Management of Cytomegalovirus, Epstein-Barr Virus, and HIV Viral Load Quality Control Data Using Unity Real Time.

Quality control (QC) rules (Westgard rules) are applied to viral load testing to identify runs that should be reviewed or repeated, but this requires balancing the patient safety benefits of error detection with the cost and inefficiency of false rejection. In this study, we identified the total allowable errors (TEa) from the literature and utilized a commercially available software program (Unity Real Time; Bio-Rad Laboratories) to manage QC data, assess assay performance, and provide QC decision support for both FDA-approved/cleared (Abbott cytomegalovirus [CMV] and HIV viral load) as well as laboratory-developed (Epstein-Barr virus [EBV] viral load) assays. Unity Real Time was used to calculate means, standard deviations (SDs), and coefficient of variation (CV; in percent) of negative, low-positive, and high-positive control data from 73 to 83 days of testing. Sigma values were calculated to measure the test performance relative to a TEa of 0.5 log10. The sigma value of 5.06 for EBV predicts ∼230 erroneous results per million individual patient tests (0.02% frequency), whereas sigma values of >6 for CMV (11.32) and HIV (7.66) indicate <4 erroneous results per million individual patient tests. The Unity Real Time QC Design module utilized these sigma values to recommend QC rules and provided objective evidence for loosening the laboratory's existing QC rules for run acceptability, potentially reducing false rejection rates by 10-fold for the assay with the most variation (EBV viral load). This study provides a framework for laboratories, with Unity Real Time as a tool, to evaluate assay performance relative to clinical decision points and establish optimal rules for routine monitoring of molecular viral load assay performance.

Polarization spectroscopy of high-order harmonic generation in gallium arsenide.

An interesting property of high harmonic generation in solids is its laser polarization dependent nature which in turn provides information about the crystal and band structure of the generation medium. Here we report on the linear polarization dependence of high-order harmonic generation from a gallium arsenide crystal. Interestingly, we observe a significant evolution of the anisotropic response of above bandgap harmonics as a function of the laser intensity. We attribute this change to fundamental microscopic effects of the emission process comprising a competition between intraband and interband dynamics. This intensity dependence of the anisotropic nature of the generation process offers the possibility to drive and control the electron current along preferred directions of the crystal, and could serve as a switching technique in an integrated all-solid-state petahertz optoelectronic device.

Self-probed ptychography from semiconductor high-harmonic generation.

We demonstrate a method to image an object using a self-probing approach based on semiconductor high-harmonic generation. On the one hand, ptychography enables high-resolution imaging from the coherent light diffracted by an object. On the other hand, high-harmonic generation from crystals is emerging as a new source of extreme-ultraviolet ultrafast coherent light. We combine these two techniques by performing ptychography measurements with nanopatterned crystals serving as the object as well as the generation medium of the harmonics. We demonstrate that this strong field in situ approach can provide structural information about an object. With the future developments of crystal high harmonics as a compact short-wavelength light source, our demonstration can be an innovative approach for nanoscale imaging of photonic and electronic devices in research and industry.

Characterization of a novel transitional group Rickettsia species (Rickettsia tillamookensis sp. nov.) from the western black-legged tick, Ixodes pacificus.

A previously unrecognized Rickettsia species was isolated in 1976 from a pool of Ixodes pacificus ticks collected in 1967 from Tillamook County, Oregon, USA. The isolate produced low fever and mild scrotal oedema following intraperitoneal injection into male guinea pigs (Cavia porcellus). Subsequent serotyping characterized this isolate as distinct from recognized typhus and spotted fever group Rickettsia species; nonetheless, the isolate remained unevaluated by molecular techniques and was not identified to species level for the subsequent 30 years. Ixodes pacificus is the most frequently identified human-biting tick in the western United States, and as such, formal identification and characterization of this potentially pathogenic Rickettsia species is warranted. Whole-genome sequencing of the Tillamook isolate revealed a genome 1.43 Mbp in size with 32.4 mol% G+C content. Maximum-likelihood phylogeny of core proteins places it in the transitional group of Rickettsia basal to both Rickettsia felis and Rickettsia asembonensis. It is distinct from existing named species, with maximum average nucleotide identity of 95.1% to R. asembonensis and maximum digital DNA-DNA hybridization score similarity to R. felis at 80.1%. The closest similarity at the 16S rRNA gene (97.9%) and sca4 (97.5%/97.6% respectively) is to Candidatus 'Rickettsia senegalensis' and Rickettsia sp. cf9, both isolated from cat fleas (Ctenocephalides felis). We characterized growth at various temperatures and in multiple cell lines. The Tillamook isolate grows aerobically in Vero E6, RF/6A and DH82 cells, and growth is rapid at 28 °C and 32 °C. Using accepted genomic criteria, we propose the name Rickettsia tillamookensis sp. nov., with the type strain Tillamook 23. Strain Tillamook 23 is available from the Centers for Disease Control and Prevention Rickettsial Isolate Reference Collection (WDCM 1093), Atlanta, GA, USA (CRIRC accession number RTI001T) and the Collection de Souches de l'Unité des Rickettsies (WDCM 875), Marseille, France (CSUR accession number R5043). Using accepted genomic criteria, we propose the name Rickettsia tillamookensis sp. nov., with the type strain Tillamook 23 (=CRIRC RTI001=R5043).

Pathogenesis, Host Innate Immune Response, and Aerosol Transmission of Influenza D Virus in Cattle.

The recently discovered influenza D virus (IDV) of the Orthomyxoviridae family has been detected in swine and ruminants with a worldwide distribution. Cattle are considered to be the primary host and reservoir, and previous studies suggested a tropism of IDV for the upper respiratory tract and a putative role in the bovine respiratory disease complex. This study aimed to characterize the pathogenicity of IDV in naive calves as well as the ability of this virus to transmit by air. Eight naive calves were infected by aerosol with a recent French isolate, D/bovine/France/5920/2014. Results show that IDV replicates not only in the upper respiratory tract but also in the lower respiratory tract (LRT), inducing moderate bronchopneumonia with restricted lesions of interstitial pneumonia. Inoculation was followed by IDV-specific IgG1 production as early as 10 days postchallenge and likely both Th1 and Th2 responses. Study of the innate immune response in the LRT of IDV-infected calves indicated the overexpression of pathogen recognition receptors and of chemokines CCL2, CCL3, and CCL4, but without overexpression of genes involved in the type I interferon pathway. Finally, virological examination of three aerosol-sentinel animals, housed 3 m apart from inoculated calves (and thus subject to infection by aerosol transmission), and IDV detection in air samples collected in different areas showed that IDV can be airborne transmitted and infect naive contact calves on short distances. This study suggests that IDV is a respiratory virus with moderate pathogenicity and probably a high level of transmission. It consequently can be considered predisposing to or a cofactor of respiratory disease.IMPORTANCE Influenza D virus (IDV), a new genus of the Orthomyxoviridae family, has a broad geographical distribution and can infect several animal species. Cattle are so far considered the primary host for IDV, but the pathogenicity and the prevalence of this virus are still unclear. We demonstrated that under experimental conditions (in a controlled environment and in the absence of coinfecting pathogens), IDV is able to cause mild to moderate disease and targets both the upper and lower respiratory tracts. The virus can transmit by direct as well as aerosol contacts. While this study evidenced overexpression of pathogen recognition receptors and chemokines in the lower respiratory tract, IDV-specific IgG1 production as early as 10 days postchallenge, and likely both Th1 and Th2 responses, further studies are warranted to better understand the immune responses triggered by IDV and its role as part of the bovine respiratory disease complex.

Orbital angular momentum from semiconductor high-order harmonics.

Light beams carrying orbital angular momentum (OAM) have led to stunning applications in various fields from quantum information to microscopy. We examine OAM from the recently observed high-harmonic generation (HHG) in semiconductor crystals. HHG from solids could be a valuable approach for integrated high-flux short-wavelength coherent light sources. First, we verify the transfer and conservation of the OAM in the strong-field regime of interaction from the generation laser to the harmonics. Secondly, we create OAM beams by etching a spiral zone structure directly at the surface of a zinc oxide crystal. Such diffractive optics act on the generated harmonics and produces focused optical vortices with sub-micrometric size.

Complete Characterization of Phase and Amplitude of Bichromatic Extreme Ultraviolet Light.

Intense, mutually coherent beams of multiharmonic extreme ultraviolet light can now be created using seeded free-electron lasers, and the phase difference between harmonics can be tuned with attosecond accuracy. However, the absolute value of the phase is generally not determined. We present a method for determining precisely the absolute phase relationship of a fundamental wavelength and its second harmonic, as well as the amplitude ratio. Only a few easily calculated theoretical parameters are required in addition to the experimental data.

Leech (Myzobdella lugubris) infestations in largemouth bass (Micropterus salmoides) in Back Bay, Virginia, USA.

Back Bay is an oligohaline, coastal bay in southeast Virginia, USA. Since 2004, leeches have been observed in the oral cavities of largemouth bass (Micropterus salmoides) in this body of water. Leeches (Myzobdella lugubris) have previously been documented in the oral cavities of largemouth bass in the Currituck Sound, which is confluent with Back Bay on its southern border. Supplemental stocking of largemouth bass in Back Bay since 2009 has resulted in an increasing population; however, concern exists that leech infestation may be negatively affecting health of larger fish, which are still less abundant than expected. Despite the wide distribution of this leech, there is little available literature regarding its health impacts on hosts. In this study, we examine potential impacts of oral leech infestations on stress markers and haematological parameters of largemouth bass in Back Bay. No significant changes in plasma glucose or cortisol were observed between leech-infested and uninfested fish, and haematological parameters were not significantly different between the groups. Further, there was no evidence of systemic infections associated with leech infestation.

Early Evidence of Sepsis-Associated Hyperperfusion-A Study of Cerebral Blood Flow Measured With MRI Arterial Spin Labeling in Critically Ill Septic Patients and Control Subjects.

OBJECTIVES: Mechanisms underlying sepsis-associated encephalopathy remain unclear, but reduced cerebral blood flow, alone or in conjunction with altered autoregulation, is reported as a potential contributor. We compared cerebral blood flow of control subjects and vasopressor-dependent septic patients. DESIGN: Randomized crossover study. SETTING: MRI with arterial spin labeling. PATIENTS: Ten sedated septic patients on mechanical ventilation (four with controlled chronic hypertension) and 12 control subjects (six with controlled chronic hypertension) were enrolled. Mean ± SD ages were 61.4 ± 10.2 and 44.2 ± 12.8 years, respectively (p = 0.003). Mean Acute Physiology and Chronic Health Evaluation II score of septic patients at ICU admission was 27.7 ± 6.6. INTERVENTIONS: To assess the potential confounding effects of sedation and mean arterial pressure, we measured cerebral blood flow with and without sedation with propofol in control subjects and at a target mean arterial pressure of 65 mm Hg and greater than or equal to 75 mm Hg in septic patients. The sequence of sedation versus no sedation and mean arterial pressure targets were randomized. MEASUREMENTS AND MAIN RESULTS: In septic patients, cerebral blood flow measured at a mean arterial pressure target of 65 mm Hg (40.4 ± 10.9 mL/100 g/min) was not different from cerebral blood flow measured at a mean arterial pressure target of greater than or equal to 75 mm Hg (41.3 ± 9.8 mL/100 g/min; p = 0.65). In control subjects, we observed no difference in cerebral blood flow measured without and with sedation (24.8 ± 4.2 vs 24.9 ± 5.9 mL/100 g/min; p = 0.93). We found no interaction between chronic hypertension and the effect of sedation or mean arterial pressure targets. Cerebral blood flow measured in sedated septic patients (mean arterial pressure target 65 mm Hg) was 62% higher than in sedated control subjects (p = 0.001). CONCLUSIONS: In septic patients, cerebral blood flow was higher than in sedated control subjects and did not vary with mean arterial pressure targets. Further research is required to understand the clinical significance of cerebral hyperperfusion in septic patients on vasopressors and to reassess the neurologic effects of current mean arterial pressure targets in sepsis.

High resolution beam profiling of X-ray free electron laser radiation by polymer imprint development.

High resolution metrology of beam profiles is presently a major challenge at X-ray free electron lasers. We demonstrate a characterization method based on beam imprints in poly (methyl methacrylate). By immersing the imprints formed at 47.8 eV into organic solvents, the regions exposed to the beam are removed similar to resist development in grayscale lithography. This allows for extending the sensitivity of the method by more than an order of magnitude compared to the established analysis of imprints created solely by ablation. Applying the Beer-Lambert law for absorption, the intensity distribution in a micron-sized focus can be reconstructed from one single shot with a high dynamic range, exceeding 103. The procedure described here allows for beam characterization at free electron lasers revealing even faint beam tails, which are not accessible when using ablation imprint methods. We demonstrate the greatly extended dynamic range on developed imprints taken in focus of conventional Fresnel zone plates and spiral zone plates producing beams with a topological charge.

Impact of disease on the survival of three commercially fished species.

Recent increases in emergent infectious diseases have raised concerns about the sustainability of some marine species. The complexity and expense of studying diseases in marine systems often dictate that conservation and management decisions are made without quantitative data on population-level impacts of disease. Mark-recapture is a powerful, underutilized, tool for calculating impacts of disease on population size and structure, even in the absence of etiological information. We applied logistic regression models to mark-recapture data to obtain estimates of disease-associated mortality rates in three commercially important marine species: snow crab (Chionoecetes opilio) in Newfoundland, Canada, that experience sporadic epizootics of bitter crab disease; striped bass (Morone saxatilis) in the Chesapeake Bay, USA, that experience chronic dermal and visceral mycobacteriosis; and American lobster (Homarus americanus) in the Southern New England stock, that experience chronic epizootic shell disease. All three diseases decreased survival of diseased hosts. Survival of diseased adult male crabs was 1% (0.003-0.022, 95% CI) that of uninfected crabs indicating nearly complete mortality of infected crabs in this life stage. Survival of moderately and severely diseased striped bass (which comprised 15% and 11% of the population, respectively) was 84% (70-100%, 95% CI), and 54% (42-68%, 95% CI) that of healthy striped bass. The disease-adjusted yearly natural mortality rate for striped bass was 0.29, nearly double the previously accepted value, which did not include disease. Survival of moderately and severely diseased lobsters was 30% (15-60%, 95% CI) that of healthy lobsters and survival of mildly diseased lobsters was 45% (27-75%, 95% CI) that of healthy lobsters. High disease mortality in ovigerous females may explain the poor recruitment and rapid declines observed in this population. Stock assessments should account for disease-related mortality when resource management options are evaluated.

ISQuest: finding insertion sequences in prokaryotic sequence fragment data.

MOTIVATION: Insertion sequences (ISs) are transposable elements present in most bacterial and archaeal genomes that play an important role in genomic evolution. The increasing availability of sequenced prokaryotic genomes offers the opportunity to study ISs comprehensively, but development of efficient and accurate tools is required for discovery and annotation. Additionally, prokaryotic genomes are frequently deposited as incomplete, or draft stage because of the substantial cost and effort required to finish genome assembly projects. Development of methods to identify IS directly from raw sequence reads or draft genomes are therefore desirable. Software tools such as Optimized Annotation System for Insertion Sequences and IScan currently identify IS elements in completely assembled and annotated genomes; however, to our knowledge no methods have been developed to identify ISs from raw fragment data or partially assembled genomes. We have developed novel methods to solve this computationally challenging problem, and implemented these methods in the software package ISQuest. This software identifies bacterial ISs and their sequence elements-inverted and direct repeats-in raw read data or contigs using flexible search parameters. ISQuest is capable of finding ISs in hundreds of partially assembled genomes within hours, making it a valuable high-throughput tool for a global search of IS elements. We tested ISQuest on simulated read libraries of 3810 complete bacterial genomes and plasmids in GenBank and were capable of detecting 82% of the ISs and transposases annotated in GenBank with 80% sequence identity. CONTACT: abiswas@cs.odu.edu.

Generation of Phase-Locked Pulses from a Seeded Free-Electron Laser.

In a coherent control experiment, light pulses are used to guide the real-time evolution of a quantum system. This requires the coherence and the control of the pulses' electric-field carrier waves. In this work, we use frequency-domain interferometry to demonstrate the mutual coherence of time-delayed pulses generated by an extreme ultraviolet seeded free-electron laser. Furthermore, we use the driving seed laser to lock and precisely control the relative phase between the two free-electron laser pulses. This new capability opens the way to a multitude of coherent control experiments, which will take advantage of the high intensity, short wavelength, and short duration of the pulses generated by seeded free-electron lasers.

Spectral-phase interferometry for direct electric-field reconstruction applied to seeded extreme-ultraviolet free-electron lasers.

We present a setup for complete characterization of femtosecond pulses generated by seeded free-electron lasers (FELs) in the extreme-ultraviolet spectral region. Two delayed and spectrally shifted replicas are produced and used for spectral phase interferometry for direct electric field reconstruction (SPIDER). We show that it can be achieved by a simple arrangement of the seed laser. Temporal shape and phase obtained in FEL simulations are well retrieved by SPIDER reconstructions, allowing to foresee the implementation of this diagnostics tool on existing and future sources. This will be a significant step towards an experimental investigation and control of FEL spectral phase.

Spectrotemporal shaping of seeded free-electron laser pulses.

We demonstrate the ability to control and shape the spectrotemporal content of extreme-ultraviolet (XUV) pulses produced by a seeded free-electron laser (FEL). The control over the spectrotemporal properties of XUV light was achieved by precisely manipulating the linear frequency chirp of the seed laser. Our results agree with existing theory, which allows us to retrieve the temporal properties (amplitude and phase) of the FEL pulse from measurements of the spectra as a function of the FEL operating parameters. Furthermore, we show the first direct evidence of the full temporal coherence of FEL light and generate Fourier limited pulses by fine-tuning the FEL temporal phase. The possibility of tailoring the spectrotemporal content of intense short-wavelength pulses represents the first step towards efficient nonlinear optics in the XUV to x-ray spectral region and will enable precise manipulation of core-electron excitations using the methods of coherent quantum control.

Multiplexing with three-primer PCR for rapid and economical microsatellite validation.

The next generation sequencing revolution has enabled rapid discovery of genetic markers, however, development of fully functioning new markers still requires a long and costly process of marker validation. This study reports a rapid and economical approach for the validation and deployment of polymorphic microsatellite markers obtained from a 454 pyrosequencing library of Atlantic cod, Gadus morhua, Linnaeus 1758. Primers were designed from raw reads to amplify specific amplicon size ranges, allowing effective PCR multiplexing. Multiplexing was combined with a three-primer PCR approach using four universal tails to label amplicons with separate fluorochromes. A total of 192 primer pairs were tested, resulting in 73 polymorphic markers. Of these, 55 loci were combined in six multiplex panels each containing between six and eleven markers. Variability of the loci was assessed on G. morhua from the Celtic Sea (n = 46) and the Scotian Shelf (n = 46), two locations that have shown genetic differentiation in previous studies. Multilocus F(ST) between the two samples was estimated at 0.067 (P = 0.001). After three loci potentially under selection were excluded, the global F(ST) was estimated at 0.043 (P = 0.001). Our technique combines three-primer and multiplex PCR techniques, allowing simultaneous screening and validation of relatively large numbers of microsatellite loci.

Temperature, hypoxia, and mycobacteriosis: effects on adult striped bass Morone saxatilis metabolic performance.

Mycobacteriosis, a chronic bacterial disease of fishes, is prevalent in adult striped bass from Chesapeake Bay (USA). Although environmental factors may play a role in disease expression, the interaction between the disease and environmental stress remains unexplored. We therefore examined the individual and interactive effects of elevated temperature, hypoxia, and mycobacteriosis on the metabolism of wild-caught adult striped bass from Chesapeake Bay using respirometry. Because the spleen is the primary target organ of mycobacteriosis in striped bass, we hypothesized that the disease interferes with the ability of fish to increase their hematocrit in the face of increasing oxygen demands. We determined standard metabolic rate (SMR), maximum metabolic rate under normoxia (MMRN), critical oxygen saturation (S(crit)), and MMR under hypoxia (3 mg O(2) l-1: MMR(H)) for healthy and visibly diseased fish (i.e. exhibiting skin lesions indicative of mycobacteriosis). Measurements were taken at a temperature within the preferred thermal range (20°C) and at an elevated temperature (28°C) considered stressful to striped bass. In addition, we calculated aerobic scope (AS(N) = MMR(N) - SMR, AS(H) = MMR(H) - SMR) and factorial scope (FS(N) = MMR(N) SMR-1, FS(H) = MMR(H) SMR-1). SMR increased with increasing temperature, and hypoxia reduced MMR, AS, and FS. Mycobacteriosis alone did not affect either MMR(N) or MMR(H). However, elevated temperature affected the ability of diseased striped bass to tolerate hypoxia (S(crit)). Overall, our data indicate that striped bass performance under hypoxia is impaired, and that elevated water temperatures, hypoxia, and severe mycobacteriosis together reduce aerobic scope more than any of these stressors acting alone. We conclude that the scope for activity of diseased striped bass in warm hypoxic waters is significantly compromised.

Two-colour generation in a chirped seeded free-electron laser: a close look.

We present the experimental demonstration of a method for generating two spectrally and temporally separated pulses by an externally seeded, single-pass free-electron laser operating in the extreme-ultraviolet spectral range. Our results, collected on the FERMI@Elettra facility and confirmed by numerical simulations, demonstrate the possibility of controlling both the spectral and temporal features of the generated pulses. A free-electron laser operated in this mode becomes a suitable light source for jitter-free, two-colour pump-probe experiments.