Food applications of bacterial cell wall hydrolases

Bacterial cell wall hydrolases (BCWHs) display a remarkable structural and functional diversity that offers perspectives for novel food applications, reaching beyond those of the archetype BCWH and established biopreservative hen egg white lysozyme. Insights in BCWHs from bacteriophages to animals have provided concepts for tailoring BCWHs to target specific pathogens or spoilage bacteria, or, conversely, to expand their working range to Gram-negative bacteria. Genetically modified foods expressing BCWHs in situ showed successful, but face regulatory and ethical concerns. An interesting spin-off development is the use of cell wall binding domains of bacteriophage BCWHs for detection and removal of foodborne pathogens. Besides for improving food safety or stability, BCWHs may also find use as functional food ingredients with specific health effects.     

Generic genetic differences between farmed and wild Atlantic salmon identified from a 7K SNP-chip

Genetic interactions between farmed and wild conspecifics are of special concern in fisheries where large numbers of domesticated individuals are released into the wild. In the Atlantic salmon (Salmo salar), selective breeding since the 1970's has resulted in rapid genetic changes in commercially important traits, such as a doubling of the growth rate. Each year, farmed salmon escape from net pens, enter rivers, and interbreed with wild salmon. Field experiments demonstrate that genetic introgression may weaken the viability of recipient populations. However, due to the lack of diagnostic genetic markers, little is known about actual rates of gene flow from farmed to wild populations. Here we present a panel of 60 single nucleotide polymorphisms (SNPs) that collectively are diagnostic in identifying individual salmon as being farmed or wild, regardless of their populations of origin. These were sourced from a pool of 7000 SNPs comparing historical wild and farmed salmon populations, and were distributed on all but two of the 29 chromosomes. We suggest that the generic differences between farmed and wild salmon at these SNPs have arisen due to domestication. The identified panel of SNPs will permit quantification of gene flow from farmed to wild salmon populations, elucidating one of the most controversial potential impacts of aquaculture. With increasing global interest in aquaculture and increasing pressure on wild populations, results from our study have implications for a wide range of species.     

BPR1K653, a novel Aurora kinase inhibitor, exhibits potent anti-proliferative activity in MDR1 (P-gp170)-mediated multidrug-resistant cancer cells

Background

Over-expression of Aurora kinases promotes the tumorigenesis of cells. The aim of this study was to determine the preclinical profile of a novel pan-Aurora kinase inhibitor, BPR1K653, as a candidate for anti-cancer therapy. Since expression of the drug efflux pump, MDR1, reduces the effectiveness of various chemotherapeutic compounds in human cancers, this study also aimed to determine whether the potency of BPR1K653 could be affected by the expression of MDR1 in cancer cells.

Principal Findings

BPR1K653 specifically inhibited the activity of Aurora-A and Aurora-B kinase at low nano-molar concentrations in vitro. Anti-proliferative activity of BPR1K653 was evaluated in various human cancer cell lines. Results of the clonogenic assay showed that BPR1K653 was potent in targeting a variety of cancer cell lines regardless of the tissue origin, p53 status, or expression of MDR1. At the cellular level, BPR1K653 induced endo-replication and subsequent apoptosis in both MDR1-negative and MDR1-positive cancer cells. Importantly, it showed potent activity against the growth of xenograft tumors of the human cervical carcinoma KB and KB-derived MDR1-positive KB-VIN10 cells in nude mice. Finally, BPR1K653 also exhibited favorable pharmacokinetic properties in rats.

Conclusions and Significance

BPR1K653 is a novel potent anti-cancer compound, and its potency is not affected by the expression of the multiple drug resistant protein, MDR1, in cancer cells. Therefore, BPR1K653 is a promising anti-cancer compound that has potential for the management of various malignancies, particularly for patients with MDR1-related drug resistance after prolonged chemotherapeutic treatments.     

Cecal ligation and puncture: the gold standard model for polymicrobial sepsis?

Sepsis is a serious medical condition characterized by dysregulated systemic inflammatory responses followed by immunosuppression. To study the pathophysiology of sepsis, diverse animal models have been developed. Polymicrobial sepsis induced by cecal ligation and puncture (CLP) is the most frequently used model because it closely resembles the progression and characteristics of human sepsis. Here we summarize the role of several immune components in the pathogenesis of sepsis induced by CLP. However, several therapies proposed on the basis of promising results obtained by CLP could not be translated to the clinic. This demonstrates that experimental sepsis models do not completely mimic human sepsis. We propose several strategies to narrow the gap between experimental sepsis models and clinical sepsis, including targeting factors that contribute to the immunosuppressive phase of sepsis, and reproducing the heterogeneity of human patients.     

Mice lacking Alkbh1 display sex-ratio distortion and unilateral eye defects

Background

Eschericia coli AlkB is a 2-oxoglutarate- and iron-dependent dioxygenase that reverses alkylated DNA damage by oxidative demethylation. Mouse AlkB homolog 1 (Alkbh1) is one of eight members of the newly discovered family of mammalian dioxygenases.

Methods and Findings

In the present study we show non-Mendelian inheritance of the Alkbh1 targeted allele in mice. Both Alkbh1^−/− and heterozygous Alkbh1^+/− offspring are born at a greatly reduced frequency. Additionally, the sex-ratio is considerably skewed against female offspring, with one female born for every three to four males. Most mechanisms that cause segregation distortion, act in the male gametes and affect male fertility. The skewing of the sexes appears to be of paternal origin, and might be set in the pachythene stage of meiosis during spermatogenesis, in which Alkbh1 is upregulated more than 10-fold. In testes, apoptotic spermatids were revealed in 5–10% of the tubules in Alkbh1^−/− adults. The deficiency of Alkbh1 also causes misexpression of Bmp2, 4 and 7 at E11.5 during embryonic development. This is consistent with the incompletely penetrant phenotypes observed, particularly recurrent unilateral eye defects and craniofacial malformations.

Conclusions

Genetic and phenotypic assessment suggests that Alkbh1 mediates gene regulation in spermatogenesis, and that Alkbh1 is essential for normal sex-ratio distribution and embryonic development in mice.     

Molecular characterization of HIV-1 CRF01_AE in Mekong Delta, Vietnam, and impact of T-cell epitope mutations on HLA recognition (ANRS 12159)

Background

To date, 11 HIV-1 subtypes and 48 circulating recombinant forms have been described worldwide. The underlying reason why their distribution is so heterogeneous is not clear. Host genetic factors could partly explain this distribution. The aim of this study was to describe HIV-1 strains circulating in an unexplored area of Mekong Delta, Vietnam, and to assess the impact of optimal epitope mutations on HLA binding.

Methods

We recruited 125 chronically antiretroviral-naive HIV-1-infected subjects from five cities in the Mekong Delta. We performed high-resolution DNA typing of HLA class I alleles, sequencing of Gag and RT-Prot genes and phylogenetic analysis of the strains. Epitope mutations were analyzed in patients bearing the HLA allele restricting the studied epitope. Optimal wild-type epitopes from the Los Alamos database were used as reference. T-cell epitope recognition was predicted using the immune epitope database tool according to three different scores involved in antigen processing (TAP and proteasome scores) and HLA binding (MHC score).

Results

All sequences clustered with CRF01_AE. HLA class I genotyping showed the predominance of Asian alleles as A*11:01 and B*46:01 with a Vietnamese specificity held by two different haplotypes. The percentage of homology between Mekong and B consensus HIV-1 sequences was above 85%. Divergent epitopes had TAP and proteasome scores comparable with wild-type epitopes. MHC scores were significantly lower in divergent epitopes with a mean of 2.4 (±0.9) versus 2 (±0.7) in non-divergent ones (p<0.0001).

Conclusions

Our study confirms the wide predominance of CRF01_AE in the Mekong Delta where patients harbor a specific HLA pattern. Moreover, it demonstrates the lower MHC binding affinity among divergent epitopes. This weak immune pressure combined with a narrow genetic diversity favors immune escape and could explain why CRF01_AE is still predominant in Vietnam, particularly in the Mekong area.     

A cell-based high-throughput screen for epidermal growth factor receptor pathway inhibitors

Epidermal growth factor receptor (EGFR) is a valid drug target for development of target-based therapeutics against non-small-cell lung cancer. In this study, we established a high-throughput cell-based assay to screen for compounds that may inhibit EGFR activation and/or EGFR-mediated downstream signaling pathway. This drug screening platform is based on the characterization of an EGFR-transfected 32D cell line (32D-EGFR). The expression of EGFR in 32D cells allowed cell proliferation in the presence of either epidermal growth factor (EGF) or interleukin 3 (IL-3) and provided a system for both screening and counterscreening of EGFR pathway-inhibitory compounds. After the completion of primary and secondary screenings in which 32D-EGFR cells were grown under the stimulation of either EGF or IL-3, 9 of 20,000 compounds were found to selectively inhibit the EGF-dependent proliferation, but not the IL-3-dependent proliferation, of 32D-EGFR cells. Subsequent analysis showed that 3 compounds of the 9 initial hits directly inhibited the kinase activity of recombinant EGFR in vitro and the phosphorylation of EGFR in H1299 cells transfected with EGFR. Thus, this 32D-EGFR assay system provides a promising approach for identifying novel EGFR and EGFR signaling pathway inhibitors with potential antitumor activity.     

In Vitro and in Vivo Protein-bound Tyrosine Nitration Characterized by Diagonal Chromatography

A new proteomics technique for analyzing 3-nitrotyrosine-containing peptides is presented here. This technique is based on the combined fractional diagonal chromatography peptide isolation procedures by which specific classes of peptides are isolated following a series of identical reverse-phase HPLC separation steps. Here dithionite is used to reduce 3-nitrotyrosine to 3-aminotyrosine peptides, which thereby become more hydrophilic. Our combined fractional diagonal chromatography technique was first applied to characterize tyrosine nitration in tetranitromethane-modified BSA and further led to a high quality list of 335 tyrosine nitration sites in 267 proteins in a peroxynitrite-treated lysate of human Jurkat cells. We then analyzed a serum sample of a C57BL6/J mouse in which septic shock was induced by intravenous Salmonella infection and identified six in vivo nitration events in four serum proteins, thereby illustrating that our technique is sufficiently sensitive to identify rare in vivo tyrosine nitration sites in a very complex background.Nitration of tyrosine to 3-nitrotyrosine is one of several protein modifications occurring during oxidative stress (1, 2). This modification is considered as a two-step process in which a tyrosine radical is first formed followed by the addition of ^•NO₂ yielding 3-nitrotyrosine. One of the mechanisms through which tyrosine can be nitrated is via the peroxynitrite radical (ONOO⁻); however, alternative pathways exist such as nitration by hemoperoxidases in the presence of hydrogen peroxide and nitrite (3) and reaction of the tyrosyl radical with nitric oxide yielding 3-nitrosotyrosine, which can be further oxidized to 3-nitrotyrosine.Nitration of protein-bound tyrosines can have important implications on the structure and activity of proteins (4–6) and is linked to a variety of pathological conditions such as Alzheimer disease (7) and atherosclerosis (8). Proteins containing 3-nitrotyrosine residues have mainly been identified by one- or two-dimensional PAGE followed by Western blotting using 3-nitrotyrosine-specific antibodies (9) or following affinity enrichment (10, 11). However, rather few in vivo tyrosine nitration sites have thus far been mapped onto proteins, and hence, the exact sites of in vivo nitration remain elusive. This is highly likely due to the overall low abundance of this modification as was recently illustrated by the identification of only 31 nitration sites in 29 proteins in a comprehensive analysis of mouse brain tissue covering 7,792 proteins (12). Furthermore, it was estimated that in diseased cells or organs the number of nitrated tyrosines should be as low as 0.00001–0.001% of all tyrosines (5), numbers that clearly indicate the need to enrich for 3-nitrotyrosine peptides prior to mass spectrometric analysis. In addition, several MS and MS/MS detection problems for 3-nitrotyrosine peptides were reported (13, 14) that also influence identification of such peptides.Recently, a procedure for enriching 3-nitrotyrosine peptides was described (10). In brief, reduced and alkylated proteins were digested after which primary amino groups were blocked by acetylation. Nitrotyrosines were then reduced to aminotyrosine using dithionite (15), unveiling novel primary amino groups on which sulfhydryl groups were coupled that allowed binding peptides to thiopropyl-Sepharose beads. In contrast to an earlier affinity-based isolation protocol (16), this improved enrichment procedure was more effective and led to the characterization of 150 tyrosine nitration sites in 102 proteins using a total of 3.1 mg of a mouse brain homogenate sample that was in vitro nitrated (10). However, this procedure requires many modification steps, which, when incomplete, will introduce artifacts (see “Results”). Among others, these explain the rather low number of identified nitrated tyrosines peptides using the high amount of starting material that was in vitro nitrated.Our laboratory adapted diagonal chromatography (17) for contemporary mass spectrometry-driven proteomics. Central in our combined fractional diagonal chromatography (COFRADIC1 (18)) approach is a chemical or enzymatic step that changes the reverse-phase column retention properties of a set of peptides such that these can be isolated. Among others, we developed COFRADIC protocols for isolating peptides carrying amino acid modifications such as phosphorylation (19), N-glycosylation (20), and sialylation (21) or peptides that are the result of protein processing (22–24). Here we describe a COFRADIC procedure for sorting peptides carrying nitrated tyrosines. Peptide sorting is based on a hydrophilic shift after reducing the nitro group to its amino counterpart. The applicability of COFRADIC for analyzing this modification is illustrated by characterization of four 3-nitrotyrosines in BSA treated with tetranitromethane, the mapping of 335 different nitration sites in 267 different proteins starting from 300 μg of an in vitro peroxynitrite (ONOO⁻)-treated Jurkat lysate, and the identification of six unique nitrated tyrosine residues in four serum proteins in a mouse sepsis model.