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The article concerns the problem of inland waterways transport of LNG. Its aim is to present the algorithm of conceptual design of inland barges for LNG transport, intended for exploitation on European waterways. The article describes the areas where LNG barges exist, depending on the allowable operating parameters on the waterways. It presents existing architectural and construction solutions of barges for inland LNG transport, as well as the necessary equipment, due to the nature of cargo. Then the article presents the procedure of the conceptual design of LNG barges, including navigation restrictions and functional and economic criteria. The conceptual design algorithm of LGN barges, presented in the article, allows to preliminary design calculations, on the basis of which, are obtained the main dimensions and parameters of unit, depending on the transport task and the class of inland waterways, on which the transport will be realized.

This paper presents the methodology of strength verification during load out of the heavy cargo, in this case Arkutun Dagi SE-Topside platform. General methodology of making calculation models and load algorithms has been presented. Paper shows results of verification of global shear forces and bending moments using self-developed algorithms to modify centre of gravity, fill tanks and hydrostatically balance a 3D finite element model with commercial hydrostatic code. The NAPA and ANSYS codes were used to calculate hydrostatic pressures and to apply to 3D-FE models and to carry out strength calculation of barge construction.

Premise: Southern Africa is a biodiversity hotspot rich in endemic plants and lichen-forming fungi. However, species-level data about lichen photobionts in this region are minimal. We focused on Trebouxia (Chlorophyta), the most common lichen photobiont, to understand how southern African species fit into the global biodiversity of this genus and are distributed across biomes and mycobiont partners., Methods: We sequenced Trebouxia nuclear ribosomal ITS and rbcL of 139 lichen thalli from diverse biomes in South Africa and Namibia. Global Trebouxia phylogenies incorporating these new data were inferred with a maximum likelihood approach. Trebouxia biodiversity, biogeography, and mycobiont-photobiont associations were assessed in phylogenetic and ecological network frameworks., Results: An estimated 43 putative Trebouxia species were found across the region, including seven potentially endemic species. Only five clades represent formally described species: T. arboricola s.l. (A13), T. cf. cretacea (A01), T. incrustata (A06), T. lynniae (A39), and T. maresiae (A46). Potential endemic species were not significantly associated with the Greater Cape Floristic Region or desert. Trebouxia species occurred frequently across multiple biomes. Annual precipitation, but not precipitation seasonality, was significant in explaining variation in Trebouxia communities. Consistent with other studies of lichen photobionts, the Trebouxia-mycobiont network had an anti-nested structure., Conclusions: Depending on the metric used, ca. 20-30% of global Trebouxia biodiversity occurs in southern Africa, including many species yet to be described. With a classification scheme for Trebouxia now well established, tree-based approaches are preferable over "barcode gap" methods for delimiting new species., (© 2024 Botanical Society of America.)

Microplastic particles have been detected in the human body. This study aimed to develop a blood digestion method that preserves microplastics during analysis. Acidic and alkaline reagents, commonly used for isolating plastic particles from organic materials, were tested on human blood samples and microplastics. Nitric acid, hydrochloric acid, potassium hydroxide, and sodium hydroxide were examined over time. Additionally, a pepsin-pancreatin combination was utilized for blood digestion. Light microscopy assessed digestion efficiency and particle count changes, while Raman microspectroscopy distinguished between plastic and cell debris. The acidic reagents were ineffective in removing the organic material, while alkaline reagents were effective without significant effects on microplastics. Blood digestion using pepsin and pancreatin demonstrated efficient digestion without negative consequences for the particles. While potassium hydroxide digestion is already established, novel use of the pepsin-pancreatin combination was introduced to digest human blood, indicating its potential for isolating plastic particles from tissue and human food., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Sloths are unusual mobile ecosystems, containing a high diversity of epibionts living and growing in their fur as they climb slowly through the canopies of tropical forests. These epibionts include poorly studied algae, arthropods, fungi, and bacteria, making sloths likely reservoirs of unexplored biodiversity. This review aims to identify gaps and eliminate misconceptions in our knowledge of sloths and their epibionts, and to identify key questions to stimulate future research into the functions and roles of sloths within a broader ecological and evolutionary context. This review also seeks to position the sloth fur ecosystem as a model for addressing fundamental questions in metacommunity and movement ecology. The conceptual and evidence-based foundation of this review aims to serve as a guide for future hypothesis-driven research into sloths, their microbiota, sloth health and conservation, and the coevolution of symbioses in general., (© 2021 The Authors. Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society.)

Purpose: To investigate image quality, and radiation dose between ECG-gated singlesource and dual-source CT Angiography (CTA) protocols for planning of Trans-catheter Aortic Valve Implantation (TAVI) with a reference non ECG-gated single-source protocol., Methods: A total of 120 patients were included in four groups: Non ECG-gated single-source (SS), ECG-gated single-source (SSECG), ECG-gated dual-source high-pitch (DSECG), or non-ECG-gated dual-source high-pitch mode (DS). Qualitative image quality of the aortic annulus, aortic valve, and coronary ostia as well as presence of motion or stair-step artefacts of the thoracic aorta were independently assessed by two readers. Quantitative image quality was assessed to calculate contrast to noise ratio., Results: Subjective and objective scoring of motion artefacts was significantly reduced in SSECG, DSECG and DS (p= 0.010). The imaging length was comparable between groups. Aortic annulus, aortic valve, and coronary ostia were reliably evaluable in all patients with SSECG, DSECG and DS protocols., Conclusion: High-pitch, dual-source CT angiography of the whole aorta with or without ECG gating is a dose-efficient and time-saving examination strategy before TAVI. However acquisition timing within the cardiac cycle needs to be taken into account., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Purpose: The aim of this study was to evaluate the diagnostic performance of virtual noncalcium (VNCa) dual-energy computed tomography (DECT) reconstructions enabling visualization of bone marrow edema for characterization of incidental thoracolumbar compression fractures in routine thoracoabdominal staging computed tomography (CT)., Materials and Methods: We retrospectively analyzed 51 oncological patients without suspected fracture or indicative complaints presenting at least 1 thoracolumbar compression fracture on routine thoracoabdominal staging DECT who had been examined between October 2015 and June 2017 using third-generation dual-source CT, had a previous CT within 3 months before, and also had undergone additional magnetic resonance imaging within 14 days, which served as the standard of reference. Three independent and blinded radiologists initially evaluated all vertebrae on conventional grayscale DECT series; after at least 8 weeks, observers reevaluated all cases using grayscale and color-coded VNCa DECT images. The age of each fracture was determined as either acute, chronic, or inconclusive. Specificity, sensitivity, and intraobserver and interobserver agreements were calculated taking into account clustering., Results: A total of 98 vertebral compression fractures were detected in 51 patients (20 women, 31 men; median of 1 fracture per patient). The reference standard defined 45 as acute and 53 as chronic. For identification of only acute fractures (cutoff 1), the combination of grayscale and VNCa image series showed a higher sensitivity (91% vs 47%; P < 0.001) but equal specificity (both 100%) compared with analysis of grayscale images alone. When defining a positive finding as a fracture considered either acute or inconclusive (cutoff 2), combined analysis of grayscale and VNCa images showed similar sensitivity (96% vs 93%; P = 0.28) but significantly higher specificity (96% vs 75%; P < 0.001) compared with evaluation of grayscale images alone. Area under the curve analysis for detection of vertebral compression fractures showed superior results for reading of grayscale and VNCa image series (0.98) compared with analysis of grayscale images alone (0.89; P < 0.001)., Conclusion: Dual-energy CT-derived color-coded VNCa reconstructions substantially improve the characterization of incidental thoracolumbar compression fractures seen on routine thoracoabdominal staging DECT by allowing for visualization of bone marrow edema.

Purpose: The aim of this study was to evaluate the impact of automated attenuation-based tube potential selection (ATPS) on image quality and radiation dose exposure parameters at a computed tomography angiography (CTA) lower-extremity runoff., Materials and Methods: Two hundred forty patients (156 men, 84 women) underwent CTA examinations of the lower-extremity runoff on a second-generation dual-source computed tomography system: 120 patients at a fixed tube potential of 120 kV and a tube current of 180 reference mAs, another 120 patients using automated ATPS. Volume computed tomography dose index (CTDIvol), dose-length-product (DLP), body diameters, noise, signal-to-noise ratio, and subjective image quality were compared., Results: In the ATPS group, 80 kV was automatically selected in 102 patients, 100 kV in 15 patients, and 120 kV in 3 patients; 140 kV was not chosen in any of the cases. The median CTDIvol of 4.81 mGy (2.2-10.6 mGy) and DLP of 568 mGy⋅cm (203-1324 mGy⋅cm) in the ATPS group were significantly lower compared with the CTDIvol of 8.1 mGy (4.4-14.4 mGy) and DLP of 1027.5 mGy⋅cm (509-1806 mGy⋅cm) in the fixed 120-kV group (P < 0.01). Image quality was comparable (P > 0.05)., Conclusion: Automated ATPS allows for significant dose savings in lower-extremity runoff CTA, whereas image quality remains constant at a high level.

Background: Most of the applied radiation dose at CT is in the lower photon energy range, which is of limited diagnostic importance., Objective: To investigate image quality and effects on radiation parameters of 100-kVp spectral filtration single-energy chest CT using a tin-filter at third-generation dual-source CT in comparison to standard 100-kVp chest CT., Materials and Methods: Thirty-three children referred for a non-contrast chest CT performed on a third-generation dual-source CT scanner were examined at 100 kVp with a dedicated tin filter with a tube current-time product resulting in standard protocol dose. We compared resulting images with images from children examined using standard single-source chest CT at 100 kVp. We assessed objective and subjective image quality and compared radiation dose parameters., Results: Radiation dose was comparable for children 5 years old and younger, and it was moderately decreased for older children when using spectral filtration (P=0.006). Effective tube current increased significantly (P=0.0001) with spectral filtration, up to a factor of 10. Signal-to-noise ratio and image noise were similar for both examination techniques (P≥0.06). Subjective image quality showed no significant differences (P≥0.2)., Conclusion: Using 100-kVp spectral filtration chest CT in children by means of a tube-based tin-filter on a third-generation dual-source CT scanner increases effective tube current up to a factor of 10 to provide similar image quality at equivalent dose compared to standard single-source CT without spectral filtration.

In selected cases, cuffed tunneled catheters via the iliac vein are implanted as a last resort access for hemodialysis. To monitor the correct position, sonography of the inferior vena cava (IVC) is sufficient in most cases. Position control using an X-ray of the abdomen is not routinely recommended when femoral catheters are implanted. In this report, we describe the case of a 59-year-old patient on chronic hemodialysis due to granulomatosis with polyangiitis and complex shunt history with multiple shunt occlusions and revisions. The implantation of an iliac-cuffed tunneled catheter led to complications because the catheter was malpositioned into the left ascending lumbar vein (ALV). It is important to be aware of potential incorrect positioning of dialysis catheters into the ALV. Due to the anatomical relation to the IVC, this happens more frequently on the left side than on the right side. In case of doubt, the correct placement of large-bore catheters via iliac access route should be verified by means of appropriate imaging before hemodialysis is performed., (© 2017 S. Karger AG, Basel.)

Background: Computed tomography (CT) low-dose (LD) imaging is used to lower radiation exposure, especially in vascular imaging; in current literature, this is mostly on latest generation high-end CT systems., Purpose: To evaluate the effects of reduced tube current on objective and subjective image quality of a 15-year-old 16-slice CT system for pulmonary angiography (CTPA)., Material and Methods: CTPA scans from 60 prospectively randomized patients (28 men, 32 women) were examined in this study on a 15-year-old 16-slice CT scanner system. Standard CT (SD) settings were 100 kV and 150 mAs, LD settings were 100 kV and 50 mAs. Attenuation of the pulmonary trunk, various anatomic landmarks, and image noise were quantitatively measured; contrast-to-noise ratios (CNR) and signal-to-noise ratios (SNR) were calculated. Three independent blinded radiologists subjectively rated each image series using a 5-point grading scale., Results: CT dose index (CTDI) in the LD series was 66.46% lower compared to the SD settings (2.49 ± 0.55 mGy versus 7.42 ± 1.17 mGy). Attenuation of the pulmonary trunk showed similar results for both series (SD 409.55 ± 91.04 HU; LD 380.43 HU ± 93.11 HU; P  = 0.768). Subjective image analysis showed no significant differences between SD and LD settings regarding the suitability for detection of central and peripheral PE (central SD/LD, 4.88; intra-class correlation coefficients [ICC], 0.894/4.83; ICC, 0.745; peripheral SD/LD, 4.70; ICC, 0.943/4.57; ICC, 0.919; all P  > 0.4)., Conclusion: The LD protocol, on a 15-year-old CT scanner system without current high-end hardware or post-processing tools, led to a dose reduction of approximately 67% with similar subjective image quality and delineation of central and peripheral pulmonary arteries.

Background: Evaluation of latest generation automated attenuation-based tube potential selection (ATPS) impact on image quality and radiation dose in contrast-enhanced chest-abdomen-pelvis computed tomography examinations for gynaecologic cancer staging., Methods: This IRB approved single-centre, observer-blinded retrospective study with a waiver for informed consent included a total of 100 patients with contrast-enhanced chest-abdomen-pelvis CT for gynaecologic cancer staging. All patients were examined with activated ATPS for adaption of tube voltage to body habitus. 50 patients were scanned on a third-generation dual-source CT (DSCT), and another 50 patients on a second-generation DSCT. Predefined image quality setting remained stable between both groups at 120 kV and a current of 210 Reference mAs. Subjective image quality assessment was performed by two blinded readers independently. Attenuation and image noise were measured in several anatomic structures. Signal-to-noise ratio (SNR) was calculated. For the evaluation of radiation exposure, CT dose index (CTDI vol ) values were compared., Results: Diagnostic image quality was obtained in all patients. The median CTDI vol (6.1 mGy, range 3.9-22 mGy) was 40 % lower when using the algorithm compared with the previous ATCM protocol (median 10.2 mGy · cm, range 5.8-22.8 mGy). A reduction in potential to 90 kV occurred in 19 cases, a reduction to 100 kV in 23 patients and a reduction to 110 kV in 3 patients of our experimental cohort. These patients received significantly lower radiation exposure compared to the former used protocol., Conclusion: Latest generation automated ATPS on third-generation DSCT provides good diagnostic image quality in chest-abdomen-pelvis CT while average radiation dose is reduced by 40 % compared to former ATPS protocol on second-generation DSCT.

Purpose To evaluate whether a dual-energy (DE) computed tomographic (CT) virtual noncalcium technique can improve the detection rate of acute thoracolumbar vertebral compression fractures in patients with osteoporosis compared with that at magnetic resonance (MR) imaging depending on the level of experience of the reading radiologist. Materials and Methods This retrospective study was approved by the institutional ethics committee. Informed consent was obtained from all patients. Forty-nine patients with osteoporosis who were suspected of having acute vertebral fracture underwent DE CT and MR imaging. Conventional linear-blended CT scans and corresponding virtual noncalcium reconstructions were obtained. Five radiologists with varying levels of experience evaluated gray-scale CT scans for the presence of fractures and their suspected age. Then, virtual noncalcium images were evaluated to detect bone marrow edema. Findings were compared with those from MR imaging (the standard of reference). Sensitivity and specificity analyses for diagnostic performance and matched pair analyses were performed on vertebral fracture and patient levels. Results Sixty-two fractures were classified as fresh and 52 as old at MR imaging. The diagnostic performance of all readers in the detection of fresh fractures improved with the addition of virtual noncalcium reconstructions compared with that with conventional CT alone. Although the diagnostic accuracy of the least experienced reader with virtual noncalcium CT (accuracy with CT alone, 61%; accuracy with virtual noncalcium technique, 83%) was within the range of that of the most experienced reader with CT alone, the latter improved his accuracy with the noncalcium technique (from 81% to 95%), coming close to that with MR imaging. The number of vertebrae rated as unclear decreased by 59%-90% or from 15-53 to 2-13 in absolute numbers across readers. The number of patients potentially referred to MR imaging decreased by 36%-87% (from 11-23 to 2-10 patients). Considering the gain in true decisions with the virtual noncalcium technique on a patient level, between 12 (most experienced reader) and 17 (least experienced reader) MR examinations could have been avoided. Conclusion The DE CT-based virtual noncalcium technique may enable depiction of bone marrow edema in thoracolumbar vertebral compression fractures in patients with osteoporosis, with good accordance with MR imaging when images are read by experienced radiologists. Although less experienced readers improved their diagnostic performance to some degree, the experienced reader's diagnostic performance approached that with MR imaging. (©) RSNA, 2016.

Objective: Evaluation of the intimal flap visibility comparing 2nd and 3rd generation dual-source high-pitch CT., Methods: Twenty-five consecutive patients with aortic dissection underwent CT angiography on a second and third generation dual-source CT scanner using prospective ECG-gated high-pitch dual-source CT acquisition mode. Contrast material, saline flush and flow rate were kept equal for optimum comparability. The visibility of the intimal flap as well as the delineation of the different vascular structures was evaluated., Results: In 3rd generation dual-source high-pitch CT we could show a significant improvement of intimal flap visibility in aortic dissection. Especially, the far end of the dissection membrane could be better evaluated in 3rd generation high-pitch CT, reaching statistical significance (P < 0.01)., Conclusion: 3rd Generation high-pitch CT angiography shows a better delineation of the aortic intimal flap in a small patient cohort, especially in the far ends of the dissection membrane. This might be due to higher tube power in this CT generation. However, to generalise these findings larger trials are needed.

Epithelial ovarian cancer (EOC) is the most frequent cause of death from all gynecological malignancies because of its late diagnosis. As N-glycosylation is modified in the course of ovarian cancer, it is a promising source of tumor biomarkers. In this work, serum glycoproteins, depleted from albumin and IgG, were separated by 2DE. Protein spots of acute-phase proteins were identified by peptide mapping and their corresponding glycan moieties were released enzymatically, fluorescently labeled and analyzed by CE-LIF. In the positive acute-phase proteins, haptoglobin, α1-antitrypsin, and α1-antichymotrypsin, an increase of antennarity and Lewis(X) motif could be measured in EOC patients on tri- and/or tetraantennary N-glycans. Tetraantennary N-glycans containing three Lewis(X) epitopes and triantennary N-glycans containing a β(1-6) branch and a Lewis(X) epitope were only present in EOC patients. We also showed for the first time that the core-fucosylated biantennary digalactosylated N-glycan of α1-acid glycoprotein is a potential biomarker for EOC. To conclude, core-fucosylated biantennary N-glycans on α1-acid glycoprotein as well as higher antennarity and increased amounts of Lewis(X) motif on haptoglobin, α1-antitrypsin, and α1-antichymotrypsin are promising biomarkers for EOC. Nevertheless, their specificity and selectivity for the early detection of EOC should be evaluated in a larger study., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objectives: To compare quantitative image quality parameters in abdominal dual-energy computed tomography angiography (DE-CTA) using an advanced image-based (Mono+) reconstruction algorithm for virtual monoenergetic imaging and standard DE-CTA., Methods: Fifty-five patients (36 men; mean age, 64.2 ± 12.7 years) who underwent abdominal DE-CTA were retrospectively included. Mono + images were reconstructed at 40, 50, 60, 70, 80, 90 and 100 keV levels and as standard linearly blended M&#95;0.6 images (60 % 100 kV, 40 % 140 kV). The contrast-to-noise ratio (CNR) and signal-to-noise ratio (SNR) of the common hepatic (CHA), splenic (SA), superior mesenteric (SMA) and left renal arteries (LRA) were objectively measured., Results: Mono+ DE-CTA series showed a statistically superior CNR for 40, 50, 60, 70 and 80 keV (P < 0.031) compared to M&#95;0.6 images for all investigated arteries except SMA at 80 keV (P = 0.08). CNR at 40 keV revealed a mean relative increase of 287.7 % compared to linearly blended images among all assessed arteries (P < 0.001). SNR of Mono+ images was consistently significantly higher at 40, 50, 60 and 70 keV compared to M&#95;0.6 for CHA and SA (P < 0.009)., Conclusions: Compared to linearly blended images, Mono+ reconstructions at low keV levels of abdominal DE-CTA datasets significantly improve quantitative image quality., Key Points: • Mono+ combines increased attenuation with reduced image noise compared to standard DE-CTA. • Mono+ shows superior contrast-to-noise ratios at low keV compared to linearly-blended images. • Contrast-to-noise ratio in monoenergetic DE-CTA peaks at 40 keV. • Mono+ reconstructions significantly improve quantitative image quality at low keV levels.

Purpose: To assess the effect of a noise-optimized image-based virtual monoenergetic imaging (VMI+) algorithm in direct comparison with the traditional VMI technique and standard linearly-blended images emulating 120-kVp acquisition (M&#95;0.3) on image quality at dual-energy CT in patients with lung cancer., Materials and Methods: Dual-source dual-energy CT examinations of 48 patients with biopsy-proven primary (n=31) or recurrent (n=20) lung cancer were evaluated. Images were reconstructed as M&#95;0.3, and VMI+ and traditional VMI series at 40, 55, and 70keV. Attenuation of tumor, descending aorta, pulmonary trunk, latissimus muscle, and noise were measured. Signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) were calculated. Five-point scales were used by three observers to subjectively evaluate general image impression, tumor delineation, image sharpness, and image noise., Results: Background noise was consistently lower with VMI+ compared to VMI at all keV levels (all p<0.0001) and M&#95;0.3 (all p≤0.0004). Tumor SNR and CNR peaked in the 40keV VMI+ series, significantly higher compared to all VMI and M&#95;0.3 series (all p<0.0008). Observers preferred the 55keV VMI+ series regarding general image impression and tumor delineation compared to all other series (all p<0.0001). Image sharpness and image noise ratings were highest in the 55keV VMI+ and 70keV VMI and VMI+ reconstructions., Conclusions: Tumor CNR peaked at 40keV VMI+ while observers preferred 55keV VMI+ series overall other series for dual-energy CT of lung cancer. The noise-optimized VMI+ technique showed significantly lower background noise and higher SNR and CNR compared to the traditional VMI technique at matching keV levels., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Objective: The aim of the study was to evaluate objective and subjective image qualities of virtual monoenergetic imaging (VMI) in dual-source dual-energy computed tomography (DECT) and optimal kiloelectron-volt (keV) levels for lung cancer., Methods: Fifty-nine lung cancer patients underwent chest DECT. Images were reconstructed as VMI series at energy levels of 40, 60, 80, and 100 keV and standard linear blending (M&#95;0.3) for comparison. Objective and subjective image qualities were assessed., Results: Lesion contrast peaked in 40-keV VMI reconstructions (2.5 ± 2.9) and 60 keV (1.9 ± 3.0), which was superior to M&#95;0.3 (0.5 ± 2.7) for both comparisons (P < 0.001). Compared with M&#95;0.3, subjective ratings were highest for 60-keV VMI series regarding general image quality (4.48 vs 4.52; P = 0.74) and increased for lesion demarcation (4.07 vs 4.84; P < 0.001), superior to all other VMI series (P < 0.001). Image sharpness was similar between both series. Image noise was rated superior in the 80-keV and M&#95;0.3 series, followed by 60 keV., Conclusions: Virtual monoenergetic imaging reconstructions at 60-keV provided the best combination of subjective and objective image qualities in DECT of lung cancer.

Background and Purpose: Dose-saving techniques in neck CT cause increased image noise that can be counteracted by iterative reconstruction. Our aim was to evaluate the image quality of advanced modeled iterative reconstruction (ADMIRE) in contrast-enhanced low-tube-voltage neck CT., Materials and Methods: Sixty-one patients underwent 90-kV(peak) neck CT by using third-generation 192-section dual-source CT. Image series were reconstructed with standard filtered back-projection and ADMIRE strength levels 1, 3, and 5. Attenuation and noise of the sternocleidomastoid muscle, internal jugular vein, submandibular gland, tongue, subscapularis muscle, and cervical fat were measured. Signal-to-noise and contrast-to-noise ratios were calculated. Two radiologists assessed image noise, image contrast, delineation of smaller structures, and overall diagnostic acceptability. Interobserver agreement was calculated., Results: Image noise was significantly reduced by using ADMIRE compared with filtered back-projection with the lowest noise observed in ADMIRE 5 (filtered back-projection, 9.4 ± 2.4 Hounsfield units [HU]; ADMIRE 1, 8.3 ± 2.8 HU; ADMIRE 3, 6.7 ± 2.0 HU; ADMIRE 5, 5.4 ± 1.7 HU; all, P < .001). Sternocleidomastoid SNR and internal jugular vein-sternocleidomastoid contrast-to-noise ratios were significantly higher for ADMIRE with the best results in ADMIRE 5 (all, P < .001). Subjective image quality and image contrast of ADMIRE 3 and 5 were consistently rated better than those for filtered back-projection and ADMIRE 1 (all, P < .001). Image noise was rated highest for ADMIRE 5 (all, P < .005). Delineation of smaller structures was voted higher in all ADMIRE strength levels compared with filtered back-projection (P < .001). Global interobserver agreement was good (0.75)., Conclusions: Contrast-enhanced 90-kVp neck CT is feasible, and ADMIRE 5 shows superior objective image quality compared with filtered back-projection. ADMIRE 3 and 5 show the best subjective image quality., (© 2016 by American Journal of Neuroradiology.)

Objectives: To define optimal keV settings for advanced monoenergetic (Mono+) dual-energy computed tomography (DECT) in patients with head and neck squamous cell carcinoma (SCC)., Methods: DECT data of 44 patients (34 men, mean age 55.5 ± 16.0 years) with histopathologically confirmed SCC were reconstructed as 40, 55, 70 keV Mono + and M&#95;0.3 (30 % 80 kV) linearly blended series. Attenuation of tumour, sternocleidomastoid muscle, internal jugular vein, submandibular gland, and noise were measured. Three radiologists with >3 years of experience subjectively assessed image quality, lesion delineation, image sharpness, and noise., Results: The highest lesion attenuation was shown for 40 keV series (248.1 ± 94.1 HU), followed by 55 keV (150.2 ± 55.5 HU; P = 0.001). Contrast-to-noise ratio (CNR) at 40 keV (19.09 ± 13.84) was significantly superior to all other reconstructions (55 keV, 10.25 ± 9.11; 70 keV, 7.68 ± 6.31; M&#95;0.3, 5.49 ± 3.28; all P < 0.005). Subjective image quality was highest for 55 keV images (4.53; κ = 0.38, P = 0.003), followed by 40 keV (4.14; κ = 0.43, P < 0.001) and 70 keV reconstructions (4.06; κ = 0.32, P = 0.005), all superior (P < 0.004) to linear blending M&#95;0.3 (3.81; κ = 0.280, P = 0.056)., Conclusions: Mono + DECT at low keV levels significantly improves CNR and subjective image quality in patients with head and neck SCC, as tumour CNR peaks at 40 keV, and 55 keV images are preferred by observers., Key Points: • Mono + DECT combines increased contrast with reduced image noise, unlike linearly blended images. • Mono + DECT imaging allows for superior CNR and subjective image quality. • Head and neck tumour contrast-to-noise ratio peaks at 40 keV. • 55 keV images are preferred over all other series by observers.

Objective: To investigate and compare the use of automated tube potential selection (ATPS) with automated tube current modulation (ATCM) in high-pitch dual-source computed tomographic angiography (CTA) for imaging the whole aorta without electrocardiogram synchronization., Methods: Two groups of 60 patients underwent CTA on a dual-source computed tomographic device in high-pitch mode: ATCM (with 100-kV fixed tube potential) was used in group 1 and ATPS (with the same image quality options) in group 2. For the evaluation of radiation exposure, CT dose index and dose-length product were analyzed. Contrast and image quality were assessed by 2 independent observers., Results: The ATPS group received a higher radiation dose than the ATCM group (P < 0.001) because in 80% of patients, the software switched to use of a 120-kV tube potential. In all cases, images of the aorta were of sufficient quality., Conclusions: High-pitch dual-source CTA of the aorta using ATPS is feasible in clinical routine, but is associated with higher radiation exposure than the ATCM protocol. This finding contradicts previously evaluations of ATPS based on single-source techniques.

Introduction: To investigate low-tube-voltage 80-kVp computed tomography (CT) of head and neck primary and recurrent squamous cell carcinoma (SCC) regarding objective and subjective image quality., Methods: We retrospectively evaluated 65 patients (47 male, 18 female; mean age: 62.1 years) who underwent head and neck dual-energy CT (DECT) due to biopsy-proven primary (n = 50) or recurrent (n = 15) SCC. Eighty peak kilovoltage and standard blended 120-kVp images were compared. Attenuation and noise of malignancy and various soft tissue structures were measured. Tumor signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) were calculated. Subjective image quality was rated by three reviewers using 5-point grading scales regarding overall image quality, lesion delineation, image sharpness, and image noise. Radiation dose was assessed as CT dose index volume (CTDIvol). Interobserver agreement was calculated using intraclass correlation coefficient (ICC)., Results: Mean tumor attenuation (153.8 Hounsfield unit (HU) vs. 97.1 HU), SNR (10.7 vs. 8.3), CNR (8.1 vs. 4.8), and subjective tumor delineation (score, 4.46 vs. 4.13) were significantly increased (all P < 0.001) with 80-kVp acquisition compared to standard blended 120-kVp images. Noise of all measured structures was increased in 80-kVp acquisition (P < 0.001). Overall interobserver agreement was good (ICC, 0.86; 95 % confidence intervals: 0.82-0.89). CTDIvol was reduced by 48.7 % with 80-kVp acquisition compared to standard DECT (4.85 ± 0.51 vs. 9.94 ± 0.81 mGy cm, P < 0.001)., Conclusions: Head and neck CT with low-tube-voltage 80-kVp acquisition provides increased tumor delineation, SNR, and CNR for CT imaging of primary and recurrent SCC compared to standard 120-kVp acquisition with an accompanying significant reduction of radiation exposure.

Introduction: The aim of this study was to compare solubility, microhardness, radiopacity, and setting time of Biodentine with ProRoot MTA., Methods: Solubility in distilled water, radioopacity, and setting time were evaluated in accordance with International Standard ISO 6876:2001. In addition, the solubility in Phosphate Buffered Saline (PBS) buffer was determined. For microhardness-testing, ten samples of each cement were produced. All samples were loaded with a diamond indenter point with a weight of 100 g for 30s. All data were analysed using the Student-t-test., Results: Both materials fulfilled the requirements of the International Standard ISO 6876:2001 and showed a solubility of <3% after 24 h. At all exposure times Biodentine was significantly more soluble than ProRoot MTA (p < 0.0001). After immersion in PBS-buffer a precipitation of hydroxyapatite was visible. The Vickers microhardness for Biodentine was significantly higher (62.35 ± 11.55HV) compared with ProRoot MTA (26.93 ± 4.66HV) (p < 0.0001). ProRoot MTA was significantly more radiopaque (6.40 ± 0.06 mm Al) than Biodentine (1.50 ± 0.10 mm Al) (p < 0.0001). The setting time for Biodentine (85.66 ± 6.03 min) was significantly lower than for ProRoot MTA (228.33 ± 2.88 min) (p < 0.0001)., Conclusions: Biodentine and ProRoot MTA displayed different material properties. The solubility of both cements was in accordance with the International Standard ISO 6876:2001, whereas ProRoot MTA showed a significantly lower solubility. With regard to microhardness, Biodentine may be used to replace dentine. The radioopacity of Biodentine did not fulfil the requirements laid down in the International Standard ISO 6876:2001. The setting time for ProRoot MTA is significantly higher. Both materials can be used in different indications where specific material properties may be favourable. Hence, the here tested material properties are of clinical relevance.

Introduction: The aim of this in vitro study was to assess an alternative method using light-curing composite for removing fractured endodontic instruments with a tube technique., Methods: Two different stainless steel endodontic instruments (ISO 20: Hedstrom files, K-files; VDW, Munich, Germany) were cut at the diameter of 0.4 mm. These fragments were fixed in a vise leaving a free end of 1 or 2 mm. Cyanoacrylate (Instant Fix; Henry Schein Dental, Melville, NY), dual-curing Rebilda DC (VOCO, Cuxhaven, Germany), and light-curing SureFil SDR (Dentsply, York, PA) were placed into microtubes (N'Durance Syringe Tips; Septodont, Saint-Maur, France) and shifted over the instruments (n = 20 in each group). After polymerization, pull-out tests were performed with a constant speed of 2 mm/min; failure load was measured digitally. Data were analyzed using the Kruskal-Wallis test followed by the Dunn test for pairwise comparison., Results: The median failure load was up to 62.5 N for SDR, 35.8 N for Rebilda, and 14.7 N for cyanoacrylate, respectively. Both tested composites yielded significantly higher values in pull-out tests than cyanoacrylate. The disconnecting force was highest when light-cured composite SDR was used for fixation. Removing Hedstrom files resulted in higher values than removing K-files. The median force when using SDR was 79.7 N (interquartile range, 66.0-86.8 N) in Hedstrom files and 53.3 N (interquartile range, 47.1-58.5 N) in K-files., Conclusions: Within the limitations of this study, the use of light-curing composite inside of the microtube was superior compared with the use of cyanoacrylate or chemically cured composite, which are being used presently., (Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: The aim of this study was to compare the shear bond strength of Biodentine, ProRoot MTA (MTA), glass ionomer cement (GIC) and composite resin (CR) on dentine., Methods: 120 extracted human third molars were embedded in cold-cured-resin and grinned down to the dentine. For each material 30 specimens were produced in standardised height and width and the materials were applied according to manufacturers´ instructions on the dentine samples. Only in the CR group a self-etching dentine-adhesive was used. In all other groups the dentine was not pre-treated. All specimens were stored at 37.5 °C and 100% humidity for 2d, 7d and 14d. With a testing device the shear bond strength was determined (separation of the specimens from the dentine surface). The statistical evaluation was performed using ANOVA and Tukey-test (p < 0.05)., Results: At all observation periods the CR showed the significant highest shear bond strength (p < 0.05). After 2d significant differences in the shear bond strength were detectable between all tested materials, whereby CR had the highest and MTA the lowest values (p < 0.05). After 7d and 14d the shear bond strengths of MTA and Biodentine increased significantly compared to the 2d investigation period (p < 0.05). Biodentine showed a significantly higher shear bond strength than MTA (p < 0.05), while the difference between Biodentine and GIC was not significant (p > 0.05)., Conclusions: After 7d Biodentine showed comparable shear bond values than GIC, whereas the shear bond values for MTA were significantly lower even after 14d. The adhesion of Biodentine to dentine surface seams to be superior compared to that of MTA.

Objectives: To evaluate software for automatic segmentation, labeling and reformation of anatomical aligned axial images of the thoracolumbar spine on CT in terms of accuracy, potential for time savings and workflow improvement., Material and Methods: 77 patients (28 women, 49 men, mean age 65.3±14.4 years) with known or suspected spinal disorders (degenerative spine disease n=32; disc herniation n=36; traumatic vertebral fractures n=9) underwent 64-slice MDCT with thin-slab reconstruction. Time for automatic labeling of the thoracolumbar spine and reconstruction of double-angulated axial images of the pathological vertebrae was compared with manually performed reconstruction of anatomical aligned axial images. Reformatted images of both reconstruction methods were assessed by two observers regarding accuracy of symmetric depiction of anatomical structures., Results: In 33 cases double-angulated axial images were created in 1 vertebra, in 28 cases in 2 vertebrae and in 16 cases in 3 vertebrae. Correct automatic labeling was achieved in 72 of 77 patients (93.5%). Errors could be manually corrected in 4 cases. Automatic labeling required 1min in average. In cases where anatomical aligned axial images of 1 vertebra were created, reconstructions made by hand were significantly faster (p<0.05). Automatic reconstruction was time-saving in cases of 2 and more vertebrae (p<0.05). Both reconstruction methods revealed good image quality with excellent inter-observer agreement., Conclusion: The evaluated software for automatic labeling and anatomically aligned, double-angulated axial image reconstruction of the thoracolumbar spine on CT is time-saving when reconstructions of 2 and more vertebrae are performed. Checking results of automatic labeling is necessary to prevent errors in labeling., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Purpose: The purpose of the study was to evaluate 70 kVp dual-source computed tomography pulmonary angiography (CTPA) with reduced iodine load in comparison with single-source 70 and 100 kVp CTPA with standard iodine load regarding image quality and radiation dose., Materials and Methods: Three groups with 40 consecutive patients each underwent either standard single-source 100 kVp (120 mAs; group A), single-source 70 kVp (208 mAs; group B), or dual-source 70 kVp CTPA (416 mAs; group C). A volume of 70 mL of contrast material with 400 mg I/mL (groups A, B) or 300 mg I/mL (group C) was administered. Chest diameter, dose-length product, intravascular signal attenuation, image noise, signal to noise ratio (SNR), and contrast to noise ratio (CNR) were compared. Two observers rated subjective image quality regarding intravascular enhancement and image noise using 5-point scales., Results: Chest diameter and age were similar (P ≥ 0.28) for all groups. Compared with group A, the average dose-length product was 59% lower in group B (67.3 ± 11.8 vs. 164.7 ± 50.6 mGy cm, P<0.001) and similar between groups A and C (167.7 ± 41.2 mGy cm, P = 0.39). Average SNR and CNR were significantly higher for group C (21.5 ± 4.7 and 19.0 ± 4.5, respectively) compared with groups A (18.3 ± 3.5 and 15.8 ± 3.4, respectively) and B (17.3 ± 5.8 and 15.6 ± 5.5, respectively; all Ps ≤ 0.001). Subjective image quality ratings regarding enhancement and noise were highest for group C (1.73 ± 0.62 and 2.03 ± 0.66, respectively)., Conclusions: Compared with standard 100 kVp CTPA, single-source 70 kVp CTPA allows for significant radiation dose savings with comparable SNR and CNR, whereas dual-source 70 kVp CTPA results in a superior objective image quality albeit a reduction of iodine concentration.

Cell surfaces are covered with a dense carbohydrate layer referred to as the glycocalyx. Because different cell types express different glycan signatures, it is of paramount importance to have robust methods to analyze the glycome of living cells. To achieve this, a common procedure involves cell lysis and extraction of membrane (glyco)proteins and yields a major proportion of high-mannose N-glycans that most likely stem from intracellular proteins derived from the ER. Using HEK 293 cells as a model system, we developed a reproducible, sensitive, and fast method to profile surface N-glycosylation from living cells. We directly released glycopeptides from cell surfaces through tryptic digestion of freshly harvested and vital cells, thereby improving the detection and quantification of complex-type N-glycans by increasing their relative amount from 14 to 85%. It was also possible to detect 25 additional structures in HEK 293, 48 in AGE1.HN, 42 in CHO-K1, and 51 in Hep G2 cells. The additional signals provided deeper insight into cell-type-specific N-glycan features such as antennarity, fucosylation, and sialylation. Thus, this protocol, which can potentially be applied to any cells, will be useful in the fields of glycobiotechnology and biomarker discovery.

Alterations in glycosylation have been observed in many human diseases and specific changes in glycosylation have been proposed as relevant diagnostic information. Capillary electrophoresis coupled with laser-induced fluorescence (CE-LIF) is a robust method to quantify desialylated N-glycans that are labeled with 8-aminopyrene-1,3,6-trisulfonic acid prior to analysis. To date, only a maximum of 12 glycan structures, the most abundant ones, have been identified by CE-LIF to characterize glycome modulations of total serum in the course of the diseases. In most forms of cancer, findings using CE-LIF were limited to the increase of triantennary structures carrying a Lewis(x) epitope. In this work, we identified 32 linkage and positional glycan isomers in healthy human serum using exoglycosidase digestions as well as standard glycoproteins, for which we report the assignment of novel structures. It was possible to identify and quantify 34 glycan isomers in the serum of primary epithelial ovarian cancer patients (EOC). Reduced levels of diantennary structures and of high-mannose 5 were statistically significant in the EOC samples, and also, elevated branching as well as increased antennary fucosylation were observed. For the first time, we could demonstrate that not only antennary fucosylation was of relevance in tetraantennary structures but also core-fucosylated tetraantennary N-glycans were statistically increased in EOC patients. The results of the current study provide an improved dataset to be used in glycan biomarker discovery.

Alterations in IgG N-glycosylation coincide with the development of a number of diseases including cancer and could potentially be used as diagnostic markers. CE-LIF of 8-aminopyrene-1,3,6-trisulfonic acid labeled N-glycans is a well-established rapid method to characterize IgG N-glycans that needs only low amounts of starting material. However,sialylated N-glycans have short migration times due to their negative charge. As a result,some of them are not well resolved and co-migrate with neutral glycans. In this work, we neutralized the negative charge of sialic acids by methylation and optimized the protocol using the commercially available disialylated biantennary oligosaccharide (A2G2S2). IgGN-glycans isolated from healthy human serum were then analyzed using this method. We could demonstrate that co-migration of A2, FA2G2S1, and FA2B[3]G1S1 was prevented,which allowed an accurate quantification of these N-glycans. Finally, we investigated the IgG N-glycan profiles of patients suffering from ovarian cancer using the conventional and methylation methods.With both methods, we observed an increase of agalactosylated structures that was accompanied by a decrease in digalactosylated structures. Finally, using the methylation protocol, we could further demonstrate an increase of A2, which was technically impossible with the conventional method.

Therapeutic efficacy of glycoproteins is affected by many factors, including molecular size and net charge; both are influenced by the presence and composition of glycan structures. Human alpha 1-antitrypsin (A1AT) was cloned and expressed in human embryonic kidney cells (HEK293) that are capable of mammalian glycosylation. Utilizing PCR-based site-directed mutagenesis, new A1AT variants were created with single, double, or triple additional N-glycosylation sites to the three naturally occurring N-glycosylation sites. Because of the supplementary N-glycans, the A1AT variants exhibited an increased molecular weight. Retention of inhibitory activity was shown via trypsin inhibitory assay. The A1AT variants were treated with PNGase F, and the resulting N-glycans were analyzed by MALDI-TOF mass spectrometry. The N-glycan profile of the recombinant A1AT variants was mostly composed of monofucosylated bi-, tri-, and tetraantennary complex-type N-glycans, with a tendency toward higher antennary structures compared to the wild-type. The relevance of N-glycosylation in A1AT for the circulatory serum half-life was demonstrated in CD1 mice. The A1AT neoglycoprotein with an additional N-glycosylation site at position N123 exhibited a 62% increase in serum half-life. Additionally, using a two-compartment model, the A1AT variants exhibited increased α-phase values, especially N123 (223%) and N201 (255%). The results suggest the recombinant A1AT neoglycoprotein as a serious alternative to A1AT derived from human plasma.

The successive events of shedding and regulated intramembrane proteolysis are known to comprise a fundamental biological process of type I and II membrane proteins (e.g. amyloid precursor protein, Notch receptor and pro-tumor necrosis factor-α). Some of the resulting fragments were shown to be involved in important intra- and extracellular signalling events. Although shedding of the human transferrin receptor-1 (TfR1) has been known for > 30 years and soluble TfR1 is an accepted diagnostic marker, the fate of the remaining N-terminal fragment (NTF) remains unknown. In the present study, we demonstrate for the first time that TfR1-NTF is subject to regulated intramembrane proteolysis and, using MALDI-TOF-TOF-MS, we have identified the cleavage site as being located C-terminal from Gly-84. We showed that the resulting C-terminal peptide is extracellularly released after regulated intramembrane proteolysis and it was detected as a monomer with an internal disulfide bridge. We further identified signal peptide peptidase-like 2a and mainly signal peptide peptidase-like 2b as being responsible for the intramembrane proteolysis of TfR1-NTF., (© 2013 The Authors Journal compilation © 2013 FEBS.)

Sample preparation is the rate-limiting step in glycan analysis workflows. Among all of the steps, enzymatic digestions, which are usually performed overnight, are the most time-consuming. In the current study, we report an economical and fast preparation of N-glycans from serum, including microwave-assisted enzymatic digestion in the absence of denaturing chemicals and solvents during the release. To this end, we used a household microwave oven to accelerate both pronase and endo-β-N-acetylglucosaminidase H (Endo H) digestions. Purification was then performed using self-made SP20SS and carbon tips. We were able to prepare samples in 55 min instead of 21 h. Finally, the method was applied in the context of oncological biomarker discovery exemplarily to ovarian and colon cancer. We observed a significant downregulation of sialylated hybrid structures in ovarian cancer samples using capillary electrophoresis-laser-induced fluorescence (CE-LIF). Furthermore, sepsis, a systemic inflammatory response syndrome, was also included in the study to understand whether the changes observed in ovarian cancer patients were due to the cancer itself or to the inflammation that usually accompanies its development. Because sialylated hybrid structures were upregulated in sepsis samples, the downregulation of these structures in ovarian cancer is specific to the cancer itself and, therefore, could be used as a biomarker., (Copyright © 2012 Elsevier Inc. All rights reserved.)

CLCA proteins (calcium-activated chloride channel regulators) have been linked to diseases involving secretory disorders, including cystic fibrosis (CF) and asthma. They have been shown to modulate endogenous chloride conductance, possibly by acting as metalloproteases. Based on the differential processing of the subunits after posttranslational cleavage, two subgroups of CLCA proteins can be distinguished. In one subgroup, both subunits are secreted, in the other group, the carboxy-terminal subunit possesses a transmembrane segment, resulting in shedding of only the amino-terminal subunit. Recent data on the post-translational cleavage and proteolytic activity of CLCA are limited to secreted CLCA. In this study, we characterized the cleavage of mCLCA6, a murine CLCA possessing a transmembrane segment. As for secreted CLCA, the cleavage in the endoplasmic reticulum was not observed for a protein with the E157Q mutation in the HEXXH motif of mCLCA6, suggesting that this mutant protein and secreted CLCA family members share a similar autoproteolytic cleavage mechanism. In contrast to secreted CLCA proteins with the E157Q mutation, the uncleaved precursor of the mCLCA6E157Q mutant reached the plasma membrane, where it was cleaved and the amino-terminal subunit was shed into the supernatant. Using crude membrane fractions, we showed that cleavage of the mCLCA6E157Q protein is zinc-dependent and sensitive to metalloprotease inhibitors, suggesting secondary cleavage by a metalloprotease. Interestingly, anchorage of mCLCA6E157Q to the plasma membrane is not essential for its secondary cleavage, because the mCLCA6(Δ™)E157Q mutant still underwent cleavage. Our data suggest that the processing of CLCA proteins is more complex than previously recognized.

Purpose: To investigate the impact of cataract surgery on the association of the ocular pulse amplitude (OPA) and intraocular pressure (IOP) with respect to the interpretation of OPA as an estimate of ocular blood flow., Methods: Twenty-four patients with cataract were included in a clinical study. OPA was measured using dynamic contour tonometry (DCT, Pascal(®), SMT Swiss Microtechnology AG, Switzerland). IOP was measured by means of Goldmann applanation tonometry (IOP GAT) and DCT (IOP DCT). All measurements were performed before and one day after cataract surgery., Results: At baseline, OPA was correlated to IOP GAT (r = 0.67, P = 0.0002) and IOP DCT (r = 0.82, P < 0.0001), but not to age or axial length. Postoperative OPA was correlated to IOP GAT (r = 0.67, P = 0.0002) and IOP DCT (r = 0.65, P = 0.0004). In 17 patients, IOP GAT decreased after surgery (IOP DCT n = 14), whereas an increase was apparent in seven patients (IOP DCT n = 10). The mean absolute deviation of IOP GAT pre- to post-surgery was 4.54 mmHg ± 2.47 (range 1-10 mmHg) and 5.4 mmHg ± 3.2 (range 1.1-13.1 mmHg) for IOP DCT. The changes of OPA were significantly correlated to changes in IOP GAT (r = 0.48, P = 0.017) and IOP DCT (r = 0.60, P = 0.001). IOP GAT and IOP DCT changes were not correlated to changes in corneal thickness., Conclusions: The OPA measured with the Pascal(®) device seems to be dependent on IOP changes. Particular caution should be taken in the interpretation of OPA in estimating pulsatile ocular blood flow.

Glycosylation is a post-translational modification that is of paramount importance in the production of recombinant pharmaceuticals as most recombinantly produced therapeutics are N- and/or O-glycosylated. Being a cell-system-dependent process, it also varies with expression systems and growth conditions, which result in glycan microheterogeneity and macroheterogeneity. Glycans have an effect on drug stability, serum half-life, and immunogenicity; it is therefore important to analyze and optimize the glycan decoration of pharmaceuticals. This review summarizes the aspects of protein glycosylation that are of interest to biotechnologists, namely, biosynthesis and biological relevance, as well as the tools to optimize and to analyze protein glycosylation.

High-mannose and hybrid-type N-glycans are present in human serum glycoproteins in low abundance but have recently been described to play an important role in immune responses. It is therefore important to find a strategy to selectively analyze their structures in the context of health and disease in order to understand their impact on disease mechanisms. We report here the characterization of high-mannose and hybrid-type N-glycans in total human serum. To this end, N-glycans were released using Endo-β-N-acetylglucosaminidase H (Endo H) and analyzed by CE-LIF and MALDI-TOF-MS. We found that the high-mannose structures Man(5-9)GlcNAc(1) represented the majority of the pool. The monoglucosylated structure Glc(1)Man(9)GlcNAc(1) as well as four hybrid structures could be identified. Then, we compared the Endo H-released serum glycome of patients suffering from rheumatoid arthritis with healthy controls as mannose-binding lectin deficiency (MBL) and modulation of α-mannosidase activity were previously associated with this disease. Interestingly, we observed that both high-mannose and hybrid structures were fairly constant, suggesting that circulating MBL and α-mannosidase may not affect significantly the levels of serum glycoproteins carrying these glycans., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Several members of the CLCA family of proteins, originally named chloride channels, calcium-activated, have been shown to modulate chloride conductance in various cell types via an unknown mechanism. Moreover, the human (h) hCLCA1 is thought to modulate the severity of disease in asthma and cystic fibrosis (CF) patients. All CLCA proteins are post-translationally cleaved into two subunits, and recently, a conserved HEXXH zinc-binding amino acid motif has been identified, suggesting a role for CLCA proteins as metalloproteases. Here, we have characterized the cleavage and autoproteolytic activity of the murine model protein mCLCA3, which represents the murine orthologue of human hCLCA1. Using crude membrane fractions from transfected HEK293 cells, we demonstrate that mCLCA3 cleavage is zinc-dependent and exclusively inhibited by cation-chelating metalloprotease inhibitors. Cellular transport and secretion were not affected in response to a cleavage defect that was introduced by the insertion of an E157Q mutation within the HEXXH motif of mCLCA3. Interspecies conservation of these key results was further confirmed with the porcine (p) orthologue of hCLCA1 and mCLCA3, pCLCA1. Importantly, the mCLCA3E157Q mutant was cleaved after co-transfection with the wild-type mCLCA3 in HEK293 cells, suggesting that an intermolecular autoproteolytic event takes place. Edman degradation and MALDI-TOF-MS of the protein fragments identified a single cleavage site in mCLCA3 between amino acids 695 and 696. The data strongly suggest that secreted CLCA proteins have zinc-dependent autoproteolytic activity and that they may cleave additional proteins.

Background: To evaluate which factors predispose to an intraoperative conversion to the usual limbal approach in minimally invasive strabismus surgery (MISS)., Methods: This study included 451 consecutive patients operated on by one surgeon at Kantonsspital St Gallen, Switzerland, with minimally invasive rectus muscle surgery between February 2003 and December 2007. We evaluated the intraoperative conversion rate to the usual limbal approach over time, and performed a retrospective determination of date of surgery, age of patient, motility of the eye, primary or revision surgery, and the type and dose of surgery in 982 consecutive rectus muscle procedures., Results: In 3.6% (35/982) of MISS procedures, an intraoperative conversion to a large, limbal approach was necessary. The overall conversion rate decreased over time, from 8.4% in 2003 to 0.4% in 2007. The multivariate regression analysis showed a significant negative influence between the date of surgery and the conversion rate (p < 0.005). Muscle resections were associated with a higher conversion rate (p < 0.001). The other evaluated factors had no significant influence on an intraoperative enlargement of the conjunctival opening., Conclusions: This study confirms the reliability of the new MISS technique, and shows a low conversion rate to the usual limbal approach. The conversion rate decreased over time with increasing surgical experience. Muscle resections were associated with a higher conversion rate, while the age of the patient, the motility of the eye, revision surgery and the dose of surgery had no significant influence on an intraoperative conversion.

Human alpha-1-antitrypsin (A1AT) is a protease inhibitor that is involved in the protection of lungs from neutrophil elastase enzyme that drastically modifies tissue functioning. The glycoprotein consists of 394 amino acids and is N-glycosylated at Asn-46, Asn-83, and Asn-247. A1AT deficiency is currently treated with A1AT that is purified from human serum. In view of therapeutic applications, rA1AT was produced using a novel human neuronal cell line (AGE1.HN®) and we investigated the N-glycosylation pattern as well as the in vitro anti-inflammatory activity of the recombinant glycoprotein. rA1AT (300 mg/L) was biologically active as analyzed using elastase assay. The N-glycan pool, released by PNGase F digestion, was characterized using 2D-HPLC, MALDI-TOF mass spectrometry, and by exoglycosidase digestions. A total of 28 N-glycan structures were identified, ranging from diantennary to tetraantennary complex-type N-glycans. Most of the N-glycans were found to be (α1-6) core-fucosylated and part of them contain the Lewis X epitope. The two major compounds are a monosialylated diantennary difucosylated glycan and a disialylated diantennary core-fucosylated glycan, representing 25% and 18% of the total N-glycan pool, respectively. Analysis of the site-specificity revealed that Asn-247 was mainly occupied by diantennary N-glycans whereas Asn-46 was occupied by di-, and triantennary N-glycans. Asn-83 was exclusively occupied by sialylated tri- and tetraantennary N-glycans. Next, we evaluated the anti-inflammatory activity of rA1AT using A1AT purified from human serum as a reference. rA1AT was found to inhibit the production of TNF-α in neutrophils and monocytes as commercial A1AT does., (Copyright © 2011 Wiley Periodicals, Inc.)

The addition of N-glycans to clinically used proteins enhances their therapeutic features. Here we report the design of a novel peptide tag with an unnatural N-glycosylation site, which may increase the N-glycan content of generally any protein. The designed GlycoTags were attached to A1AT, EPO and AGP and constructs were expressed in HEK293 or CHO cells. Hereby we could prove that the attached unnatural N-glycosylation site is decorated with complex-type N-glycans and that the spacer as well as the C-terminal "tail" sequence are critical for the usage of the novel N-glycosylation site. This demonstrates that the novel GlycoTag is a convenient tool to provide proteins with extra N-glycan moieties by simply adding a peptide tag sequence as small as 22 amino acids., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The leukocytic adhesion receptor L-selectin plays a crucial role in the first step of the adhesion cascade, enabling leukocytes to migrate into surrounding tissues during inflammation and immune surveillance. We analyzed the site-specific N-glycosylation of the lectin and EGF-like domain of L-selectin using recombinant variants ("LEHis"). The three glycosylation sites of LEHis were mutated to obtain singly glycosylated variants that were expressed in HEK293F cells. alpha1-Acid glycoprotein (AGP), expressed in the same system, was used to distinguish between cell type- and protein-specific glycosylation. Using mass spectrometry and exoglycosidase digestions, we established that LEHis was mostly bearing multifucosylated diantennary N-glycans with a major fraction terminating with GalNAc residues replacing the more common Gal. We could also show that parts of the GalNAc residues were sulfated. Furthermore, we identified novel diantennary glycan structures terminating with the motif GalNAc-GalNAc or SO(4)-GalNAc-GalNAc, which have not been described for N-glycans yet. Interestingly, none of these specific features were found in the N-glycan profile of AGP. This indicates that protein intrinsic information of L-selectin leads to decoration with specific N-glycans, which in turn may be related to L-selectin function.

Among the large set of cell surface glycan structures, the carbohydrate polymer polysialic acid (polySia) plays an important role in vertebrate brain development and synaptic plasticity. The main carrier of polySia in the nervous system is the neural cell adhesion molecule NCAM. As polySia with chain lengths of more than 40 sialic acid residues was still observed in brain of newborn Ncam(-/-) mice, we performed a glycoproteomics approach to identify the underlying protein scaffolds. Affinity purification of polysialylated molecules from Ncam(-/-) brain followed by peptide mass fingerprinting led to the identification of the synaptic cell adhesion molecule SynCAM 1 as a so far unknown polySia carrier. SynCAM 1 belongs to the Ig superfamily and is a powerful inducer of synapse formation. Importantly, the appearance of polysialylated SynCAM 1 was not restricted to the Ncam(-/-) background but was found to the same extent in perinatal brain of WT mice. PolySia was located on N-glycans of the first Ig domain, which is known to be involved in homo- and heterophilic SynCAM 1 interactions. Both polysialyltransferases, ST8SiaII and ST8SiaIV, were able to polysialylate SynCAM 1 in vitro, and polysialylation of SynCAM 1 completely abolished homophilic binding. Analysis of serial sections of perinatal Ncam(-/-) brain revealed that polySia-SynCAM 1 is expressed exclusively by NG2 cells, a multifunctional glia population that can receive glutamatergic input via unique neuron-NG2 cell synapses. Our findings sug-gest that polySia may act as a dynamic modulator of SynCAM 1 functions during integration of NG2 cells into neural networks.

Purpose: To identify and quantify the time course of fluorescein leakage of the optic nerve head in primary open-angle glaucoma (POAG) and controls., Methods: Twenty patients with POAG (aged 58 +/- 10 years) and 14 controls (aged 51 +/- 12 years, p = 0.07) were included in a prospective study. Fluorescein leakage of the optic disc was quantified using digital image analysis. A new leakage ratio (fluorescence of the optic disc divided by fluorescence of the surrounding retina) was defined and fluorescein leakage was quantified at 7-8, 9-10, 11-12, and 13-14 min after injection of 2.5 cc sodium fluorescein (10%)., Results: The fluorescein leakage exhibited a significantly different time course with higher leakage ratio values in POAG compared to controls (7-8 min: 1.24 +/- 0.32 vs 1.16 +/- 0.12; 9-10 min: 1.37 +/- 0.37 vs 1.19 +/- 0.1; 11-12 min: 1.38 +/- 0.36 vs 1.24 +/- 0.13; 13-14 min: 1.44 +/- 0.36 vs 1.27 +/- 0.13; p = 0.004). The change in optic disc fluorescence from 7-8 min to 9-10 min was significantly higher in POAG compared to controls (0.13 +/- 0.09 vs 0.03 +/- 0.07; p = 0.002)., Conclusion: The time course of fluorescein leakage is significantly different in POAG compared to controls. This might reflect damage of the optic disc vasculature related to increased vascular permeability.

Purpose: The aim of this study was to evaluate the sensitivity to detect glaucoma by observer-dependent nerve fibre layer imaging and evaluation using a scanning laser ophthalmoscope., Patients and Methods: Fifty-four patients with primary open angle glaucoma (POAG), 41 patients with normal tension glaucoma (NTG) and 65 eyes of control subjects were included in a retrospective study. Nerve fibre layer imaging was performed using a confocal scanning laser ophthalmoscope (SLO, Rodenstock Instr., argon laser 488 nm). Digitised images of the nerve fibre layer were graded off-line by two masked experienced observers. The nerve fibre layer was graded for every single degree (defect or no defect) at 1.7 and 3.4 mm distance to the optic nerve head omitting the nasal 110 degrees. Sensitivity and specificity levels were calculated using ROC analysis., Results: Patients with POAG exhibited nerve fibre layer defects of 100 degrees +/- 94 degrees (1.7 mm and 3.4 mm distance) and patients with NTG of 94 degrees +/- 94 degrees (1.7 mm distance) and 103 degrees +/- 92 degrees (3.4 mm distance), significantly larger compared to controls (1 degrees +/- 3 degrees [1.7 mm, p < 0.0001] and 1 degrees +/- 4 degrees [3.4 mm, p < 0.0001]). Sensitivity at > 90 % specificity was 80 % (1.7 mm) and 82 % (3.4 mm) for POAG. In NTG, sensitivity at > 90 % specifity was 81 % (1.7 mm) and 85 % (3.4 mm). In early glaucoma (MD < -6 dB, n = 39) sensitivity decreased to 75 % (1.7 mm) and 81 % (3.4 mm)., Conclusion: Observer-dependent evaluation and grading of the nerve fibre layer using a scanning laser ophthalmoscope reaches considerably high sensitivity levels to detect glaucoma with negligible differences between the central and peripheral regions., ((c) Georg Thieme Verlag KG Stuttgart New York.)