MALDI–MS Profiling to Address Honey Bee Health Status under Bacterial Challenge through Computational Modeling

Honey bees play a critical role in the maintenance of plant biodiversity and sustainability of food webs. In the past few decades, bees have been subjected to biotic and abiotic threats causing various colony disorders. Therefore, monitoring solutions to help beekeepers to improve bee health are necessary. Matrix‐assisted laser desorption ionization–mass spectrometry (MALDI–MS) profiling has emerged within this decade as a powerful tool to identify in routine micro‐organisms and is currently used in real‐time clinical diagnosis. MALDI BeeTyping is developed to monitor significant hemolymph molecular changes in honey bees upon infection with a series of entomopathogenic Gram‐positive and ‐negative bacteria. A Serratia marcescens strain isolated from one naturally infected honey bee collected from the field is also considered. A series of hemolymph molecular mass fingerprints is individually recorded and to the authors' knowledge, the first computational model harboring a predictive score of 97.92% and made of nine molecular signatures that discriminate and classify the honey bees’ systemic response to the bacteria is built. Hence, the model is challenged by classifying a training set of hemolymphs and an overall recognition of 91.93% is obtained. Through this work, a novel, time and cost saving high‐throughput strategy that addresses honey bee health on an individual scale is introduced.     

PEGylation of lysine residues improves the proteolytic stability of fibronectin while retaining biological activity

Excessive proteolysis of fibronectin (FN) impairs tissue repair in chronic wounds. Since FN is essential in wound healing, our goal is to improve its proteolytic stability and at the same time preserve its biological activity. We have previously shown that reduced FN conjugated with polyethylene glycol (PEG) at cysteine residues is more proteolytically stable than native FN. Cysteine‐PEGylated FN supported cell adhesion and migration to the same extent as native FN. However, unlike native FN, cysteine‐PEGylated FN was not assembled into an extracellular matrix (ECM) when immobilized. Here, we present an alternative approach in which FN is preferentially PEGylated at lysine residues using different molecular weight PEGs. We show that lysine PEGylation does not perturb FN secondary structure. PEG molecular weight, from 2 to 10 kDa, positively correlates with FN–PEG proteolytic stability. Cell adhesion, cell spreading, and gelatin binding decrease with increasing molecular weight of PEG. The 2‐kDa FN–PEG conjugate shows comparable cell adhesion to native FN and binds gelatin. Moreover, immobilized FN–PEG is assembled into ECM fibrils. In summary, lysine PEGylation of FN can be used to stabilize FN against proteolytic degradation with minimal perturbation to FN structure and retained biological activity.     

Localization of phosphorylation sites with respect to the functional domains of the mouse L cell glucocorticoid receptor

Digestion of the rat liver glucocorticoid receptor with chymotrypsin results in the generation of a 42-kDa fragment which contains the steroid-binding and DNA-binding domains and the antigenic site for the BuGR anti-glucocorticoid receptor monoclonal antibody, while digestion with trypsin generates a 15-kDa receptor fragment containing only the DNA-binding function and the BuGR epitope (Eisen, L.P., Reichman, M.E., Thompson, E.B., Gametchu, B., Harrison, R. W., and Eisen, H.J. (1985) J. Biol. Chem. 260, 11805-11810). In this paper, glucocorticoid receptor of mouse L cells that were grown in the presence of [32P]orthophosphate was digested with trypsin or chymotrypsin (either before or after immune purification with BuGR antibody) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and Western blotting. The receptor is endogenously phosphorylated only on serine residues. Chymotrypsin digestion results in a 32P-labeled 42-kDa receptor fragment which contains steroid-binding, DNA-binding, and BuGR-reactive sites. Trypsin digestion generates a 27-kDa steroid-bound fragment (meroreceptor) which is not labeled with 32P and a 32P-labeled 15-kDa fragment which contains both the DNA-binding domain and the BuGR epitope. We have calculated that there are 4 times as many phosphate residues in the intact receptor than in the 42-kDa chymotrypsin fragment. From examination of 32P-labeled receptor fragments, we have deduced that one phosphate is located between amino acids 398 and 447, a region containing the BuGR epitope and about one-third of the DNA-binding domain, and the remaining three phosphates appear to be clustered just to the amino-terminal side of the BuGR epitope in a region defined by amino acids 313 to 369. Treatment of intact 32P-labeled receptor in cytosol with alkaline phosphatase removes these three phosphates, but it does not remove the phosphate from the DNA-binding-BuGR-reactive fragment and it does not affect the ability of the transformed receptor to bind to DNA-cellulose.     

Genome‐wide association study identifies loci associated with liability to alcohol and drug dependence that is associated with variability in reward‐related ventral striatum activity in African‐ and European‐Americans

Genetic influences on alcohol and drug dependence partially overlap, however, specific loci underlying this overlap remain unclear. We conducted a genome‐wide association study (GWAS) of a phenotype representing alcohol or illicit drug dependence (ANYDEP) among 7291 European‐Americans (EA; 2927 cases) and 3132 African‐Americans (AA: 1315 cases) participating in the family‐based Collaborative Study on the Genetics of Alcoholism. ANYDEP was heritable (h ² in EA = 0.60, AA = 0.37). The AA GWAS identified three regions with genome‐wide significant (GWS; P < 5E‐08) single nucleotide polymorphisms (SNPs) on chromosomes 3 (rs34066662, rs58801820) and 13 (rs75168521, rs78886294), and an insertion‐deletion on chromosome 5 (chr5:141988181). No polymorphisms reached GWS in the EA. One GWS region (chromosome 1: rs1890881) emerged from a trans‐ancestral meta‐analysis (EA + AA) of ANYDEP, and was attributable to alcohol dependence in both samples. Four genes (AA: CRKL, DZIP3, SBK3; EA: P2RX6) and four sets of genes were significantly enriched within biological pathways for hemostasis and signal transduction. GWS signals did not replicate in two independent samples but there was weak evidence for association between rs1890881 and alcohol intake in the UK Biobank. Among 118 AA and 481 EA individuals from the Duke Neurogenetics Study, rs75168521 and rs1890881 genotypes were associated with variability in reward‐related ventral striatum activation. This study identified novel loci for substance dependence and provides preliminary evidence that these variants are also associated with individual differences in neural reward reactivity. Gene discovery efforts in non‐European samples with distinct patterns of substance use may lead to the identification of novel ancestry‐specific genetic markers of risk.     

Altered profile of secondary metabolites in the root exudates of Arabidopsis ATP-binding cassette transporter mutants

Following recent indirect evidence suggesting a role for ATP-binding cassette (ABC) transporters in root exudation of phytochemicals, we identified 25 ABC transporter genes highly expressed in the root cells most likely to be involved in secretion processes. Of these 25 genes, we also selected six full-length ABC transporters and a half-size transporter for in-depth molecular and biochemical analyses. We compared the exuded root phytochemical profiles of these seven ABC transporter mutants to those of the wild type. There were three nonpolar phytochemicals missing in various ABC transporter mutants compared to the wild type when the samples were analyzed by high-performance liquid chromatography-mass spectrometry. These data suggest that more than one ABC transporter can be involved in the secretion of a given phytochemical and that a transporter can be involved in the secretion of more than one secondary metabolite. The primary and secondary metabolites present in the root exudates of the mutants were also analyzed by gas chromatography-mass spectrometry, which allowed for the identification of groups of compounds differentially found in some of the mutants compared to the wild type. For instance, the mutant Atpdr6 secreted a lower level of organic acids and Atmrp2 secreted a higher level of amino acids as compared to the wild type. We conclude that the release of phytochemicals by roots is partially controlled by ABC transporters.     

HADDOCK versus HADDOCK: new features and performance of HADDOCK2.0 on the CAPRI targets

Here we present version 2.0 of HADDOCK, which incorporates considerable improvements and new features. HADDOCK is now able to model not only protein-protein complexes but also other kinds of biomolecular complexes and multi-component (N > 2) systems. In the absence of any experimental and/or predicted information to drive the docking, HADDOCK now offers two additional ab initio docking modes based on either random patch definition or center-of-mass restraints. The docking protocol has been considerably improved, supporting among other solvated docking, automatic definition of semi-flexible regions, and inclusion of a desolvation energy term in the scoring scheme. The performance of HADDOCK2.0 is evaluated on the targets of rounds 4-11, run in a semi-automated mode using the original information we used in our CAPRI submissions. This enables a direct assessment of the progress made since the previous versions. Although HADDOCK performed very well in CAPRI (65% and 71% success rates, overall and for unbound targets only, respectively), a substantial improvement was achieved with HADDOCK2.0.     

LOX-1 inhibition in myocardial ischemia-reperfusion injury: modulation of MMP-1 and inflammation

A recently identified lectin-like oxidized low-density lipoprotein receptor (LOX-1) mediates endothelial cell injury and facilitates inflammatory cell adhesion. We studied the role of LOX-1 in myocardial ischemia-reperfusion (I/R) injury. Anesthetized Sprague-Dawley rats were subjected to 60 min of left coronary artery (LCA) ligation, followed by 60 min of reperfusion. Rats were treated with saline, LOX-1 blocking antibody JXT21 (10 mg/kg), or nonspecific anti-goat IgG (10 mg/kg) before I/R. Ten other rats underwent surgery without LCA ligation and served as a sham control group. LOX-1 expression was markedly increased during I/R (P < 0.01 vs. sham control group). Simultaneously, the expression of matrix metalloproteinase-1 (MMP-1) and adhesion molecules (P-selectin, VCAM-1, and ICAM-1) was also increased in the I/R area (P < 0.01 vs. sham control group). There was intense leukocyte accumulation in the I/R area in the saline-treated group. Treatment of rats with the LOX-1 antibody prevented I/R-induced upregulation of LOX-1 and reduced MMP-1 and adhesion molecule expression as well as leukocyte recruitment. LOX-1 antibody, but not nonspecific IgG, also reduced myocardial infarct size (P < 0.01 vs. saline-treated I/R group). To explore the link between LOX-1 and adhesion molecule expression, we measured expression of oxidative stress-sensitive p38 mitogen-activated protein kinase (p38 MAPK). The activity of p38 MAPK was increased during I/R (P < 0.01 vs. sham control), and use of LOX-1 antibody inhibited p38 MAPK activation (P < 0.01). These findings indicate that myocardial I/R upregulates LOX-1 expression, which through p38 MAPK activation increases the expression of MMP-1 and adhesion molecules. Inhibition of LOX-1 exerts an important protective effect against myocardial I/R injury.