Positive Role of Delta Neutrophil Index (DNI) as a Prodiagnostic Marker in Cecal Ligation and Puncture (CLP)-Induced Sepsis Murine Model

Sepsis is an emergent infectious disease and a leading cause of death despite immediate intervention. While Delta neutrophil index (DNI) and myeloperoxidase (MPO) are known as a prodiagnostic marker of sepsis, the preclinical evidence of the best marker of sepsis is unclear. For this, using a well-designed cecal ligation and puncture (CLP)-induced sepsis mouse model, we comparatively measured the level and cost-effectiveness of sepsis biomarkers such as DNI, myeloperoxidase (MPO), procalcitonin (PCT), and tumor necrosis factor-alpha (TNF-α). First, we found that the optimal time point for early detection is at 6 h, 24 h post-CLP. Strikingly, the peak level and fold change of DNI was revealed at 24 h, further showing the best fold change as compared with other biomarker levels. Given the fold change at 6, 24 h, PCT was next to DNI. Third, a cost-effectiveness survey showed that DNI was the best, with PCT next. Further, DNI level was moderate positively associated with PCT (ρ = 0.697, p = 0.012) and TNF-α (ρ = 0.599, p = 0.040). Collectively, these data indicate that DNI in CLP-induced sepsis mice is as effective as the existent inflammatory biomarkers such as MPO, PCT and TNF-α to predict the prognosis of sepsis. This might have clinically important implications that DNI is cost effective, thus quickly and rationally applying to diverse types of imminent sepsis regardless of species. This might be the first report on the validity of DNI in preclinical CLP-induced murine sepsis

Use of Enrichment Culture for Directed Evolution of the Vibrio fluvialis JS17 ω-Transaminase, Which Is Resistant to Product Inhibition by Aliphatic Ketones

A novel high-throughput screening method that overcame product inhibition was used to isolate a mutant ω-transaminase from Vibrio fluvialis JS17. An enzyme library was generated using error-prone PCR mutagenesis and then enriched on minimal medium containing 2-aminoheptane as the sole nitrogen source and 2-butanone as an inhibitory ketone. An identified mutant enzyme, ω-TAmla, showed significantly reduced product inhibition by aliphatic ketone. The product inhibition constants of the mutant with 2-butanone and 2-heptanone were 6- and 4.5-fold higher than those of the wild type, respectively. Using ω-TAmla (50 U/ml) overexpressed in Escherichia coli BL21, 150 mM 2-aminoheptane was successfully resolved to (R)-2-aminoheptane (enantiomeric excess, >99%) with 53% conversion with an enantioselectivity of >100

Deletional Protein Engineering Based on Stable Fold

<div><p>Diversification of protein sequence-structure space is a major concern in protein engineering. Deletion mutagenesis can generate a protein sequence-structure space different from substitution mutagenesis mediated space, but it has not been widely used in protein engineering compared to substitution mutagenesis, because it causes a relatively huge range of structural perturbations of target proteins which often inactivates the proteins. In this study, we demonstrate that, using green fluorescent protein (GFP) as a model system, the drawback of the deletional protein engineering can be overcome by employing the protein structure with high stability. The systematic dissection of N-terminal, C-terminal and internal sequences of GFPs with two different stabilities showed that GFP with high stability (s-GFP), was more tolerant to the elimination of amino acids compared to a GFP with normal stability (n-GFP). The deletion studies of s-GFP enabled us to achieve three interesting variants viz. s-DL4, s-N14, and s-C225, which could not been obtained from n-GFP. The deletion of 191–196 loop sequences led to the variant s-DL4 that was expressed predominantly as insoluble form but mostly active. The s-N14 and s-C225 are the variants without the amino acid residues involving secondary structures around N- and C-terminals of GFP fold respectively, exhibiting comparable biophysical properties of the n-GFP. Structural analysis of the variants through computational modeling study gave a few structural insights that can explain the spectral properties of the variants. Our study suggests that the protein sequence-structure space of deletion mutants can be more efficiently explored by employing the protein structure with higher stability.</p> </div

Importance of expression system in the production of unnatural recombinant proteins in Escherichia coli

In this study, we investigated the efficiencies by which the pET and pQE expression systems produce unnatural recombinant proteins by residue-specific incorporation of unnatural amino acids, a method through which it was found that type of gene expression system tremendously influences the production yield of unnatural proteins in Escherichia coli. Green fluorescent protein (GFP) and a single-chain Fv antibody against c-Met were utilized as model recombinant proteins while L-homopropargylglycine (Hpg), a methionine analogue that incorporates into the methionine residues of a recombinant protein, was used as model unnatural amino acid. The pET system produced an almost negligible amount of Hpg-incorporated unnatural protein compared to the amount of methionine-incorporated natural protein. However, comparable amounts of unnatural and natural protein were produced by the pQE expression system. The amount of unnatural GFP protein produced through pET expression was not increased despite the over-expression of methionyl tRNA synthetase, which can enhance the activation rate of methionyl-tRNA with a methionine analogue. Incorporation of Hpg decreased the productivity of active GFP by approximately 2.5 fold, possibly caused by the inefficient folding of Hpg-incorporated GFP. Conversely, the productivity of functional anti-c-Met sc-Fv was not influenced by incorporation of Hpg. We confirmed through LC-MS and LCMS/MS that Hpg was incorporated into the methionine residues of the recombinant proteins produced by the pQE expression system

Comparison of relative whole cell fluorescence of s-GFP, n-GFP and deletion variants.

<p>The relative fluorescence (in arbitrary units) is defined as the whole cell fluorescence compared with fluorescence of cells expressing s-GFP^[a] and n-GFP^[b] respectively. All the fluorescence values were normalized by the O.D_{600 nm} of expressed cells. ^[c]Deletion of preceding residues in primary structure of n-GFP resulted in loss of fluorescence and experiments were not carried out. ^[+]Reported elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051510#pone.0051510-Li1" target="_blank">[6]</a> that these loop deletions makes the protein non-functional.</p

The negative impact of long working hours on mental health in young Korean workers.

Long working hours are known to have a negative effect on health. However, there is no clear evidence for a direct link between mental health and long working hours in the young adult populations. Therefore, we aimed to determine whether long working hours are associated with mental health in young adult workers. Data were collected from a 2012 follow-up survey of the Youth Panel 2007. A total of 3,332 young adult employees (aged 20 to 35) were enrolled in the study. We analyzed stress, depression, and suicidal thoughts by multivariate logistic regression analysis based on working hours (41 to 50, 51 to 60 and over 60 hours, compared to 31 to 40 hours per week), which was adjusted for sex, age, marriage status, region, and educational level. From the 3,332 young adult employees, about 60% of the workers worked more than 40 hours and 17% of the workers worked more than 50 hours per week. In a Chi-square test, stress level, depression, and suicidal thoughts increased with increasing working hours (p-value <0.001, 0.007, and 0.018, respectively). The multivariate logistic regression model showed that, compared to the 31 to 40 hours per week group, the adjusted odds ratios of the 41 to 50, 51 to 60, and over 60 hours per week groups for stress were 1.46(1.23-1.74), 2.25(1.79-2.83) and 2.55(1.72-3.77), respectively. A similar trend was shown in depression [odds ratios: 2.08(1.23-3.53), 2.79(1.44-5.39) and 4.09(1.59-10.55), respectively] and suicidal ideation [odds ratios: 1.98(0.95-4.10), 3.48(1.48-8.19) and 5.30(1.61-17.42), respectively]. We concluded that long working hours were associated with stress, depression, and suicidal ideation in young employees, aged 20 to 35

Computational analysis of changes in structural interactions of s-GFP variants.

<p>Comparison of lost hydrogen bonding network between s-GFP and deleted variants a) In s-N11 variant, deletion of F8 disrupted the hydrogen bond interaction with G4 along with loss of other interactions as compared with s-GFP. In s-N14 mutants, deletion of V12, P13 and I14 were shown clearly by comparing with s-GFP which resulted in loss of hydrogen bond interaction with G35, L119 and G33 respectivley. b) The interaction map of s-C225 showed the deleted residues T225 and A227, and the lost hydrogen bonds. Deletion of V224, T225 and A227 were shown in the interaction map of s-C224. c) The combined deletion variant s-N11C227 interaction map shows the deleted residues Phe8, Gly4, Gly10 and Ala227 and the interactions they were involved.</p

Computational modeling and molecular dynamic simulation of s-DL4.

<p>a) Lost hydrogen bonding between R80–L195 is shown in the interaction map with additional interaction surrounding the chromophore. Comparison of b) root mean square deviation (rmsd) and c) root mean square fluctuation (rmsf) of the main chain atoms of s-GFP and s-DL4.</p

Fluorescence excitation and emission spectra.

<p>a) Excitation and b) Emission spectra of s-GFP, s-N14, s-C225 and n-GFP, measured using fluorescence spectrometer. All amplitudes were arbitrarily normalized to a maximum value of 1.0 to show the difference in spectral wavelength not the spectral intensity.</p

Fluorescence microscope image of s-GFP and deletion variants.

<p>s-GFP, s-N14 and s-C225 were employed in live cell imaging by expressing in <i>E. coli</i>. The images were captured using fluorescence microscope equipped with digital image analyzer.</p