Semi-autonomous real-time programmable fluorescence lifetime segmentation with a digital micromirror device.

Time-correlated single-photon counting (TCSPC) is the gold standard for performing lifetime spectroscopy in biological assays. Traditional fluorescence lifetime imaging (FLIM) using laser scanning microscopes are inherently slow due to point scanning all pixels in the field-of-view. Wide-field implementations of TCSPC spectroscopy using microchannel plates benefit from particularly fast acquisition times at the expense of temporal resolution, and are fundamentally limited by photon counting rates. Here, we introduce programmable lifetime imaging (PLI), combining the advantages of wide-field imaging using total internal reflection excitation with state-of-the-art TCSPC detector technology for accurate lifetime determination in an object-oriented manner using a digital micromirror device (DMD). The fluorescent emission is projected onto the DMD to facilitate the sequential segmentation of fluorescence from individual objects in the field-of-view, allowing for both image acquisition and fluorescence lifetime determination of the assay. The sensitivity of PLI is demonstrated by manually segmenting fluorescence from fixed cell assays. We also demonstrate an automated implementation of PLI, using a camera as a feedback mechanism to segment fluorescence produced by emitting objects of interest in the imaging field-of-view, highlighting the advantages of measurement only in areas where valuable information exists. As a result, PLI is able to reduce acquisition time of fluorescence lifetime data by at least an order of magnitude compared to laser scanning implementations.

Liver Transplantation for Acute Liver Failure Attributed to Leptospirosis: A Report of Two Cases.

BACKGROUND: Leptospirosis is a zoonosis caused by pathogenic spirochetes of the genus Leptospira. Although it may be limited to nonspecific fever, leptospirosis may also be responsible for neurological symptoms or fulminant diseases such as Weil's disease. Diagnosis is challenging due to the difficulty in isolating the organism and the delays required for performing the serological test. CASE PRESENTATION: Two cases of leptospirosis are presented here. The clinical picture differed from a real Weil's disease in the first case and from a neuro-leptospirosis in the second. However, both patients underwent liver transplantation because of the severity of the associated acute liver failure. Unfortunately, one of the cases had a fatal outcome. CONCLUSION: Antibiotic treatment for leptospirosis should not be delayed by the lack of a positive serology test for this potentially lethal disease. In the context of a history of exposure to risk factors for leptospirosis, a negative serology must be repeated 7 days to 2 weeks following the first test. Although not always present, acute liver injury may, in rare cases, require liver transplantation.

TNFR1 membrane reorganization promotes distinct modes of TNFα signaling.

Signaling by the ubiquitously expressed tumor necrosis factor receptor 1 (TNFR1) after ligand binding plays an essential role in determining whether cells exhibit survival or death. TNFR1 forms distinct signaling complexes that initiate gene expression programs downstream of the transcriptional regulators NFκB and AP-1 and promote different functional outcomes, such as inflammation, apoptosis, and necroptosis. Here, we investigated the ways in which TNFR1 was organized at the plasma membrane at the nanoscale level to elicit different signaling outcomes. We confirmed that TNFR1 forms preassembled clusters at the plasma membrane of adherent cells in the absence of ligand. After trimeric TNFα binding, TNFR1 clusters underwent a conformational change, which promoted lateral mobility, their association with the kinase MEKK1, and activation of the JNK/p38/NFκB pathway. These phenotypes required a minimum of two TNFR1-TNFα contact sites; fewer binding sites resulted in activation of NFκB but not JNK and p38. These data suggest that distinct modes of TNFR1 signaling depend on nanoscale changes in receptor organization.

Limited proteolysis and peptide mapping for comparability of biopharmaceuticals: An evaluation of repeatability, intra-assay precision and capability to detect structural change.

The use of limited proteolysis followed by peptide mapping for the comparability of the higher-order structure of biopharmaceuticals was investigated. In this approach the proteolysis is performed under non-reducing and non-denaturing conditions, and the resulting peptide map is determined by the samples primary and higher order structures. This allows comparability of biopharmaceuticals to be made in terms of their higher order structure, using a method that is relatively simple to implement. The digestion of a monoclonal antibody under non-denaturing conditions was analyzed using peptide mapping, circular dichroism (CD) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This allowed an optimal digestion time to be chosen. This method was then assessed for its ability to detect structural change using a monoclonal antibody, which had been subjected to a range of stresses; deglycosylation, mild denaturation and a batch that had failed specifications due to in-process reduction. The repeatability and inter-assay precision were assessed. It was demonstrated that the limited proteolysis peptide maps of the three stressed samples were significantly different to control samples and that the differences observed were consistent between the occasions when the assays were run. A combination of limited proteolysis and CD or SDS-PAGE analysis was shown to enhance the capacity of these techniques to detect structural change, which otherwise would not have been observed.