The Arabidopsis Plastidial Glucose-6-Phosphate Transporter GPT1 is Dually Targeted to Peroxisomes via the Endoplasmic Reticulum.

Studies on Glucose-6-phosphate (G6P)/phosphate translocator isoforms GPT1 and GPT2 reported the viability of Arabidopsis (Arabidopsis thaliana) gpt2 mutants, whereas heterozygous gpt1 mutants exhibited a variety of defects during fertilization/seed set, indicating that GPT1 is essential for this process. Among other functions, GPT1 was shown to be important for pollen and embryo-sac development. Because our previous work on the irreversible part of the oxidative pentose phosphate pathway (OPPP) revealed comparable effects, we investigated whether GPT1 may dually localize to plastids and peroxisomes. In reporter fusions, GPT2 localized to plastids, but GPT1 also localized to the endoplasmic reticulum (ER) and around peroxisomes. GPT1 contacted two oxidoreductases and also peroxins that mediate import of peroxisomal membrane proteins from the ER, hinting at dual localization. Reconstitution in yeast (Saccharomyces cerevisiae) proteoliposomes revealed that GPT1 preferentially exchanges G6P for ribulose-5-phosphate (Ru5P). Complementation analyses of heterozygous +/gpt1 plants demonstrated that GPT2 is unable to compensate for GPT1 in plastids, whereas GPT1 without the transit peptide (enforcing ER/peroxisomal localization) increased gpt1 transmission significantly. Because OPPP activity in peroxisomes is essential for fertilization, and immunoblot analyses hinted at the presence of unprocessed GPT1-specific bands, our findings suggest that GPT1 is indispensable in both plastids and peroxisomes. Together with its G6P-Ru5P exchange preference, GPT1 appears to play a role distinct from that of GPT2 due to dual targeting.

Vaccine effectiveness against severe COVID-19 during the Omicron wave in Germany: results from the COViK study.

PURPOSE: COViK, a prospective hospital-based multicenter case-control study in Germany, aims to assess the effectiveness of COVID-19 vaccines against severe disease. Here, we report vaccine effectiveness (VE) against COVID-19-caused hospitalization and intensive care treatment during the Omicron wave. METHODS: We analyzed data from 276 cases with COVID-19 and 494 control patients recruited in 13 hospitals from 1 December 2021 to 5 September 2022. We calculated crude and confounder-adjusted VE estimates. RESULTS: 21% of cases (57/276) were not vaccinated, compared to 5% of controls (26/494; p < 0.001). Confounder-adjusted VE against COVID-19-caused hospitalization was 55.4% (95% CI: 12-78%), 81.5% (95% CI: 68-90%) and 95.6% (95%CI: 88-99%) after two, three and four vaccine doses, respectively. VE against hospitalization due to COVID-19 remained stable up to one year after three vaccine doses. CONCLUSION: Three vaccine doses remained highly effective in preventing severe disease and this protection was sustained; a fourth dose further increased protection.

Left Ventricular Wall Reconstruction with Autologous Vascularized Tunica Muscularis of Stomach in a Porcine Pilot Model.

INTRODUCTION: Surgical replacement of dysfunctional cardiac muscle with regenerative tissue is an important option to combat heart failure. But, current available myocardial prostheses like a Dacron or a pericardium patch neither have a regenerative capacity nor do they actively contribute to the heart's pump function. This study aimed to show the feasibility of utilizing a vascularized stomach patch for transmural left ventricular wall reconstruction. METHODS: A left ventricular transmural myocardial defect was reconstructed by performing transdiaphragmatic autologous transplantation of a vascularized stomach segment in six Lewe minipigs. Three further animals received a conventional Dacron patch as a control treatment. The first 3 animals were followed up for 3 months until planned euthanasia, whereas the observation period for the remaining 3 animals was scheduled 6 months following surgery. Functional assessment of the grafts was carried out via cardiac magnetic resonance tomography and angiography. Physiological remodeling was evaluated histologically and immunohistochemically after heart explantation. RESULTS: Five out of six test animals and all control animals survived the complex surgery and completed the follow-up without clinical complications. One animal died intraoperatively due to excessive bleeding. No animal experienced rupture of the stomach graft. Functional integration of the heterotopically transplanted stomach into the surrounding myocardium was observed. Angiography showed development of connections between the gastric graft vasculature and the coronary system of the host cardiac tissue. CONCLUSIONS: The clinical results and the observed physiological integration of gastric grafts into the cardiac structure demonstrate the feasibility of vascularized stomach tissue as myocardial prosthesis. The physiological remodeling indicates a regenerative potential of the graft. Above all, the connection of the gastric vessels with the coronary system constitutes a rationale for the use of vascularized and, therefore, viable stomach tissue for versatile tissue engineering applications.

Biocompatibility of an apical ring plug for left ventricular assist device explantation: Results of a feasibility pre-clinical study.

BACKGROUND: Patients receiving left ventricle assist devices (LVADs) as bridge to recovery remain a minority with 1%-5% of LVADs explanted after improvement of myocardial function. Nevertheless, considering the growing population of patients supported with LVADs, an increasing demand of new explantation strategies is expected in the near future. A novel plug for LVAD explantation has been developed and its biocompatibility profile needs to be proved. This study tested the biocompatibility of this novel plug in an in vivo ovine model. METHODS: Six adult Blackhead Persian female sheep received plug implantation on the cardiac apex via minimally invasive approach and were clinically observed up to 90 days. Echocardiography was performed to detect thrombus formation or further plug-related complications. After the observation period, euthanasia was performed and samples including the plug and the surrounding tissues were obtained to be analyzed with correlative light and electron microscopy. Organ necrosis, ischemia and peripheral embolism were investigated. RESULTS: Three animals survived surgery and completed the follow-up time without experiencing clinical complications. Echocardiographic controls excluded the presence of an intracavitary thrombus in the left ventricle (LV). Autopsy confirmed no signs of local infection, LV thrombus or peripheral embolism. Light and electron microscopy revealed an intact epithelium covering a layer of connective tissue on the plug surface facing the heart lumen. CONCLUSIONS: This novel apical plug for LVAD explantation allows for endothelial and connective tissue growth on its ventricular side within 90 days from surgery. Further studies are required to fully demonstrate the biocompatibility of this apical plug and investigate the optimal anticoagulation regimen to be applied after implantation.

In vivo proteomics identifies the competence regulon and AliB oligopeptide transporter as pathogenic factors in pneumococcal meningitis.

Streptococcus pneumoniae (pneumococci) is a leading cause of severe bacterial meningitis in many countries worldwide. To characterize the repertoire of fitness and virulence factors predominantly expressed during meningitis we performed niche-specific analysis of the in vivo proteome in a mouse meningitis model, in which bacteria are directly inoculated into the cerebrospinal fluid (CSF) cisterna magna. We generated a comprehensive mass spectrometry (MS) spectra library enabling bacterial proteome analysis even in the presence of eukaryotic proteins. We recovered 200,000 pneumococci from CSF obtained from meningitis mice and by MS we identified 685 pneumococci proteins in samples from in vitro filter controls and 249 in CSF isolates. Strikingly, the regulatory two-component system ComDE and substrate-binding protein AliB of the oligopeptide transporter system were exclusively detected in pneumococci recovered from the CSF. In the mouse meningitis model, AliB-, ComDE-, or AliB-ComDE-deficiency resulted in attenuated meningeal inflammation and disease severity when compared to wild-type pneumococci indicating the crucial role of ComDE and AliB in pneumococcal meningitis. In conclusion, we show here mechanisms of pneumococcal adaptation to a defined host compartment by a proteome-based approach. Further, this study provides the basis of a promising strategy for the identification of protein antigens critical for invasive disease caused by pneumococci and other meningeal pathogens.

Defects in Peroxisomal 6-Phosphogluconate Dehydrogenase Isoform PGD2 Prevent Gametophytic Interaction in Arabidopsis thaliana.

We studied the localization of 6-phosphogluconate dehydrogenase (PGD) isoforms of Arabidopsis (Arabidopsis thaliana). Similar polypeptide lengths of PGD1, PGD2, and PGD3 obscured which isoform may represent the cytosolic and/or plastidic enzyme plus whether PGD2 with a peroxisomal targeting motif also might target plastids. Reporter-fusion analyses in protoplasts revealed that, with a free N terminus, PGD1 and PGD3 accumulate in the cytosol and chloroplasts, whereas PGD2 remains in the cytosol. Mutagenesis of a conserved second ATG enhanced the plastidic localization of PGD1 and PGD3 but not PGD2. Amino-terminal deletions of PGD2 fusions with a free C terminus resulted in peroxisomal import after dimerization, and PGD2 could be immunodetected in purified peroxisomes. Repeated selfing of pgd2 transfer (T-)DNA alleles yielded no homozygous mutants, although siliques and seeds of heterozygous plants developed normally. Detailed analyses of the C-terminally truncated PGD2-1 protein showed that peroxisomal import and catalytic activity are abolished. Reciprocal backcrosses of pgd2-1 suggested that missing PGD activity in peroxisomes primarily affects the male gametophyte. Tetrad analyses in the quartet1-2 background revealed that pgd2-1 pollen is vital and in vitro germination normal, but pollen tube growth inside stylar tissues appeared less directed. Mutual gametophytic sterility was overcome by complementation with a genomic construct but not with a version lacking the first ATG. These analyses showed that peroxisomal PGD2 activity is required for guided growth of the male gametophytes and pollen tube-ovule interaction. Our report finally demonstrates an essential role of oxidative pentose-phosphate pathway reactions in peroxisomes, likely needed to sustain critical levels of nitric oxide and/or jasmonic acid, whose biosynthesis both depend on NADPH provision.

Toxin exposure and HLA alleles determine serum antibody binding to toxic shock syndrome toxin 1 (TSST-1) of Staphylococcus aureus.

Life-threatening toxic shock syndrome is often caused by the superantigen toxic shock syndrome toxin-1 (TSST-1) produced by Staphylococcus aureus. A well-known risk factor is the lack of neutralizing antibodies. To identify determinants of the anti-TSST-1 antibody response, we examined 976 participants of the German population-based epidemiological Study of Health in Pomerania (SHIP-TREND-0). We measured anti-TSST-1 antibody levels, analyzed the colonization with TSST-1-encoding S. aureus strains, and performed a genome-wide association analysis of genetic risk factors. TSST-1-specific serum IgG levels varied over a range of 4.2 logs and were elevated by a factor of 12.3 upon nasal colonization with TSST-1-encoding S. aureus. Moreover, the anti-TSST-1 antibody levels were strongly associated with HLA class II gene loci. HLA-DRB1*03:01 and HLA-DQB1*02:01 were positively, and HLA-DRB1*01:01 as well as HLA-DQB1*05:01 negatively associated with the anti-TSST-1 antibody levels. Thus, both toxin exposure and HLA alleles affect the human antibody response to TSST-1.

Effectiveness of vaccines in preventing hospitalization due to COVID-19: A multicenter hospital-based case-control study, Germany, June 2021 to January 2022.

We included 852 patients in a prospectively recruiting multicenter matched case-control study in Germany to assess vaccine effectiveness (VE) in preventing COVID-19-associated hospitalization during the Delta-variant dominance. The two-dose VE was 89 % (95 % CI 84-93 %) overall, 79 % in patients with more than two comorbidities and 77 % in adults aged 60-75 years. A third dose increased the VE to more than 93 % in all patient-subgroups.

A bead-based multiplex assay covering all coronaviruses pathogenic for humans for sensitive and specific surveillance of SARS-CoV-2 humoral immunity.

Serological assays measuring antibodies against SARS-CoV-2 are key to describe the epidemiology, pathobiology or induction of immunity after infection or vaccination. Of those, multiplex assays targeting multiple antigens are especially helpful as closely related coronaviruses or other antigens can be analysed simultaneously from small sample volumes, hereby shedding light on patterns in the immune response that would otherwise remain undetected. We established a bead-based 17-plex assay detecting antibodies targeting antigens from all coronaviruses pathogenic for humans: SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV strains 229E, OC43, HKU1, and NL63. The assay was validated against five commercial serological immunoassays, a commercial surrogate virus neutralisation test, and a virus neutralisation assay, all targeting SARS-CoV-2. It was found to be highly versatile as shown by antibody detection from both serum and dried blot spots and as shown in three case studies. First, we followed seroconversion for all four endemic HCoV strains and SARS-CoV-2 in an outbreak study in day-care centres for children. Second, we were able to link a more severe clinical course to a stronger IgG response with this 17-plex-assay, which was IgG1 and IgG3 dominated. Finally, our assay was able to discriminate recent from previous SARS-CoV-2 infections by calculating the IgG/IgM ratio on the N antigen targeting antibodies. In conclusion, due to the comprehensive method comparison, thorough validation, and the proven versatility, our multiplex assay is a valuable tool for studies on coronavirus serology.

Alternative targeting of Arabidopsis plastidic glucose-6-phosphate dehydrogenase G6PD1 involves cysteine-dependent interaction with G6PD4 in the cytosol.

Arabidopsis peroxisomes contain an incomplete oxidative pentose-phosphate pathway (OPPP), consisting of 6-phosphogluconolactonase and 6-phosphogluconate dehydrogenase isoforms with peroxisomal targeting signals (PTS). To start the pathway, glucose-6-phosphate dehydrogenase (G6PD) is required; however, G6PD isoforms with obvious C-terminal PTS1 or N-terminal PTS2 motifs are lacking. We used fluorescent reporter fusions to explore possibly hidden peroxisomal targeting information. Among the six Arabidopsis G6PD isoforms only plastid-predicted G6PD1 with free C-terminal end localized to peroxisomes. Detailed analyses identified SKY as an internal PTS1-like signal; however, in a medial G6PD1 reporter fusion with free N- and C-terminal ends this cryptic information was overruled by the transit peptide. Yeast two-hybrid analyses revealed selective protein-protein interactions of G6PD1 with catalytically inactive G6PD4, and of both G6PD isoforms with plastid-destined thioredoxin m2 (Trx(m2) ). Serine replacement of redox-sensitive cysteines conserved in G6PD4 abolished the G6PD4-G6PD1 interaction, albeit analogous changes in G6PD1 did not. In planta bimolecular fluorescence complementation (BiFC) demonstrated that the G6PD4-G6PD1 interaction results in peroxisomal import. BiFC also confirmed the interaction of Trx(m2) with G6PD4 (or G6PD1) in plastids, but co-expression analyses revealed Trx(m2) -mediated retention of medial G6PD4 (but not G6PD1) reporter fusions in the cytosol that was stabilized by CxxC¹¹³S exchange in Trx(m2) . Based on preliminary findings with plastid-predicted rice G6PD isoforms, we dismiss Arabidopsis G6PD4 as non-functional. G6PD4 orthologs (new P0 class) apparently evolved to become cytosolic redox switches that confer thioredoxin-relayed alternative targeting to peroxisomes.

A Comprehensive View on the Human Antibody Repertoire Against Staphylococcus aureus Antigens in the General Population.

Our goal was to provide a comprehensive overview of the antibody response to Staphylococcus aureus antigens in the general population as a basis for defining disease-specific profiles and diagnostic signatures. We tested the specific IgG and IgA responses to 79 staphylococcal antigens in 996 individuals from the population-based Study of Health in Pomerania. Using a dilution-based multiplex suspension array, we extended the dynamic range of specific antibody detection to seven orders of magnitude, allowing the precise quantification of high and low abundant antibody specificities in the same sample. The observed IgG and IgA antibody responses were highly heterogeneous with differences between individuals as well as between bacterial antigens that spanned several orders of magnitude. Some antigens elicited significantly more IgG than IgA and vice versa. We confirmed a strong influence of colonization on the antibody response and quantified the influence of sex, smoking, age, body mass index, and serum glucose on anti-staphylococcal IgG and IgA. However, all host parameters tested explain only a small part of the extensive variability in individual response to the different antigens of S. aureus.

Comparison of sequential and helical scanning for radiation dose and image quality: results of the Prospective Multicenter Study on Radiation Dose Estimates of Cardiac CT Angiography (PROTECTION) I Study.

OBJECTIVE: Concerns have been raised about the radiation exposure of coronary CT angiography (CTA). Recently, a prospective ECG-triggered sequential coronary CTA technique was developed to reduce exposure to ionizing radiation. The purpose of this analysis was to determine the impact of a sequential scanning technique on image quality and radiation dose in a prespecified subgroup analysis of the Prospective Multicenter Study on Radiation Dose Estimates of Cardiac CT Angiography I (PROTECTION I) Study when compared with a standard helical scanning technique. MATERIALS AND METHODS: This analysis comprises 685 64-MDCT coronary angiography studies of 47 international study sites in which the image quality was assessed by an experienced coronary CTA investigator using a 4-point score (1 = nondiagnostic, 4 = excellent image quality). Image quality was analyzed in all patients studied with the sequential scanning mode (n = 99) and in randomly selected patients of the population studied with the helical acquisition mode (n = 586). Radiation dose estimates were derived from the dose-length product (DLP) and a conversion coefficient for the chest (0.014 mSv x mGy(-1) x cm(-1)). RESULTS: Although the sequential scanning mode significantly reduced radiation dose estimates by 68% from 11.2 mSv for the helical mode to 3.6 mSv for the sequential mode (p < 0.001), the median diagnostic image quality scores were comparable in both groups. The median diagnostic score for both scanning modes was 3.5 (interquartile range: sequential vs helical mode, 3.25-3.75 vs 3.0-3.75, respectively; p = 0.62). CONCLUSION: The results of the PROTECTION I Study suggest that the prospective ECG-triggered sequential coronary CTA technique significantly reduces radiation dose without impairing image quality when compared with the standard retrospective helical data acquisition in patients with a low and stable heart rate.

Technical report: xMAPr - High-dynamic-range (HDR) quantification of antigen-specific antibody binding.

Here, we demonstrate a high-dynamic-range quantification of antibody binding to single antigens in a multiplexed suspension bead array format. Using a dilution-based approach and the newly developed data analysis tool the quantitative dynamic range is increased by three orders of magnitude in the selected samples. The strong increase in dynamic range results in a more robust data basis for downstream analyses.

Estimated radiation dose associated with cardiac CT angiography.

CONTEXT: Cardiac computed tomography (CT) angiography (CCTA) has emerged as a useful diagnostic imaging modality in the assessment of coronary artery disease. However, the potential risks due to exposure to ionizing radiation associated with CCTA have raised concerns. OBJECTIVES: To estimate the radiation dose of CCTA in routine clinical practice as well as the association of currently available strategies with dose reduction and to identify the independent factors contributing to radiation dose. DESIGN, SETTING, AND PATIENTS: A cross-sectional, international, multicenter, observational study (50 study sites: 21 university hospitals and 29 community hospitals) of estimated radiation dose in 1965 patients undergoing CCTA between February and December 2007. Linear regression analysis was used to identify independent predictors associated with dose. MAIN OUTCOME MEASURE: Dose-length product (DLP) of CCTA. RESULTS: The median DLP of 1965 CCTA examinations performed at 50 study sites was 885 mGy x cm (interquartile range, 568-1259 mGy x cm), which corresponds to an estimated radiation dose of 12 mSv (or 1.2 x the dose of an abdominal CT study or 600 chest x-rays). A high variability in DLP was observed between study sites (range of median DLPs per site, 331-2146 mGy x cm). Independent factors associated with radiation dose were patient weight (relative effect on DLP, 5%; 95% confidence interval [CI], 4%-6%), absence of stable sinus rhythm (10%; 95% CI, 2%-19%), scan length (5%; 95% CI, 4%-6%), electrocardiographically controlled tube current modulation (-25%; 95% CI, -23% to -28%; applied in 73% of patients), 100-kV tube voltage (-46%; 95% CI, -42% to -51%; applied in 5% of patients), sequential scanning (-78%; 95% CI, -77% to -79%; applied in 6% of patients), experience in cardiac CT (-1%; 95% CI, -1% to 0%), number of CCTAs per month (0%; 95% CI, 0%-1%), and type of 64-slice CT system (for highest vs lowest dose system, 97%; 95% CI, 88%-106%). Algorithms for dose reduction were not associated with deteriorated diagnostic image quality in this observational study. CONCLUSIONS: Median doses of CCTA differ significantly between study sites and CT systems. Effective strategies to reduce radiation dose are available but some strategies are not frequently used. The comparable diagnostic image quality may support an increased use of dose-saving strategies in adequately selected patients.

Reduction of radiation dose estimates in cardiac 64-slice CT angiography in patients after coronary artery bypass graft surgery.

OBJECTIVES: The objectives of this prospective investigation in patients after bypass graft surgery were (1) to estimate radiation dose for routine bypass graft computed tomography (CT) angiography, (2) to study the impact of anatomically adapted and ECG-controlled tube current modulation on radiation dose estimates, and (3) effects on qualitative and quantitative image quality parameters. METHODS: Radiation dose was estimated for 194 consecutive patients undergoing 64-slice CT angiography (Somatom Sensation 64 Cardiac, Siemens Medical Solutions). The impact of anatomically adapted tube current modulation was studied in 2 consecutive patients groups. Furthermore, the impact of ECG-controlled tube current modulation, applied as indicated, was evaluated in both groups. RESULTS: Mean radiation dose estimate for a 64-slice CT bypass graft study was 18.9 +/- 6.0 mSv (CTDIvol 42.3 +/- 12.9 mGy). The application of anatomically adapted tube current modulation had no effect on dose parameters (CTDIvol 43.3 +/- 13.2 vs. 40.1 +/- 12.1 mGy for those with versus those without anatomically adapted tube current modulation, P = 0.1). Additional implementation of ECG-controlled tube current modulation resulted in reduced dose parameters (CTDIvol of 32.9 +/- 2.6 vs. 58.9 +/- 3.9 mGy and radiation dose estimates: 14.7 +/- 1.9 mSv vs. 26.5 +/- 2.1 mSv for those with versus those without ECG pulsing, both P < 0.01). There was no deterioration in image quality with use of tube current modulation algorithms. CONCLUSIONS: The radiation burden associated with 64-slice bypass graft CT angiographies is substantial. Anatomically adapted tube current modulation does not reduce radiation dose parameters, whereas ECG-controlled tube current modulation was associated with a 45% reduction in dose estimates. Application of both tube current modulation algorithms did not result in reduced image quality.

Intranasal Vaccination With Lipoproteins Confers Protection Against Pneumococcal Colonisation.

Streptococcus pneumoniae is endowed with a variety of surface-exposed proteins representing putative vaccine candidates. Lipoproteins are covalently anchored to the cell membrane and highly conserved among pneumococcal serotypes. Here, we evaluated these lipoproteins for their immunogenicity and protective potential against pneumococcal colonisation. A multiplex-based immunoproteomics approach revealed the immunogenicity of selected lipoproteins. High antibody titres were measured in sera from mice immunised with the lipoproteins MetQ, PnrA, PsaA, and DacB. An analysis of convalescent patient sera confirmed the immunogenicity of these lipoproteins. Examining the surface localisation and accessibility of the lipoproteins using flow cytometry indicated that PnrA and DacB were highly abundant on the surface of the bacteria. Mice were immunised intranasally with PnrA, DacB, and MetQ using cholera toxin subunit B (CTB) as an adjuvant, followed by an intranasal challenge with S. pneumoniae D39. PnrA protected the mice from pneumococcal colonisation. For the immunisation with DacB and MetQ, a trend in reducing the bacterial load could be observed, although this effect was not statistically significant. The reduction in bacterial colonisation was correlated with the increased production of antigen-specific IL-17A in the nasal cavity. Immunisation induced high systemic IgG levels with a predominance for the IgG1 isotype, except for DacB, where IgG levels were substantially lower compared to MetQ and PnrA. Our results indicate that lipoproteins are interesting targets for future vaccine strategies as they are highly conserved, abundant, and immunogenic.

Radiation dose estimates from cardiac multislice computed tomography in daily practice: impact of different scanning protocols on effective dose estimates.

BACKGROUND: Multislice computed tomography angiography (CTA) is a promising technology for imaging patients with suspected coronary artery disease. Compared with 16-slice CTA, the improved spatial and temporal resolution of 64-slice CTA (0.6- versus 1.0-mm slice thickness and 330- versus 420-ms gantry rotation time) is associated with an increase in radiation dose. The objective of this retrospective investigation was to compare the estimated dose received during 16- and 64-slice CTA in daily practice and to investigate the impact of different scan protocols on dose and image quality. METHODS AND RESULTS: Radiation dose was estimated for 1035 patients undergoing coronary CTA. Scanning algorithms with and without an ECG-dependent dose modulation and with a reduced tube voltage were investigated on dose estimates and image quality. In the entire patient cohort, radiation dose estimates were 6.4+/-1.9 and 11.0+/-4.1 mSv for 16- and 64-slice CTA, respectively (P<0.01). The reduction in radiation dose estimates ranged between 37% and 40% and between 53% and 64% with the use of ECG-dependent dose modulation and with the combined use of the dose modulation and a reduced tube voltage, respectively. The reduction in dose estimates was not associated with a reduction in diagnostic image quality as assessed by the signal-to-noise ratio and by the frequency of coronary segments with diagnostic image quality. CONCLUSIONS: The increase in spatial and temporal resolution with 64-slice CTA is associated with an increased radiation dose for coronary CTA. Dose-saving algorithms are very effective in reducing radiation exposure and should be used whenever possible.

* Six-Year-Old Sheep as a Clinically Relevant Large Animal Model for Aortic Valve Replacement Using Tissue-Engineered Grafts Based on Decellularized Allogenic Matrix.

Tissue-engineered (TE) grafts based on decellularized grafts have shown very promising results in preclinical and clinical studies. However, in animal models valves have either been tested in juvenile models or in the clinically less relevant pulmonary valve position. In this study, we tested the grafts in the aortic valve (AV) position of 6-year-old sheep, as geriatric patients in need of an AV substitute due to calcification are the largest patient group benefiting from TE grafts. Decellularized AV (DAV; n = 4) and DAV additionally re-endothelialized with autologous cells (n = 3) were implanted in the AV position of 6-year-old female sheep. Function was investigated at implantation and explantation 12 months later. Regeneration capacity was analyzed by the repopulation degree of the graft with recipient's cells, by the generation of a new endothelial layer and by intracellular staining against pro-collagen type I. DAV and re-endothelialized AV demonstrated excellent function with only two valves developing mild insufficiencies (1°). Of the repopulating cells only few cells were identified as inflammation cells, while the majority was found to be interstitial cells producing procollagen type I. Endothelial coverage was found, but seemed to be reduced. The regenerative capacity of decellularized matrix is not only a feature exhibited when implanted in juvenile individuals but also is evident when implanted in the high-pressure AV position of older sheep, revealing the potential of TE grafts in age-advanced patients.

Characterization of human and Staphylococcus aureus proteins in respiratory mucosa by in vivo- and immunoproteomics.

Staphylococcus aureus is a Gram-positive opportunistic bacterium which can be found as a commensal in the nares of about 50% of the human population. Besides asymptomatic carriage, S. aureus has also been found to colonize nasal polyps, a subform of chronic rhinosinusitis, in 60 to 100% of cases, and even reside intracellularly in nasal polyp tissue. The aim of this study was to shed light on the behavior of S. aureus in the human airways by analyzing S. aureus-specific proteins in nasal polyp tissue from patients with chronic rhinosinusitis and to characterize the immunogenic potential of the identified (mainly secreted) proteins. As a result, in total >600 S. aureus proteins were identified by high resolution mass spectrometry or multiple reaction monitoring. Of those roughly 180 are typically localized in the membrane, surface exposed or secreted. For 115 S. aureus proteins, partially also detected in vivo by mass spectrometry, IgA- and IgG-specific antibody signals were profiled. Strong antibody signals were predominantly found for surface expose or secreted proteins. SIGNIFICANCE: In this study, we used high resolution mass spectrometry to identify S. aureus proteins directly in infected nasal polyp tissue. We discovered bacterial proteins involved in invasion of tissue, virulence, bacterial signal transduction or acquisition of nutrients. Some of the detected superantigens and Spls are known to provoke secretion of a broad spectrum of cytokines. Therefore, our manuscript contains new information about the invasion of S. aureus in nasal polyp tissue and its protein-specific immunogenicity.

A global Staphylococcus aureus proteome resource applied to the in vivo characterization of host-pathogen interactions.

Data-independent acquisition mass spectrometry promises higher performance in terms of quantification and reproducibility compared to data-dependent acquisition mass spectrometry methods. To enable high-accuracy quantification of Staphylococcus aureus proteins, we have developed a global ion library for data-independent acquisition approaches employing high-resolution time of flight or Orbitrap instruments for this human pathogen. We applied this ion library resource to investigate the time-resolved adaptation of S. aureus to the intracellular niche in human bronchial epithelial cells and in a murine pneumonia model. In epithelial cells, abundance changes for more than 400 S. aureus proteins were quantified, revealing, e.g., the precise temporal regulation of the SigB-dependent stress response and differential regulation of translation, fermentation, and amino acid biosynthesis. Using an in vivo murine pneumonia model, our data-independent acquisition quantification analysis revealed for the first time the in vivo proteome adaptation of S. aureus. From approximately 2.15 × 105 S. aureus cells, 578 proteins were identified. Increased abundance of proteins required for oxidative stress response, amino acid biosynthesis, and fermentation together with decreased abundance of ribosomal proteins and nucleotide reductase NrdEF was observed in post-infection samples compared to the pre-infection state.