A Peptidisc-Based Survey of the Plasma Membrane Proteome of a Mammalian Cell.

Membrane proteins play critical roles at the cell surface and their misfunction is a hallmark of many human diseases. A precise evaluation of the plasma membrane proteome is therefore essential for cell biology and for discovering novel biomarkers and therapeutic targets. However, the low abundance of this proteome relative to soluble proteins makes it difficult to characterize, even with the most advanced proteomics technologies. Here, we apply the peptidisc membrane mimetic to purify the cell membrane proteome. Using the HeLa cell line as a reference, we capture 500 different integral membrane proteins, with half annotated to the plasma membrane. Notably, the peptidisc library is enriched with several ABC, SLC, GPCR, CD, and cell adhesion molecules that generally exist at low to very low copy numbers in the cell. We extend the method to compare two pancreatic cell lines, Panc-1 and hPSC. Here we observe a striking difference in the relative abundance of the cell surface cancer markers L1CAM, ANPEP, ITGB4, and CD70. We also identify two novel SLC transporters, SLC30A1 and SLC12A7, that are highly present in the Panc-1 cell only. The peptidisc library thus emerges as an effective way to survey and compare the membrane proteome of mammalian cells. Furthermore, since the method stabilizes membrane proteins in a water-soluble state, members of the library, here SLC12A7, can be specifically isolated.

Francisella opportunistica sp. nov., isolated from human blood and cerebrospinal fluid.

Two isolates of a Gram-negative, non-spore-forming coccobacillus cultured from the blood and cerebrospinal fluid of immunocompromised patients in the United States were described previously. Biochemical and phylogenetic analyses revealed that they belong to a novel species within the Francisella genus. Here we describe a third isolate of this species, recovered from blood of a febrile patient with renal failure, and formally name the Francisella species. Whole genome comparisons indicated the three isolates display greater than 99.9 % average nucleotide identity (ANI) to each other and are most closely related to the tick endosymbiont F. persica, with only 88.6-88.8 % ANI to the type strain of F. persica. Based on biochemical, metabolic and genomic comparisons, we propose that these three isolates should be recognized as Francisella opportunistica sp. nov, with the type strain of the species, PA05-1188T, available through the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM 107100) and the American Type Culture Collection (ATCC BAA-2974).

A second-generation virtual-pinhole PET device for enhancing contrast recovery and improving lesion detectability of a whole-body PET/CT scanner.

PURPOSE: We have developed a second-generation virtual-pinhole (VP) positron emission tomography (PET) device that can position a flat-panel PET detector around a patient's body using a robotic arm to enhance the contrast recovery coefficient (CRC) and detectability of lesions in any region-of-interest using a whole-body PET/computed tomography (CT) scanner. METHODS: We constructed a flat-panel VP-PET device using 32 high-resolution detectors, each containing a 4  ×  4 MPPC array and 16  ×  16 LYSO crystals of 1.0  ×  1.0  ×  3.0 mm3 each. The flat-panel detectors can be positioned around a patient's body anywhere in the imaging field-of-view (FOV) of a Siemens Biograph 40 PET/CT scanner by a robotic arm. New hardware, firmware and software have been developed to support the additional detector signals without compromising a scanner's native functions. We stepped a 22 Na point source across the axial FOV of the scanner to measure the sensitivity profile of the VP-PET device. We also recorded the coincidence events measured by the scanner detectors and by the VP-PET detectors when imaging phantoms of different sizes. To assess the improvement in the CRC of small lesions, we imaged an elliptical torso phantom measuring 316  ×  228  ×  162 mm3 that contains spherical tumors with diameters ranging from 3.3 to 11.4 mm with and without the VP-PET device. Images were reconstructed using a list mode Maximum-Likelihood Estimation-Maximization algorithm implemented on multiple graphics processing units (GPUs) to support the unconventional geometries enabled by a VP-PET system. The mean and standard deviation of the CRC were calculated for tumors of different sizes. Monte Carlo simulation was also conducted to image clusters of lesions in a torso phantom using a PET/CT scanner alone or the same scanner equipped with VP-PET devices. Receiver operating characteristic (ROC) curves were analyzed for three system configurations to evaluate the improvement in lesion detectability by the VP-PET device over the native PET/CT scanner. RESULTS: The repeatability in positioning the flat-panel detectors using a robotic arm is better than 0.15 mm in all three directions. Experimental results show that the average CRC of 3.3, 4.3, and 6.0 mm diameter tumors was 0.82%, 2.90%, and 5.25%, respectively, when measured by the native scanner. The corresponding CRC was 2.73%, 6.21% and 10.13% when imaged by the VP-PET insert device with the flat-panel detector under the torso phantom. These values may be further improved to 4.31%, 9.65% and 18.01% by a future dual-panel VP-PET insert device if DOI detectors are employed to triple its detector efficiency. Monte Carlo simulation results show that the tumor detectability can be improved by a VP-PET device that has a single flat-panel detector. The improvement is greater if the VP-PET device employs a dual-panel design. CONCLUSIONS: We have developed a prototype flat-panel VP-PET device and integrated it with a clinical PET/CT scanner. It significantly enhances the contrast of lesions, especially for those that are borderline detectable by the native scanner, within regions-of-interest specified by users. Simulation demonstrated the enhancement in lesion detectability with the VP-PET device. This technology may become a cost-effective solution for organ-specific imaging tasks.

A Bead-Based Flow Cytometric Assay for Monitoring Yersinia pestis Exposure in Wildlife.

Yersinia pestis is the causative agent of plague and is considered a category A priority pathogen due to its potential for high transmissibility and the significant morbidity and mortality it causes in humans. Y. pestis is endemic to the western United States and much of the world, necessitating programs to monitor for this pathogen on the landscape. Elevated human risk of plague infection has been spatially correlated with spikes in seropositive wildlife numbers, particularly rodent-eating carnivores, which are frequently in contact with the enzootic hosts and the associated arthropod vectors of Y. pestis In this study, we describe a semiautomated bead-based flow cytometric assay developed for plague monitoring in wildlife called the F1 Luminex plague assay (F1-LPA). Based upon Luminex/Bio-Plex technology, the F1-LPA targets serological responses to the F1 capsular antigen of Y. pestis and was optimized to analyze antibodies eluted from wildlife blood samples preserved on Nobuto filter paper strips. In comparative evaluations with passive hemagglutination, the gold standard tool for wildlife plague serodiagnosis, the F1-LPA demonstrated as much as 64× improvement in analytical sensitivity for F1-specific IgG detection and allowed for unambiguous classification of IgG status. The functionality of the F1-LPA was demonstrated for coyotes and other canids, which are the primary sentinels in wildlife plague monitoring, as well as felids and raccoons. Additionally, assay formats that do not require species-specific immunological reagents, which are not routinely available for several wildlife species used in plague monitoring, were determined to be functional in the F1-LPA.

Collection and characterization of samples for establishment of a serum repository for lyme disease diagnostic test development and evaluation.

Serological assays and a two-tiered test algorithm are recommended for laboratory confirmation of Lyme disease. In the United States, the sensitivity of two-tiered testing using commercially available serology-based assays is dependent on the stage of infection and ranges from 30% in the early localized disease stage to near 100% in late-stage disease. Other variables, including subjectivity in reading Western blots, compliance with two-tiered recommendations, use of different first- and second-tier test combinations, and use of different test samples, all contribute to variation in two-tiered test performance. The availability and use of sample sets from well-characterized Lyme disease patients and controls are needed to better assess the performance of existing tests and for development of improved assays. To address this need, the Centers for Disease Control and Prevention and the National Institutes of Health prospectively collected sera from patients at all stages of Lyme disease, as well as healthy donors and patients with look-alike diseases. Patients and healthy controls were recruited using strict inclusion and exclusion criteria. Samples from all included patients were retrospectively characterized by two-tiered testing. The results from two-tiered testing corroborated the need for novel and improved diagnostics, particularly for laboratory diagnosis of earlier stages of infection. Furthermore, the two-tiered results provide a baseline with samples from well-characterized patients that can be used in comparing the sensitivity and specificity of novel diagnostics. Panels of sera and accompanying clinical and laboratory testing results are now available to Lyme disease serological test users and researchers developing novel tests.

Virulence difference between the prototypic Schu S4 strain (A1a) and Francisella tularensis A1a, A1b, A2 and type B strains in a murine model of infection.

BACKGROUND: The use of prototypic strains is common among laboratories studying infectious agents as it promotes consistency for data comparability among and between laboratories. Schu S4 is the prototypic virulent strain of Francisella tularensis and has been used extensively as such over the past six decades. Studies have demonstrated virulence differences among the two clinically relevant subspecies of F. tularensis, tularensis (type A) and holarctica (type B) and more recently between type A subpopulations (A1a, A1b and A2). Schu S4 belongs to the most virulent subspecies of F. tularensis, subspecies tularensis. METHODS: In this study, we investigated the relative virulence of Schu S4 in comparison to A1a, A1b, A2 and type B strains using a temperature-based murine model of infection. Mice were inoculated intradermally and a hypothermic drop point was used as a surrogate for death. Survival curves and the length of temperature phases were compared for all infections. Bacterial burdens were also compared between the most virulent type A subpopulation, A1b, and Schu S4 at drop point. RESULTS: Survival curve comparisons demonstrate that the Schu S4 strain used in this study resembles the virulence of type B strains, and is significantly less virulent than all other type A (A1a, A1b and A2) strains tested. Additionally, when bacterial burdens were compared between mice infected with Schu S4 or MA00-2987 (A1b) significantly higher burdens were present in the blood and spleen of mice infected with MA00-2987. CONCLUSIONS: The knowledge gained from using Schu S4 as a prototypic virulent strain has unquestionably advanced the field of tularemia research. The findings of this study, however, indicate that careful consideration of F. tularensis strain selection must occur when the overall virulence of the strain used could impact the outcome and interpretation of results.

Use of temperature for standardizing the progression of Francisella tularensis in mice.

The study of infectious agents, their pathogenesis, the host response and the evaluation of newly developed countermeasures often requires the use of a living system. Murine models are frequently used to undertake such investigations with the caveat that non-biased measurements to assess the progression of infection are underutilized. Instead, murine models predominantly rely on symptomology exhibited by the animal to evaluate the state of the animal's health and to determine when euthanasia should be performed. In this study, we used subcutaneous temperature as a non-subjective measurement to follow and compare infection in mice inoculated with Francisella tularensis, a Gram-negative pathogen that produces an acute and fatal illness in mice. A reproducible temperature pattern defined by three temperature phases (normal, febrile and hypothermic) was identified in all mice infected with F. tularensis, regardless of the infecting strain. More importantly and for the first time a non-subjective, ethical, and easily determined surrogate endpoint for death based on a temperature, termed drop point, was identified and validated with statistical models. In comparative survival curve analyses for F. tularensis strains with differing virulence, the drop point temperature yielded the same results as those obtained using observed time to death. Incorporation of temperature measurements to evaluate F. tularensis was standardized based on statistical models to provide a new level of robustness for comparative analyses in mice. These findings should be generally applicable to other pathogens that produce acute febrile disease in animal models and offers an important tool for understanding and following the infection process.

Virulence differences among Francisella tularensis subsp. tularensis clades in mice.

Francisella tularensis subspecies tularensis (type A) and holarctica (type B) are of clinical importance in causing tularemia. Molecular typing methods have further separated type A strains into three genetically distinct clades, A1a, A1b and A2. Epidemiological analyses of human infections in the United States suggest that A1b infections are associated with a significantly higher mortality rate as compared to infections caused by A1a, A2 and type B. To determine if genetic differences as defined by molecular typing directly correlate with differences in virulence, A1a, A1b, A2 and type B strains were compared in C57BL/6 mice. Here we demonstrate significant differences between survival curves for infections caused by A1b versus A1a, A2 and type B, with A1b infected mice dying earlier than mice infected with A1a, A2 or type B; these results were conserved among multiple strains. Differences were also detected among type A clades as well as between type A clades and type B with respect to bacterial burdens, and gross anatomy in infected mice. Our results indicate that clades defined within F. tularensis subsp. tularensis by molecular typing methods correlate with virulence differences, with A1b strains more virulent than A1a, A2 and type B strains. These findings indicate type A strains are not equivalent with respect to virulence and have important implications for public health as well as basic research programs.

Acute inflammatory encephalomyelitis following Campylobacter enteritis associated with high titre antiganglioside GM1 IgG antibodies.

Campylobacter enteritis is commonly associated with various forms of the Guillain-Barré syndrome but not central nervous system (CNS) inflammation. We present a case of Campylobacter enteritis associated with acute inflammatory encephalomyelitis and high titre antiganglioside GM1 IgG antibodies. The finding of antiganglioside antibodies in inflammatory demyelination of the CNS may identify avenues for research into pathogenesis. The relationship between antiganglioside antibodies and CNS inflammation is discussed.