Urinary desmosine as a biomarker in acute lung injury

Acute lung injury (ALI) is a complex disorder associated with an acute inflammatory response thought to contribute to tissue injury. Desmosine, a cross-linking amino acid present in elastin, is released during matrix degradation and cleared by the kidney. Results from animal models and human disease studies have suggested that ALI is associated with the release of desmosine, resulting in increased urinary desmosine. A radioimmunoassay was used to monitor urinary desmosine levels over 10 days in ten patients with ALI. The concentration of desmosine was measured with and without acid hydrolysis. Baseline urinary desmosine was increased in two of ten patients. The concentration of desmosine at baseline did not appear to be related to age, gender, neutrophil elastase (NE)/α₁-antiprotease complex concentration or P_aO₂/F_iO₂ ratio. No meaningful changes in desmosine levels were noted after removal from mechanical ventilation. Baseline desmosine concentrations did not appear to correlate with the risk of death. The limited sensitivity, predictive correlations and dynamic modulation would suggest that urine desmosine has a limited role as a biomarker for ALI. Hydrolysis of urine samples appears necessary for optimal measurement of urine desmosine.     

Filamentous Basidiomycetes in the Clinical Laboratory

Filamentous basidiomycetes are difficult to identify in the clinical laboratory, mostly due to lack of sporulation, and their role as agents of fungal infection can be difficult to assess. More cases of infection with these agents are being reported as more laboratories gain proficiency with the recognition of their subtle morphologic features and the use of DNA-based methods for identification. Most infections occur in the respiratory tract and sinuses, although brain infection has been reported. Susceptibility testing suggests that these agents will respond well to azole drugs other than fluconazole.     

Molecular cross-talk between sponge host and associated microbes

Marine organisms especially those that live sessile, as sponges, are well known to have specific relationships with a great variety of microorganisms including bacteria and fungi. As most simple metazoan phylum, the Porifera, which emerged first during the transition from the non-Metazoa to the Metazoa from the common ancestor, comprise wide arrays of recognition molecules, both for Gram-negative bacteria and for Gram-positive bacteria as well as for fungi. They react specifically with effector molecules to inhibit or kill the invading microorganisms. The elicitation and the subsequent effector reactions of the sponges towards these microbes are outlined. However, besides of the elimination of bacteria and fungi, some of those taxa are kept as symbionts of the sponges, allowing them, for example, to accumulate the essential element manganese or to synthesize carotinoids. The sponges produce low-molecular-weight bioactive compounds, secondary metabolites, to eliminate the microorganisms. In addition, they are armed with cationic antimicrobial peptides allowing them to defend against invasive microorganisms and, in parallel, to kill or repel also metazoan invaders. The broad range of chemically and functionally different compounds qualifies the Porifera as the most important animal phylum to be exploited as a source for the isolation of new potential drugs. First molecular biological strategies have been outlined to obtain those compounds in a sustainable way, by producing them recombinantly.     

Side-by-side comparability of batch and continuous downstream for the production of monoclonal antibodies

Continuous processing is the future production method for monoclonal antibodies (mAbs). A fully continuous, fully automated downstream process based on disposable equipment was developed and implemented inside the MoBiDiK pilot plant. However, a study evaluating the comparability between batch and continuous processing based on product quality attributes was not conducted before. The work presented fills this gap comparing both process modes experimentally by purifying the same harvest material (side-by-side comparability). Samples were drawn at different time points and positions in the process for batch and continuous mode. Product quality attributes, product-related impurities, as well as process-related impurities were determined. The resulting polished material was processed to drug substance and further evaluated regarding storage stability and degradation behavior. The in-process control data from the continuous process showed the high degree of accuracy in providing relevant process parameters such as pH, conductivity, and protein concentration during the entire process duration. Minor differences between batch and continuous samples are expected as different processing conditions are unavoidable due to the different nature of batch and continuous processing. All tests revealed no significant differences in the intermediates and comparability in the drug substance between the samples of both process modes. The stability study of the final product also showed no differences in the stability profile during storage and forced degradation. Finally, online data analysis is presented as a powerful tool for online-monitoring of chromatography columns during continuous processing.     

Precision mapping of the human O‐GalNAc glycoproteome through SimpleCell technology

Glycosylation is the most abundant and diverse posttranslational modification of proteins. While several types of glycosylation can be predicted by the protein sequence context, and substantial knowledge of these glycoproteomes is available, our knowledge of the GalNAc‐type O‐glycosylation is highly limited. This type of glycosylation is unique in being regulated by 20 polypeptide GalNAc‐transferases attaching the initiating GalNAc monosaccharides to Ser and Thr (and likely some Tyr) residues. We have developed a genetic engineering approach using human cell lines to simplify O‐glycosylation (SimpleCells) that enables proteome‐wide discovery of O‐glycan sites using ‘bottom‐up’ ETD‐based mass spectrometric analysis. We implemented this on 12 human cell lines from different organs, and present a first map of the human O‐glycoproteome with almost 3000 glycosites in over 600 O‐glycoproteins as well as an improved NetOGlyc4.0 model for prediction of O‐glycosylation. The finding of unique subsets of O‐glycoproteins in each cell line provides evidence that the O‐glycoproteome is differentially regulated and dynamic. The greatly expanded view of the O‐glycoproteome should facilitate the exploration of how site‐specific O‐glycosylation regulates protein function.     

Detoxification of Mercury by Methanobactin from Methylosinus trichosporium OB3b

Many methanotrophs have been shown to synthesize methanobactin, a novel biogenic copper-chelating agent or chalkophore. Methanobactin binds copper via two heterocyclic rings with associated enethiol groups. The structure of methanobactin suggests that it can bind other metals, including mercury. Here we report that methanobactin from Methylosinus trichosporium OB3b does indeed bind mercury when added as HgCl₂ and, in doing so, reduced toxicity associated with Hg(II) for both Alphaproteobacteria methanotrophs, including M. trichosporium OB3b, M. trichosporium OB3b ΔmbnA (a mutant defective in methanobactin production), and Methylocystis sp. strain SB2, and a Gammaproteobacteria methanotroph, Methylomicrobium album BG8. Mercury binding by methanobactin was evident in both the presence and absence of copper, despite the fact that methanobactin had a much higher affinity for copper due to the rapid and irreversible binding of mercury by methanobactin. The formation of a gray precipitate suggested that Hg(II), after being bound by methanobactin, was reduced to Hg(0) but was not volatilized. Rather, mercury remained associated with methanobactin and was also found associated with methanotrophic biomass. It thus appears that although the mercury-methanobactin complex was cell associated, mercury was not removed from methanobactin. The amount of biomass-associated mercury in the presence of methanobactin from M. trichosporium OB3b was greatest for M. trichosporium wild-type strain OB3b and the ΔmbnA mutant and least for M. album BG8, suggesting that methanotrophs may have selective methanobactin uptake systems that may be based on TonB-dependent transporters but that such uptake systems exhibit a degree of infidelity.     

Syntheses,biological evaluation and SAR of ingenol mebutate analogues for treatment of actinic keratosis and non-melanoma skin cancer

Ingenol mebutate is the active ingredient in Picato® a new drug for the treatment of actinic keratosis. A number of derivatives related to ingenol mebutate were prepared by chemical synthesis from ingenol with the purpose of investigating the SAR and potency in assays relating to pro-inflammatory effects (induction of PMN oxidative burst and keratinocyte cytokine release), the potential of cell death induction, as well as the chemical stability. By modifications of the ingenol scaffold several prerequisites for activity were identified. The chemical stability of the compounds could be linked to an acyl migration mechanism. We were able to find analogues of ingenol mebutate with comparable in vitro properties. Some key features for potent and more stable ingenol derivatives have been identified.