Spectral studies on aluminum ion binding to the ligands with phenolic group(s): implications for the differences between N- and C-terminal binding sites of human serum apotransferrin

Both the binding and releasing of ferric ions in C-, and N-terminal binding sites of human serum transferrin are different. To understand the difference here the interactions of aluminum with the ligands containing phenolic group(s), including 8-hydroxyquinoline, salicylic acid, N,N'-di(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid, N,N'-ethylenebis[2-(o-hydroxyphenolic)glycine], and human serum apotransferrin, respectively, are investigated by using UV difference and fluorescence spectra methods in 0.1 M N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid at pH 7.4. Aluminum binding produces a UV difference peak near 235 nm that is characteristic of phenolic groups binding to aluminum. The peak at 235 nm has been used to determine conditional binding constants of log K(Al-HBED)=8.88+/-0.74 and log K(Al-EHPG)=9.38+/-0.03. However, the effects of aluminum binding on the fluorescence intensity of N,N'-ethylenebis[2-(o-hydroxyphenolic)glycine], salicylic acid and N,N'-di(2-hydroxybenzyl) ethylenediamine-N,N'-diacetic acid, 8-hydroxyquinoline are disparate, the former showing a decrease and the latter an increase. At pH 7.4, there is N cdots, three dots, centered H-O type intramolecular hydrogen bond in 8-hydroxyquinoline, N,N'-di(2-hydroxybenzyl) ethylenediamine-N,N'-diacetic acid and O cdots, three dots, centered H-O type intramolecular hydrogen bond in salicylic acid, N,N'-ethylenebis[2-(o-hydroxyphenolic)glycine]. The effects of salts on the fluorescence intensity of the ligands containing phenolic group(s) show that fluorescence emission increases with the breaking of an N cdots, three dots, centered H-O type intramolecular hydrogen bond and fluorescence emission decreases with the breaking of an O cdots, three dots, centered H-O type intramolecular hydrogen bond. Fluorescence titrations of apotransferrin and both forms of monoferric transferrin with aluminum indicated that there is O cdots, three dots, centered H-O type intramolecular hydrogen bonds for the phenolic groups of Tyr426 and Tyr517 in the C-terminal binding site. While N cdots, three dots, centered H-O type intramolecular hydrogen bonds are found for the phenolic groups of Tyr95 and Tyr188 in the N-terminal binding site.     

Modified apoptotic molecule (BID) reduces hepatitis C virus infection in mice with chimeric human livers

Hepatitis C virus (HCV) encodes a polyprotein consisting of core, envelope (E1, E2, p7), and nonstructural polypeptides (NS2, NS3, NS4A, NS4B, NS5A, NS5B). The serine protease (NS3/NS4A), helicase (NS3), and polymerase (NS5B) constitute valid targets for antiviral therapy. We engineered BH3 interacting domain death agonist (BID), an apoptosis-inducing molecule, to contain a specific cleavage site recognized by the NS3/NS4A protease. Cleavage of the BID precursor molecule by the viral protease activated downstream apoptotic molecules of the mitochondrial pathway and triggered cell death. We extended this concept to cells transfected with an infectious HCV genome, hepatocytes containing HCV replicons, a Sindbis virus model for HCV, and finally HCV-infected mice with chimeric human livers. Infected mice injected with an adenovirus vector expressing modified BID exhibited HCV-dependent apoptosis in the human liver xenograft and considerable declines in serum HCV titers.     

A novel far-red bimolecular fluorescence complementation system that allows for efficient visualization of protein interactions under physiological conditions

Fluorescent protein (FP) has enabled the analysis of biomolecular interactions in living cells, and bimolecular fluorescence complementation (BiFC) represents one of the newly developed imaging technologies to directly visualize protein–protein interactions in living cells. Although 10 different FPs that cover a broad range of spectra have been demonstrated to support BiFC, only Cerulean (cyan FP variant), Citrine and Venus (yellow FP variants)-based BiFC systems can be used under 37 °C physiological temperature. The sensitivity of two mRFP-based red BiFC systems to higher temperatures (i.e., 37 °C) limits their applications in most mammalian cell-based studies. Here we report that mLumin, a newly isolated far-red fluorescent protein variant of mKate with an emission maximum of 621 nm, enables BiFC analysis of protein–protein interactions at 37 °C in living mammalian cells. Furthermore, the combination of mLumin with Cerulean- and Venus-based BiFC systems allows for simultaneous visualization of three pairs of protein–protein interactions in the same cell. The mLumin-based BiFC system will facilitate simultaneous visualization of multiple protein–protein interactions in living cells and offer the potential to visualize protein–protein interactions in living animals.     

Prediction of Perioperative Cardiac Events through Preoperative NT-pro-BNP and cTnI after Emergent Non-Cardiac Surgery in Elderly Patients

Objectives

Clinical risk stratification has an important function in preoperative evaluation of patients at risk for cardiac events prior to non-cardiac surgery. The aim of this study was to determine whether the combined measurement of pre-operative N-terminal pro-brain natriuretic peptide (NT-pro-BNP) and cardiac troponin I (cTnI) could provide useful prognostic information about postoperative major adverse cardiac events (MACE) within 30 days in patients aged over 60 years undergoing emergent non-cardiac surgery.

Methods

The study group comprised 2519 patients aged over 60 years that were undergoing emergent non-cardiac surgery between December 2007 and December 2013. NT-pro-BNP and cTnI were measured during hospital admission. The patients were monitored for MACE (cardiac death, non-fatal myocardial infarction, or cardiac arrest) during the 30-day postoperative follow-up period.

Results

MACE occurred in 251 patients (10.0%). Preoperative NT-pro-BNP and cTNI level were significantly higher in the individuals that experienced MACE than in those who did not (P < 0.001). The confounding factors of age, sex, co-morbidities and preoperative medications were adjusted in a multivariate logistic regression analysis. This analysis showed that preoperative NT-proBNP level > 917 pg/mL (OR 4.81, 95% CI 3.446–6.722, P < 0.001) and cTnI ≥ 0.07 ng/mL (OR 8.74, 95% CI 5.881–12.987, P < 0.001) remained significantly and independently associated with MACE after the adjustment of the confounding factors. Kaplan-Meier event-free survival curves demonstrated that patients with preoperative simultaneous NT-proBNP level > 917 pg/mL and cTnT ≥0.07 ng/mL had worse event-free survival than individual assessments of either biomarker.

Conclusion

Preoperative plasma NT-proBNP and cTnI are both independently associated with an increased risk of MACE in elderly patients after emergent non-cardiac surgery. The combination of these biomarkers provides better prognostic information than using either biomarker separately.     

Simultaneous determination of didanosine and its amino acid prodrug,valdidanosine by hydrophilic interaction chromatography coupled with electrospray ionization tandem mass spectrometry: Application to a pharmacokinetic study in rats

A rapid, sensitive and selective ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method with hydrophilic interaction chromatography has been developed and validated for the simultaneous determination of didanosine and valdidanosine (L-valine amino acid ester prodrug of didanosine) in rat plasma. Solid-phase extraction (SPE) column was employed to extract the analytes from rat plasma, with high extraction recovery (>85%) for both didanosine and valdidanosine. The analytes were then separated by hydrophilic interaction chromatography (HILIC column) and detected by a triple-quadrupole mass spectrometry equipped with an electrospray ionization (ESI) source. The method was linear over the concentration ranges of 2–20,000 ng/mL for didanosine and 4–300 ng/mL for valdidanosine. The lower limit of quantitation (LLOQ) of didanosine and valdidanosine was 2 and 4 ng/mL, respectively. The intra-day and inter-day relative standard deviation (RSD) were less than 15% and the relative errors (RE) were all within 15%. Finally, the validated UPLC–MS/MS method was successfully applied to the pharmacokinetic study after either didanosine or valdidanosine orally administrated to the Sprague–Dawley rats.     

Broadening the avenue of intersubgenomic heterosis in oilseed Brassica

Accumulated evidence has shown that each of the three basic Brassica genomes (A, B and C) has undergone profound changes in different species, and has led to the concept of the “subgenome”. Significant intersubgenomic heterosis was observed in hybrids between traditional Brassica napus and first generation lines of new type B. napus. The latter were produced by the partial introgression of subgenomic components from different species into B. napus. To increase the proportion of exotic subgenomic components and thus achieve stronger heterosis, lines of first generation new type B. napus were intercrossed with each other, and subjected to intensive marker-assisted selection to develop the second generation of new type B. napus. The second generation showed better agronomic traits and a higher proportion of introgression of subgenomic components than did the first generation. Compared with the commercial hybrid and the hybrids produced with the first generation new type B. napus, the novel hybrids showed stronger heterosis for seed yield during the 2 years of field trials. The extent of heterosis showed a significant positive correlation with the introgressed subgenomic components in the parental new type B. napus. To increase the content of the exotic subgenomic components further and to allow sustainable breeding of novel lines of new type B. napus, we initiated the development of a gene pool for new type B. napus that contained a substantial amount of genetic variation in the A^r and C^c genome. We discuss new approaches to broaden the avenue of intersubgenomic heterosis in oilseed Brassica.     

L(p) approximation capabilities of sum-of-product and sigma-pi-sigma neural networks

This paper studies the L(p) approximation capabilities of sum-of-product (SOPNN) and sigma-pi-sigma (SPSNN) neural networks. It is proved that the set of functions that are generated by the SOPNN with its activation function in $L_{loc};p(\mathcal{R})$ is dense in $L;p(\mathcal{K})$ for any compact set $\mathcal{K}\subset \mathcal{R};N$, if and only if the activation function is not a polynomial almost everywhere. It is also shown that if the activation function of the SPSNN is in ${L_{loc};\infty(\mathcal{R})}$, then the functions generated by the SPSNN are dense in $L;p(\mathcal{K})$ if and only if the activation function is not a constant (a.e.).