Community-Driven Data Analysis Training for Biology

The primary problem with the explosion of biomedical datasets is not the data, not computational resources, and not the required storage space, but the general lack of trained and skilled researchers to manipulate and analyze these data. Eliminating this problem requires development of comprehensive educational resources. Here we present a community-driven framework that enables modern, interactive teaching of data analytics in life sciences and facilitates the development of training materials. The key feature of our system is that it is not a static but a continuously improved collection of tutorials. By coupling tutorials with a web-based analysis framework, biomedical researchers can learn by performing computation themselves through a web browser without the need to install software or search for example datasets. Our ultimate goal is to expand the breadth of training materials to include fundamental statistical and data science topics and to precipitate a complete re-engineering of undergraduate and graduate curricula in life sciences. This project is accessible at https://training.galaxyproject.org. We developed an infrastructure that facilitates data analysis training in life sciences. It is an interactive learning platform tuned for current types of data and research problems. Importantly, it provides a means for community-wide content creation and maintenance and, finally, enables trainers and trainees to use the tutorials in a variety of situations, such as those where reliable Internet access is unavailable

Proteomic analysis of silenced cathepsin B expression suggests non-proteolytic cathepsin B functionality.

A list of human protein uniprot IDs. The proteins were identified by LC-MS/MS in the cellular supernatant of MDA-MB-231 cells, originally published in: F.C. Sigloch, J.D. Knopf, J. Weißer, A. Gomez-Auli, M.L. Biniossek, A. Petrera, et al., Proteomic analysis of silenced cathepsin B expression suggests non-proteolytic cathepsin B functionality, Biochim. Biophys. Acta - Mol. Cell Res. 1863 (2016) 2700–2709. doi:10.1016/j.bbamcr.2016.08.005. https://www.ncbi.nlm.nih.gov/pubmed/2752667

Proteomics LC-MS/MS test dataset for protein quantitation via stable isotope labelling

<p>The provided mzML file can be used as a test dataset for protein identification and quantitation software. It was generated from human embryonic kidney (HEK) cells that were either unlabelled or labelled with heavy SILAC (K6R6, unimod accession 188, PSI-MS Name: "Label:13C(6)"). Apart from different labelling, the HEK cells were kept in exactly the same conditions and harvested simultaneously. Light and heavy labelled proteins from HEK cell lysate were mixed in a certain ratio, digested with Trypsin and measured on a ThermoFisher QExactive mass spectrometer. A more detailed description on the generation of the dataset will soon be accessible at PRIDE.</p> <p>The provided mzML file has been converted from Thermo RAW and slightly modified via msConvert (ProteoWizard). To reduce the filesize and to speed up analysis, it has further been filtered to contain only the data measured between 2,000 sec and 3,000 sec of the original LC-MS/MS run.</p

Transmembrane Collagen XVII Modulates Integrin Dependent Keratinocyte Migration via PI3K/Rac1 Signaling

<div><p>The hemidesmosomal transmembrane component collagen XVII (ColXVII) plays an important role in the anchorage of the epidermis to the underlying basement membrane. However, this adhesion protein seems to be also involved in the regulation of keratinocyte migration, since its expression in these cells is strongly elevated during reepithelialization of acute wounds and in the invasive front of squamous cell carcinoma, while its absence in ColXVII-deficient keratinocytes leads to altered cell motility. Using a genetic model of murine <i>Col17a1⁻</i>^{<b><i>/</i></b><i>−</i>} keratinocytes we elucidated ColXVII mediated signaling pathways in cell adhesion and migration. <i>Col17a1⁻</i>^{<b><i>/</i></b><i>−</i>} keratinocytes exhibited increased spreading on laminin 332 and accelerated, but less directed cell motility. These effects were accompanied by increased expression of the integrin subunits β4 and β1. The migratory phenotype, as evidenced by formation of multiple unstable lamellipodia, was associated with enhanced phosphoinositide 3-kinase (PI3K) activity. Dissection of the signaling pathway uncovered enhanced phosphorylation of the β4 integrin subunit and the focal adhesion kinase (FAK) as activators of PI3K. This resulted in elevated Rac1 activity as a downstream consequence. These results provide mechanistic evidence that ColXVII coordinates keratinocyte adhesion and directed motility by interfering integrin dependent PI3K activation and by stabilizing lamellipodia at the leading edge of reepithelializing wounds and in invasive squamous cell carcinoma.</p></div

Loss of ColXVII leads to phosphorylation of FAK.

<p><b>A</b>, Keratinocytes derived from wild type (Ctrl) and <i>Col17a1⁻</i>^{<b><i>/</i></b><i>−</i>} mice were allowed to adhere to LN332 for 2 hours, lysed and immunoblotted with antibodies to phospho-FAK (Y397) and total FAK. <b>B</b>, Indirect immunofluorescence staining of Ctrl and <i>Col17a1⁻</i>^{<b><i>/</i></b><i>−</i>} keratinocytes with phospho-FAK (Y397) antibody. <i>Scale bar  = 10 µm</i>. <b>C</b> and <b>D</b>, <i>Col17a1⁻</i>^{<b><i>/</i></b><i>−</i>} keratinocytes were treated with DMSO (Ctrl) and two different phospho-FAK inhibitors (PF 573228 [5 µM] and Inhibitor 14 [1 µM]) for 6 hours. The cells were lysed and equal amounts of total protein were immunoblotted with antibodies to phospho-FAK (Y397) and total FAK (<b>C</b>, representative immunoblot) and to phospho-Akt and total Akt (<b>D</b>). The graph shows quantification of phospho-Akt relative to total Akt. Individuals are indicated by different symbols (number of independent measurements  = 2); **<i>p</i><0.01. <b>E</b>, <i>Col17a1⁻</i>^{<b><i>/</i></b><i>−</i>} keratinocytes with comprising β4 integrin subunit knockdown were treated with DMSO (Ctrl) or phospho-FAK inhibitor (PF 573228 [5 µM] for 6 hours. The cells were lysed and equal amounts of total protein were immunoblotted with antibodies to phospho-Akt and total Akt.</p

Altered cell detachment in the absence of ColXVII.

<p><b>A</b>, Trypsin-based cell detachment assay. Confluent cell layers of keratinocytes derived from wild type (Ctrl) and <i>Col17a1⁻</i>^{<b><i>/</i></b><i>−</i>} mice were treated with trypsin/EDTA (0.05%/0.02%) for indicated time points (cells of three individuals per genotype have been analyzed; number of independent measurements  = 5). <b>B</b>, For the centrifugal force-based cell detachment assay cells were allowed to adhere on laminin 332 (LN332) for 10 minutes before measuring the strength of the adhesion at indicated centrifugal forces (cells of three individuals per genotype have been analyzed; number of independent measurements  = 3). For both assays adherent cells were stained with 0.5% crystal-violet, lysed with 1% SDS, and the percentage of adherent cells was determined spectrophotometrically at 540 nm. Data are shown as mean ± SEM; **<i>p</i><0.01.</p

Upregulation of α6β4 and β1 integrins in <i>Col17a1^−/−</i> skin.

<p><b>A</b> and <b>B</b>, Skin lysates from WT and <i>Col17a1⁻</i>^{<b><i>/</i></b><i>−</i>} mice were immunoblotted with indicated antibodies. The graphs combine the quantification for β4 protein expression of four individuals per genotype (<b>A</b>) and β1 protein expression of three individuals (<b>B</b>). *<i>p</i><0.05. <b>C</b>, Quantitative RT-PCR of primary wild type and <i>Col17a1⁻</i>^{<b><i>/</i></b><i>−</i>} keratinocytes (cells of four individuals per genotype have been analyzed; number of independent measurements  = 3). Data are shown as mean ± SEM; *<i>p</i><0.05.</p

Enhanced PI3K signaling in <i>Col17a1^−/−</i> keratinocytes.

<p><b>A</b>, Keratinocytes derived from wild type (Ctrl) and <i>Col17a1⁻</i>^{<b><i>/</i></b><i>−</i>} mice were lysed and equal amounts of total protein were immunoblotted with phospho-Akt and total Akt antibodies. The graph shows quantification of phospho-Akt relative to total Akt (cells of three individuals per genotype have been analyzed in 3 independent measurements); <i>*p</i><0.05. <b>B</b>, <i>Col17a1⁻</i>^{<b><i>/</i></b><i>−</i>} keratinocytes were grown on glass-bottom culture dishes and treaded with either DMSO or LY294002 [50 µM]. Cell migration was recorded by time-lapse imaging every 5 minutes during 4 hours. The distance migrated is indicated on the x-axis, the processive index (PI) on the y-axis.</p

Migration properties of control (Ctrl) and <i>Col17a1⁻</i>^{<b><i>/</i></b><i>−</i>} keratinocytes (see also Figure 5A, 6B and S1B).

<p>PI  =  processive index. The data are shown as mean ± SEM.</p><p><b>(##)  = </b><i>p<</i>0.01; <b>(#)  = </b><i>p</i><0.05; <i>(§§)  =  p<</i>0.001; <i>(§)</i>  =  <i>p</i><0.01.</p

β4 integrin subunit is functionally involved in cell adhesion and spreading of <i>Col17a1^−/−</i> cells.

<p><i>Col17a1⁻</i>^{<b><i>/</i></b><i>−</i>} keratinocytes were transduced with empty pLKO vector (mock) and two different shRNA to β4 integrin subunit (β4kd#1 and β4kd#2). <b>A</b>, The cells were lysed and equal amounts of total protein were immunoblotted with indicated antibodies. <b>B</b>, Confluent layers of <i>Col17a1⁻</i>^{<b><i>/</i></b><i>−</i>} cells were subjected to trypsin/EDTA detachment assay (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087263#pone-0087263-g001" target="_blank">Figure 1</a>). The percentage of adherent cells are shown as mean ± SEM (number of independent measurements  = 3); <i>*p</i><0.05. <b>C</b>, Cells were grown on LN332 coated chamber slides for 30 minutes, fixed and processed for indirect immunofluorescence with an actin antibody. The graph shows the cell area in µm² (<i>n = </i>35). The data are shown as mean ± SEM. *<i>p</i><0.05. <b>D</b>, <i>Col17a1⁻</i>^{<b><i>/</i></b><i>−</i>} keratinocytes were treated with DMSO (Ctrl) or different phospho-FAK inhibitors (PF 573228 [5 µM] and Inhibitor 14 [1 µM]) for 6 hours and thereafter subjected to trypsin/EDTA detachment assay. The data are shown as mean ± SEM (cells of three individuals have been analyzed; number of independent measurements  = 5).</p