ATR autophosphorylation as a molecular switch for checkpoint activation

The ataxia telangiectasia-mutated and Rad3-related (ATR) kinase is a master checkpoint regulator safeguarding the genome. Upon DNA damage, the ATR-ATRIP complex is recruited to sites of DNA damage by RPA-coated single-stranded DNA and activated by an elusive process. Here, we show that ATR is transformed into a hyperphosphorylated state after DNA damage, and that a single autophosphorylation event at Thr 1989 is crucial for ATR activation. Phosphorylation of Thr 1989 relies on RPA, ATRIP, and ATR kinase activity, but unexpectedly not on the ATR stimulator TopBP1. Recruitment of ATR-ATRIP to RPA-ssDNA leads to congregation of ATR-ATRIP complexes and promotes Thr 1989 phosphorylation in trans. Phosphorylated Thr 1989 is directly recognized by TopBP1 via the BRCT domains 7 and 8, enabling TopBP1 to engage ATR-ATRIP, to stimulate the ATR kinase, and to facilitate ATR substrate recognition. Thus, ATR autophosphorylation on RPA-ssDNA is a molecular switch to launch robust checkpoint response.     

A recombinant vaccine of H5N1 HA1 fused with foldon and human IgG Fc induced complete cross-clade protection against divergent H5N1 viruses

Development of effective vaccines to prevent influenza, particularly highly pathogenic avian influenza (HPAI) caused by influenza A virus (IAV) subtype H5N1, is a challenging goal. In this study, we designed and constructed two recombinant influenza vaccine candidates by fusing hemagglutinin 1 (HA1) fragment of A/Anhui/1/2005(H5N1) to either Fc of human IgG (HA1-Fc) or foldon plus Fc (HA1-Fdc), and evaluated their immune responses and cross-protection against divergent strains of H5N1 virus. Results showed that these two recombinant vaccines induced strong immune responses in the vaccinated mice, which specifically reacted with HA1 proteins and an inactivated heterologous H5N1 virus. Both proteins were able to cross-neutralize infections by one homologous strain (clade 2.3) and four heterologous strains belonging to clades 0, 1, and 2.2 of H5N1 pseudoviruses as well as three heterologous strains (clades 0, 1, and 2.3.4) of H5N1 live virus. Importantly, immunization with these two vaccine candidates, especially HA1-Fdc, provided complete cross-clade protection against high-dose lethal challenge of different strains of H5N1 virus covering clade 0, 1, and 2.3.4 in the tested mouse model. This study suggests that the recombinant fusion proteins, particularly HA1-Fdc, could be developed into an efficacious universal H5N1 influenza vaccine, providing cross-protection against infections by divergent strains of highly pathogenic H5N1 virus.     

Ezrin Interacts with the SARS Coronavirus Spike Protein and Restrains Infection at the Entry Stage

Background

Entry of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and its envelope fusion with host cell membrane are controlled by a series of complex molecular mechanisms, largely dependent on the viral envelope glycoprotein Spike (S). There are still many unknowns on the implication of cellular factors that regulate the entry process.

Methodology/Principal Findings

We performed a yeast two-hybrid screen using as bait the carboxy-terminal endodomain of S, which faces the cytosol during and after opening of the fusion pore at early stages of the virus life cycle. Here we show that the ezrin membrane-actin linker interacts with S endodomain through the F1 lobe of its FERM domain and that both the eight carboxy-terminal amino-acids and a membrane-proximal cysteine cluster of S endodomain are important for this interaction in vitro. Interestingly, we found that ezrin is present at the site of entry of S-pseudotyped lentiviral particles in Vero E6 cells. Targeting ezrin function by small interfering RNA increased S-mediated entry of pseudotyped particles in epithelial cells. Furthermore, deletion of the eight carboxy-terminal amino acids of S enhanced S-pseudotyped particles infection. Expression of the ezrin dominant negative FERM domain enhanced cell susceptibility to infection by SARS-CoV and S-pseudotyped particles and potentiated S-dependent membrane fusion.

Conclusions/Significance

Ezrin interacts with SARS-CoV S endodomain and limits virus entry and fusion. Our data present a novel mechanism involving a cellular factor in the regulation of S-dependent early events of infection.     

High Resolution Size Analysis of Fetal DNA in the Urine of Pregnant Women by Paired-End Massively Parallel Sequencing

Background

Fetal DNA in maternal urine, if present, would be a valuable source of fetal genetic material for noninvasive prenatal diagnosis. However, the existence of fetal DNA in maternal urine has remained controversial. The issue is due to the lack of appropriate technology to robustly detect the potentially highly degraded fetal DNA in maternal urine.

Methodology

We have used massively parallel paired-end sequencing to investigate cell-free DNA molecules in maternal urine. Catheterized urine samples were collected from seven pregnant women during the third trimester of pregnancies. We detected fetal DNA by identifying sequenced reads that contained fetal-specific alleles of the single nucleotide polymorphisms. The sizes of individual urinary DNA fragments were deduced from the alignment positions of the paired reads. We measured the fractional fetal DNA concentration as well as the size distributions of fetal and maternal DNA in maternal urine.

Principal Findings

Cell-free fetal DNA was detected in five of the seven maternal urine samples, with the fractional fetal DNA concentrations ranged from 1.92% to 4.73%. Fetal DNA became undetectable in maternal urine after delivery. The total urinary cell-free DNA molecules were less intact when compared with plasma DNA. Urinary fetal DNA fragments were very short, and the most dominant fetal sequences were between 29 bp and 45 bp in length.

Conclusions

With the use of massively parallel sequencing, we have confirmed the existence of transrenal fetal DNA in maternal urine, and have shown that urinary fetal DNA was heavily degraded.     

Chemical xenobiotics and mitochondrial autoantigens in primary biliary cirrhosis: identification of antibodies against a common environmental, cosmetic, and food additive, 2-octynoic acid

Emerging evidence has suggested environmental factors as causative agents in the pathogenesis of primary biliary cirrhosis (PBC). We have hypothesized that in PBC the lipoyl domain of the immunodominant E2 component of pyruvate dehydrogenase (PDC-E2) is replaced by a chemical xenobiotic mimic, which is sufficient to break self-tolerance. To address this hypothesis, based upon our quantitative structure-activity relationship data, a total of 107 potential xenobiotic mimics were coupled to the lysine residue of the immunodominant 15 amino acid peptide of the PDC-E2 inner lipoyl domain and spotted on microarray slides. Sera from patients with PBC (n = 47), primary sclerosing cholangitis (n = 15), and healthy volunteers (n = 20) were assayed for Ig reactivity. PBC sera were subsequently absorbed with native lipoylated PDC-E2 peptide or a xenobiotically modified PDC-E2 peptide, and the remaining reactivity analyzed. Of the 107 xenobiotics, 33 had a significantly higher IgG reactivity against PBC sera compared with control sera. In addition, 9 of those 33 compounds were more reactive than the native lipoylated peptide. Following absorption, 8 of the 9 compounds demonstrated cross-reactivity with lipoic acid. One compound, 2-octynoic acid, was unique in both its quantitative structure-activity relationship analysis and reactivity. PBC patient sera demonstrated high Ig reactivity against 2-octynoic acid-PDC-E2 peptide. Not only does 2-octynoic acid have the potential to modify PDC-E2 in vivo but importantly it was/is widely used in the environment including perfumes, lipstick, and many common food flavorings.     

Vancomycin resistance: Modeling backbone variants with d-Ala-d-Ala and d-Ala-d-Lac peptides

To seek vancomycin analogs with broader antibacterial activity, effects of backbone modifications for the agylcon 2 on binding with d-Ala-d-Ala- and d-Ala-d-Lac-containing peptides were investigated by Monte Carlo/free energy perturbation (MC/FEP) calculations. The experimental trend in binding affinities for 2 with three tripeptides was well reproduced. Possible modifications of the peptide bond between residues 4 and 5 were then considered, specifically for conversion of the OCNH linkage to CH₂NH₂⁺ (6), FCCH (7), HCCH (8), and HNCO (9). The MC/FEP results did not yield binding improvements for 7, 8, and 9, though the fluorovinyl replacement is relatively benign. The previously reported analog 6 remains as the only variant that exhibits improved affinity for the d-Ala-d-Lac sequence and acceptable affinity for the d-Ala-d-Ala sequence.     

Antitumor activity of diethynylfluorene derivatives of gold(I)

A list of diethynylfluorenes and their gold(I) derivatives have been studied for their antitumor activity as a function of their structure–activity relationships. End-capping the fluoren-9-one unit with gold(I) moieties could significantly strengthen the cytotoxic activity in vitro on three human cancer cell lines with induction of reactive oxygen species generation on Hep3B hepatocellular carcinoma cells and exhibit attractive antitumor activity from in vivo nude mice Hep3B xenograft model with limited adverse effects on vital organs including liver and kidney.     

Dissecting sites important for complement regulatory activity in membrane cofactor protein (MCP; CD46)

Membrane cofactor protein (MCP; CD46), a widely distributed regulator of complement activation, is a cofactor for the factor I-mediated degradation of C3b and C4b deposited on host cells. MCP possesses four extracellular, contiguous complement control protein modules (CCPs) important for this inhibitory activity. The goal of the present study was to delineate functional sites within these modules. We employed multiple approaches including mutagenesis, epitope mapping, and comparisons to primate MCP to make the following observations. First, functional sites were located to each of the four CCPs. Second, some residues were important for both C3b and C4b interactions while others were specific for one or the other. Third, while a reduction in ligand binding was invariably accompanied by a parallel reduction in cofactor activity (CA), other mutants lost or had reduced CA but retained ligand binding. Fourth, two C4b-regulatory domains overlapped measles virus interactive regions, indicating that the hemagglutinin docks to a site important for complement inhibition. Fifth, several MCP regulatory areas corresponded to functionally critical, homologous positions in other CCP-bearing C3b/C4b-binding proteins. Based on these data and the recently derived crystal structure of repeats one and two, computer modeling was employed to predict MCP structure and examine active sites.     

An ounce of prevention or a pound of cure: bioeconomic risk analysis of invasive species

Numbers of non-indigenous species--species introduced from elsewhere - are increasing rapidly worldwide, causing both environmental and economic damage. Rigorous quantitative risk-analysis frameworks, however, for invasive species are lacking. We need to evaluate the risks posed by invasive species and quantify the relative merits of different management strategies (e.g. allocation of resources between prevention and control). We present a quantitative bioeconomic modelling framework to analyse risks from non-indigenous species to economic activity and the environment. The model identifies the optimal allocation of resources to prevention versus control, acceptable invasion risks and consequences of invasion to optimal investments (e.g. labour and capital). We apply the model to zebra mussels (Dreissena polymorpha), and show that society could benefit by spending up to US$324 000 year(-1) to prevent invasions into a single lake with a power plant. By contrast, the US Fish and Wildlife Service spent US$825 000 in 2001 to manage all aquatic invaders in all US lakes. Thus, greater investment in prevention is warranted.