What would you do if you could sequence everything?

It could be argued that the greatest transformative aspect of the Human Genome Project has been not the sequencing of the genome itself, but the resultant development of new technologies. A host of new approaches has fundamentally changed the way we approach problems in basic and translational research. Now, a new generation of high-throughput sequencing technologies promises to again transform the scientific enterprise, potentially supplanting array-based technologies and opening up many new possibilities. By allowing DNA/RNA to be assayed more rapidly than previously possible, these next-generation platforms promise a deeper understanding of genome regulation and biology. Significantly enhancing sequencing throughput will allow us to follow the evolution of viral and bacterial resistance in real time, to uncover the huge diversity of novel genes that are currently inaccessible, to understand nucleic acid therapeutics, to better integrate biological information for a complete picture of health and disease at a personalized level and to move to advances that we cannot yet imagine.     

Online immunoaffinity LC/MS/MS. A general method to increase sensitivity and specificity: How do you do it and what do you need?

There is an increased emphasis on hyphenated techniques such as immunoaffinity LC/MS/MS (IA-LC/MS/MS) or IA-LC/MRM. These techniques offer competitive advantages with respect to sensitivity and selectivity over traditional LC/MS and are complementary to ligand binding assays (LBA) or ELISA's. However, these techniques are not entirely straightforward and there are several tips and tricks to routine sample analysis. We describe here our methods and procedures for how to perform online IA-LC/MS/MS including a detailed protocol for the preparation of antibody (Ab) enrichment columns. We have included sample trapping and Ab methods. Furthermore, we highlight tips, tricks, minimal and optimal approaches. This technology has been shown to be viable for several applications, species and fluids from small molecules to proteins and biomarkers to PK assays.     

Cracking the folding code. Why do some proteins adopt partially folded conformations, whereas other don't?

Many, but not all, globular proteins have been shown to have compact intermediate state(s) under equilibrium conditions in vitro, giving rise to the question: why do some proteins adopt partially folded conformations, whereas other do not? Here we show that charge to hydrophobicity ratio of a polypeptide chain may represent a key determinant in this respect, as proteins known to form equilibrium partially folded intermediates are specifically localized within a unique region of charge-hydrophobicity space. Thus, the competence of a protein to form equilibrium intermediate(s) may be determined by the bulk content of hydrophobic and charged amino acid residues rather than by the positioning of amino acids within the sequence.     

What do you think you are quantifying? An appraisal of histochemical methods in the measurements of the activities of lysosomal enzymes.

The effect of a standard method of fixation (formalin-calcium 24 hr at 4 degrees C, followed by washing in gum-sucrose) on the activity of three lysosomal enzymes (N-acetyl-beta-D-glucosaminidase, acid phosphatase, beta-D-galactosidase) in the liver of three species (human, rabbit, lamb) was studied by biochemical methods, and the results were compared with staining intensities in histochemical preparations of the same tissues. The following conclusions were reached: (1) Fixation by formaldehyde changes the characteristics of the enzymes and makes comparisons between biochemistry and histochemistry difficult to interpret; (2) The intensity of staining in fixed or unfixed tissue sections bears no relation to absolute levels of enzyme activity; (3) Changes in staining intensity of a particular enzyme activity in a particular organ of a particular species prepared in a particular way are significant. Quantifying these changes is useful, as long as absolute values are not considered, and it is realized that it is only the difference that is being quantified.     

Where do you come from; where do you go? Pluripotency,differentiation and malfunction of stem cells

The international conference 'Stem Cells in Development and Disease' took place in September 2011 at the Max-Delbrück-Center for Molecular Medicine (MDC) in Berlin. It brought together scientists working on different types of stem cell and covered the latest findings in stem cell biology, including the genetic and epigenetic mechanisms of reprogramming, maintenance of pluripotency and differentiation.     

Where do hamsters go after cereal harvest? A case study.

Agricultural intensification in recent decades has led to a major loss of biodiversity in agricultural landscapes. Special agri-environmental schemes aim at restoring and protecting biodiversity. In Central Europe, the common hamster Cricetus cricetus (Linnaeus, 1758) is a prominent victim of changes in agricultural management and land-use. After cereal-harvest, common hamsters are deprived from shelter and food sources and little is known on how individuals cope with these changes. We investigated how a population of common hamsters use agricultural landscapes after cereal harvest in 45 fields within a distribution hotspot of the common hamster in Central Germany. We recorded hamster burrow densities before and after cereal harvest in the winter cereals as well as in the two most important specialty crops in the study region, red cabbage and carrot. Common hamster burrows were more abundant in cereals than in the specialty crops before harvest. After cereal harvest, common hamster burrow densities decreased in cereal fields and increased in carrot but not red cabbage. Carrot fields thus provide alternative habitat for common hamsters after cereal harvest. Since many carrot cultivars are harvested in late autumn, these fields potentially provide a safe habitat for hamsters until hibernation. We recommend a targeted subsidizing of the cultivation of late harvest carrot cultivars in regions where common hamster conservation is a priority. These fields should be placed strategically and in sufficient quantities in focus landscapes. Other specialty crops could potentially also help in targeted protection schemes but whether and how they can benefit common hamsters after the cereal harvest needs further investigation.