Vascular Immunotargeting to Endothelial Determinant ICAM-1 Enables Optimal Partnering of Recombinant scFv-Thrombomodulin Fusion with Endogenous Cofactor

The use of targeted therapeutics to replenish pathologically deficient proteins on the luminal endothelial membrane has the potential to revolutionize emergency and cardiovascular medicine. Untargeted recombinant proteins, like activated protein C (APC) and thrombomodulin (TM), have demonstrated beneficial effects in acute vascular disorders, but have failed to have a major impact on clinical care. We recently reported that TM fused with an scFv antibody fragment to platelet endothelial cell adhesion molecule-1 (PECAM-1) exerts therapeutic effects superior to untargeted TM. PECAM-1 is localized to cell-cell junctions, however, whereas the endothelial protein C receptor (EPCR), the key co-factor of TM/APC, is exposed in the apical membrane. Here we tested whether anchoring TM to the intercellular adhesion molecule (ICAM-1) favors scFv/TM collaboration with EPCR. Indeed: i) endothelial targeting scFv/TM to ICAM-1 provides ∼15-fold greater activation of protein C than its PECAM-targeted counterpart; ii) blocking EPCR reduces protein C activation by scFv/TM anchored to endothelial ICAM-1, but not PECAM-1; and iii) anti-ICAM scFv/TM fusion provides more profound anti-inflammatory effects than anti-PECAM scFv/TM in a mouse model of acute lung injury. These findings, obtained using new translational constructs, emphasize the importance of targeting protein therapeutics to the proper surface determinant, in order to optimize their microenvironment and beneficial effects.     

An ERP Study of the Processing of Common and Decimal Fractions: How Different They Are

This study explored event-related potential (ERP) correlates of common fractions (1/5) and decimal fractions (0.2). Thirteen subjects performed a numerical magnitude matching task under two conditions. In the common fraction condition, a nonsymbolic fraction was asked to be judged whether its magnitude matched the magnitude of a common fraction; in the decimal fraction condition, a nonsymbolic fraction was asked to be matched with a decimal fraction. Behavioral results showed significant main effects of condition and numerical distance, but no significant interaction of condition and numerical distance. Electrophysiological data showed that when nonsymbolic fractions were compared to common fractions, they displayed larger N1 and P3 amplitudes than when they were compared to decimal fractions. This finding suggested that the visual identification for nonsymbolic fractions was different under the two conditions, which was not due to perceptual differences but to task demands. For symbolic fractions, the condition effect was observed in the N1 and P3 components, revealing stimulus-specific visual identification processing. The effect of numerical distance as an index of numerical magnitude representation was observed in the P2, N3 and P3 components under the two conditions. However, the topography of the distance effect was different under the two conditions, suggesting stimulus specific semantic processing of common fractions and decimal fractions.     

The rate of opening and closing of the DNA gate for topoisomerase II

Type II DNA topoisomerases can catalyze the transport of one DNA segment through a transient break in another DNA segment by a complex mechanism of ATP hydrolysis. According to the hydrolysis process of two ATPs, a multi-state model is proposed to investigate the work cycle of DNA topoisomerase II. The rate of the opening and closing of the DNA topoisomerase gate is evaluated by determining the release rate of inorganic phosphates. The calculated results show that, under the condition of the high concentration of ATP, the work cycle of DNA topoisomerase II is about 0.84 s which is in agreement with the experimental data.     

Cytochrome bc1 complex [2Fe-2S] cluster and its interaction with ubiquinone and ubihydroquinone at the Qo site: a double-occupancy Qo site model.

The ubiquinone complement of Rhodobacter capsulatus chromatophore membranes has been characterized by its isooctane solvent extractability and electrochemistry; we find that the main ubiquinone pool (Qpool) amounts to about 80% of the total ubiquinone and has an Em7 value close to 90 mV. To investigate the interactions of ubiquinone with the cyt bc1 complex, we have examined the distinctive EPR line shapes of the [2Fe-2S] cluster of the cyt bc1 complex when the Qpool-cyt bc1 complex interactions are modulated by changing the numbers of Q or QH2 present (by solvent extraction and reconstitution), by the exposure of the [2Fe-2S] to the Qpool in different redox states, by the presence of inhibitors specific for the Qo site (myxothiazol and stigmatellin) and Qi site (antimycin), and by site-specific mutations of side chains of the cyt b polypeptide (mutants F144L and F144G) previously identified as important for Qo site structure. Evidence suggests that the Qo site can accommodate two ubiquinone molecules. One (designated Qos) is bound relatively strongly and is second only to the ubiquinone of the QA site of the reaction center in its resistance to solvent extraction. In this strong interaction, the Qo site binds Q and QH2 with approximately equal affinities. Their bound states are distinguished by their effects on the [2Fe-2S] cluster spectral feature at gx at 1.783 (Q) and gx at 1.777 (QH2); titration of the line-shape change reveals an Em7 value of approximately 95 mV. The other molecule (Qow) is bound more weakly, in the same range as the ubiquinone of the QB site of the reaction center. Again, the affinities of the Q form (gx at 1.800) and QH2 form (gx at 1.777) are nearly equal, and the Em7 value measured is approximately 80 mV. These results are discussed in terms of earlier EPR analyses of the cyt bc1 complexes of other systems. A Qo site double-occupancy model is considered that builds on the previous model based on Qo site mutants [Robertson, D. E., Daldal, F.,& Dutton, P. L. (1990) Biochemistry 29, 11249-11260] and includes the recent suggestion that two of the [2F3-2S] cluster ligands of the R. capsulatus cyt bc1 complex are histidines [Gurbiel, R. J. Ohnishi, T., Robertson, D. E. Daldal, F., & Hoffman, B. M. (1991) Biochemistry 30, 11579-11584]. We speculate that the cyt bc1 complex complexes a full enzymatic turnover without necessary exchange of ubiquinone with the Qpool.     

SKIL facilitates tumorigenesis and immune escape of NSCLC via upregulating TAZ/autophagy axis

Immune escape is an important mechanism in tumorigenesis. The aim of this study was to investigate roles of SKIL in tumorigenesis and immune escape of non-small-cell lung cancer (NSCLC). SKIL expression levels in NSCLC cell line, clinical sample, and adjacent normal tissue were measured by quantitative PCR, western blot, or immunohistochemistry. Lentivirus was used to overexpress/silence SKIL or TAZ expression. Malignant phenotypes of NSCLC cells were evaluated by colony formation, transwell, and MTT assays, and in xenograft mice model. Syngeneic mice model and flow cytometry were used to evaluate T cell infiltration. Quantitative PCR and western blot were applied to evaluate relevant mRNA and protein levels, respectively. Co-immunoprecipitation was applied to unveil the interaction between SKIL and TAZ. SKIL expression was higher in NSCLC tissue compared to adjacent normal tissue. Silencing of SKIL inhibited malignant phenotypes of NSCLC cells and promoted T cell infiltration. SKIL-knockdown inhibited autophagy and activated the STING pathway in NSCLC cells through down-regulation of TAZ. Silencing of TAZ cancelled the effects of SKIL overexpression on malignant phenotypes and autophagy of NSCLC cells. Inhibition of autophagy reversed the effects of SKIL/TAZ overexpression on the STING pathway. In conclusion, SKIL promoted tumorigenesis and immune escape of NSCLC cells through upregulation of TAZ/autophagy axis and inhibition on downstream STING pathway.Subject terms: Immunology, Cancer     

Identification of compound heterozygous DNAH11 variants in a Han-Chinese family with primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is a group of genetically and clinically heterogeneous disorders with motile cilia dysfunction. It is clinically characterized by oto-sino-pulmonary diseases and subfertility, and half of the patients have situs inversus (Kartagener syndrome). To identify the genetic cause in a Han-Chinese pedigree, whole-exome sequencing was conducted in the 37-year-old proband, and then, Sanger sequencing was performed on available family members. Minigene splicing assay was applied to verify the impact of the splice-site variant. Compound heterozygous variants including a splice-site variant (c.1974-1G>C, rs1359107415) and a missense variant (c.7787G>A, p.(Arg2596Gln), rs780492669), in the dynein axonemal heavy chain 11 gene (DNAH11) were identified and confirmed as the disease-associated variants of this lineage. The minigene expression in vitro revealed that the c.1974-1G>C variant could cause skipping over exon 12, predicted to result in a truncated protein. This discovery may enlarge the DNAH11 variant spectrum of PCD, promote accurate genetic counselling and contribute to PCD diagnosis.