High-throughput multi-antigen microfluidic fluorescence immunoassays

Here we describe the development of a high-throughput multi-antigen microfluidic fluorescence immunoassay system. A 100-chamber polydimethylsiloxane (PDMS) chip performs up to 5 tests for each of 10 samples. In this particular study system, the specificity of detection was demonstrated, and calibration curves were produced for C-reactive protein (CRP), prostate-specific antigen (PSA), ferritin, and vascular endothelial growth factor (VEGF). The measurements show sensitivity at and below clinically normal levels (with a signal-to-noise ratio >8 at as low as 10 pM antigen concentration). The chip uses 100 nL per sample for all tests. The developed system is an important step toward derivative immunoassay applications in scientific research and "point-of-care" testing in medicine.     

A DNA barcode library for Germany′s mayflies,stoneflies and caddisflies (Ephemeroptera,Plecoptera and Trichoptera)

Mayflies, stoneflies and caddisflies (Ephemeroptera, Plecoptera and Trichoptera) are prominent representatives of aquatic macroinvertebrates, commonly used as indicator organisms for water quality and ecosystem assessments. However, unambiguous morphological identification of EPT species, especially their immature life stages, is a challenging, yet fundamental task. A comprehensive DNA barcode library based upon taxonomically well‐curated specimens is needed to overcome the problematic identification. Once available, this library will support the implementation of fast, cost‐efficient and reliable DNA‐based identifications and assessments of ecological status. This study represents a major step towards a DNA barcode reference library as it covers for two‐thirds of Germany's EPT species including 2,613 individuals belonging to 363 identified species. As such, it provides coverage for 38 of 44 families (86%) and practically all major bioindicator species. DNA barcode compliant sequences (≥500 bp) were recovered from 98.74% of the analysed specimens. Whereas most species (325, i.e., 89.53%) were unambiguously assigned to a single Barcode Index Number (BIN) by its COI sequence, 38 species (18 Ephemeroptera, nine Plecoptera and 11 Trichoptera) were assigned to a total of 89 BINs. Most of these additional BINs formed nearest neighbour clusters, reflecting the discrimination of geographical subclades of a currently recognized species. BIN sharing was uncommon, involving only two species pairs of Ephemeroptera. Interestingly, both maximum pairwise and nearest neighbour distances were substantially higher for Ephemeroptera compared to Plecoptera and Trichoptera, possibly indicating older speciation events, stronger positive selection or faster rate of molecular evolution.     

Intravital microscopy of the murine pulmonary microcirculation.

Intravital microscopy (IVM) is considered as the gold standard for in vivo investigations of dynamic microvascular regulation. The availability of transgenic and knockout animals has propelled the development of murine IVM models for various organs, but technical approaches to the pulmonary microcirculation are still scarce. In anesthetized and ventilated BALB/c mice, we established a microscopic access to the surface of the right upper lung lobe by surgical excision of a window of 7- to 10-mm diameter from the right thoracic wall. The window was covered by a transparent polyvinylidene membrane and sealed with alpha-cyanoacrylate. Removal of intrathoracic air via a trans-diaphragmal intrapleural catheter coupled the lung surface to the window membrane. IVM preparations were hemodynamically stable for at least 120 min, with mean arterial blood pressure above 70 mmHg, and mean arterial Po(2) and arterial Pco(2) in the range of 90-100 Torr and 30-40 Torr, respectively. Imaged lungs did not show any signs of acute lung injury or edema. Following infusion of FITC dextran, subpleural pulmonary arterioles and venules of up to 50-microm diameter and alveolar capillary networks could be visualized during successive expiratory plateau phases over a period of at least 2 h. Vasoconstrictive responses to hypoxia (11% O(2)) or infusion of the thromboxane analog U-46619 were prominent in medium-sized arterioles (30- to 50-microm diameter), minor in small arterioles <30 microm, and absent in venules. The presented IVM model may constitute a powerful new tool for investigations of pulmonary microvascular responses in mice.     

Single-molecule studies of synaptotagmin and complexin binding to the SNARE complex

The assembly of multiprotein complexes at the membrane interface governs many signaling processes in cells. However, very few methods exist for obtaining biophysical information about protein complex formation at the membrane. We used single molecule fluorescence resonance energy transfer to study complexin and synaptotagmin interactions with the SNARE complex in deposited lipid bilayers. Using total internal reflectance microscopy, individual binding events at the membrane could be resolved despite an excess of unbound protein in solution. Fluorescence resonance energy transfer (FRET)-efficiency derived distances for the complexin-SNARE interaction were consistent with the crystal structure of the complexin-SNARE complex. The unstructured N-terminal region of complexin showed broad distributions of FRET efficiencies to the SNARE complex, suggesting that information on conformational variability can be obtained from FRET efficiency distributions. The low-affinity interaction of synaptotagmin with the SNARE complex changed dramatically upon addition of Ca2+ with high FRET efficiency interactions appearing between the C2B domain and linker domains of synaptotagmin and the membrane proximal portion of the SNARE complex. These results demonstrate that single molecule FRET can be used as a "spectroscopic ruler" to simultaneously gain structural and kinetic information about transient multiprotein complexes at the membrane interface.     

Short bioactive Spiegelmers to migraine-associated calcitonin gene-related peptide rapidly identified by a novel approach: tailored-SELEX

We developed an integrated method to identify aptamers with only 10 fixed nucleotides through ligation and removal of primer binding sites within the systematic evolution of ligands by exponential enrichment (SELEX) process. This Tailored-SELEX approach was validated by identifying a Spiegelmer (‘mirror-image aptamer’) that inhibits the action of the migraine-associated target calcitonin gene-related peptide 1 (α-CGRP) with an IC₅₀ of 3 nM at 37°C in cell culture. Aptamers are oligonucleotide ligands that can be generated to bind to targets with high affinity and specificity. Stabilized aptamers and Spiegelmers have shown activity in vivo and may be used as therapeutics. Aptamers are isolated by in vitro selection from combinatorial nucleic acid libraries that are composed of a central randomized region and additional fixed primer binding sites with ~30–40 nt. The identified sequences are usually not short enough for efficient chemical Spiegelmer synthesis, post-SELEX stabilization of aptamers and economical production. If the terminal primer binding sites are part of the target recognizing domain, truncation of aptamers has proven difficult and laborious. Tailored-SELEX results in short sequences that can be tested more rapidly in biological systems. Currently, our identified CGRP binding Spiegelmer serves as a lead compound for in vivo studies.     

ImShot: An Open-Source Software for Probabilistic Identification of Proteins In Situ and Visualization of Proteomics Data

Imaging mass spectrometry (IMS) has developed into a powerful tool allowing label-free detection of numerous biomolecules in situ. In contrast to shotgun proteomics, proteins/peptides can be detected directly from biological tissues and correlated to its morphology leading to a gain of crucial clinical information. However, direct identification of the detected molecules is currently challenging for MALDI–IMS, thereby compelling researchers to use complementary techniques and resource intensive experimental setups. Despite these strategies, sufficient information could not be extracted because of lack of an optimum data combination strategy/software. Here, we introduce a new open-source software ImShot that aims at identifying peptides obtained in MALDI–IMS. This is achieved by combining information from IMS and shotgun proteomics (LC–MS) measurements of serial sections of the same tissue. The software takes advantage of a two-group comparison to determine the search space of IMS masses after deisotoping the corresponding spectra. Ambiguity in annotations of IMS peptides is eliminated by introduction of a novel scoring system that identifies the most likely parent protein of a detected peptide in the corresponding IMS dataset. Thanks to its modular structure, the software can also handle LC–MS data separately and display interactive enrichment plots and enriched Gene Ontology terms or cellular pathways. The software has been built as a desktop application with a conveniently designed graphic user interface to provide users with a seamless experience in data analysis. ImShot can run on all the three major desktop operating systems and is freely available under Massachusetts Institute of Technology license.     

Beyond genetics. Influence of dietary factors and gut microbiota on type 1 diabetes

Type 1 diabetes (T1D) is an autoimmune disease ultimately leading to destruction of insulin secreting β-cells in the pancreas. Genetic susceptibility plays an important role in T1D etiology, but even mono-zygotic twins only have a concordance rate of around 50%, underlining that other factors than purely genetic are involved in disease development. Here we review the influence of dietary and environmental factors on T1D development in humans as well as animal models. Even though data are still inconclusive, there are strong indications that gut microbiota dysbiosis plays an important role in T1D development and evidence from animal models suggests that gut microbiota manipulation might prove valuable in future prevention of T1D in genetically susceptible individuals.