De novo transcriptome assembly of the oak processionary moth Thaumetopoea processionea.

OBJECTIVES: The oak processionary moth (OPM) (Thaumetopoea processionea) is a species of moth (order: Lepidoptera) native to parts of central Europe. However, in recent years, it has become an invasive species in various countries, particularly in the United Kingdom and the Netherlands. The larvae of the OPM are covered with urticating barbed hairs (setae) causing irritating and allergic reactions at the three last larval stages (L3-L5). The aim of our study was to generate a de novo transcriptomic assembly for OPM larvae by including one non-allergenic stage (L2) and two allergenic stages (L4 and L5). A transcriptomic assembly will help identify potential allergenic peptides produced by OPM larvae, providing valuable information for developing novel therapeutic strategies and allergic immunodiagnostic assays. DATA: Transcriptomes of three larval stages of the OPM were de novo assembled and annotated using Trinity and Trinotate, respectively. A total of 145,251 transcripts from 99,868 genes were identified. Bench-marking universal single-copy orthologues analysis indicated high completeness of the assembly. About 19,600 genes are differentially expressed between the non-allergenic and allergenic larval stages. The data provided here contribute to the characterization of OPM, which is both an invasive species and a health hazard.

Single-material beam hardening correction via an analytical energy response model for diagnostic CT.

BACKGROUND: Various clinical studies show the potential for a wider quantitative role of diagnostic X-ray computed tomography (CT) beyond size measurements. Currently, the clinical use of attenuation values is, however, limited due to their lack of robustness. This issue can be observed even on the same scanner across patient size and positioning. There are different causes for the lack of robustness in the attenuation values; one possible source of error is beam hardening of the X-ray source spectrum. The conventional and well-established approach to address this issue is a calibration-based single material beam hardening correction (BHC) using a water cylinder. PURPOSE: We investigate an alternative approach for single-material BHC with the aim of producing a more robust result for the attenuation values. The underlying hypothesis of this investigation is that calibration-based BHC automatically corrects for scattered radiation in a manner that is suboptimal in terms of bias as soon as the scanned object strongly deviates from the water cylinder used for calibration. METHODS: The approach we propose performs BHC via an analytical energy response model that is embedded into a correction pipeline that efficiently estimates and subtracts scattered radiation in a patient-specific manner prior to BHC. The estimation of scattered radiation is based on minimizing, in average, the squared difference between our corrected data and the vendor-calibrated data. The used energy response model is considering the spectral effects of the detector response and the prefiltration of the source spectrum, including a beam-shaping bowtie filter. The performance of the correction pipeline is first characterized with computer simulated data. Afterward, it is tested using real 3-D CT data sets of two different phantoms, with various kV settings and phantom positions, assuming a circular data acquisition. The results are compared in the image domain to those from the scanner. RESULTS: For experiments with a water cylinder, the proposed correction pipeline leads to similar results as the vendor. For reconstructions of a QRM liver phantom with extension ring, the proposed correction pipeline achieved a more uniform and stable outcome in the attenuation values of homogeneous materials within the phantom. For example, the root mean squared deviation between centered and off-centered phantom positioning was reduced from 6.6 to 1.8 HU in one profile. CONCLUSIONS: We have introduced a patient-specific approach for single-material BHC in diagnostic CT via the use of an analytical energy response model. This approach shows promising improvements in terms of robustness of attenuation values for large patient sizes. Our results contribute toward improving CT images so as to make CT attenuation values more reliable for use in clinical practice.

Human Lentiviral Gene Therapy Restores the Cellular Phenotype of Autosomal Recessive Complete IFN-γR1 Deficiency.

Autosomal recessive (AR) complete interferon-γ receptor 1 (IFN-γR1) deficiency, also known as one genetic etiology of Mendelian susceptibility to mycobacterial disease (MSMD), is a life-threatening congenital disease leading to premature death. Affected patients present a pathognomonic predisposition to recurrent and severe infections with environmental mycobacteria or the Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine. Current therapeutic options are limited to antibiotic treatment and hematopoietic stem cell transplantation, however with poor outcome. Given the clinical success of gene therapy, we introduce the first lentiviral-based gene therapy approach to restore expression and function of the human IFN-γR-downstream signaling cascade. In our study, we developed lentiviral vectors constitutively expressing the human IFN-γR1 and demonstrate stable transgene expression without interference with cell viability and proliferation in transduced human hematopoietic cells. Using an IFN-γR1-deficient HeLa cell model, we show stable receptor reconstitution and restored IFN-γR1 signaling without adverse effect on cell functionality. Transduction of both SV40-immortalized and primary fibroblasts derived from IFN-γR1-deficient MSMD patients was able to recover IFN-γR1 expression and restore type II IFN signaling upon stimulation with IFN-γ. In summary, we highlight lentiviral vectors to correct the IFN-γ mediated immunity and present the first gene therapy approach for patients suffering from AR complete IFN-γR1 deficiency.

Impact of the non-negativity constraint in model-based iterative reconstruction from CT data.

PURPOSE: Model-based iterative reconstruction is a promising approach to achieve dose reduction without affecting image quality in diagnostic x-ray computed tomography (CT). In the problem formulation, it is common to enforce non-negative values to accommodate the physical non-negativity of x-ray attenuation. Using this a priori information is believed to be beneficial in terms of image quality and convergence speed. However, enforcing non-negativity imposes limitations on the problem formulation and the choice of optimization algorithm. For these reasons, it is critical to understand the value of the non-negativity constraint. In this work, we present an investigation that sheds light on the impact of this constraint. METHODS: We primarily focus our investigation on the examination of properties of the converged solution. To avoid any possibly confounding bias, the reconstructions are all performed using a provably converging algorithm started from a zero volume. To keep the computational cost manageable, an axial CT scanning geometry with narrow collimation is employed. The investigation is divided into five experimental studies that challenge the non-negativity constraint in various ways, including noise, beam hardening, parametric choices, truncation, and photon starvation. These studies are complemented by a sixth one that examines the effect of using ordered subsets to obtain a satisfactory approximate result within 50 iterations. All studies are based on real data, which come from three phantom scans and one clinical patient scan. The reconstructions with and without the non-negativity constraint are compared in terms of image similarity and convergence speed. In select cases, the image similarity evaluation is augmented with quantitative image quality metrics such as the noise power spectrum and closeness to a known ground truth. RESULTS: For cases with moderate inconsistencies in the data, associated with noise and bone-induced beam hardening, our results show that the non-negativity constraint offers little benefit. By varying the regularization parameters in one of the studies, we observed that sufficient edge-preserving regularization tends to dilute the value of the constraint. For cases with strong data inconsistencies, the results are mixed: the constraint can be both beneficial and deleterious; in either case, however, the difference between using the constraint or not is small relative to the overall level of error in the image. The results with ordered subsets are encouraging in that they show similar observations. In terms of convergence speed, we only observed one major effect, in the study with data truncation; this effect favored the use of the constraint, but had no impact on our ability to obtain the converged solution without constraint. CONCLUSIONS: Our results did not highlight the non-negativity constraint as being strongly beneficial for diagnostic CT imaging. Altogether, we thus conclude that in some imaging scenarios, the non-negativity constraint could be disregarded to simplify the optimization problem or to adopt other forward projection models that require complex optimization machinery to be used together with non-negativity.

[Carpal tunnel syndrome and ATTR-amyloidosis]. / Karpaltunnelsyndrom und ATTR-Amyloidose.

BACKGROUND: Carpal tunnel syndrome is the most common compression syndrome of a peripheral nerve. It mostly affects patients older than 50 years. One cause for a carpal tunnel syndrome is transthyretin (ATTR) amyloid, which deposits in the carpal tunnel tissue. Carpal tunnel syndrome can be the first symptom of ATTR amyloidosis, which in the worst case leads to amyloid cardiomyopathy with the symptoms of heart failure with reduction in quality and time of life. For this article, all histological tissue samples of carpal tunnel tissue, collected from 2010 to 2018, were evaluated according to age and gender of each patient. Evaluation of the ATTR amyloid content in different regions of the carpal tunnel has enabled a recommendation for resection for optimal histological diagnosis. MATERIAL AND METHODS: In the Amyloid Registry Kiel, all cases are archived according to type of amyloid and available tissue. We evaluated 582 resected tissue samples of patients with ATTR amyloid in the carpal tunnel, collected from 2010 to the beginning of 2018. In addition, amyloid load of two different regions of the carpal tunnel (synovial tissue and tissue of the flexor retinaculum) were compared. RESULTS: The majority of resections came from women (53 %). The median age was 78 years for the entire collective, 77 years for men and 79 years for women. Specimens of the flexor retinaculum contained significantly more amyloid (9.66 % amyloid) than specimens of the synovial tissue (2.10 % amyloid). The prevalence of ATTR amyloid in carpal tunnel syndrome is 11.66 %. CONCLUSIONS: Both men and women develop a carpal tunnel syndrome caused by ATTR amyloid. In particular, at the age of over 50 years, amyloid deposits should be considered in the context of the etiological clarification of the carpal tunnel syndrome. Early histological diagnosis is highly relevant to identify the risk of cardiac amyloidosis. For an early and correct diagnosis of ATTR amyloidosis, histological examination of the flexor retinaculum is particularly necessary, and a sample excision should always be obtained and examined histologically.

Activation of Recombinantly Expressed l-Amino Acid Oxidase from Rhizoctonia solani by Sodium Dodecyl Sulfate.

l-Amino acid oxidases (l-AAO) catalyze the oxidative deamination of l-amino acids to the corresponding α-keto acids. The non-covalently bound cofactor FAD is reoxidized by oxygen under formation of hydrogen peroxide. We expressed an active l-AAO from the fungus Rhizoctonia solani as a fusion protein in E. coli. Treatment with small amounts of the detergent sodium dodecyl sulfate (SDS) stimulated the activity of the enzyme strongly. Here, we investigated whether other detergents and amphiphilic molecules activate 9His-rsLAAO1. We found that 9His-rsLAAO1 was also activated by sodium tetradecyl sulfate. Other detergents and fatty acids were not effective. Moreover, effects of SDS on the oligomerization state and the protein structure were analyzed. Native and SDS-activated 9His-rsLAAO1 behaved as dimers by size-exclusion chromatography. SDS treatment induced an increase in hydrodynamic radius as observed by size-exclusion chromatography and dynamic light scattering. The activated enzyme showed accelerated thermal inactivation and an exposure of additional protease sites. Changes in tryptophan fluorescence point to a more hydrophilic environment. Moreover, FAD fluorescence increased and a lower concentration of sulfites was sufficient to form adducts with FAD. Taken together, these data point towards a more open conformation of SDS-activated l-amino acid oxidase facilitating access to the active site.

Establishing and validating the fluorescent amyloid ligand h-FTAA (heptamer formyl thiophene acetic acid) to identify transthyretin amyloid deposits in carpal tunnel syndrome.

Transthyretin-derived (ATTR) amyloidosis is a frequent finding in carpal tunnel syndrome. We tested the following hypotheses: the novel fluorescent amyloid ligand heptameric formic thiophene acetic acid (h-FTAA) has a superior sensitivity for the detection of amyloid compared with Congo red-staining; Amyloid load correlates with patient gender and/or patient age. We retrieved 208 resection specimens obtained from 184 patients with ATTR amyloid in the carpal tunnel. Serial sections were stained with Congo red, h-FTAA and an antibody directed against transthyretin (TTR). Stained sections were digitalized and forwarded to computational analyses. The amount of amyloid was correlated with patient demographics. Amyloid stained intensely with h-FTAA and an anti-TTR-antibody. Congo red-staining combined with fluorescence microscopy was significantly less sensitive than h-FTAA-fluorescence and TTR-immunostaining: the highest percentage area was found in TTR-immunostained sections, followed by h-FTAA and Congo red. The Pearson correlation coefficient was .8 (Congo red vs. h-FTAA) and .9 (TTR vs. h-FTAA). Amyloid load correlated with patient gender, anatomical site and patient age. h-FTAA is a highly sensitive method to detect even small amounts of ATTR amyloid in the carpal tunnel. The staining protocol is easy and h-FTAA may be a much more sensitive procedure to detect amyloid at an earlier stage.

Recombinant expression and characterization of a L-amino acid oxidase from the fungus Rhizoctonia solani.

L-Amino acid oxidases (L-AAOs) catalyze the oxidative deamination of L-amino acids to the corresponding α-keto acids, ammonia, and hydrogen peroxide. L-AAOs are homodimeric enzymes with FAD as a non-covalently bound cofactor. They are of potential interest for biotechnological applications. However, heterologous expression has not succeeded in producing large quantities of active recombinant L-AAOs with a broad substrate spectrum so far. Here, we report the heterologous expression of an active L-AAO from the fungus Rhizoctonia solani in Escherichia coli as a fusion protein with maltose-binding protein (MBP) as a solubility tag. After purification, it was possible to remove the MBP-tag proteolytically without influencing the enzyme activity. MBP-rsLAAO1 and 9His-rsLAAO1 converted basic and large hydrophobic L-amino acids as well as methyl esters of these L-amino acids. The progress of the conversion of L-phenylalanine and L-leucine into the corresponding α-keto acids was determined by HPLC and 1H-NMR analysis of reaction mixtures, respectively. Enzymatic activity was stimulated 50-100-fold by SDS treatment. K m values ranging from 0.9-10 mM and v max values from 3 to 10 U mg-1 were determined after SDS activation of 9His-rsLAAO1 for the best substrates. The enzyme displayed a broad pH optimum between pH 7.0 and 9.5. In summary, a successful overexpression of recombinant L-AAO in E. coli was established that results in a promising enzymatic activity and a broad substrate spectrum for biotechnological application.

Noise performance of low-dose CT: comparison between an energy integrating detector and a photon counting detector using a whole-body research photon counting CT scanner.

Photon counting detector (PCD)-based computed tomography (CT) is an emerging imaging technique. Compared to conventional energy integrating detector (EID)-based CT, PCD-CT is able to exclude electronic noise that may severely impair image quality at low photon counts. This work focused on comparing the noise performance at low doses between the PCD and EID subsystems of a whole-body research PCD-CT scanner, both qualitatively and quantitatively. An anthropomorphic thorax phantom was scanned, and images of the shoulder portion were reconstructed. The images were visually and quantitatively compared between the two subsystems in terms of streak artifacts, an indicator of the impact of electronic noise. Furthermore, a torso-shaped water phantom was scanned using a range of tube currents. The product of the noise and the square root of the tube current was calculated, normalized, and compared between the EID and PCD subsystems. Visual assessment of the thorax phantom showed that electronic noise had a noticeably stronger degrading impact in the EID images than in the PCD images. The quantitative results indicated that in low-dose situations, electronic noise had a noticeable impact (up to a 5.8% increase in magnitude relative to quantum noise) on the EID images, but negligible impact on the PCD images.

A comparison of linear interpolation models for iterative CT reconstruction.

PURPOSE: Recent reports indicate that model-based iterative reconstruction methods may improve image quality in computed tomography (CT). One difficulty with these methods is the number of options available to implement them, including the selection of the forward projection model and the penalty term. Currently, the literature is fairly scarce in terms of guidance regarding this selection step, whereas these options impact image quality. Here, the authors investigate the merits of three forward projection models that rely on linear interpolation: the distance-driven method, Joseph's method, and the bilinear method. The authors' selection is motivated by three factors: (1) in CT, linear interpolation is often seen as a suitable trade-off between discretization errors and computational cost, (2) the first two methods are popular with manufacturers, and (3) the third method enables assessing the importance of a key assumption in the other methods. METHODS: One approach to evaluate forward projection models is to inspect their effect on discretized images, as well as the effect of their transpose on data sets, but significance of such studies is unclear since the matrix and its transpose are always jointly used in iterative reconstruction. Another approach is to investigate the models in the context they are used, i.e., together with statistical weights and a penalty term. Unfortunately, this approach requires the selection of a preferred objective function and does not provide clear information on features that are intrinsic to the model. The authors adopted the following two-stage methodology. First, the authors analyze images that progressively include components of the singular value decomposition of the model in a reconstructed image without statistical weights and penalty term. Next, the authors examine the impact of weights and penalty on observed differences. RESULTS: Image quality metrics were investigated for 16 different fan-beam imaging scenarios that enabled probing various aspects of all models. The metrics include a surrogate for computational cost, as well as bias, noise, and an estimation task, all at matched resolution. The analysis revealed fundamental differences in terms of both bias and noise. Task-based assessment appears to be required to appreciate the differences in noise; the estimation task the authors selected showed that these differences balance out to yield similar performance. Some scenarios highlighted merits for the distance-driven method in terms of bias but with an increase in computational cost. Three combinations of statistical weights and penalty term showed that the observed differences remain the same, but strong edge-preserving penalty can dramatically reduce the magnitude of these differences. CONCLUSIONS: In many scenarios, Joseph's method seems to offer an interesting compromise between cost and computational effort. The distance-driven method offers the possibility to reduce bias but with an increase in computational cost. The bilinear method indicated that a key assumption in the other two methods is highly robust. Last, strong edge-preserving penalty can act as a compensator for insufficiencies in the forward projection model, bringing all models to similar levels in the most challenging imaging scenarios. Also, the authors find that their evaluation methodology helps appreciating how model, statistical weights, and penalty term interplay together.

Dose-efficient ultrahigh-resolution scan mode using a photon counting detector computed tomography system.

An ultrahigh-resolution (UHR) data collection mode was enabled on a whole-body, research photon counting detector (PCD) computed tomography system. In this mode, 64 rows of [Formula: see text] detector pixels were used, which corresponded to a pixel size of [Formula: see text] at the isocenter. Spatial resolution and image noise were quantitatively assessed for the UHR PCD scan mode, as well as for a commercially available UHR scan mode that uses an energy-integrating detector (EID) and a set of comb filters to decrease the effective detector size. Images of an anthropomorphic lung phantom, cadaveric swine lung, swine heart specimen, and cadaveric human temporal bone were qualitatively assessed. Nearly equivalent spatial resolution was demonstrated by the modulation transfer function measurements: 15.3 and [Formula: see text] spatial frequencies were achieved at 10% and 2% modulation, respectively, for the PCD system and 14.2 and [Formula: see text] for the EID system. Noise was 29% lower in the PCD UHR images compared to the EID UHR images, representing a potential dose savings of 50% for equivalent image noise. PCD UHR images from the anthropomorphic phantom and cadaveric specimens showed clear delineation of small structures.