All-in-One digital microfluidics pipeline for proteomic sample preparation and analysis.

Highly sensitive and reproducible analysis of samples containing low amounts of protein is restricted by sample loss and the introduction of contaminants during processing. Here, we report an All-in-One digital microfluidic (DMF) pipeline for proteomic sample reduction, alkylation, digestion, isotopic labeling and analysis. The system features end-to-end automation, with integrated thermal control for digestion, optimized droplet additives for sample manipulation and analysis, and an automated interface to liquid chromatography with tandem mass spectrometry (HPLC-MS/MS).

Heterogeneity of osteoclast activity and bone turnover in different skeletal sites.

Objective: To compare osteoclasts and bone turnover in the cranial and appendicular skeletons of mice and determine whether estrogen depletion has an impact on these differences.

Design: In vitro osteoclastogenesis (OCG) was performed on osteoclasts precursors derived from calvarial, mandibular and femoral bone marrow. In vitro, mature osteoclasts were stained with TRAP in plastic petri dishes and with DAPI and Phalloidin on glass coverslips to identify mature osteoclasts and compare osteoclast surface area and nuclei number in the different bone sites, respectively.

Proximity biotinylation and affinity purification are complementary approaches for the interactome mapping of chromatin-associated protein complexes.

Unlabelled: Mapping protein-protein interactions for chromatin-associated proteins remains challenging. Here we explore the use of BioID, a proximity biotinylation approach in which a mutated biotin ligase (BirA*) is fused to a bait of interest, allowing for the local activation of biotin and subsequent biotinylation of proteins in the bait vicinity. BioID allowed for successful interactome mapping of core histones and members of the mediator complex.

Microbiome analysis - from technical advances to biological relevance.

The development of culture-independent techniques and next-generation sequencing has led to a staggering rise in the number of microbiome studies over the last decade. Although it remains important to identify the taxa of microbes present in a variety of environmental samples, including the gut microbiomes of healthy and diseased individuals, the next stage of microbiome research will need to focus on uncovering the role of the microbiome rather than its mere composition. Here, we introduce techniques that go beyond identifying the taxa present within a sample and examine the biological function of the microbiome or the host-microbiome interaction.

Conditional and inducible transgene expression in mice through the combinatorial use of Cre-mediated recombination and tetracycline induction.

Here we describe a triple transgenic mouse system, which combines the tissue specificity of any Cre-transgenic line with the inducibility of the reverse tetracycline transactivator (rtTA)/tetracycline-responsive element (tet-O)-driven transgenes. To ensure reliable rtTA expression in a broad range of cell types, we have targeted the rtTA transgene into the ROSA26 locus. The rtTA expression, however, is conditional to a Cre recombinase-mediated excision of a STOP region from the ROSA26 locus.

Regulation of the mating pheromone and invasive growth responses in yeast by two MAP kinase substrates.

Background: In the budding yeast Saccharomyces cerevisiae, components of a single mitogen-activated protein (MAP) kinase pathway transduce two distinct signals, each of which activates an independent developmental programme: peptide mating pheromones initiate the mating response, whereas nutrient limitation initiates filamentous growth. One of the MAP kinases in this pathway, Fus3, triggers mating but antagonizes filamentous growth, while the other, Kss 1, preferentially triggers filamentous growth. Both kinases activate the same transcription factor, Ste 12, which can stimulate gene expression specific to each of the developmental programmes.

The geometric osteotomy: joint preservation in juxta-articular surface bone neoplasms.

Purpose. To present the oncologic and functional results of a consecutive series of patients treated by geometric osteotomy and allograft reconstruction for juxta-articular surface bone neoplasms.Patients.