Thrombin and Plasmin Alter the Proteome of Neutrophil Extracellular Traps

Neutrophil extracellular traps (NETs) consist of a decondensed DNA scaffold decorated with neutrophil-derived proteins. The proteome of NETs, or “NETome,” has been largely elucidated in vitro. However, components such as plasma and extracellular matrix proteins may affect the NETome under physiological conditions. Here, using a reductionistic approach, we explored the effects of two proteases active during injury and wounding, human thrombin and plasmin, on the NETome. Using high-resolution mass spectrometry, we identified a total of 164 proteins, including those previously not described in NETs. The serine proteases, particularly thrombin, were also found to interact with DNA and bound to NETs in vitro. Among the most abundant proteins were those identified previously, including histones, neutrophil elastase, and antimicrobial proteins. We observed reduced histone (H2B, H3, and H4) and neutrophil elastase levels upon the addition of the two proteases. Analyses of NET-derived tryptic peptides identified subtle changes upon protease treatments. Our results provide evidence that exogenous proteases, present during wounding and inflammation, influence the NETome. Taken together, regulation of NETs and their proteins under different physiological conditions may affect their roles in infection, inflammation, and the host response

Effects of thrombin and its related peptide fragments on neutrophils

Wounds are highly proteolytic environments containing proteases from host cells as well as colonising bacteria. In this milieu, novel host defence peptides derived from host proteins such as cytokines and coagulation pathway proteins are being generated. For example, proteolysis of the serum protein thrombin forms C-terminal peptides that have antibacterial and anti-endotoxic properties. In addition, these peptides were shown to have immunomodulatory effects on monocytes, but whether they have any effects on the other major innate immune cells, neutrophils, was not known. Hence, we aimed to address the immunomodulatory potential of these C-terminal truncations on neutrophil functions. We show that the prototypic thrombin C-terminal peptide GKY25 can attenuate neutrophil responses to lipopolysaccharides (LPS) and the cytokine interleukin-8 in vitro. Independently, GKY25 induced the shedding of neutrophil adhesion molecule CD62L, thus reducing neutrophil surface tethering capacity in vitro. Furthermore, subcutaneous or intraperitoneal GKY25 reduced neutrophil response to subcutaneous LPS administration in vivo. This demonstrates the therapeutic potential of GKY25 in modulating host response in endotoxin-mediated conditions. Of interest was to investigate whether endogenous C-terminal peptides can also modulate neutrophils’ activities. Using two C-terminal truncated peptides – HVF18 and FYT21 – generated by the proteolysis of thrombin by neutrophil and Pseudomonas aeruginosa elastases respectively, we show that FYT21 was more effective at inhibiting LPS-induced neutrophil response at physiologically relevant concentrations. Furthermore, FYT21, even more effectively than GKY25, reduced neutrophil surface CD62L. These indicate a possible host evasion mechanism where FYT21 reduces inflammatory responses to facilitate P. aeruginosa colonisation. Neutrophils’ presence in wounds extend beyond their inflammatory functions; they produce neutrophil extracellular traps (NETs), an extracellular network of DNA decorated with neutrophil-derived proteins. NET-associated proteins have been argued to trap and kill bacteria, as well as being implicated in autoimmune complications. However, in vitro assessments of its proteome (or NETome) had excluded external factors’ influence and non-neutrophil proteins from analyses. Using a reductionistic approach, we applied physiological concentrations of thrombin and plasmin, two of the more abundant serum proteins representing the different stages of wounding, to NETs. Using a proteomic approach, we identified 164 NET-associated proteins, where majority of the proteins were shared between treatments but were different in abundances. We show that most classical NET-proteins were reduced in the presence of thrombin and plasmin, possibly through direct proteolytic effects (i.e. on histones) or other regulatory mechanism (i.e. for neutrophil elastase). This indicates that the NETome, under various inflammatory conditions, can be dynamically different. We propose that by elucidating these differences in a physiological context, it could potentially lead to discovery of novel diagnostic or therapeutic markers relevant to specific diseases where NETs are implicated.Doctor of Philosoph

Antecedents of job and life satisfaction and the moderating role of employee engagement.

This study examines meaning of work, psychological availability, supportive supervisor relations, distributive justice and employee engagement in relation to job and life satisfaction. One hundred working adults, coming from different industrial and organizational settings, completed self report questionnaires. Results indicate that meaning of work, psychological availability and distributive justice has significant positive relationships with job satisfaction. Psychological availability has further shown a positive association with life satisfaction. Employee engagement is found to be effective in moderating the relationship between psychological availability and life satisfaction; and that of distributive justice and job satisfaction. Implications, strengths and limitations of the study are discussed.BUSINES

Effect of microchannel junction angle on two-phase liquid-gas Taylor flow

Two-phase liquid-gas Taylor flow triggered by blocking-squeezing mechanism was studied with different junction angle h microchannels, i.e. 20 , 45 , 90 , 135 and 160 , at various liquid (ethanol) and gas (He) flow rates. We experimentally investigated the effects of flow rates and h on the gas bubble VB and liquid slug VS volumes. A theoretical model was formulated for the quantitative predictions of bubble and slug sizes for different h and flow rates. Good agreements were obtained between theoretical predictions and experimental observations. The unit cell volume VU (VB + VS) decreased pronouncedly for the 20 channel with decreasing liquid or increasing gas flow rate, due to the slight increase in VB and large decrease in VS. In comparison, for the 45 , 90 , 135 and 160 channels with increasing liquid or decreasing gas flow rate, VU were less sensitive to fluid flow rate changes, due to the approximate cancellation between VB decrease and VS increase. For the 20 and 45 channels, it produced larger VU, due to larger VB and VS, when compared to the 90 channel. This is caused by the larger gas bubble throat width DN at the junction when h 90 ), DN is approximately equal to the gas channel width, with VB, VS and VU approximately the same as the 90 channel. With h 90 (i.e. 90 , 135 and 160 channels), as evident from the smaller VU, higher gas bubble density can be obtained when compared to h < 90 (i.e. 20 and 45 channels). Hitherto, this observation has not been realized, and the mechanics is first investigated here with the employment of extreme h (i.e. 20 and 160 ). A thorough understanding of the underlying mechanics affecting Taylor flow can facilitate its exploitation for controlled gas bubble and liquid slug generation. Our theoretical model facilitates the tuning of the channel designs and fluid flow rates to achieve the desired gas bubble and liquid slug sizes for specific applications.NMRC (Natl Medical Research Council, S’pore)Accepted versio

Effect of corporate name changes on value of companies

This study has three purposes, viz., ( 1) to identify the reasons for changing a corporate name and the types of expenditures incurred in the name change process; (2) to identify the perception of general investors towards the companies with regards to their name changes; and (3) to analyse how the stock market in Singapore reacts to announcements of corporate name change. Two research methods, v1z., questionnaire surveys (on general investors and companies changing their names during the period January 1989 to June 1994) and event study (using data collected from January 1989 to July 1994) have been applied in this study. This study revealed that corporate name changes would not affect the value of companies. The stock prices would not react significantly to name change announcements. The five functional characteristics (Distinctive, Relevant, Memorable, Flexible and Positive) of corporate names were also found to have no significant impact on investors' investment decisions. This study concludes that name change cannot be used as an effective tool for market repositioning. Due to our time and financial constraints, this study is restricted to companies listed on the Stock Exchange of Singapore.BUSINES

The Inland Revenue Authority of Singapore : recent service innovations and their effects on taxpayers

Intense market competition and the increasing expectations of the public have encouraged many government bodies to become statutory boards, or even private corporations. This conversion enabled them to adopt corporate philosophies and become more service-oriented in their operations.BUSINES

Cell-Free DNA Promotes Thrombin Autolysis and Generation of Thrombin-Derived C-Terminal Fragments

10.3389/fimmu.2021.593020Frontiers in Immunology1259302

The Hong Kong government's role in the SAR's future after China's WTO accession.

This study starts with a question: "How may the special administrative region (SAR) government play a role in preventing Hong Kong from losing her economic significance following China's recession to the WTO?" and proceeds with a quanlitative case study approach, focusing on the broad macro strategic issues to the Hong Kong economy.Master of Business Administration (Business Law

Thrombin and Plasmin Alter the Proteome of Neutrophil Extracellular Traps

Neutrophil extracellular traps (NETs) consist of a decondensed DNA scaffold decorated with neutrophil-derived proteins. The proteome of NETs, or “NETome,” has been largely elucidated in vitro. However, components such as plasma and extracellular matrix proteins may affect the NETome under physiological conditions. Here, using a reductionistic approach, we explored the effects of two proteases active during injury and wounding, human thrombin and plasmin, on the NETome. Using high-resolution mass spectrometry, we identified a total of 164 proteins, including those previously not described in NETs. The serine proteases, particularly thrombin, were also found to interact with DNA and bound to NETs in vitro. Among the most abundant proteins were those identified previously, including histones, neutrophil elastase, and antimicrobial proteins. We observed reduced histone (H2B, H3, and H4) and neutrophil elastase levels upon the addition of the two proteases. Analyses of NET-derived tryptic peptides identified subtle changes upon protease treatments. Our results provide evidence that exogenous proteases, present during wounding and inflammation, influence the NETome. Taken together, regulation of NETs and their proteins under different physiological conditions may affect their roles in infection, inflammation, and the host response.MOE (Min. of Education, S’pore)Published versio

Thrombin-derived host defence peptide modulates neutrophil rolling and migration in vitro and functional response in vivo

Host defence peptides (HDPs) derived from the C-terminus of thrombin are proteolytically generated by enzymes released during inflammation and wounding. In this work, we studied the effects of the prototypic peptide GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE), on neutrophil functions. In vitro, GKY25 was shown to decrease LPS-induced neutrophil activation. In addition, the peptide induced CD62L shedding on neutrophils without inducing their activation. Correspondingly, GKY25-treated neutrophils showed reduced attachment and rolling behaviour on surfaces coated with the CD62L ligand E-selectin. The GKY25-treated neutrophils also displayed a dampened chemotactic response against the chemokine IL-8. Furthermore, in vivo, mice treated with GKY25 exhibited a reduced local ROS response against LPS. Taken together, our results show that GKY25 can modulate neutrophil functions in vitro and in vivo.MOE (Min. of Education, S’pore)Published versio