Recent advances in covalent, site-specific protein immobilization [version 1; referees:3 approved]

The properties of biosensors, biomedical implants, and other materials based on immobilized proteins greatly depend on the method employed to couple the protein molecules to their solid support. Covalent, site-specific immobilization strategies are robust and can provide the level of control that is desired in this kind of application. Recent advances include the use of enzymes, such as sortase A, to couple proteins in a site-specific manner to materials such as microbeads, glass, and hydrogels. Also, self-labeling tags such as the SNAP-tag can be employed. Last but not least, chemical approaches based on bioorthogonal reactions, like the azide–alkyne cycloaddition, have proven to be powerful tools. The lack of comparative studies and quantitative analysis of these immobilization methods hampers the selection process of the optimal strategy for a given application. However, besides immobilization efficiency, the freedom in selecting the site of conjugation and the size of the conjugation tag and the researcher’s expertise regarding molecular biology and/or chemical techniques will be determining factors in this regard

Carbohydrate chemistry: synthetic and structural challenges towards the end of the 20th century*

Abstract: Carbohydrate chemistry has acquired considerable interest in many different facets during the last decades of the century. Particularly, the synthesis of complex carbohydrates in the form of large oligosaccharides has paved the way for a better appreciation of the function of carbohydrates in biological systems. The impressive advances associated with the structural determination and analysis of oligosaccharides has furthermore contributed to the increased focus on the role of carbohydrates particularly in the form of glycoproteins but also as signal molecules in general. The primary goal of this work is to discuss the advancement of structural assessment of large oligosaccharides as typi®ed by the structural determination of the 22-mer saccharide from the LPS of Salmonella enterica ssp. typhimurium strain 1135 using very high ®eld strength NMR spectroscopy. The challenges of synthesizing and analysing the structure of glycopeptides will also be discussed. Finally, the latest developments in glycopeptide libraries generated by solidphase combinatorial chemistry will be presented in addition to examples of new techniques where the increased sensitivity using nanoprobe technology in combination with MALDI-TOF MS spectroscopy was used in the structural assignment of such complex glycopeptides; a methodology of invaluable importance for the analysis of glycopeptides on single beads

Development of selective ADAMTS-5 peptide substrates to monitor proteinase activity

The dysregulation of proteinase activity is a hallmark of osteoarthritis (OA), a disease characterized by progressive degradation of articular cartilage by catabolic proteinases such as a disintegrin and metalloproteinase with thrombospondin type I motifs-5 (ADAMTS-5). The ability to detect such activity sensitively would aid disease diagnosis and the evaluation of targeted therapies. Förster resonance energy transfer (FRET) peptide substrates can detect and monitor disease-related proteinase activity. To date, FRET probes for detecting ADAMTS-5 activity are nonselective and relatively insensitive. We describe the development of rapidly cleaved and highly selective ADAMTS-5 FRET peptide substrates through in silico docking and combinatorial chemistry. The lead substrates 3 and 26 showed higher overall cleavage rates (∼3–4-fold) and catalytic efficiencies (∼1.5–2-fold) compared to the best current ADAMTS-5 substrate ortho-aminobenzoyl(Abz)-TESE↓SRGAIY-N-3-[2,4-dinitrophenyl]-l-2,3-diaminopropionyl(Dpa)-KK-NH2. They exhibited high selectivity for ADAMTS-5 over ADAMTS-4 (∼13–16-fold), MMP-2 (∼8–10-fold), and MMP-9 (∼548–2561-fold) and detected low nanomolar concentrations of ADAMTS-5

Vocational Rehabilitation in Mild Traumatic Brain Injury: Supporting Return to Work and Daily Life Functioning

Persisting post-concussive symptoms are challenging to treat and may delay return-to-work (RTW). The aims of this study were to describe a multidisciplinary and holistic vocational rehabilitation (VR) program for individuals with mild traumatic brain injury (mTBI) and to explore course and predictors of employment outcome during VR. The VR program was described using the Standard Operating Procedures (SOPs) framework. Further, a retrospective, cohort study on individuals with mTBI receiving VR was conducted based on clinical records (n = 32; 22% males; mean age 43.2 years; 1.2 years since injury on average). The primary outcome was difference in hours at work per week from pre- to post-VR, and the secondary outcome was change in a three-level RTW-status. Time since injury, age, sex, and loss of consciousness were investigated as predictors of the outcomes. The VR intervention is individually tailored and targets patients' individual needs. Thus, it may combine a variety of methods based on a biopsychosocial theoretical model. During VR, hours at work, 17.0 ± 2.2, p < 0.001, and RTW-status, OR = 14.0, p < 0.001, improved significantly with 97% having returned to work after VR. Shorter length of time since injury and male sex were identified as predictors of a greater gain of working hours. Time since injury was the strongest predictor; double the time was associated with a reduction in effect by 4.2 ± 1.4 h after adjusting for working hours at start of VR. In sum, these results suggest that individuals facing persistent problems following mTBI may still improve employment outcomes and RTW after receiving this multidisciplinary and holistic VR intervention, even years after injury. While results are preliminary and subject to bias due to the lack of a control group, this study warrants further research into employment outcomes and VR following mTBI, including who may benefit the most from treatment

Click-based synthesis and proteomic profiling of lipstatin analogues

Using click chemistry to enable both structural diversity and proteome profiling within a natural product derived library, two out of nineteen lipstatin analogues showed similar activity to Orlistat against fatty acid synthase (FAS), but with an improved ability to induce tumour cell death