Direct Cleavage Research Articles

Clinical significance of intercellular contact at the four-cell stage of human embryos, and the use of abnormal cleavage patterns to identify embryos with low implantation potential: a time-lapse study

To investigate the clinical significance of intercellular contact point (ICCP) in four-cell stage human embryos and the effectiveness of morphology and abnormal cleavage patterns in identifying embryos with low implantation potential. Retrospective cohort study. Private IVF center. A total of 223 consecutive IVF and intracytoplasmic sperm injection treatment cycles, with all resulting embryos cultured in the Embryoscope, and a subset of 207 cycles analyzed for ICCP number where good-quality four-cell embryos were available on day 2 (n = 373 IVF and n = 392 intracytoplasmic sperm injection embryos). None. Morphologic score on day 3, embryo morphokinetic parameters, incidence of abnormal biological events, and known implantation results. Of 765 good-quality four-cell embryos, 89 (11.6%) failed to achieve six ICCPs; 166 of 765 (21.7%) initially had fewer than six ICCPs but were able to establish six ICCPs before subsequent division. Embryos with fewer than six ICCPs at the end of four-cell stage had a lower implantation rate (5.0% vs. 38.5%), with lower embryology performance in both conventional and morphokinetic assessments, compared with embryos achieving six ICCPs by the end of four-cell stage. Deselecting embryos with poor morphology, direct cleavage, reverse cleavage, and fewer than six ICCPs at the four-cell stage led to a significantly improved implantation rate (33.6% vs. 22.4%). Embryos with fewer than six ICCPs at the end of the four-cell stage show compromised subsequent development and reduced implantation potential. Deselection of embryos with poor morphology and abnormal cleavage revealed via time-lapse imaging could provide the basis of a qualitative algorithm for embryo selection.

Targeting hepatitis B virus cccDNA by CRISPR/Cas9 nuclease efficiently inhibits viral replication

Chronic hepatitis B virus (HBV) infection causes liver cirrhosis and hepatocellular carcinoma and remains a serious health problem worldwide. Covalently closed circular DNA (cccDNA) in the liver cell nucleus sustains HBV infection. Major treatments for HBV infection include the use of interferon-α and nucleotide analogs, but they cannot eradicate cccDNA. As a novel tool for genome editing, clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system developed from bacteria can be used to accurately and efficiently engineer and modify genomic DNA. In this study, the CRISPR/Cas9 system was used to target the HBV genome and efficiently inhibit HBV infection. We synthesized four single-guide RNAs (sgRNAs) targeting the conserved regions of HBV. The expression of these sgRNAS with Cas9 reduced the viral production in Huh7 cells as well as in HBV-replication cell HepG2.2.15. We further demonstrated that CRISPR/Cas9 direct cleavage and cleavage-mediated mutagenesis occurred in HBV cccDNA of transfected cells. In the new mouse model carrying HBV cccDNA, injection of sgRNA–Cas9 plasmids via rapid tail vein resulted in the low level of cccDNA and HBV protein. In conclusion, the designed CRISPR/Cas9 system can accurately and efficiently target HBV cccDNA and inhibit HBV replication. This system may be used as a novel therapeutic strategy against chronic HBV infection.

Granzyme B Mediates Both Direct and Indirect Cleavage of Extracellular Matrix in Skin After Chronic Low‐Dose Ultraviolet Light Irradiation

Extracellular matrix (ECM) degradation is a hallmark of tissue aging, as well as many other chronic inflammatory diseases that can lead to a loss of function. Granzyme B (GzmB), a serine protease that is expressed by a variety cells, has been shown to accumulate in the extracellular space during chronic inflammation and cleave a number of proteins. Using a chronic low‐grade UV‐irradiation murine model, we hypothesized that GzmB contributes to skin damage and ECM degradation through processes involving both direct ECM cleavage and indirect ECM cleavage through the induction of other proteinases. Wild‐type and GzmB‐knockout (KO) mice were repeatedly exposed to minimal erythemal doses of solar simulated UV‐irradiation for up to 20 weeks. GzmB expression was significantly increased in wild‐type UV‐treated skin compared to non‐irradiated controls. GzmB deficiency significantly protected against the formation of wrinkles and the loss of dermal collagen density. GzmB‐KO mice were also protective against the loss of fibronectin (FN) in vivo, and FN fragments were released from the cell‐derived matrix of cultured fibroblasts after GzmB treatment. GzmB‐mediated FN fragments increased the release of metalloproteinase (MMP)‐1 and MMP‐3 from fibroblasts. Furthermore, GzmB cleavage of decorin predisposed collagen fibrils more susceptible to attack by MMP‐1. Finally, GzmB reduced the barrier function of a monolayer of keratinocytes in vitro as measured by electrical impedance. Collectively, these findings indicate a significant role for GzmB in ECM degradation, which may have implications in many aged‐related dermatological indications.

ChemInform Abstract: Convenient Formation of Triarylphosphines by Nickel‐Catalyzed C—P Cross‐Coupling with Aryl Chlorides.

A convenient strategy has been developed for the preparation of various phosphine ligands in good to excellent yields through a nickel-catalyzed C-P bond-forming step. This reaction proceeded smoothly and tolerated a variety of functional groups to provide a new method for the synthesis of important phosphine ligands through the direct cleavage of a C-Cl bond.

A serine protease isolated from the bristles of the Amazonic caterpillar, Premolis semirufa, is a potent complement system activator.

BackgroundThe caterpillar of the moth Premolis semirufa, commonly named pararama, is found in the Brazilian Amazon region. Accidental contact with the caterpillar bristles causes an intense itching sensation, followed by symptoms of an acute inflammation, which last for three to seven days after the first incident. After multiple accidents a chronic inflammatory reaction, called “Pararamose”, characterized by articular synovial membrane thickening with joint deformities common to chronic synovitis, frequently occurs. Although complement mediated inflammation may aid the host defense, inappropriate or excessive activation of the complement system and generation of anaphylatoxins can lead to inflammatory disorder and pathologies. The aim of the present study was to evaluate, in vitro, whether the Premolis semirufa’s bristles extract could interfere with the human complement system.ResultsThe bristles extract was able to inhibit the haemolytic activity of the alternative pathway, as well as the activation of the lectin pathway, but had no effect on the classical pathway, and this inhibition seemed to be caused by activation and consumption of complement components. The extract induced the production of significant amounts of all three anaphylatoxins, C3a, C4a and C5a, promoted direct cleavage of C3, C4 and C5 and induced a significant generation of terminal complement complexes in normal human serum. By using molecular exclusion chromatography, a serine protease of 82 kDa, which activates complement, was isolated from P. semirufa bristles extract. The protease, named here as Ps82, reduced the haemolytic activity of the alternative and classical pathways and inhibited the lectin pathway. In addition, Ps82 induced the cleavage of C3, C4 and C5 and the generation of C3a and C4a in normal human serum and it was capable to cleave human purified C5 and generate C5a. The use of Phenanthroline, metalloprotease inhibitor, in the reactions did not significantly interfere with the activity of the Ps82, whereas the presence of PMSF, serine protease inhibitor, totally blocked the activity.ConclusionThese data show that a serine protease present in the Premolis semirufa’s bristles extract has the ability to activate the complement system, which may contribute to the inflammatory process presented in humans after envenomation.

Direct mechanochemical cleavage of functional groups from graphene

Mechanical stress can drive chemical reactions and is unique in that the reaction product can depend on both the magnitude and the direction of the applied force. Indeed, this directionality can drive chemical reactions impossible through conventional means. However, unlike heat- or pressure-driven reactions, mechanical stress is rarely applied isometrically, obscuring how mechanical inputs relate to the force applied to the bond. Here we report an atomic force microscope technique that can measure mechanically induced bond scission on graphene in real time with sensitivity to atomic-scale interactions. Quantitative measurements of the stress-driven reaction dynamics show that the reaction rate depends both on the bond being broken and on the tip material. Oxygen cleaves from graphene more readily than fluorine, which in turn cleaves more readily than hydrogen. The technique may be extended to study the mechanochemistry of any arbitrary combination of tip material, chemical group and substrate.

CF3+ fragmentation by electron impact ionization of perfluoro-propyl-vinyl-ethers, C5F10O, in gas phase

The gas phase fragmentations of perfluoro-propyl-vinyl ether (PPVE, C5F10O) are studied experimentally. Dominant fragmentations of PPVE are found to be the result of a dissociative ionization reaction, i.e., CF3+ via direct bond cleavage, and C2F3O− and C3F7O− via electron attachment. Regardless of the appearance energy of around 14.5 eV for the dissociative ionization of CF3+, the observed ion efficiency for the CF3+ ion was extremely large the order of 10−20 cm−2, compared with only 10−21 cm−2 for the other channels. PPVE characteristically generated CF3+ as the largest abundant ion are advantageous for use of feedstock gases in plasma etching processes.

Electronic structure and noncovalent interactions within ion-radical complexes of N-(2-furylmethyl)aniline molecular ions.

We investigate the electronic structure and noncovalent interactions within cation-radical complexes that are relevant in the electron impact mass spectrometry of N-(2-furylmethyl)anilines, 4-R-C6H4-NH-CH2-C4H3O with (R = -H, -OCH3, -CH3, -F, -Cl, -Br). In particular, we consider the reactive intermediates that precede the final products of two previously suggested dissociation pathways for these systems, i.e., (i) a direct cleavage of the NH-CH2 bond and (ii) an isomerization/fragmentation mechanism. The study is performed by means of correlated calculations (UCCSD and UMP2) together with density functionals (UM06 an UM06-2x) along with the triple-ζ quality basis set 6-311++G(2d,2p). In addition, we carried out a topological analysis of the electron density in accordance with the quantum theory of atoms in molecules (QTAIM) together with the examination of the noncovalent interaction (NCI) index. In contrast with previous studies based on the UB3LYP approximation, we could determine the transition states associated with both fragmentation pathways. The Rice-Ramsperger-Kassel-Marcus theory, used to determine the relative importance of these dissociation mechanisms, indicates that whereas the direct cleavage and the isomerization/fragmentation reaction routes have similar constant rates at low energy, the former prevails when the energy of the system is increased. The QTAIM analysis reveals that the charge of the cation-radical complex is mainly located on either a furfuryl (direct cleavage mechanism) or a pyrylium (isomerization/fragmentation pathway) ion and that these units interact with a neutral radical aniline moiety. The localization of the positive charge in either a furfuryl or pyrylium cation is in agreement with the preminecence of the m/z = 81 fragment in the mass spectrometry of N-(2-furylmethyl)anilines. Moreover, the QTAIM properties indicate that the α unpaired electron of the system is principally distributed over the nitrogen and the ortho and para carbon atoms with respect to the -NH group in the R-C6H4-NH unit. The investigation of the NCI index and the intermolecular bond critical points and bond paths gives an account of the NCIs linking the radical cation clusters under consideration. Finally, we found correlations which indicate that the concentration of the m/z = 81 fragment in a mass spectrum is reduced with the interaction energy of the radical complex from which it is originated. Altogether, this work shows how the combination of suitable electronic structure calculations along with post wave function analyses can yield important insights about the formation and properties of cation-radical complexes relevant in mass spectrometry.

Cleavage of Lignin-Derived 4-O-5 Aryl Ethers over Nickel Nanoparticles Supported on Niobic Acid-Activated Carbon Composites

Cleavage of lignin-derived 4-O-5 aryl ethers has been conducted over nickel nanoparticles supported on niobic acid-activated carbon composite under mild conditions. The hydrated niobic acid has been successfully supported and well dispersed on activated carbon. Due to the coexisting Bronsted and Lewis acid sites on the hydrated niobic oxide, the Ni/xNbAC catalysts exhibited higher activities for cleavage of C–O ether bonds and dehydration than those of the Ni/AC catalyst. With increasing content of niobic acid, a larger amount of O-free alkane is obtained owing to niobic acid-promoted removal of oxygen from lignin-derived aryl ethers. The cleavage of C–O ether bonds and dehydration of cyclohexanol to cyclohexane are both favored at high temperature. The direct cleavage of the 4-O-5 aryl ether bond can also be achieved under low H2 pressure, forming phenol and benzene as intermediates, followed by hydrodeoxygenation of phenol to cyclohexane.

Synthesis of EFdA via a Diastereoselective Aldol Reaction of a Protected 3-Keto Furanose

An efficient enantioselective total synthesis of EFdA, a remarkably potent anti-HIV nucleoside analogue with various favorable pharmacological profiles, has been achieved in 37% overall yield from diacetone-D-glucose by a 14-step sequence that features a highly diastereoselective installation of the tetrasubstituted stereogenic center at the C4' position, direct oxidative cleavage of an acetonide-protected diol derivative to an aldehyde, and one-pot 2'-deoxygenation of a ribonucleoside intermediate.

The Serine Protease Pic From Enteroaggregative Escherichia coli Mediates Immune Evasion by the Direct Cleavage of Complement Proteins.

Enteroaggregative and uropathogenic Escherichia coli, Shigella flexneri 2a, and the hybrid enteroaggregative/Shiga toxin-producing E. coli strain (O104:H4) are important pathogens responsible for intestinal and urinary tract infections, as well as sepsis and hemolytic uremic syndrome. They have in common the production of a serine protease called Pic. Several biological roles for Pic have been described, including protection of E. coli DH5α from complement-mediated killing. Hereby we showed that Pic significantly reduces complement activation by all 3 pathways. Pic cleaves purified C3/C3b and other proteins from the classic and lectin pathways, such as C4 and C2. Cleavage fragments of C3, C4, and C2 were also observed with HB101(pPic1) culture supernatants, and C3 cleavage sites were mapped by fluorescence resonance energy transfer peptides. Experiments using human serum as a source of complement proteins confirmed Pic proteolytic activity on these proteins. Furthermore, Pic works synergistically with the human complement regulators factor I and factor H, promoting inactivation of C3b. In the presence of both regulators, further degradation of C3 α' chain was observed. Therefore, Pic may contribute to immune evasion of E. coli and S. flexneri, favoring invasiveness and increasing the severity of the disorders caused by these pathogens.

Proof-of-concept Studies for siRNA-mediated Gene Silencing for Coagulation Factors in Rat and Rabbit.

The present study aimed at establishing feasibility of delivering short interfering RNA (siRNA) to target the coagulation cascade in rat and rabbit, two commonly used species for studying thrombosis and hemostasis. siRNAs that produced over 90% mRNA knockdown of rat plasma prekallikrein and rabbit Factor X (FX) were identified from in vitro screens. An ionizable amino lipid based lipid nanoparticle (LNP) formulation for siRNA in vivo delivery was characterized as tolerable and exerting no appreciable effect on coagulability at day 7 postdosing in both species. Both prekallikrein siRNA-LNP and FX siRNA-LNP resulted in dose-dependent and selective knockdown of target gene mRNA in the liver with maximum reduction of over 90% on day 7 following a single dose of siRNA-LNP. Knockdown of plasma prekallikrein was associated with modest clot weight reduction in the rat arteriovenous shunt thrombosis model and no increase in the cuticle bleeding time. Knockdown of FX in the rabbit was accompanied with prolongation in ex vivo clotting times. Results fit the expectations with both targets and demonstrate for the first time, the feasibility of targeting coagulation factors in rat, and, more broadly, targeting a gene of interest in rabbit, via systemic delivery of ionizable LNP formulated siRNA.

Study on the effect of stress state and crystal orientation on micro-crack tip propagation behavior in phase field crystal method

A nanometer scale mechanism for micro crack propagation under uniaxial tension in single crystals is investigated using phase field crystal (PFC) simulation. The uniaxial tensile loading is strain controlled. And three initial typical stresses of pre-existing center crack in (111) crystal plane of face centered cubic structure are chosen to study the effects of initial stress state on micro-crack propagation. Moreover, the influences of different crystal orientations, when the crystal suffers from uniaxial tension, are also investigated. Due to the influence of time scale and length scale in the PFC method, the motion of dislocations, vacancies, shear band and twinning structure should be observed and described during the propagation process of micro cracks. In addition, the free energy curves of different processes are drawn and discussed in order to explain the different behaviors of the crystal in the propagation of cracks. Simulation results show that the propagation behavior of micro cracks can be closely associated with the initial stress state. It is found that the propagation behavior mainly occurs in the 11>(111) slip system. Besides, the crystal orientation has a significant effect on the mechanism of activation and evolution. In the pre-stretching system, slip dislocation is induced near the micro-crack tip, and then its slide in [011] direction will cause the cleavage of a certain crystal plane, and promote the micro cracks to extend. However, to a certain level, the propagating direction of the micro-crack tip will turn to another slip direction [101]. As a result, zigzag edge appears. By contrast, in the pre-shear system, the tip of the micro crack propagates in a cleavage mode, and results in the appearance of slip dislocation [101] near the micro-crack tip. Afterwards, the motion of slip dislocation promotes the production of vacancies. And owing to the aggregation and combination of vacancies, secondary cracks form and propagate in the process that follows. At the same time, in a pre-deviatoric system, the micro crack propagates forward with direct cleavage of [101] slip direction near the micro-crack tip until the single crystal sample fractures. Furthermore, no slip dislocation appears during the whole process. The mechanism of micro-crack tip propagating behavior varies with crystal orientation. When the crystal orientation angle is lower, the micro-crack tip prefers to produce slip dislocation around it, and the following dislocation slide will induce vacancies, then a secondary crack also forms because of the aggregation and combination of vacancies. On the other hand, when the aggregation degree is higher, the micro-crack tip is inclined to directly propagate in a cleavage mode, and its propagating direction is nearly perpendicular to the stretching direction.

RNase L targets distinct sites in influenza A virus RNAs.

Influenza A virus (IAV) infections are influenced by type 1 interferon-mediated antiviral defenses and by viral countermeasures to these defenses. When IAV NS1 protein is disabled, RNase L restricts virus replication; however, the RNAs targeted for cleavage by RNase L under these conditions have not been defined. In this study, we used deep-sequencing methods to identify RNase L cleavage sites within host and viral RNAs from IAV PR8ΔNS1-infected A549 cells. Short hairpin RNA knockdown of RNase L allowed us to distinguish between RNase L-dependent and RNase L-independent cleavage sites. RNase L-dependent cleavage sites were evident at discrete locations in IAV RNA segments (both positive and negative strands). Cleavage in PB2, PB1, and PA genomic RNAs suggests that viral RNPs are susceptible to cleavage by RNase L. Prominent amounts of cleavage mapped to specific regions within IAV RNAs, including some areas of increased synonymous-site conservation. Among cellular RNAs, RNase L-dependent cleavage was most frequent at precise locations in rRNAs. Our data show that RNase L targets specific sites in both host and viral RNAs to restrict influenza virus replication when NS1 protein is disabled. RNase L is a critical component of interferon-regulated and double-stranded-RNA-activated antiviral host responses. We sought to determine how RNase L exerts its antiviral activity during influenza virus infection. We enhanced the antiviral activity of RNase L by disabling a viral protein, NS1, that inhibits the activation of RNase L. Then, using deep-sequencing methods, we identified the host and viral RNAs targeted by RNase L. We found that RNase L cleaved viral RNAs and rRNAs at very precise locations. The direct cleavage of IAV RNAs by RNase L highlights an intimate battle between viral RNAs and an antiviral endonuclease.

Phenol Deoxygenation Mechanisms on Fe(110) and Pd(111)

The catalytic deoxygenation of phenolic compounds has become a major area of interest in recent years because they are produced during the pyrolysis of lignin and are present in biofuels. Our previous work showed that a PdFe bimetallic catalyst was catalytically active for the deoxygenation of phenolics. To better understand and control the catalytic deoxygenation reaction of phenolics, the detailed surface reaction mechanisms are needed for phenol, a key intermediate in phenolic deoxygeantion. Here, we have examined five distinct reaction mechanisms for the deoxygenation of phenol on the Fe(110) and Pd(111) surfaces so as to identify the most likely deoxygenation mechanism on these surfaces. Our results show that the elementary phenol deoxygenation reaction step for each mechanism was highly endothermic on Pd(111), whereas the same mechanisms are exothermic on Fe(110). On the basis of the reaction energy studies, detailed mechanistic studies were performed on the Fe(110) surface, and it was found that the most energetically and kinetically favorable reaction mechanism occurs via the direct cleavage of the C–O bond.

The use of morphokinetics as a predictor of implantation: a multicentric study to define and validate an algorithm for embryo selection.

Can we use morphokinetic markers to select the embryos most likely to implant and are the results likely to be consistent across different clinics? Yes, morphokinetic markers can be used to select the embryos most likely to implant and the results were similar in different IVF clinics that share methods and organization to some extent. With the introduction of time-lapse technology several authors have proposed the use of kinetic markers to improve embryo selection. The majority of these markers can be detected as early as Day 2 of development. Morphology remains the gold standard but kinetic markers have been proven as excellent tools to complement our decisions. Nevertheless, the majority of time-lapse studies are based on small data sets deriving from one single clinic. Retrospective multicentric study of 1664 cycles of which 799 were used to develop an algorithm (Phase 1 of the study) and 865 to test its predictive power (Phase 2 of the study). University-affiliated infertility centres patients undergoing first or second ICSI cycle using their own or donated oocytes. Embryo development was analysed with a time-lapse imaging system. Variables studied included the timing to two cells (t2), three cells (t3), four cells (t4) and five cells (t5) as well as the length of the second cell cycle (cc2 = t3 - t2) and the synchrony in the division from two to four cells (s2 = t4 - t3). Implantation (IR) and clinical pregnancy (CPR) rates were also analysed. During Phase 1 of the study we identified three variables most closely related to implantation: t3 (34-40 h), followed by cc2 (9-12 h) and t5 (45-55 h). Based on these results we elaborated an algorithm that classified embryos from A to D according to implantation potential. During Phase 2 of the study the algorithm was validated in a different group of patients that included 865 cycles and 1620 embryos transferred. In this phase of the study, embryos were categorized based on the algorithm and significant differences in IR were observed between the different categories ('A' 32%, 'B' 28%, 'C' 26%, 'D' 20% and 'E' 17%, P < 0.001). In addition we identified three quality criteria: direct cleavage from one to three cells, uneven blastomere size in second cell cycle and multinucleation in third cell cycle. The retrospective nature of the study limits its potential value, although the use of one database to generate the algorithm (embryos from this database were not selected by any morphokinetic criteria) and one database to validate it reinforces our conclusions. The elaboration of an algorithm based on a larger database derived from different (albeit related) clinics raises the possibility that such algorithms could be applied in different clinical settings.

Granzyme B mediates both direct and indirect cleavage of extracellular matrix in skin after chronic low-dose ultraviolet light irradiation.

Extracellular matrix (ECM) degradation is a hallmark of many chronic inflammatory diseases that can lead to a loss of function, aging, and disease progression. Ultraviolet light (UV) irradiation from the sun is widely considered as the major cause of visible human skin aging, causing increased inflammation and enhanced ECM degradation. Granzyme B (GzmB), a serine protease that is expressed by a variety of cells, accumulates in the extracellular milieu during chronic inflammation and cleaves a number of ECM proteins. We hypothesized that GzmB contributes to ECM degradation in the skin after UV irradiation through both direct cleavage of ECM proteins and indirectly through the induction of other proteinases. Wild-type and GzmB-knockout mice were repeatedly exposed to minimal erythemal doses of solar-simulated UV irradiation for 20 weeks. GzmB expression was significantly increased in wild-type treated skin compared to nonirradiated controls, colocalizing to keratinocytes and to an increased mast cell population. GzmB deficiency significantly protected against the formation of wrinkles and the loss of dermal collagen density, which was related to the cleavage of decorin, an abundant proteoglycan involved in collagen fibrillogenesis and integrity. GzmB also cleaved fibronectin, and GzmB-mediated fibronectin fragments increased the expression of collagen-degrading matrix metalloproteinase-1 (MMP-1) in fibroblasts. Collectively, these findings indicate a significant role for GzmB in ECM degradation that may have implications in many age-related chronic inflammatory diseases.

Cigarette Smoke and the Induction of Urokinase Plasminogen Activator Receptor In Vivo: Selective Contribution of Isoforms to Bronchial Epithelial Phenotype.

The urokinase plasminogen activator receptor (uPAR) gene (PLAUR) has been identified as an asthma susceptibility gene, with polymorphisms within that gene being associated with baseline lung function, lung function decline, and lung function in a smoking population. Soluble cleaved uPAR (scuPAR), a molecule identified as a marker of increased morbidity and mortality in a number of diseases, has been shown to be elevated in the airways of patients with asthma and in patients with chronic obstructive pulmonary disease. However, the functionality of soluble receptor isoforms and their relationship with an important initiator for obstructive lung disease, cigarette smoke, remains undefined. In this study, we set out to determine the effect of cigarette smoke on soluble uPAR isoforms, its regulatory pathway and the resultant effect on bronchial epithelial cell function. We identified a positive association between cigarette pack-years and uPAR expression in the airway bronchial epithelium of biopsies from patients with asthma (n = 27; P = 0.0485). In vitro, cigarette smoke promoted cleavage of uPAR from the surface of bronchial epithelial cells (1.5× induction; P < 0.0001) and induced the soluble spliced isoform through changes in messenger RNA expression (∼2× change; P < 0.001), driven by loss of endogenous 3' untranslated region suppression. Elevated expression of the soluble isoforms resulted in a proremodeling cell phenotype, characterized by increased proliferation and matrix metalloproteinase-9 expression in primary bronchial epithelial cells. This suggests that cigarette smoke elevates soluble receptor isoforms in bronchial epithelial cells through direct (cleavage) and indirect (messenger RNA expression) means. These findings provide further insight into how cigarette smoke may influence changes in the airways of importance to airway remodeling and obstructive lung disease progression.

Direct cleavage of sorbitol from oligosaccharides via a sequential hydrogenation-hydrolysis pathway

The production of sorbitol from polysaccharides is widely believed to proceed via hydrolysis to glucose and subsequent hydrogenation. Nevertheless, our previous study on the hydrolytic hydrogenation of cellobiose confirmed simultaneous hydrolysis and hydrogenation with a higher kinetic selectivity of hydrogenation over hydrolysis. Herein, kinetics of hydrogenolysis of trisaccharides with α-1,4 and β-1,4 glycosidic linkages were studied using Ru/C in combination with a molecular acid as catalyst system. Kinetic analysis emphasises a fast hydrogenation of the reducing end of trisaccharides followed by a facilitated cleavage of the terminal sorbitol unit. Considering the obtained reaction rate constants, hydrogenation compared to hydrolysis proceeds up to 24 and 15 times faster for maltotriose and cellotriose, respectively. Additionally, superior reaction rate constants and decreased activation energies for hydrolytic cleavage of sorbitol can be observed. Hence, a sequential hydrogenation-hydrolysis pathway clearly contributes to sorbitol formation based on oligosaccharides.

The Eph Tyrosine Kinase Receptors EphB2 and EphA2 Are Novel Proteolytic Substrates of Tissue Factor/Coagulation Factor VIIa

Tissue factor (TF) binds the serine protease factor VIIa (FVIIa) to form a proteolytically active complex that can trigger coagulation or activate cell signaling. Here we addressed the involvement of tyrosine kinase receptors (RTKs) in TF/FVIIa signaling by antibody array analysis and subsequently found that EphB2 and EphA2 of the Eph RTK family were cleaved in their ectodomains by TF/FVIIa. We used N-terminal Edman sequencing and LC-MS/MS analysis to characterize the cleaved Eph isoforms and identified a key arginine residue at the cleavage site, in agreement with the tryptic serine protease activity of FVIIa. Protease-activated receptor 2 (PAR2) signaling and downstream coagulation activity was non-essential in this context, in further support of a direct cleavage by TF/FVIIa. EphB2 was cleaved by FVIIa concentrations in the subnanomolar range in a number of TF expressing cell types, indicating that the active cellular pool of TF was involved. FVIIa caused potentiation of cell repulsion by the EphB2 ligand ephrin-B1, demonstrating a novel proteolytical event to control Eph-mediated cell segregation. These results define Eph RTKs as novel proteolytical targets of TF/FVIIa and provide new insights into how TF/FVIIa regulates cellular functions independently of PAR2.