Add-On Prostaglandin E1 in Venovenous Extracorporeal Membrane Oxygenation: A Randomized, Double-Blind, Placebo-controlled Pilot Trial.

Rationale: Prostaglandin E1 (alprostadil; PGE1), in addition to low-dose unfractionated heparin, increases the biocompatibility of extracorporeal systems and enhances the efficacy of artificial organs without increasing bleeding risk. Objectives: We investigated the safety and efficacy of PGE1 in adults receiving venovenous extracorporeal membrane oxygenation (ECMO). Methods: This study was a randomized, double-blind, placebo-controlled phase II pilot trial at two medical intensive care units at the Medical University of Vienna, Austria. Adults with venovenous ECMO were randomly assigned to receive an intravenous infusion of 5 ng/kg/min PGE1 or placebo (0.9% saline) in addition to standard anticoagulation with unfractionated heparin. Measurements and Main Results: The primary outcome was the rate of transfused packed red blood cells per ECMO day. Secondary outcomes were the incidence of and time to clinically overt bleeding and thromboembolic events. A post hoc subgroup analysis included only patients with coronavirus disease (COVID-19). Between September 2016 and April 2021, of 133 screened patients, 50 patients were randomized, of whom 48 received the assigned study medication (24 per group). The transfusion rate was similar between groups (0.41 vs. 0.39; P = 0.733). PGE1 was associated with fewer thromboembolic events (7 vs. 16; P = 0.020) and longer thromboembolism-free time (hazard ratio [HR], 0.302; P = 0.01), fewer clinically overt bleeding events (2 vs. 11; P = 0.017), and longer bleeding-free time (HR, 0.213; P = 0.047). In patients with COVID-19 (n = 25), the HRs for clinically overt bleeding and thromboembolism were 0.276 (95% confidence interval, 0.035-2.186) and 0.521 (95% confidence interval, 0.149-1.825), respectively. Conclusions: Add-on treatment with PGE1 was safe but did not meet the primary endpoint of reducing the rate of red blood cell transfusions in patients receiving venovenous ECMO. Larger studies need to evaluate the safety and efficacy of additional PGE1 in ECMO. Clinical trial registered with EudraCT (2015-005014-30) and www.clinicaltrials.gov (NCT02895373).

EGF and BMPs Govern Differentiation and Patterning in Human Gastric Glands.

BACKGROUND & AIMS: The homeostasis of the gastrointestinal epithelium relies on cell regeneration and differentiation into distinct lineages organized inside glands and crypts. Regeneration depends on Wnt/ß-catenin pathway activation, but to understand homeostasis and its dysregulation in disease, we need to identify the signaling microenvironment governing cell differentiation. By using gastric glands as a model, we have identified the signals inducing differentiation of surface mucus-, zymogen-, and gastric acid-producing cells. METHODS: We generated mucosoid cultures from the human stomach and exposed them to different growth factors to obtain cells with features of differentiated foveolar, chief, and parietal cells. We localized the source of the growth factors in the tissue of origin. RESULTS: We show that epidermal growth factor is the major fate determinant distinguishing the surface and inner part of human gastric glands. In combination with bone morphogenetic factor/Noggin signals, epidermal growth factor controls the differentiation of foveolar cells vs parietal or chief cells. We also show that epidermal growth factor is likely to underlie alteration of the gastric mucosa in the precancerous condition atrophic gastritis. CONCLUSIONS: Use of our recently established mucosoid cultures in combination with analysis of the tissue of origin provided a robust strategy to understand differentiation and patterning of human tissue and allowed us to draw a new, detailed map of the signaling microenvironment in the human gastric glands.

Prevalence and Clinical Impact of Reduced Coagulation Factor XII Activity in Patients Receiving Extracorporeal Membrane Oxygenation.

OBJECTIVES: Extracorporeal membrane oxygenation provides large surface exposure to human blood leading to coagulation activation. Only limited clinical data are available on contact activation and coagulation factor XII activity in extracorporeal membrane oxygenation patients. DESIGN: Prospective cohort study. SETTING: Three medical ICUs at the Medical University of Vienna. PATIENTS: Adult patients receiving venovenous or venoarterial extracorporeal membrane oxygenation. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The primary outcome was the change in coagulation factor XII activity in response to extracorporeal membrane oxygenation. Secondary outcomes included the prevalence of reduced coagulation factor XII activity (< 60%) among patients receiving extracorporeal membrane oxygenation and association of coagulation factor XII activity with thromboembolic and bleeding complications. An exploratory endpoint was the association of coagulation factor XII activity and activated partial thromboplastin time in heparinase-treated samples in vitro. Fifty-one patients with a total of 117 samples were included in the study between July 2018 and February 2020. Fifty patients (98%) had reduced coagulation factor XII activity at any timepoint during extracorporeal membrane oxygenation. Median coagulation factor XII activity during extracorporeal membrane oxygenation treatment was 30% (interquartile range, 21.5-41%) and increased after discontinuation (p = 0.047). Patients with thromboembolic complications had higher median coagulation factor XII activity during extracorporeal membrane oxygenation (34% vs 23%; p = 0.023). The odds of a thromboembolic event increased by 200% per tertile of median coagulation factor XII activity (crude odds ratio, 3.034; 95% CI, 1.21-7.63). No association with bleeding was observed. In heparinase-treated samples, coagulation factor XII activity correlated well with activated partial thromboplastin time (r = -0.789; p = 0.007). CONCLUSIONS: We observed a high prevalence of reduced coagulation factor XII activity in adult patients on extracorporeal membrane oxygenation, which may confound activated partial thromboplastin time measurements and limit its clinical usefulness for monitoring and titrating anticoagulation with unfractionated heparin. Lower coagulation factor XII activity was associated with less thromboembolic complications, which may highlight the potential of coagulation factor XII to serve as a target for anticoagulation in extracorporeal membrane oxygenation.

ADP heptose, a novel pathogen-associated molecular pattern identified in Helicobacter pylori.

The gastric pathogen Helicobacter pylori activates the NF-κB pathway in human epithelial cells via the recently discovered α-kinase 1 TRAF-interacting protein with forkhead-associated domain (TIFA) axis. We and others showed that this pathway can be triggered by heptose 1,7-bisphosphate (HBP), an LPS intermediate produced in gram-negative bacteria that represents a new pathogen-associated molecular pattern (PAMP). Here, we report that our attempts to identify HBP in lysates of H. pylori revealed surprisingly low amounts, failing to explain NF-κB activation. Instead, we identified ADP-glycero-ß-D-manno-heptose (ADP heptose), a derivative of HBP, as the predominant PAMP in lysates of H. pylori and other gram-negative bacteria. ADP heptose exhibits significantly higher activity than HBP, and cells specifically sensed the presence of the ß-form, even when the compound was added extracellularly. The data lead us to conclude that ADP heptose not only constitutes the key PAMP responsible for H. pylori-induced NF-κB activation in epithelial cells, but it acts as a general gram-negative bacterial PAMP.-Pfannkuch, L., Hurwitz, R., Traulsen, J., Sigulla, J., Poeschke, M., Matzner, L., Kosma, P., Schmid, M., Meyer, T. F. ADP heptose, a novel pathogen-associated molecular pattern identified in Helicobacter pylori.

Polarised epithelial monolayers of the gastric mucosa reveal insights into mucosal homeostasis and defence against infection.

OBJECTIVE: Helicobacter pylori causes life-long colonisation of the gastric mucosa, leading to chronic inflammation with increased risk of gastric cancer. Research on the pathogenesis of this infection would strongly benefit from an authentic human in vitro model. DESIGN: Antrum-derived gastric glands from surgery specimens served to establish polarised epithelial monolayers via a transient air-liquid interface culture stage to study cross-talk with H. pylori and the adjacent stroma. RESULTS: The resulting 'mucosoid cultures', so named because they recapitulate key characteristics of the gastric mucosa, represent normal stem cell-driven cultures that can be passaged for months. These highly polarised columnar epithelial layers encompass the various gastric antral cell types and secrete mucus at the apical surface. By default, they differentiate towards a foveolar, MUC5AC-producing phenotype, whereas Wnt signalling stimulates proliferation of MUC6-producing cells and preserves stemness-reminiscent of the gland base. Stromal cells from the lamina propria secrete Wnt inhibitors, antagonising stem-cell niche signalling and inducing differentiation. On infection with H. pylori, a strong inflammatory response is induced preferentially in the undifferentiated basal cell phenotype. Infection of cultures for several weeks produces foci of viable bacteria and a persistent inflammatory condition, while the secreted mucus establishes a barrier that only few bacteria manage to overcome. CONCLUSION: Gastric mucosoid cultures faithfully reproduce the features of normal human gastric epithelium, enabling new approaches for investigating the interaction of H. pylori with the epithelial surface and the cross-talk with the basolateral stromal compartment. Our observations provide striking insights in the regulatory circuits of inflammation and defence.

High resolution quantitative proteomics of HeLa cells protein species using stable isotope labeling with amino acids in cell culture(SILAC), two-dimensional gel electrophoresis(2DE) and nano-liquid chromatograpohy coupled to an LTQ-OrbitrapMass spectrometer.

The proteomics field has shifted over recent years from two-dimensional gel electrophoresis (2-DE)-based approaches to SDS-PAGE or gel-free workflows because of the tremendous developments in isotopic labeling techniques, nano-liquid chromatography, and high-resolution mass spectrometry. However, 2-DE still offers the highest resolution in protein separation. Therefore, we combined stable isotope labeling with amino acids in cell culture of controls and apoptotic HeLa cells with 2-DE and the subsequent analysis of tryptic peptides via nano-liquid chromatography coupled to an LTQ-Orbitrap mass spectrometer to obtain quantitative data using the methods with the highest resolving power on all levels of the proteomics workflow. More than 1,200 proteins with more than 2,700 protein species were identified and quantified from 816 Coomassie Brilliant Blue G-250 stained 2-DE spots. About half of the proteins were identified and quantified only in single 2-DE spots. The majority of spots revealed one to five proteins; however, in one 2-DE spot, up to 23 proteins were identified. Only half of the 2-DE spots represented a dominant protein with more than 90% of the whole protein amount. Consequently, quantification based on staining intensities in 2-DE gels would in approximately half of the spots be imprecise, and minor components could not be quantified. These problems are circumvented by quantification using stable isotope labeling with amino acids in cell culture. Despite challenges, as shown in detail for lamin A/C and vimentin, the quantitative changes of protein species can be detected. The combination of 2-DE with high-resolution nano-liquid chromatography-mass spectrometry allowed us to identify proteomic changes in apoptotic cells that would be unobservable using any of the other previously employed proteomic workflows.

Use of the CytoSorb adsorber in patients with acute-on-chronic liver failure.

CytoSorb is a hemoadsorptive column used to remove high concentrations of proinflammatory cytokines in septic shock. Data on CytoSorb application in acute-on-chronic liver failure (ACLF) is lacking. This retrospective observational study analyzed 21 ACLF patients admitted to ICUs at the Vienna General Hospital who received CytoSorb adsorber therapy between 2017 and 2023. Median ICU length of stay was 8 days (IQR: 3-13), the ICU survival rate was 23.8% (n = 5). Significant decreases in bilirubin (median peak: 20.7 mg/dL to median post-treatment: 10.8 mg/dL; - 47.8%; p < 0.001), procalcitonin (1.34 to 0.74 pg/mL; - 44.6%; p < 0.001), interleukin-6 (385 to 131 ng/mL; - 66.0%; p = 0.0182)-but also of platelets (72 to 31 G/L; - 56.9%; p = 0.0014) and fibrinogen (230 to 154 mg/dL; - 33.0%; p = 0.0297) were detected. ICU survivors had a trend towards a stronger relative decrease in bilirubin (- 76.1% vs. - 48.2%), procalcitonin (- 90.6% vs. - 23.5%), and IL-6 (- 54.6% vs. - 17.8%) upon CytoSorb treatment. Moreover, no serious CytoSorb-attributed complications were detected. In conclusion, use of CytoSorb adsorber in ACLF patients results in a significant decrease in bilirubin and proinflammatory cytokines, while platelets and fibrinogen were also lowered. Prospective trials are warranted to investigate the impact of CytoSorb on clinical outcomes of ACLF patients with high proinflammatory cytokine levels.

Comparative genomics reveals distinct host-interacting traits of three major human-associated propionibacteria.

BACKGROUND: Propionibacteria are part of the human microbiota. Many studies have addressed the predominant colonizer of sebaceous follicles of the skin, Propionibacterium acnes, and investigated its association with the skin disorder acne vulgaris, and lately with prostate cancer. Much less is known about two other propionibacterial species frequently found on human tissue sites, Propionibacterium granulosum and Propionibacterium avidum. Here we analyzed two and three genomes of P. granulosum and P. avidum, respectively, and compared them to two genomes of P. acnes; we further highlight differences among the three cutaneous species with proteomic and microscopy approaches. RESULTS: Electron and atomic force microscopy revealed an exopolysaccharide (EPS)-like structure surrounding P. avidum cells, that is absent in P. acnes and P. granulosum. In contrast, P. granulosum possesses pili-like appendices, which was confirmed by surface proteome analysis. The corresponding genes were identified; they are clustered with genes encoding sortases. Both, P. granulosum and P. avidum lack surface or secreted proteins for predicted host-interacting factors of P. acnes, including several CAMP factors, sialidases, dermatan-sulphate adhesins, hyaluronidase and a SH3 domain-containing lipoprotein; accordingly, only P. acnes exhibits neuraminidase and hyaluronidase activities. These functions are encoded on previously unrecognized island-like regions in the genome of P. acnes. CONCLUSIONS: Despite their omnipresence on human skin little is known about the role of cutaneous propionibacteria. All three species are associated with a variety of diseases, including postoperative and device-related abscesses and infections. We showed that the three organisms have evolved distinct features to interact with their human host. Whereas P. avidum and P. granulosum produce an EPS-like surface structure and pili-like appendices, respectively, P. acnes possesses a number of unique surface-exposed proteins with host-interacting properties. The different surface properties of the three cutaneous propionibacteria are likely to determine their colonizing ability and pathogenic potential on the skin and at non-skin sites.

Neuter is not common in Dutch: eye movements reveal asymmetrical gender processing.

Native speakers of languages with transparent gender systems can use gender cues to anticipate upcoming words. To examine whether this also holds true for a non-transparent two-way gender system, i.e. Dutch, eye movements were monitored as participants followed spoken instructions to click on one of four displayed items on a screen (e.g., Klik op [Formula: see text] rode appel [Formula: see text], 'Click on the[Formula: see text] red apple[Formula: see text]'). The items contained the target, a colour- and/or gender-matching competitor, and two unrelated distractors. A mixed-effects regression analysis revealed that the presence of a colour-matching and/or gender-matching competitor significantly slowed the process of finding the target. The gender effect, however, was only observed for common nouns, reflecting the fact that neuter gender-marking cannot disambiguate as all Dutch nouns become neuter when used as diminutives. The gender effect for common nouns occurred before noun onset, suggesting that gender information is, at least partially, activated automatically before encountering the noun.

Biomarkers for differentiation of coronavirus disease 2019 or extracorporeal membrane oxygenation related inflammation and bacterial/fungal infections in critically ill patients: A prospective observational study.

Secondary infections in coronavirus disease 2019 (COVID-19) patients are difficult to distinguish from inflammation associated with COVID-19 and/or extracorporeal membrane oxygenation (ECMO). Therefore, highly specific and sensitive biomarkers are needed to identify patients in whom antimicrobial therapy can be safely withheld. In this prospective monocentric study, 66 COVID-19 patients admitted to the intensive care unit (ICU) for ECMO evaluation were included. A total of 46 (70%) patients with secondary infections were identified by using broad microbiological and virological panels and standardized diagnostic criteria. Various laboratory parameters including C-reactive protein (CRP), interleukin (IL)-6, procalcitonin (PCT), and IL-10 were determined at time of study inclusion. The best test performance for differentiating bacterial/fungal secondary infections and COVID-19 and/or ECMO associated inflammation was achieved by IL-10 (ROC-AUC 0.84) and a multivariant step-wise regression model including CRP, IL-6, PCT, and IL-10 (ROC-AUC 0.93). Data obtained in the present study highlights the use of IL-10 to differentiate secondary bacterial/fungal infections from COVID-19 and/or ECMO associated inflammation in severely ill COVID-19 patients.

Histone H3 clipping is a novel signature of human neutrophil extracellular traps.

Neutrophils are critical to host defence, executing diverse strategies to perform their antimicrobial and regulatory functions. One tactic is the production of neutrophil extracellular traps (NETs). In response to certain stimuli, neutrophils decondense their lobulated nucleus and release chromatin into the extracellular space through a process called NETosis. However, NETosis, and the subsequent degradation of NETs, can become dysregulated. NETs are proposed to play a role in infectious as well as many non-infection related diseases including cancer, thrombosis, autoimmunity and neurological disease. Consequently, there is a need to develop specific tools for the study of these structures in disease contexts. In this study, we identified a NET-specific histone H3 cleavage event and harnessed this to develop a cleavage site-specific antibody for the detection of human NETs. By microscopy, this antibody distinguishes NETs from chromatin in purified and mixed cell samples. It also detects NETs in tissue sections. We propose this antibody as a new tool to detect and quantify NETs.

Quantitative phosphoproteomics reveals link between Helicobacter pylori infection and RNA splicing modulation in host cells.

The Gram-negative, spiral-shaped bacterium Helicobacter pylori is a common human pathogen that causes chronic inflammation of the human gastric mucosa, leading to peptic ulceration and/or gastric cancer. Here, we analyzed changes in the phosphoproteome of gastric epithelial cells (AGS) upon infection with H. pylori using a combination of SILAC, phosphoprotein enrichment, 2-DE, and MALDI TOF/TOF-MS. From a total of 526 spots we identified 391 protein species (143 proteins) and quantified 332 (127 proteins). Nearly, one-third of the identified proteins (40/143) were associated with the spliceosome or RNA splicing. The abundance of 20 proteins was altered by H. pylori infection, in particular, a number of serine arginine-rich (SR) proteins involved in the regulation and control of alternative splicing. Importantly, the combined methodologies enabled the detection of infection-dependent protein species-specific regulation, suggesting functional modulation of individual protein species. These findings reveal unexpected new insights into the mechanisms of host cell manipulation by H. pylori, which are likely associated with gastric pathologies, including gastric cancer.

Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans.

Neutrophils are the first line of defense at the site of an infection. They encounter and kill microbes intracellularly upon phagocytosis or extracellularly by degranulation of antimicrobial proteins and the release of Neutrophil Extracellular Traps (NETs). NETs were shown to ensnare and kill microbes. However, their complete protein composition and the antimicrobial mechanism are not well understood. Using a proteomic approach, we identified 24 NET-associated proteins. Quantitative analysis of these proteins and high resolution electron microscopy showed that NETs consist of modified nucleosomes and a stringent selection of other proteins. In contrast to previous results, we found several NET proteins that are cytoplasmic in unstimulated neutrophils. We demonstrated that of those proteins, the antimicrobial heterodimer calprotectin is released in NETs as the major antifungal component. Absence of calprotectin in NETs resulted in complete loss of antifungal activity in vitro. Analysis of three different Candida albicans in vivo infection models indicated that NET formation is a hitherto unrecognized route of calprotectin release. By comparing wild-type and calprotectin-deficient animals we found that calprotectin is crucial for the clearance of infection. Taken together, the present investigations confirmed the antifungal activity of calprotectin in vitro and, moreover, demonstrated that it contributes to effective host defense against C. albicans in vivo. We showed for the first time that a proportion of calprotectin is bound to NETs in vitro and in vivo.

Influence of RET/PTC1 and RET/PTC3 oncoproteins in radiation-induced papillary thyroid carcinomas on amounts of cytoskeletal protein species.

Radiation-induced human papillary thyroid carcinomas (PTCs) show a high prevalence of fusions of the RET proto-oncogene to heterologous genes H4 (RET/PTC1) and ELE1 (RET/PTC3), respectively. In contrast to the normal membrane-bound RET protein, aberrant RET fusion proteins are constitutively active oncogenic cytosolic proteins that can lead to malignant transformation of thyroid epithelia. To detect specific tumor-associated protein changes that reflect the effect of RET/PTC fusion proteins, we analyzed normal thyroid tissues, thyroid tumors of the RET/PTC1 and RET/PTC3 type and their respective lymph node metastases by a combination of high-resolution two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-mass spectrometry. PTCs without RET rearrangements served as controls. Several cytoskeletal protein species showed quantitative changes in tumors and lymph node metastases harboring RET/PTC1 or RET/PTC3. We observed prominent C-terminal actin fragments assumedly generated by protease cleavages induced due to enhanced amounts of the active actin-binding protein cofilin-1. In addition, three truncated vimentin species, one of which was proven to be headless, were shown to be highly abundant in tumors and metastases of both RET/PTC types. The observed protein changes are closely connected with the constitutive activation of RET-rearranged oncoproteins and reflect the importance to elucidate disease-related typical signatures on the protein species level.

Role of Premycofactocin Synthase in Growth, Microaerophilic Adaptation, and Metabolism of Mycobacterium tuberculosis.

Mycofactocin is a new class of peptide-derived redox cofactors present in a selected group of bacteria including Mycobacterium tuberculosis. Mycofactocin biosynthesis requires at least six genes, including mftD, encoding putative lactate dehydrogenase, which catalyzes the penultimate biosynthetic step. Cellular functions remained unknown until recent reports on the significance of mycofactocin in primary alcohol metabolism. Here, we show that mftD transcript levels were increased in hypoxia-adapted M. tuberculosis; however, mftD functionality was found likely dispensable for l-lactate metabolism. Targeted deletion of mftD reduced the survival of M. tuberculosis in in vitro and in vivo hypoxia models but increased the bacterial growth in glucose-containing broth as well as in the lungs and spleens, albeit modestly, of aerosol-infected C57BL/6J mice. The cause of this growth advantage remains unestablished; however, the mftD-deficient M. tuberculosis strain had reduced NAD(H)/NADP(H) levels and glucose-6-phosphate dehydrogenase activity with no impairment in phthiocerol dimycocerosate lipid synthesis. An ultrastructural examination of parental and mycofactocin biosynthesis gene mutants in M. tuberculosis, M. marinum, and M. smegmatis showed no altered cell morphology and size except the presence of outer membrane-bound fibril-like features only in a mutant subpopulation. A cell surface-protein analysis of M. smegmatis mycofactocin biosynthesis mutants with trypsin revealed differential abundances of a subset of proteins that are known to interact with mycofactocin and their homologs that can enhance protein aggregation or amyloid-like fibrils in riboflavin-starved eukaryotic cells. In sum, phenotypic analyses of the mutant strain implicate the significance of MftD/mycofactocin in M. tuberculosis growth and persistence in its host. IMPORTANCE Characterization of proteins with unknown functions is a critical research priority as the intracellular growth and metabolic state of Mycobacterium tuberculosis, the causative agent of tuberculosis, remain poorly understood. Mycofactocin is a peptide-derived redox cofactor present in almost all mycobacterial species; however, its functional relevance in M. tuberculosis pathogenesis and host survival has never been studied experimentally. In this study, we examine the phenotypes of an M. tuberculosis mutant strain lacking a key mycofactocin biosynthesis gene in in vitro and disease-relevant mouse models. Our results pinpoint the multifaceted role of mycofactocin in M. tuberculosis growth, hypoxia adaptation, glucose metabolism, and redox homeostasis. This evidence strongly implies that mycofactocin could fulfill specialized biochemical functions that increase the survival fitness of mycobacteria within their specific niche.

Incidence and Etiology of System Exchanges in Patients Receiving Extracorporeal Membrane Oxygenation.

Extracorporeal membrane oxygenation (ECMO) has established as a cornerstone therapy in severe acute respiratory distress syndrome and refractory hemodynamic failure. As circuit integrity is crucial for adequate organ support, component failure may necessitate a system exchange. In this retrospective study, incidence and etiology of system exchanges during applications of venovenous, venoarterial ECMO, and extracorporeal CO2 removal were examined. Sixty-three (44.4%) of 142 patients were affected by one or more exchanges, totaling 105 replaced circuits. The predominant exchange reason was clotting (n = 20), followed by hemolysis (n = 19), systemic coagulation disorders (n = 13), reconfiguration (n = 13), impaired gas exchange (n = 10), mechanical complications (n = 8), bleeding (n = 6), failed weaning (n = 5), prophylactic exchange (n = 3), and undocumented/other (n = 8). Nineteen (18.1%) events were classified as acute and 70 (66.7%) events as elective exchanges. Patients with circuit exchanges more frequently underwent renal replacement therapy at ECMO initiation (49.2% vs. 29.1%; p = 0.023), had a longer ECMO treatment duration (18 vs. 7.5 days, p < 0.001), and lower hospital survival (29.5% vs. 57.1%; p = 0.002). Considering the high occurrence of coagulation complications, further optimization of coagulation management is deemed necessary.

Helicobacter pylori proteomics by 2-DE/MS, 1-DE-LC/MS and functional data mining.

With its predicted proteome of 1550 proteins (data set Etalon) Helicobacter pylori 26695 represents a perfect model system of medium complexity for investigating basic questions in proteomics. We analyzed urea-solubilized proteins by 2-DE/MS (data set 2-DE) and by 1-DE-LC/MS (Supprot); proteins insoluble in 9 M urea but solubilized by SDS (Pellet); proteins precipitating in the Sephadex layer at the application side of IEF (Sephadex) by 1-DE-LC/MS; and proteins precipitating close to the application side within the IEF gel by LC/MS (Startline). The experimental proteomics data of H. pylori comprising 567 proteins (protein coverage: 36.6%) were stored in the Proteome Database System for Microbial Research (http://www.mpiib-berlin.mpg.de/2D-PAGE/), which gives access to raw mass spectra (MALDI-TOF/TOF) in T2D format, as well as to text files of peak lists. For data mining the protein mapping and comparison tool PROMPT (http://webclu.bio.wzw.tum.de/prompt/) was used. The percentage of proteins with transmembrane regions, relative to all proteins detected, was 0, 0.2, 0, 0.5, 3.8 and 6.3% for 2-DE, Supprot, Startline, Sephadex, Pellet, and Etalon, respectively. 2-DE does not separate membrane proteins because they are insoluble in 9 M urea/70 mM DTT and 2% CHAPS. SDS solubilizes a considerable portion of the urea-insoluble proteins and makes them accessible for separation by SDS-PAGE and LC. The 2-DE/MS analysis with urea-solubilized proteins and the 1-DE-LC/MS analysis with the urea-insoluble protein fraction (Pellet) are complementary procedures in the pursuit of a complete proteome analysis. Access to the PROMPT-generated diagrams in the Proteome Database allows the mining of experimental data with respect to other functional aspects.

Proteomic identification of secreted proteins of Propionibacterium acnes.

BACKGROUND: The anaerobic Gram-positive bacterium Propionibacterium acnes is a human skin commensal that resides preferentially within sebaceous follicles; however, it also exhibits many traits of an opportunistic pathogen, playing roles in a variety of inflammatory diseases such as acne vulgaris. To date, the underlying disease-causing mechanisms remain ill-defined and knowledge of P. acnes virulence factors remains scarce. Here, we identified proteins secreted during anaerobic cultivation of a range of skin and clinical P. acnes isolates, spanning the four known phylogenetic groups. RESULTS: Culture supernatant proteins of P. acnes were separated by two-dimensional electrophoresis (2-DE) and all Coomassie-stained spots were subsequently identified by MALDI mass spectrometry (MALDI-MS). A set of 20 proteins was secreted in the mid-exponential growth phase by the majority of strains tested. Functional annotation revealed that many of these common proteins possess degrading activities, including glycoside hydrolases with similarities to endoglycoceramidase, ß-N-acetylglucosaminidase and muramidase; esterases such as lysophospholipase and triacylglycerol lipase; and several proteases. Other secreted factors included Christie-Atkins-Munch-Petersen (CAMP) factors, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and several hypothetical proteins, a few of which are unique to P. acnes. Strain-specific differences were apparent, mostly in the secretion of putative adhesins, whose genes exhibit variable phase variation-like sequence signatures. CONCLUSIONS: Our proteomic investigations have revealed that the P. acnes secretome harbors several proteins likely to play a role in host-tissue degradation and inflammation. Despite a large overlap between the secretomes of all four P. acnes phylotypes, distinct differences between predicted host-tissue interacting proteins were identified, providing potential insight into the differential virulence properties of P. acnes isolates. Thus, our data presents a rich resource for guiding much-needed investigations on P. acnes virulence factors and host interacting properties.

A double-blind, randomized, placebo-controlled trial of intravenous L-ornithine-L-aspartate on postural control in patients with cirrhosis.

INTRODUCTION: Hepatic encephalopathy (HE) is a complication of liver disease. Several treatments have been introduced but only L-ornithine-L-aspartate (LOLA) shows proven efficacy. This double-blind, randomized, placebo-controlled trial evaluated the effect of LOLA on postural control in cirrhotics. METHODS: Forty patients were randomized to either LOLA or a placebo. HE was evaluated by psychometric testing (PSE Syndrome Test) and critical flicker frequency (CFF). Posturography [equilibrium score (ES)] provided information regarding postural control. Peripheral blood was analysed for ammonia concentration (NH(3)) and the partial pressure of ammonia (pNH(3)). RESULTS: Both groups were comparable regarding baseline variables. Posturography and PSE Syndrome Test improved in both groups; improvement was greater in the LOLA group (ES: 5.3%; PSE: 1.9) compared with the placebo (ES: 3.9%; PSE: 1.3) but did not reach significance (ES: P=0.3; PSE: P=0.5). CFF remained unchanged during treatment and between groups (P=NS). NH(3) decreased in the LOLA group (Delta: -15 micromol/L) and slightly increased in the placebo group (Delta: 11.1 micromol/L), but the differences did not reach statistical significance (P=0.07). pNH(3) remained largely unchanged (LOLA Delta: -1.2 x 10(-5) mmHg vs. placebo Delta: -0.3 x 10(-5) mmHg; P=0.21). CONCLUSION: In the LOLA group, an improvement of posturographic control and PSE Syndrome Test was observed, but a similar improvement was also achieved by the placebo. In LOLA, ammonia levels tended to decrease while they tended to increase in the placebo group. LOLA might augment the improvement achieved by intravenous fluids alone but a larger cohort will be needed to show this effect with statistical significance.

Transgenic, fluorescent Leishmania mexicana allow direct analysis of the proteome of intracellular amastigotes.

Investigating the proteome of intracellular pathogens is often hampered by inadequate methodologies to purify the pathogen free of host cell material. This has also precluded direct proteome analysis of the intracellular, amastigote form of Leishmania spp., protozoan parasites that cause a spectrum of diseases that affect some 12 million patients worldwide. Here a method is presented that combines classic, isopycnic density centrifugation with fluorescent particle sorting for purification by exploiting transgenic, fluorescent parasites to allow direct proteome analysis of the purified organisms. By this approach the proteome of intracellular Leishmania mexicana amastigotes was compared with that of extracellular promastigotes that are transmitted by insect vectors. In total, 509 different proteins were identified by mass spectrometry and database search. This number corresponds to approximately 6% of gene products predicted from the reference genome of Leishmania major. Intracellular amastigotes synthesized significantly more proteins with basic pI and showed a greater abundance of enzymes of fatty acid catabolism, which may reflect their living in acidic habitats and metabolic adaptation to nutrient availability, respectively. Bioinformatics analyses of the genes corresponding to the protein data sets produced clear evidence for skewed codon usage and translational bias in these organisms. Moreover analysis of the subset of genes whose products were more abundant in amastigotes revealed characteristic sequence motifs in 3'-untranslated regions that have been linked to translational control elements. This suggests that proteome data sets may be used to identify regulatory elements in mRNAs. Last but not least, at 6% coverage the proteome identified all vaccine antigens tested to date. Thus, the present data set provides a valuable resource for selection of candidate vaccine antigens.