact and truncated SDF-1α at endogenous levels in blood, we coupled nano-flow tandem mass spectrometry with antibody-based affinity enrichment. The assay has a quantitative range of 20 pmol/L to 20 nmol/L in human plasma as well as in rhesus monkey plasma. With only slight modification, the same assay can be used to quantify SDF-1α in mice. Using two in vivo animal studies as examples, we demonstrated that it was critical to differentiate intact SDF-1α from its truncated form in the analysis of biomarkers for pharmacologic inhibition of DPP-IV activity. These novel methods enable translational research on suppression of SDF-1 inactivation with DPP-IV inhibition and can be applied to relevant clinical samples in the future to yield new insights on change of SDF-1α levels in disease settings and in response to therapeutic interventions.

A flow system for hydrogen peroxide production at reticulated vitreous carbon via electroreduction of oxygen

In this work, a reticulated vitreous carbon electrode (RVCE, 96.5 % porosity, 24 cm^?1) was modified with 2-anthraquinonyl groups to electrocatalytically reduce dissolved oxygen in neutral aqueous solution (0.1 M phosphate buffer solution supported with 3 M potassium chloride, pH of 6.7) to hydrogen peroxide (H₂O₂) at 25 °C under atmospheric pressure. The obtained current density was ca. 3 mA cm^?2. For the first time, the oxygen reduction was investigated on a novelly designed RVCE housed in a gravity-feed flow system. Fractional current conversions obtained on the RVC flow cell were compared and contrasted with those on a two-dimensional electrode, viz. a tubular flow electrode. The modified-on catalyst has the benefit in terms of easy separation of the product from the catalyst. The in situ generated low concentration of H₂O₂ provides potential applications to water purification processes and disinfection for water and food.     

Long‐Wavelength Fluorescent Reporters for Monitoring Protein Kinase Activity

In vivo optical imaging must contend with the limitations imposed by the optical window of tissue (600–1000 nm). Although a wide array of fluorophores are available that are visualized in the red and near‐IR region of the spectrum, with the exception of proteases, there are few long wavelength probes for enzymes. This situation poses a particular challenge for studying the intracellular biochemistry of erythrocytes, the high hemoglobin content of which optically obscures subcellular monitoring at wavelengths less than 600 nm. To address this, tunable fluorescent reporters for protein kinase activity were developed. The probing wavelength is preprogrammed by using readily available fluorophores, thereby enabling detection within the optical window of tissue, specifically in the far‐red and near‐IR region. These agents were used to monitor endogenous cAMP‐dependent protein kinase activity in erythrocyte lysates and in intact erythrocytes when using a light‐activatable reporter.     

Probing the CH⋅⋅⋅π Weak Hydrogen Bond in Anesthetic Binding: The Sevoflurane–Benzene Cluster

Cooperativity between weak hydrogen bonds can be revealed in molecular clusters isolated in the gas phase. Here we examine the structure, internal dynamics, and origin of the weak intermolecular forces between sevoflurane and a benzene molecule, using multi‐isotopic broadband rotational spectra. This heterodimer is held together by a primary C? H???π hydrogen bond, assisted by multiple weak C? H???F interactions. The multiple nonbonding forces hinder the internal rotation of benzene around the isopropyl C? H bond in sevoflurane, producing detectable quantum tunneling effects in the rotational spectrum.     

Alkene–Tetrazine Ligation for Imaging Cellular DNA

5‐Vinyl‐2′‐deoxyuridine (VdU) is the first reported metabolic probe for cellular DNA synthesis that can be visualized by using an inverse electron demand Diels–Alder reaction with a fluorescent tetrazine. VdU is incorporated by endogenous enzymes into the genomes of replicating cells, where it exhibits reduced genotoxicity compared to 5‐ethynyl‐2′‐deoxyuridine (EdU). The VdU–tetrazine ligation reaction is rapid (k≈0.02 M ^?1 s^?1) and chemically orthogonal to the alkyne–azide “click” reaction of EdU‐modified DNA. Alkene–tetrazine ligation reactions provide the first alternative to azide–alkyne click reactions for the bioorthogonal chemical labeling of nucleic acids in cells and facilitate time‐resolved, multicolor labeling of DNA synthesis.     

Hydrogen Bond Cooperativity and the Three‐Dimensional Structures of Water Nonamers and Decamers

Broadband rotational spectroscopy of water clusters produced in a pulsed molecular jet expansion has been used to determine the oxygen atom geometry in three isomers of the nonamer and two isomers of the decamer. The isomers for each cluster size have the same nominal geometry but differ in the arrangement of their hydrogen bond networks. The nearest neighbor O? O distances show a characteristic pattern for each hydrogen bond network isomer that is caused by three‐body effects that produce cooperative hydrogen bonding. The observed structures are the lowest energy cluster geometries identified by quantum chemistry and the experimental and theoretical O? O distances are in good agreement. The cooperativity effects revealed by the hydrogen bond O? O distance variations are shown to be consistent with a simple model for hydrogen bonding in water that takes into account the cooperative and anticooperative bonding effects of nearby water molecules.     

Blind prediction of HIV integrase binding from the SAMPL4 challenge

Here, we give an overview of the protein-ligand binding portion of the Statistical Assessment of Modeling of Proteins and Ligands 4 (SAMPL4) challenge, which focused on predicting binding of HIV integrase inhibitors in the catalytic core domain. The challenge encompassed three components—a small “virtual screening” challenge, a binding mode prediction component, and a small affinity prediction component. Here, we give summary results and statistics concerning the performance of all submissions at each of these challenges. Virtual screening was particularly challenging here in part because, in contrast to more typical virtual screening test sets, the inactive compounds were tested because they were thought to be likely binders, so only the very top predictions performed significantly better than random. Pose prediction was also quite challenging, in part because inhibitors in the set bind to three different sites, so even identifying the correct binding site was challenging. Still, the best methods managed low root mean squared deviation predictions in many cases. Here, we give an overview of results, highlight some features of methods which worked particularly well, and refer the interested reader to papers in this issue which describe specific submissions for additional details.