Rehydration of freeze substituted brain tissue for preembedding immuno-electron microscopy

Electron microscopy (EM) volume reconstruction is a powerful tool for investigating the fundamental structure of brain circuits, but the full potential of this technique is limited by the difficulty of integrating molecular information. High quality ultrastructural preservation is necessary for EM reconstruction, and intact, highly contrasted cell membranes are essential for following small neuronal processes through serial sections. Unfortunately, the antibody labeling methods used to identify most endogenous molecules result in compromised morphology, especially of membranes. Cryofixation can produce superior morphological preservation and has the additional advantage of allowing indefinite storage of valuable samples. We have developed a method based on cryofixation that allows sensitive immunolabeling of endogenous molecules, preserves excellent ultrastructure, and is compatible with high-contrast staining for serial EM reconstruction.

LAP2alpha maintains a mobile and low assembly state of A-type lamins in the nuclear interior

Lamins form stable filaments at the nuclear periphery in metazoans. Unlike B-type lamins, lamins A and C localize also in the nuclear interior, where they interact with lamin-associated polypeptide 2 alpha (LAP2α). Using antibody labeling, we previously observed a depletion of nucleoplasmic A-type lamins in mouse cells lacking LAP2α. Here, we show that loss of LAP2α actually causes formation of larger, biochemically stable lamin A/C structures in the nuclear interior that are inaccessible to lamin A/C antibodies. While nucleoplasmic lamin A forms from newly expressed pre-lamin A during processing and from soluble mitotic lamins in a LAP2α-independent manner, binding of LAP2α to lamin A/C during interphase inhibits formation of higher order structures, keeping nucleoplasmic lamin A/C in a mobile state independent of lamin A/C S22 phosphorylation. We propose that LAP2α is essential to maintain a mobile lamin A/C pool in the nuclear interior, which is required for proper nuclear functions.

Robust, sensitive, and quantitative single-cell proteomics based on ion mobility filtering

Unbiased single-cell proteomics (scProteomics) promises to advance our understanding of the cellular composition of complex biological systems. However, a major challenge for current methods is their ability to identify and provide accurate quantitative information for low abundance proteins. Herein, we describe an ion mobility-enhanced mass spectrometry acquisition method, TIFF (Transferring Identification based on FAIMS Filtering), designed to improve the sensitivity and accuracy of label-free scProteomics. The TIFF method enabled unbiased proteome analysis to a depth of >1,700 proteins in single HeLa cells with >1,100 proteins consistently quantified, a significant improvement in overall performance. We applied the TIFF method to obtain temporal proteome profiles of >150 single murine macrophage cells during a lipopolysaccharide stimulation experiment and uncovered unanticipated temporal response trajectories. Further, we demonstrated, to our knowledge, the first application of scProteomics to classify cell populations of a human organ (the lung) without prior antibody labeling.

Interaction of Intestinal Bacteria with Human Rotavirus during Infection in Children

The gut microbiota has emerged as a key factor in the pathogenesis of intestinal viruses, including enteroviruses, noroviruses and rotaviruses (RVs), where stimulatory and inhibitory effects on infectivity have been reported. With the aim of determining whether members of the microbiota interact with RVs during infection, a combination of anti-RV antibody labeling, fluorescence-activated cell sorting and 16S rRNA amplicon sequencing was used to characterize the interaction between specific bacteria and RV in stool samples of children suffering from diarrhea produced by G1P[8] RV. The genera Ruminococcus and Oxalobacter were identified as RV binders in stools, displaying enrichments between 4.8- and 5.4-fold compared to samples nonlabeled with anti-RV antibodies. In vitro binding of the G1P[8] Wa human RV strain to two Ruminococcus gauvreauii human isolates was confirmed by fluorescence microscopy. Analysis in R. gauvreauii with antibodies directed to several histo-blood group antigens (HBGAs) indicated that these bacteria express HBGA-like substances on their surfaces, which can be the target for RV binding. Furthermore, in vitro infection of the Wa strain in differentiated Caco-2 cells was significantly reduced by incubation with R. gauvreauii. These data, together with previous findings showing a negative correlation between Ruminococcus levels and antibody titers to RV in healthy individuals, suggest a pivotal interaction between this bacterial group and human RV. These results reveal likely mechanisms of how specific bacterial taxa of the intestinal microbiota could negatively affect RV infection and open new possibilities for antiviral strategies.

Novel Time-Resolved Fluorescence Immunochromatography Paper-Based Sensor with Signal Amplification Strategy for Detection of Deoxynivalenol

Immunoassay has the advantages of high sensitivity, high specificity, and simple operation, and has been widely used in the detection of mycotoxins. For several years, time-resolved fluorescence immunochromatography (TRFIA) paper-based sensors have attracted much attention as a simple and low-cost field detection technology. However, a traditional TRFIA paper-based sensor is based on antibody labeling, which cannot easily meet the current detection requirements. A second antibody labeling method was used to amplify the fluorescence signal and improve the detection sensitivity. Polystyrene fluorescent microspheres were combined with sheep anti-mouse IgG to prepare fluorescent probes (Eu-IgGs). After the probe fully reacted with the antibody (Eu-IgGs-Abs) in the sample cell, it was deployed on the paper-based sensor using chromatography. Eu-IgGs-Abs that were not bound to the target were captured on the T-line, while those that were bound were captured on the C-line. The paper-based sensor reflected the corresponding fluorescence intensity change. Because a single molecule of the deoxynivalenol antibody could bind to multiple Eu-IgGs, this method could amplify the fluorescence signal intensity on the unit antibody and improve the detection sensitivity. The working standard curve of the sensor was established under the optimum working conditions. It showed the lower limit of detection and higher recovery rate when it was applied to actual samples and compared with other methods. This sensor has the advantages of high sensitivity, good accuracy, and good specificity, saving the amount of antibody consumed and being suitable for rapid field detection of deoxynivalenol.

Label-free lymphocytes reconstitution using side scatter for optimal T cell manufacturing

SUMMARYLymphocyte biology research commonly involves purification of lymphocyte subpopulations by fluorescence-activated cell sorting (FACS) or immunomagnetic separation (IMS), both of which typically rely on antibody labeling of validated cell markers. Methods enabling label-free segregation of lymphocyte subpopulations would be invaluable with regard to less-perturbation, simplicity and cost-effectiveness. Here, we introduce TRuST, a label-free approach for T cell reconstitution using side-scatter (SSC). TRuST-sorted SSClow cells enrich for CD4+ T and naïve T cells, while SSChigh cells enrich for CD8+ T, NK and differentiated T cells. Enrichment purity can be improved by computational gate design. SSClow cells have superior expansion capacity and generate more central memory precursors with naïve-resembling cytokine responses. Moreover, we find that both T cell differentiation status and CD4/CD8 T ratio in the starting cellular material are critical attributes predicting T cell product quality and quantity. TRuST presents an effective and reliable technique for label-free lymphocytes selection and reconstitution.

Distal convoluted tubule sexual dimorphism revealed by advanced 3D imaging

The thiazide-sensitive Na+-Cl− cotransporter (NCC) is more abundant in kidneys of female subjects than of male subjects. Because morphological remodeling of the distal convoluted tubule (DCT) is dependent on NCC activity, it has been generally assumed that there is a corresponding sexual dimorphism in the structure of the DCT, leading to a larger female DCT. Until now, this has never been directly examined. Here, optical clearing techniques were combined with antibody labeling of DCT segment markers, state-of-the-art high-speed volumetric imaging, and analysis tools to visualize and quantify DCT morphology in male and female mice and study the DCT remodeling response to furosemide. We found an unexpected sex difference in the structure of the DCT. Compared with the male mice, female mice had a shorter DCT, a higher cellular density of NCC, and a greater capacity to elongate in response to loop diuretics. Our study revealed a sexual dimorphism of the DCT. Female mice expressed a greater density of NCC transporters in a shorter structure to protect Na+ balance in the face of greater basal distal Na+ delivery yet have a larger reserve and structural remodeling capacity to adapt to unique physiological stresses. These observations provide insight into mechanisms that may drive sex differences in the therapeutic responses to diuretics.