Using TLC-MALDI-TOF to Interrogate In Vitro Peptidyl Proximal Preferences of PARP14 and Glycohydrolase Specificity.

The transfer of ADP-ribose (ADPr) from nicotinamide adenine dinucleotide (NAD+) to target proteins is mediated by a class of human diphtheria toxin-like ADP-ribosyltransferases (ARTDs; previously referred to as poly-ADP-ribose polymerases or PARPs) and the removal of ADPr is catalyzed by a family of glycohydrolases. Although thousands of potential ADPr modification sites have been identified using high-throughput mass-spectrometry, relatively little is known about the sequence specificity encoded near the modification site. Herein, we present a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) method that facilitates the in vitro analysis of proximal factors that guide ARTD target selection. We identify a minimal 5-mer peptide sequence that is necessary and sufficient to drive glutamate/aspartate targeting using PARP14 while highlighting the importance of the adjacent residues in PARP14 targeting. We measure the stability of the resultant ester bond and show that non-enzymatic removal is pH and temperature dependent, sequence independent, and occurs within hours. Finally, we use the ADPr-peptides to highlight differential activities within the glycohydrolase family and their sequence preferences. Our results highlight (1) the utility of MALDI-TOF in analyzing proximal ARTD-substrate interactions and (2) the importance of peptide sequences in governing ADPr transfer and removal.

Identification of a Novel PARP14 Site Motif and Glycohydrolase Specificity Using TLC-MALDI-TOF.

Transfer of ADP-ribose (ADPr) from nicotinamide adenine dinucleotide (NAD+) to target proteins is mediated by a class of human poly-ADP-ribose polymerases, PARPs, and removal of ADPr is catalyzed by a family of glycohydrolases. Although thousands of potential ADPr modification sites have been identified using high-throughput mass-spectrometry, relatively little is known about sequence specificity encoded near the modification site. Herein, we present a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) method that facilitates the discovery and validation of ADPr site motifs. We identify a minimal 5-mer peptide sequence that is sufficient to drive PARP14 specific activity while highlighting the importance of the adjacent residues in PARP14 targeting. We measure the stability of the resultant ester bond and show that non-enzymatic removal is sequence independent and occurs within hours. Finally, we use the ADPr-peptide to highlight differential activities within the glycohydrolase family and their sequence specificities. Our results highlight: 1) the utility of MALDI-TOF in motif discovery and 2) the importance of peptide sequence in governing ADPr transfer and removal.

Widespread occurrence of quaternary alkylammonium disinfectants in soils of Hesse, Germany.

Quaternary alkylammonium compounds (QAACs) are cationic organic compounds with amphiphilic properties that are widely used as surfactants and disinfectants in industry, households and agriculture. Several studies suggest that QAACs co-select for antibiotic resistant microorganisms and thus may contribute to the spread of antibiotic resistance in the environment. Data on QAAC occurrence in soil are scarce and limited to soils that are prone to direct exposure to QAACs. Therefore, we conducted a comprehensive study on the occurrence of QAACs in soils of Hesse, a federal state in Germany, covering an area of 21,115 km2. Sixty-five soil samples that comprised different land uses (arable, grassland, forest, vineyard) and area types (rural, agglomeration) were analysed for concentrations of alkyltrimethylammonium (ATMACs, with alkyl chain lengths C8-C16), benzylalkyldimethylammonium (BACs, C8-C18) and dialkyldimethylammonium compounds (DADMACs, C8-C18) via HPLC-MS/MS after ultrasonic-assisted extraction with acidified acetonitrile. QAACs were detected in 97 % of the soil samples irrespective of land use and area type. The most abundant QAAC homologues were DADMACs > BACs > ATMACs. The highest total QAAC concentrations were detected in alluvial soils influenced by the deposition of suspended particles during flood events, with DADMAC-C16 and -C18 as the dominant homologues. The high abundance of long-chain DADMACs suggests that legacy pollution and accumulation govern QAAC concentrations in soils. The presence of QAACs in forest soils points to a potential input via atmospheric deposition. Our work highlights the widespread occurrence of QAACs in soils of Hesse and the need for more research on their entry paths and fate in the soil ecosystem.

Scientific data from precipitation driver response model intercomparison project.

This data descriptor reports the main scientific values from General Circulation Models (GCMs) in the Precipitation Driver and Response Model Intercomparison Project (PDRMIP). The purpose of the GCM simulations has been to enhance the scientific understanding of how changes in greenhouse gases, aerosols, and incoming solar radiation perturb the Earth's radiation balance and its climate response in terms of changes in temperature and precipitation. Here we provide global and annual mean results for a large set of coupled atmospheric-ocean GCM simulations and a description of how to easily extract files from the dataset. The simulations consist of single idealized perturbations to the climate system and have been shown to achieve important insight in complex climate simulations. We therefore expect this data set to be valuable and highly used to understand simulations from complex GCMs and Earth System Models for various phases of the Coupled Model Intercomparison Project.

A Mini-Review of Ixodes Ticks Climate Sensitive Infection Dispersion Risk in the Nordic Region.

Climate change in the Nordic countries is projected to lead to both wetter and warmer seasons. This, in combination with associated vegetation changes and increased animal migration, increases the potential incidence of tick-borne diseases (TBD) where already occurring, and emergence in new places. At the same time, vegetation and animal management influence tick habitat and transmission risks. In this paper, we review the literature on Ixodes ricinus, the primary vector for TBD. Current and projected distribution changes and associated disease transmission risks are related to climate constraints and climate change, and this risk is discussed in the specific context of reindeer management. Our results indicate that climatic limitations for vectors and hosts, and environmental and societal/institutional conditions will have a significant role in determining the spreading of climate-sensitive infections (CSIs) under a changing climate. Management emerges as an important regulatory "tool" for tick and/or risk for disease transfer. In particular, shrub encroachment, and pasture and animal management, are important. The results underscore the need to take a seasonal view of TBD risks, such as (1) grazing and migratory (host) animal presence, (2) tick (vector) activity, (3) climate and vegetation, and (4) land and animal management, which all have seasonal cycles that may or may not coincide with different consequences of climate change on CSI migration. We conclude that risk management must be coordinated across the regions, and with other land-use management plans related to climate mitigation or food production to understand and address the changes in CSI risks.

Fourier transform and Vernier spectroscopy using an optical frequency comb at 3-5.4 µm.

We present a versatile mid-infrared frequency comb spectroscopy system based on a doubly resonant optical parametric oscillator tunable in the 3-5.4 µm range and two detection methods: a Fourier transform spectrometer (FTS) and a continuous-filtering Vernier spectrometer (CF-VS). Using the FTS with a multipass cell, we measure high precision broadband absorption spectra of CH4 at 3.3 µm and NO at 5.25 µm, the latter for the first time with comb spectroscopy, and we detect atmospheric species (CH4, CO, CO2, and H2O) in air in the signal and idler ranges. Multiline fitting yields minimum detectable concentrations of 10-20 ppb Hz-1/2 for CH4, NO, and CO. For the first time in the mid-infrared, we perform CF-VS using an enhancement cavity, a grating, and a single detector, and we measure the absorption spectrum of CH4 and H2O in ambient air at ∼3.3 µm, reaching a 40 ppb concentration detection limit for CH4 in 2 ms.

Association between Thigh Muscle Volume and Leg Muscle Power in Older Women.

The construct of sarcopenia is still discussed with regard to best appropriate measures of muscle volume and muscle function. The aim of this post-hoc analysis of a cross-sectional experimental study was to investigate and describe the hierarchy of the association between thigh muscle volume and measurements of functional performance in older women. Thigh muscle volume of 68 independently living older women (mean age 77.6 years) was measured via magnetic resonance imaging. Isometric strength was assessed for leg extension in a movement laboratory in sitting position with the knee flexed at 90° and for hand grip. Maximum and habitual gait speed was measured on an electronic walk way. Leg muscle power was measured during single leg push and during sit-to-stand performance. Thigh muscle volume was associated with sit-to-stand performance power (r = 0.628), leg push power (r = 0.550), isometric quadriceps strength (r = 0.442), hand grip strength (r = 0.367), fast gait speed (r = 0.291), habitual gait speed (r = 0.256), body mass index (r = 0.411) and age (r = -0.392). Muscle power showed the highest association with thigh muscle volume in healthy older women. Sit-to-stand performance power showed an even higher association with thigh muscle volume compared to single leg push power.

Processes and their explanatory factors governing distribution of organic phosphorous pools in lake sediments.

The amount of organic phosphorus (OP) and its distribution among different pools in lake sediments depends on biotic and abiotic processes driving the OP fractionation. Key environmental factors governing these transformations processes between OP fractionations in sediments were studied on the basis of geochemical characteristics of OP pools in relation to environmental factors in the sediments. The results illustrate that the factors influencing the accumulation or depletion of different OP pools were intrinsically dependent on the composition of the deposited organic matter (OM). During the mineralization of the OM the microorganisms excrete the enzyme alkaline phosphatase, accelerating the OP hydrolysis, and thereby setting the grounds for the bacterially-mediated oxidation of OM. There are two main degradation products of the labile OP pool (LOP) and the moderately labile OP pool (MLOP): Either the OP is transformed to a dissolved organic or inorganic P form, and thereby released to water column, or OP is transformed to a non-labile OP pool and stored in the sediments. A comparative study showed that oxy-hydroxides of iron (Fe) and aluminum (Al) only played an important role in influencing OP fractionation in Lake Wuliangsuhai, while the complexation reactions of OP with calcium ions and sorption to its minerals are key factors governing the OP fractionation in the two alkaline lakes. It is worth noting that a significant correlation between the Fe-P pool and the pools of LOP and MLOP indicates that the degradation of the rather labile OP pools are highly dependent on the iron redox reaction.

Land use as an explanatory factor for potential phosphorus loss risk, assessed by P indices and their governing parameters.

The total level of phosphorus (P) and the distribution of P pools in the topsoil are significantly affected by the excessive application of mineral and organic fertilizers connected with intensive agriculture. This leads to an increased potential risk for P loss, and then contributes to freshwater eutrophication. Soil test P (STP), P sorption index (PSI) and degree of P saturation (DPS) are commonly applied as proxies for assessing the risk of P loss. Although conceptually based, the empirical relationships between these operationally defined proxies and the actual P flux exhibit large spatial variations. Herein, a comprehensive synoptic study and monitoring of soil has been conducted in a watershed in north-eastern China. A set of conventional indicators for soil P loss risk were measured along with the main P pools, P sorption indices, texture, organic matter, as well as Fe and Al oxides and other mineral compositions. Moreover, detailed soil P speciation was conducted using phosphorus nuclear magnetic resonance ((31)P NMR) spectroscopy. In addition, phosphatase activities in the soils were determined for each land use soil category. The results reflected that the soil content of total P, total inorganic P and STP increased significantly following the order of increasing management intensity. STP, being strongly coupled to the application of P fertilizers, was a strong explanatory factor for the spatial differences in DPS - both between and within different land uses. The dominant inorganic and organic P species in the soils were orthophosphate and monoester-P, respectively. Their contents were oppositely correlated with the degree of management influence, with the amount of orthophosphate positively related. Alkaline phosphomonoesterase (AlP) represented the highest activities among the four representative phosphatases, i.e. enzymes that hydrolyze organic P - releasing labile orthophosphate. Orchard soils were found to contain the highest levels of monoester P as well as high AlP activities. This indicates a strong capacity to produce labile orthophosphate. Our results suggest that the type of land use can be employed as a general explanatory factor for considering the potential high risk of loss of P. Regionalized P loss parameters will further improve the accuracy of risk assessment.

An in-depth assessment into simultaneous monitoring of dissolved reactive phosphorus (DRP) and low-molecular-weight organic phosphorus (LMWOP) in aquatic environments using diffusive gradients in thin films (DGT).

Long-term laborious and thus costly monitoring of phosphorus (P) fractions is required in order to provide reasonable estimates of the levels of bioavailable phosphorus for eutrophication studies. A practical solution to this problem is the application of passive samplers, known as Diffusive Gradient in Thin films (DGTs), providing time-average concentrations. DGT, with the phosphate adsorbent Fe-oxide based binding gel, is capable of collecting both orthophosphate and low molecular weight organic phosphorus (LMWOP) compounds, such as adenosine monophosphate (AMP) and myo-inositol hexakisphosphate (IP6). The diffusion coefficient (D) is a key parameter relating the amount of analyte determined from the DGT to a time averaged ambient concentration. D at 20 °C for AMP and IP6 were experimentally determined to be 2.9 × 10(-6) cm(2) s(-1) and 1.0 × 10(-6) cm(2) s(-1), respectively. Estimations by conceptual models of LMWOP uptake by DGTs indicated that this fraction constituted more than 75% of the dissolved organic phosphorus (DOP) accumulated. Since there is no one D for LMWOP, a D range was estimated through assessment of D models. The models tested for estimating D for a variety of common LMWOP molecules proved to be still too uncertain for practical use. The experimentally determined D for AMP and IP6 were therefore used as upper and lower D, respectively, in order to estimate minimum and maximum ambient concentrations of LMWOP. Validation of the DGT data was performed by comparing concentrations of P fractions determined in natural water samples with concentration of P fractions determined using DGT. Stream water draining three catchments with different land-use (forest, mixed and agriculture) showed clear differences in relative and absolute concentrations of dissolved reactive phosphorus (DRP) and dissolved organic P (DOP). There was no significant difference between water sample and DGT DRP (p > 0.05). Moreover, the upper and lower limit D for LMWOP proved reasonable as water sample determined DOP was found to lie in-between the limits of DGT LMWOP concentrations, indicating that on average DOP consists mainly of LMWOP. "Best fit" D was determined for each stream in order to practically use the DGTs for estimating time average DOP. Applying DGT in a eutrophic lake provided insight into P cycling in the water column.

Grading system for the selection of patients with congenital aural atresia for active middle ear implants.

INTRODUCTION: Active middle ear implants (aMEI) are being increasingly used for hearing restoration in congenital aural atresia. The existing gradings used for CT findings do not meet the requirements for these implants. Some items are expendable, whereas other important imaging factors are missing. We aimed to create a new grading system that could describe the extent of the malformation and predict the viability and challenges of implanting an aMEI. METHODS: One hundred three malformed ears were evaluated using HRCT of the temporal bone. The qualitative items middle ear and mastoid pneumatization, oval window, stapes, round window, tegmen mastoideum displacement and facial nerve displacement were included. An anterior- and posterior round window corridor, oval window and stapes corridor were quantified and novelly included. They describe the size of the surgical field and the sight towards the windows. RESULTS: The ears were graded on a 16-point scale (16-13 easy, 12-9 moderate, 8-5 difficult, 4-0 high risk). The strength of agreement between the calculated score and the performed implantations was good. The comparison of the new 16-point scale with the Jahrsdoerfer score showed that both were able to conclusively detect the high-risk group; however, the new 16-point scale was able to further determine which malformed ears were favorable for aMEI, which the Jahrsdoerfer score could not do. CONCLUSION: The Active Middle Ear Implant Score for aural atresia (aMEI score) allows more precise risk stratification and decision making regarding the implantation. The use of operative corridors seems to have significantly better prognostic accuracy than the Jahrsdoerfer score.

Coherent phase lock of a 9 µm quantum cascade laser to a 2 µm thulium optical frequency comb.

We demonstrate coherent phase locking of a room-temperature continuous-wave quantum cascade laser (QCL) at 9.1 µm to a Tm-fiber laser frequency comb centered at 2 µm, with an integrated residual phase error of 0.9 rad (30 mHz to 1.5 MHz). This resulted in a QCL linewidth reduction from 525 to 25 kHz at 1 ms observation time, limited by the linewidth of the free-running frequency comb.

Biophysical modeling of brain tumor progression: from unconditionally stable explicit time integration to an inverse problem with parabolic PDE constraints for model calibration.

PURPOSE: A novel unconditionally stable, explicit numerical method is introduced to the field of modeling brain cancer progression on a tissue level together with an inverse problem (IP) based on optimal control theory that allows for automated model calibration with respect to observations in clinical imaging data. METHODS: Biophysical models of cancer progression on a tissue level are in general based on the assumption that the spatiotemporal spread of cancerous cells is determined by cell division and net migration. These processes are typically described in terms of a parabolic partial differential equation (PDE). In the present work a parallelized implementation of an unconditionally stable, explicit Euler (EE(⋆)) time integration method for the solution of this PDE is detailed. The key idea of the discussed EE(⋆) method is to relax the strong stability requirement on the spectral radius of the coefficient matrix by introducing a subdivision regime for a given outer time step. The performance is related to common implicit numerical methods. To quantify the numerical error, a simplified model that has a closed form solution is considered. To allow for a systematic, phenomenological validation a novel approach for automated model calibration on the basis of observations in medical imaging data is developed. The resulting IP is based on optimal control theory and manifests as a large scale, PDE constrained optimization problem. RESULTS: The numerical error of the EE(⋆) method is at the order of standard implicit numerical methods. The computing times are well below those obtained for implicit methods and by that demonstrate efficiency. Qualitative and quantitative analysis in 12 patients demonstrates that the obtained results are in strong agreement with observations in medical imaging data. Rating simulation success in terms of the mean overlap between model predictions and manual expert segmentations yields a success rate of 75% (9 out of 12 patients). CONCLUSIONS: The discussed EE(⋆) method provides desirable features for image-based model calibration or hybrid image registration algorithms in which the model serves as a biophysical prior. This is due to (i) ease of implementation, (ii) low memory requirements, (iii) efficiency, (iv) a straightforward interface for parameter updates, and (v) the fact that the method is inherently matrix-free. The explicit time integration method is confirmed via experiments for automated model calibration. Qualitative and quantitative analysis demonstrates that the proposed framework allows for recovering observations in medical imaging data and by that phenomenological model validity.

Cytomegalovirus replicon-based regulation of gene expression in vitro and in vivo.

There is increasing evidence for a connection between DNA replication and the expression of adjacent genes. Therefore, this study addressed the question of whether a herpesvirus origin of replication can be used to activate or increase the expression of adjacent genes. Cell lines carrying an episomal vector, in which reporter genes are linked to the murine cytomegalovirus (MCMV) origin of lytic replication (oriLyt), were constructed. Reporter gene expression was silenced by a histone-deacetylase-dependent mechanism, but was resolved upon lytic infection with MCMV. Replication of the episome was observed subsequent to infection, leading to the induction of gene expression by more than 1000-fold. oriLyt-based regulation thus provided a unique opportunity for virus-induced conditional gene expression without the need for an additional induction mechanism. This principle was exploited to show effective late trans-complementation of the toxic viral protein M50 and the glycoprotein gO of MCMV. Moreover, the application of this principle for intracellular immunization against herpesvirus infection was demonstrated. The results of the present study show that viral infection specifically activated the expression of a dominant-negative transgene, which inhibited viral growth. This conditional system was operative in explant cultures of transgenic mice, but not in vivo. Several applications are discussed.

The role of cell types in cytomegalovirus infection in vivo.

Human cytomegalovirus (HCMV) is the major viral cause of morbidity in immune compromised patients and of pre- and perinatal pathology in newborns. The clinical manifestations are highly variable and the principles which govern these differences cannot be understood without detailed knowledge on tissue specific aspects of HCMV infection. For decades the role of individual cell types during cytomegalovirus infection and disease has been discussed. The pathogenesis of mouse cytomegalovirus (MCMV) mirrors the human infection in many aspects. Although only MCMV infection is studied extensively at the level of organs, the relative contribution of specific cell types to viral pathogenesis in vivo has remained enigmatic. Here we discuss new approaches based on the cre/loxP-system to label nascent virus progeny or to lift a replication block. The salient aspect of this technique is the change of viral genome properties selectively in cells that express cre during infection in vivo. This allowed us to study the role of endothelial cells and hepatocytes for virus dissemination and will permit detailed studies on innate and adaptive immune responses to CMV.

M94 is essential for the secondary envelopment of murine cytomegalovirus.

The gene M94 of murine cytomegalovirus (MCMV) as well as its homologues UL16 in alphaherpesviruses is involved in viral morphogenesis. For a better understanding of its role in the viral life cycle, a library of random M94 mutants was generated by modified transposon-based linker scanning mutagenesis. A comprehensive set of M94 mutants was reinserted into the MCMV genome and tested for their capacity to complement the M94 null mutant. Thereby, 34 loss-of-function mutants of M94 were identified, which were tested in a second screen for their capacity to inhibit virus replication. This analysis identified two N-terminal insertion mutants of M94 with a dominant negative effect. We compared phenotypes induced by the conditional expression of these dominant negative M94 alleles with the null phenotype of the M94 deletion. The viral gene expression cascade and the nuclear morphogenesis steps were not affected in either setting. In both cases, however, secondary envelopment did not proceed in the absence of functional M94, and capsids subsequently accumulated in the center of the cytoplasmic assembly complex. In addition, deletion of M94 resulted in a block of cell-to-cell spread. Moreover, the dominant negative mutant of M94 demonstrated a defect in interacting with M99, the UL11 homologue of MCMV.

Metabolic changes in the visual cortex are linked to retinal nerve fiber layer thinning in multiple sclerosis.

OBJECTIVE: To investigate the damage to the retinal nerve fiber layer as part of the anterior visual pathway as well as an impairment of the neuronal and axonal integrity in the visual cortex as part of the posterior visual pathway with complementary neuroimaging techniques, and to correlate our results to patients' clinical symptoms concerning the visual pathway. DESIGN, SUBJECTS AND METHODS: Survey of 86 patients with relapsing-remitting multiple sclerosis that were subjected to retinal nerve fiber layer thickness (RNFLT) measurement by optical coherence tomography, to a routine MRI scan including the calculation of the brain parenchymal fraction (BPF), and to magnetic resonance spectroscopy at 3 tesla, quantifying N-acetyl aspartate (NAA) concentrations in the visual cortex and normal-appearing white matter. RESULTS: RNFLT correlated significantly with BPF and visual cortex NAA, but not with normal-appearing white matter NAA. This was connected with the patients' history of a previous optic neuritis. In a combined model, both BPF and visual cortex NAA were independently associated with RNFLT. CONCLUSIONS: Our data suggest the existence of functional pathway-specific damage patterns exceeding global neurodegeneration. They suggest a strong interrelationship between damage to the anterior and the posterior visual pathway.

Structural brain changes following peripheral vestibulo-cochlear lesion may indicate multisensory compensation.

BACKGROUND: Do central mechanisms account for the variability of clinical recovery following peripheral vestibulo-cochlear lesions? OBJECTIVE: To investigate structural (morphological) plasticity in the human brain following unilateral vestibulo-cochlear lesions which might contribute to central vestibular compensation. METHODS: The authors compared regional grey matter volume (GMV) changes in patients after surgical removal of unilateral acoustic neuroma with age-matched control subjects, and hypothesised morphometric changes in the vestibular and auditory cortices which may be related to functional disability scores. Patients were examined with a battery of neuro-otological tests and clinical scores to assess vestibular and auditory disability. RESULTS: Voxel-based morphometry was used for categorical comparison between patients and age- and gender-matched controls. GMV increase was found bilaterally in primary somatosensory cortices and motion-sensitive areas in the medial temporal gyrus (MT). Simple regression analysis revealed a GMV increase (1) in the contralesional superior temporal gyrus/posterior insula to be correlated with decreasing clinically assessed vestibular deficits; (2) in the contralesional inferior parietal lobe with decreasing functional impairment of daily living activities; and (3) in the contralesional auditory cortex (Heschl gyrus) with decreasing hearing impairment. CONCLUSIONS: These data may suggest structural cortical plasticity in multisensory vestibular cortex areas of patients with unilateral peripheral vestibulo-cochlear lesion after surgical removal of acoustic neuroma. As changes of GMV were related to vestibular function, structural brain changes may reflect central mechanisms of vestibular compensation.

Cavity-enhanced resonant photoacoustic spectroscopy with optical feedback cw diode lasers: A novel technique for ultratrace gas analysis and high-resolution spectroscopy.

Cavity-enhanced resonant photoacoustic spectroscopy with optical feedback cw diode lasers (OF-CERPAS) is introduced as a novel technique for ultratrace gas analysis and high-resolution spectroscopy. In the scheme, a single-mode cw diode laser (3 mW, 635 nm) is coupled into a high-finesse linear cavity and stabilized to the cavity by optical feedback. Inside the cavity, a build-up of laser power to at least 2.5 W occurs. Absorbing gas phase species inside the cavity are detected with high sensitivity by the photoacoustic effect using a microphone embedded in the cavity. To increase sensitivity further, coupling into the cavity is modulated at a frequency corresponding to a longitudinal resonance of an organ pipe acoustic resonator (f=1.35 kHz and Q approximately 10). The technique has been characterized by measuring very weak water overtone transitions near 635 nm. Normalized noise-equivalent absorption coefficients are determined as alpha approximately 4.4x10(-9) cm(-1) s(1/2) (1 s integration time) and 2.6x10(-11) cm(-1) s(1/2) W (1 s integration time and 1 W laser power). These sensitivities compare favorably with existing state-of-the-art techniques. As an advantage, OF-CERPAS is a "zero-background" method which increases selectivity and sensitivity, and its sensitivity scales with laser power.

A spread-deficient cytomegalovirus for assessment of first-target cells in vaccination.

Human cytomegalovirus (HCMV) is a human pathogen that causes severe disease primarily in the immunocompromised or immunologically immature individual. To date, no vaccine is available. We describe use of a spread-deficient murine CMV (MCMV) as a novel approach for betaherpesvirus vaccination. To generate a spread-deficient MCMV, the conserved, essential gene M94 was deleted. Immunization with MCMV-DeltaM94 is apathogenic and protective against wild-type challenge even in highly susceptible IFNalphabetaR(-/-) mice. MCMV-DeltaM94 was able to induce a robust CD4(+) and CD8(+) T-cell response as well as a neutralizing antibody response comparable to that induced by wild-type infection. Endothelial cells were identified as activators of CD8(+) T cells in vivo. Thus, the vaccination with a spread-deficient betaherpesvirus is a safe and protective strategy and allows the linkage between cell tropism and immunogenicity. Furthermore, genomes of MCMV-DeltaM94 were present in lungs 12 months after infection, revealing first-target cells as sites of genome maintenance.