A Smartphone-Based Rapid Telemonitoring System for Ebola and Marburg Disease Surveillance.

We have developed a digital and multiplexed platform for the rapid detection and telemonitoring of infections caused by Ebola and Marburg filoviruses. The system includes a flow cell assay cartridge that captures specific antibodies with microarrayed recombinant antigens from all six species of filovirus, and a smartphone fluorescent reader for high-performance interpretation of test results. Multiplexed viral proteins, which are expandable to include greater numbers of probes, were incorporated to obtain highest confidence results by cross-correlation, and a custom smartphone application was developed for data analysis, interpretation, and communication. The smartphone reader utilizes an opto-electro-mechanical hardware attachment that snaps at the back of a Motorola smartphone and provides a user interface to manage the operation, acquire test results, and communicate with cloud service. The application controls the hardware attachment to turn on LEDs and digitally record the optically enhanced images. Assay processing time is approximately 20 min for microliter amounts of blood, and test results are digitally processed and displayed within 15 s. Furthermore, a secure cloud service was developed for the telemonitoring of test results generated by the smartphone readers in the field. Assay system results were tested with sera from nonhuman primates that received a live attenuated EBOV vaccine. This integrated system will provide a rapid, reliable, and digital solution to prevent the rapid overwhelming of medical systems and resources during EVD or MVD outbreaks. Further, this disease-monitoring system will be useful in resource-limited countries where there is a need for dispersed laboratory analysis of recent or active infections.

High-resolution crystal structures of the D1 and D2 domains of protein tyrosine phosphatase epsilon for structure-based drug design.

Here, new crystal structures are presented of the isolated membrane-proximal D1 and distal D2 domains of protein tyrosine phosphatase epsilon (PTPℇ), a protein tyrosine phosphatase that has been shown to play a positive role in the survival of human breast cancer cells. A triple mutant of the PTPℇ D2 domain (A455N/V457Y/E597D) was also constructed to reconstitute the residues of the PTPℇ D1 catalytic domain that are important for phosphatase activity, resulting in only a slight increase in the phosphatase activity compared with the native D2 protein. The structures reported here are of sufficient resolution for structure-based drug design, and a microarray-based assay for high-throughput screening to identify small-molecule inhibitors of the PTPℇ D1 domain is also described.

Calprotectin as a Biomarker for Melioidosis Disease Progression and Management.

Melioidosis is a neglected tropical disease that is caused by the bacterium Burkholderia pseudomallei and is underreported in many countries where the disease is endemic. A long and costly administration of antibiotics is needed to clear infections, and there is an unmet need for biomarkers to guide antibiotic treatment and increase the number of patients that complete therapy. We identified calprotectin as a lead biomarker of B. pseudomallei infections and examined correlations between this serum protein and the antibiotic treatment outcomes of patients with melioidosis. Serum levels of calprotectin and C-reactive protein were significantly higher in patients with melioidosis and nonmelioidosis sepsis than in healthy controls. Median calprotectin levels were higher in patients with melioidosis than in those with nonmelioidosis sepsis, whereas C-reactive protein levels were similar in both groups. Notably, intensive intravenous antibiotic treatment of patients with melioidosis resulted in lower levels of calprotectin and C-reactive protein (P < 0.0001), coinciding with recovery. The median percent reduction of calprotectin and C-reactive protein was 71% for both biomarkers after antibacterial therapy. In contrast, we found no significant differences in calreticulin levels between the two melioidosis treatment phases. Thus, reductions in serum calprotectin levels were linked to therapeutic responses to antibiotics. Our results suggest that calprotectin may be a sensitive indicator of melioidosis disease activity and illustrate the potential utility of this biomarker in guiding the duration of antibiotic therapy.

Povidone Iodine Ointment Application to the Vaccination Site Does Not Alter Immunoglobulin G Antibody Response to Smallpox Vaccine.

U.S. military personnel deployed to high-risk areas receive the live vaccinia virus (VACV) smallpox vaccine ACAM2000. VACV shedding from the vaccination site can result in autoinoculation and contact transmission. We previously found that the application of povidone iodine ointment (PIO) to the scarification site reduced viral shedding without altering the antibody response, as measured by plaque reduction neutralization or enzyme-linked immunosorbent assays. In this study, we used protein microarray assays to measure the amount of immunoglobulin G antibody bound to (1) ACAM2000 itself and (2) individual VACV antigens that are present within ACAM2000. We assessed antibody binding in sera from primary smallpox vaccinees who applied PIO to the scarification site beginning on day 7 (PIO group) and from those who did not apply PIO (control group). In both cohorts, the postvaccination antibody response-in terms of antibody binding, both to ACAM2000 and to 11 individual VACV antigens-was significantly greater than the prevaccination response (all p < 0.0001). The postvaccination antibody binding levels of vaccinees in the PIO group did not differ from those of control vaccinees. These findings further support the topical application of PIO, starting on day 7, to reduce the viral shedding associated with smallpox vaccination.

Phosphotyrosine Substrate Sequence Motifs for Dual Specificity Phosphatases.

Protein tyrosine phosphatases dephosphorylate tyrosine residues of proteins, whereas, dual specificity phosphatases (DUSPs) are a subgroup of protein tyrosine phosphatases that dephosphorylate not only Tyr(P) residue, but also the Ser(P) and Thr(P) residues of proteins. The DUSPs are linked to the regulation of many cellular functions and signaling pathways. Though many cellular targets of DUSPs are known, the relationship between catalytic activity and substrate specificity is poorly defined. We investigated the interactions of peptide substrates with select DUSPs of four types: MAP kinases (DUSP1 and DUSP7), atypical (DUSP3, DUSP14, DUSP22 and DUSP27), viral (variola VH1), and Cdc25 (A-C). Phosphatase recognition sites were experimentally determined by measuring dephosphorylation of 6,218 microarrayed Tyr(P) peptides representing confirmed and theoretical phosphorylation motifs from the cellular proteome. A broad continuum of dephosphorylation was observed across the microarrayed peptide substrates for all phosphatases, suggesting a complex relationship between substrate sequence recognition and optimal activity. Further analysis of peptide dephosphorylation by hierarchical clustering indicated that DUSPs could be organized by substrate sequence motifs, and peptide-specificities by phylogenetic relationships among the catalytic domains. The most highly dephosphorylated peptides represented proteins from 29 cell-signaling pathways, greatly expanding the list of potential targets of DUSPs. These newly identified DUSP substrates will be important for examining structure-activity relationships with physiologically relevant targets.

Zinc binding and dimerization of Streptococcus pyogenes pyrogenic exotoxin C are not essential for T-cell stimulation.

Streptococcal pyrogenic enterotoxin C (Spe-C) is a superantigen virulence factor produced by Streptococcus pyogenes that activates T-cells polyclonally. The biologically active form of Spe-C is thought to be a homodimer containing an essential zinc coordination site on each subunit, consisting of the residues His(167), His(201), and Asp(203). Crystallographic data suggested that receptor specificity is dependent on contacts between the zinc coordination site of Spe-C and the beta-chain of the major histocompatibility complex type II (MHCII) molecule. Our results indicate that only a minor fraction of dimer is present at T-cell stimulatory concentrations of Spe-C following mutation of the unpaired side chain of cysteine at residue 27 to serine. Mutations of amino acid residues His(167), His(201), or Asp(203) had only minor effects on protein stability but resulted in greatly diminished MHCII binding, as measured by surface plasmon resonance with isolated receptor/ligand pairs and flow cytometry with MHCII-expressing cells. However, with the exception of the mutants D203A and D203N, mutation of the zinc-binding site of Spe-C did not significantly impact T-cell activation. The mutation Y76A, located in a polar pocket conserved among most superantigens, resulted in significant loss of T-cell stimulation, although no effect was observed on the overall binding to human MHCII molecules, perhaps because of the masking of this lower affinity interaction by the dominant zinc-dependent binding. To a lesser extent, mutations of side chains found in a second conserved MHCII alpha-chain-binding site consisting of a hydrophobic surface loop decreased T-cell stimulation. Our results demonstrate that dimerization and zinc coordination are not essential for biological activity of Spe-C and suggest the contribution of an alternative MHCII binding mode to T-cell activation.

Immune protection against staphylococcal enterotoxin-induced toxic shock by vaccination with a Venezuelan equine encephalitis virus replicon.

A candidate vaccine against staphylococcal enterotoxin B (SEB) was developed using a Venezuelan equine encephalitis (VEE) virus vector. This vaccine is composed of a self-replicating RNA, termed "replicon," containing the VEE nonstructural genes and cis-acting elements and a gene encoding mutagenized SEB (mSEB). Cotransfection of baby hamster kidney cells with the mSEB replicon and 2 helper RNA molecules resulted in the release of propagation-deficient mSEB-VEE replicon particles (mSEB-VRPs). Mice inoculated subcutaneously with mSEB-VRPs were protected (15 of 20 mice) from a challenge with 5 median lethal dose units of wild-type (wt) SEB. T cells from mice vaccinated with mSEB-VRP responded normally both in vitro to wt SEB and in recall response to the inactivated mSEB polypeptide. The profile of cytokines measured after challenge with wt SEB suggested that the mode of protection was predominantly Th1 dependent. Our results suggest that the VEE replicon is a practical and convenient model system for evaluating efficacy of vaccines for the control of bacterial diseases.