Glycosylation is a ubiquitous modification of lipids and proteins. Despite the essential contribution of glycoconjugates to the viability of all living organisms, diseases of glycosylation in humans have only been identified over the past few decades. The recent development of next-generation DNA sequencing techniques has accelerated the pace of discovery of novel glycosylation defects. The description of multiple mutations across glycosylation pathways not only revealed tremendous diversity in functional impairments, but also pointed to phenotypic similarities, emphasizing the interconnected flow of substrates underlying glycan assembly. The current list of 100 known glycosylation disorders provides an overview of the significance of glycosylation in human development and physiology. Congenital disorders of glycosylation -a consice chart of glycocalyx dysfunctionThierry Hennet, Jürg Cabalzar Institute of Physiology, University of Zurich, CH-8057 Zurich, Switzerland AbstractGlycosylation is a ubiquitous modification of lipids and proteins. Despite the essential contribution of glycoconjugates to the viability of all living organisms, diseases of glycosylation in humans have only been identified over the past few decades. The recent development of next-generation DNA sequencing techniques has accelerated the pace of discovery of novel glycosylation defects. The description of multiple mutations across glycosylation pathways has revealed a tremendous diversity of functional impairments bus also pointed to phenotypic similarities emphasizing the interconnected flow of substrates underlying glycan assembly. The current list of 100 known glycosylation disorders provides an overview on the significance of glycosylation in human development and physiology. Highlights Congenital disorders underline the role of glycosylation in human development.  Next-gen sequencing techniques expanded the discovery of glycosylation gene defects.  The clinical variability of glycosylation disorders implies they are underdiagnosed. Glycosylation disordersGlycosylation is by far the most complex form of protein [1,2] and lipid modification [3,4] in all domains of life. The tremendous diversity of glycoconjugate structures resulting from intricate biosynthetic pathways is a major factor hampering the assignment of functions to glycans chains. Much has been learnt from the study of disrupted glycosylation genes in model organisms, thereby establishing numerous essential contributions of glycans in regulating cell and organ functions [5]. The study of human diseases of glycosylation brings additional insights by providing a more differentiated view on glycan functions. Indeed, most human mutations are hypomorphic, thus causing partial loss of glycosylation reactions that lead to variable clinical manifestations.Diseases of glycosylation are also referred to as congenital disorders of glycosylation (CDG). Given the heterogeneity of glycans, the clinical scope of CDG is considerable, ranging from nearly normal phenotypes to sever...

Collagen is the most abundant protein in the human body and thereby a structural protein of considerable biotechnological interest. The complex maturation process of collagen, including essential post-translational modifications such as prolyl and lysyl hydroxylation, has precluded large-scale production of recombinant collagen featuring the biophysical properties of endogenous collagen. The characterization of new prolyl and lysyl hydroxylase genes encoded by the giant virus mimivirus reveals a method for production of hydroxylated collagen. The coexpression of a human collagen type III construct together with mimivirus prolyl and lysyl hydroxylases in Escherichia coli yielded up to 90 mg of hydroxylated collagen per liter culture. The respective levels of prolyl and lysyl hydroxylation reaching 25 % and 26 % were similar to the hydroxylation levels of native human collagen type III. The distribution of hydroxyproline and hydroxylysine along recombinant collagen was also similar to that of native collagen as determined by mass spectrometric analysis of tryptic peptides. The triple helix signature of recombinant hydroxylated collagen was confirmed by circular dichroism, which also showed that hydroxylation increased the thermal stability of the recombinant collagen construct. Recombinant hydroxylated collagen produced in E. coli supported the growth of human umbilical endothelial cells, underlining the biocompatibility of the recombinant protein as extracellular matrix. The high yield of recombinant protein expression and the extensive level of prolyl and lysyl hydroxylation achieved indicate that recombinant hydroxylated collagen can be produced at large scale for biomaterials engineering in the context of biomedical applications.

The aim of the present study was to quantify a large number of analytes including opioids, stimulants, benzodiazepines, z-drugs, antidepressants and neuroleptics within a single sample workup followed by a single analytical measurement. Expected drug concentrations in hair are strongly substance-dependent. Therefore, three different calibration ranges were implemented: 0.5–600 pg/mg (group 1), 10–12,000 pg/mg (group 2), and 50–60,000 pg/mg (group 3). In order to avoid saturation effects, different strategies were applied for selected transitions including the use of parent mass ions containing one or two 13C-isotopes and detuning of the declustering potential and/or collision energy. Drugs were extracted from pulverized hair by a two-step extraction protocol and measured by LC–MS-MS using Scheduled MRM™ Algorithm Pro. In total, 275 MRM transitions including 43 deuterated standards were measured. The method has been fully validated according to international guidelines. A MultiQuant™ software based tool for task-oriented data evaluation was established which allows extracting selected information from the measured data sets. The matrix effects and recoveries were within the allowed ranges for the majority of the analytes. The lower limits of quantification (LLOQs) were for approx. 54% of the analytes in the low-pg/mg-range (0.5–5 pg/mg) and for approximately 25% of the analytes between 10 and 50 pg/mg. These LLOQs considered cut-offs by the SoHT, if recommended. The herein established multi-analyte approach meets the specific requirements of forensic hair testing and can be used for the rapid and robust measurement of a wide range of psychoactive substances. The analyte-specific wide concentration ranges open up a wide field of applications.

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