Single cell analysis reveals satellite cell heterogeneity for proinflammatory chemokine expression.

Background: The expression of proinflammatory signals at the site of muscle injury are essential for efficient tissue repair and their dysregulation can lead to inflammatory myopathies. Macrophages, neutrophils, and fibroadipogenic progenitor cells residing in the muscle are significant sources of proinflammatory cytokines and chemokines. However, the inducibility of the myogenic satellite cell population and their contribution to proinflammatory signaling is less understood. Methods: Mouse satellite cells were isolated and exposed to lipopolysaccharide (LPS) to mimic sterile skeletal muscle injury and changes in the expression of proinflammatory genes was examined by RT-qPCR and single cell RNA sequencing. Expression patterns were validated in skeletal muscle injured with cardiotoxin by RT-qPCR and immunofluorescence. Results: Satellite cells in culture were able to express Tnfa, Ccl2, and Il6, within 2 h of treatment with LPS. Single cell RNA-Seq revealed seven cell clusters representing the continuum from activation to differentiation. LPS treatment led to a heterogeneous pattern of induction of C-C and C-X-C chemokines (e.g., Ccl2, Ccl5, and Cxcl0) and cytokines (e.g., Tgfb1, Bmp2, Il18, and Il33) associated with innate immune cell recruitment and satellite cell proliferation. One cell cluster was enriched for expression of the antiviral interferon pathway genes under control conditions and LPS treatment. Activation of this pathway in satellite cells was also detectable at the site of cardiotoxin induced muscle injury. Conclusion: These data demonstrate that satellite cells respond to inflammatory signals and secrete chemokines and cytokines. Further, we identified a previously unrecognized subset of satellite cells that may act as sensors for muscle infection or injury using the antiviral interferon pathway.

Myxomavirus Serp-1 Protein Ameliorates Inflammation in a Mouse Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy is an X-linked disease afflicting 1 in 3500 males that is characterized by muscle weakness and wasting during early childhood, and loss of ambulation and death by early adulthood. Chronic inflammation due to myofiber instability leads to fibrosis, which is a primary cause of loss of ambulation and cardiorespiratory insufficiency. Current standard of care focuses on reducing inflammation with corticosteroids, which have serious adverse effects. It is imperative to identify alternate immunosuppressants as treatments to reduce fibrosis and mortality. Serp-1, a Myxoma virus-derived 55 kDa secreted glycoprotein, has proven efficacy in a range of animal models of acute inflammation, and its safety and efficacy has been shown in a clinical trial. In this initial study, we examined whether pegylated Serp-1 (PEGSerp-1) treatment would ameliorate chronic inflammation in a mouse model for Duchenne muscular dystrophy. Our data revealed a significant reduction in diaphragm fibrosis and increased myofiber diameter, and significantly decreased pro-inflammatory M1 macrophage infiltration. The M2a macrophage and overall T cell populations showed no change. These data demonstrate that treatment with this new class of poxvirus-derived immune-modulating serpin has potential as a therapeutic approach designed to ameliorate DMD pathology and facilitate muscle regeneration.

Antibody variable sequences have a pronounced effect on cellular transport and plasma half-life.

Monoclonal IgG antibodies are the fastest growing class of biologics, but large differences exist in their plasma half-life in humans. Thus, to design IgG antibodies with favorable pharmacokinetics, it is crucial to identify the determinants of such differences. Here, we demonstrate that the variable region sequences of IgG antibodies greatly affect cellular uptake and subsequent recycling and rescue from intracellular degradation by endothelial cells. When the variable sequences are masked by the cognate antigen, it influences both their transport behavior and binding to the neonatal Fc receptor (FcRn), a key regulator of IgG plasma half-life. Furthermore, we show how charge patch differences in the variable domains modulate both binding and transport properties and that a short plasma half-life, due to unfavorable charge patches, may partly be overcome by Fc-engineering for improved FcRn binding.

A neutron scattering perspective on the structure, softness and dynamics of the ligand shell of PbS nanocrystals in solution.

Small-angle neutron and X-ray scattering, neutron backscattering and neutron time-of-flight spectroscopy are applied to reveal the structure of the ligand shell, the temperature-dependent diffusion properties and the phonon spectrum of PbS nanocrystals functionalized with oleic acid in deuterated hexane. The nanocrystals decorated with oleic acid as well as the desorbed ligand molecules exhibit simple Brownian diffusion with a Stokes-Einstein temperature-dependence and inhibited freezing. Ligand molecules desorbed from the surface show strong spatial confinement. The phonon spectrum of oleic acid adsorbed to the nanocrystal surface exhibits hybrid modes with a predominant Pb-character. Low-energy surface modes of the NCs are prominent and indicate a large mechanical softness in solution. This work provides comprehensive insights into the ligand-particle interaction of colloidal nanocrystals in solution and highlights its effect on the diffusion and vibrational properties as well as their mechanical softness.

Revealing Grain Boundaries and Defect Formation in Nanocrystal Superlattices by Nanodiffraction.

X-ray nanodiffraction is applied to study the formation and correlation of domain boundaries in mesocrystalline superlattices of PbS nanocrystals with face-centered cubic structure. Each domain of the superlattice can be described with one of two mesocrystalline polymorphs with different orientational orders. Close to a grain boundary, the lattice constant decreases and the superlattice undergoes an out-of-plane rotation, while the orientation of the nanocrystals with respect to the superlattice remains unchanged. These findings are explained with the release of stress on the expense of specific nanocrystal-substrate interactions. The fact that correlations between adjacent nanocrystals are found to survive the structural changes at most grain boundaries implies that the key to nanocrystal superlattices with macroscopic domain sizes are strengthened interactions with the substrate.

Revealing Structure and Crystallographic Orientation of Soft Epitaxial Assembly of Nanocrystals by Grazing Incidence X-ray Scattering.

We study the structural coherence of a self-assembled overlayer of PbS nanocrystal (NC) superlattice onto an underlying PbS NC monolayer, which acts as a template. We explore the effect of the templating layer on the structure of the overlayer asemblies by varying interfacial strain and determine the impact of new ligands on their superlattice structure. The overlayers and templates are analyzed by grazing-incidence X-ray scattering and microscopy. We find that differences in the lattice parameters of 7.7% between the two layers are tolerated in terms of a "soft epitaxial" assembly into the body-centered tetragonal superstucture and lead to structural registry within the overlayer. Conversely, at the interface, a lattice mismatch of 24.4% is too large for soft epitaxy and invokes a change in the superlattice. Upon ligand treatment, the overlayer superlattices transform their orientation axis and the NCs orient preferentially. These results provide new insights into mitigating defects in layered, heterostructured NC assemblies.

Electron-Conducting PbS Nanocrystal Superlattices with Long-Range Order Enabled by Terthiophene Molecular Linkers.

PbS nanocrystals are surface-functionalized with the organic semiconductor 5,5″-dithiol-[2,2':5,2″-terthiophene] and assembled to afford hybrid nanostructured thin films with a large structural coherence and an electron mobility of 0.2 cm2/(V s). Electrochemistry, optical spectroscopy, and quantum mechanical calculations are applied to elucidate the electronic structure at the inorganic/organic interface, and it is established that electron injection into the molecule alters its (electronic) structure, which greatly facilitates coupling of the neighboring PbS 1Se states. This is verified by field-effect and electrochemically gated transport measurements, and evidence is provided that carrier transport occurs predominantly via the 1Se states. The presented material allows studying structure-transport correlations and exploring transport anisotropies in semiconductor nanocrystal superlattices.

Monitoring Self-Assembly and Ligand Exchange of PbS Nanocrystal Superlattices at the Liquid/Air Interface in Real Time.

We investigate in situ the structural changes during self-assembly of PbS nanocrystals from colloidal solution into superlattices, solvent evaporation, and ligand exchange at the acetonitrile/air interface by grazing incidence small-angle X-ray scattering (GISAXS). We simulate and fit the diffraction peaks under the distorted wave Born approximation (DWBA) to determine the lattice parameters. We observe a continuous isotropic contraction of the superlattice in two different assembly steps, preserving the body-centered cubic lattice with an overall decrease in the lattice constants of 11%. We argue that the first contraction period is due to a combination of solvent evaporation/annealing and capillary forces acting on the superlattice, whereas the second period is dominated by the effect of replacing oleic acid on the nanocrystal surface with the short and rigid cross-linker tetrathiafulvalene dicarboxylate. This work provides guidelines for optimized ligand exchange conditions and highlights the structural particularities arising from assembling NCs on liquid surfaces.

Quantifying Angular Correlations between the Atomic Lattice and the Superlattice of Nanocrystals Assembled with Directional Linking.

We show that the combination of X-ray scattering with a nanofocused beam and X-ray cross correlation analysis is an efficient way for the full structural characterization of mesocrystalline nanoparticle assemblies with a single experiment. We analyze several hundred diffraction patterns at individual sample locations, that is, individual grains, to obtain a meaningful statistical distribution of the superlattice and atomic lattice ordering. Simultaneous small- and wide-angle X-ray scattering of the same sample location allows us to determine the structure and orientation of the superlattice as well as the angular correlation of the first two Bragg peaks of the atomic lattices, their orientation with respect to the superlattice, and the average orientational misfit due to local structural disorder. This experiment is particularly advantageous for synthetic mesocrystals made by the simultaneous self-assembly of nanocrystals and surface-functionalization with conductive ligands. While the structural characterization of such materials has been challenging so far, the present method now allows correlating the mesocrystalline structure with optoelectronic properties.

Site-Specific Ligand Interactions Favor the Tetragonal Distortion of PbS Nanocrystal Superlattices.

We analyze the structure and morphology of mesocrystalline, body-centered tetragonal (bct) superlattices of PbS nanocrystals functionalized with oleic acid. On the basis of combined scattering and real space imaging, we derive a three-dimensional (3D) model of the superlattice and show that the bct structure benefits from a balanced combination of {100}PbS-{100}PbS and {111}PbS-{111}PbS interactions between neighboring layers of nanocrystals, which uniquely stabilizes this structure. These interactions are enabled by the coaxial alignment of the atomic lattices of PbS with the superlattice. In addition, we find that this preferential orientation is already weakly present within isolated monolayers. By adding excess oleic acid to the nanocrystal solution, tetragonal distortion is suppressed, and we observe assembly into a bilayered hexagonal lattice reminiscent of a honeycomb with grain sizes of several micrometers.

Automated prostate tissue referencing for cancer detection and diagnosis.

BACKGROUND: The current practice of histopathology review is limited in speed and accuracy. The current diagnostic paradigm does not fully describe the complex and complicated patterns of cancer. To address these needs, we develop an automated and objective system that facilitates a comprehensive and easy information management and decision-making. We also develop a tissue similarity measure scheme to broaden our understanding of tissue characteristics. RESULTS: The system includes a database of previously evaluated prostate tissue images, clinical information and a tissue retrieval process. In the system, a tissue is characterized by its morphology. The retrieval process seeks to find the closest matching cases with the tissue of interest. Moreover, we define 9 morphologic criteria by which a pathologist arrives at a histomorphologic diagnosis. Based on the 9 criteria, true tissue similarity is determined and serves as the gold standard of tissue retrieval. Here, we found a minimum of 4 and 3 matching cases, out of 5, for ~80 % and ~60 % of the queries when a match was defined as the tissue similarity score ≥5 and ≥6, respectively. We were also able to examine the relationship between tissues beyond the Gleason grading system due to the tissue similarity scoring system. CONCLUSIONS: Providing the closest matching cases and their clinical information with pathologists will help to conduct consistent and reliable diagnoses. Thus, we expect the system to facilitate quality maintenance and quality improvement of cancer pathology.

A robust high throughput platform to generate functional recombinant monoclonal antibodies using rabbit B cells from peripheral blood.

We have developed a robust platform to generate and functionally characterize rabbit-derived antibodies using B cells from peripheral blood. The rapid high throughput procedure generates a diverse set of antibodies, yet requires only few animals to be immunized without the need to sacrifice them. The workflow includes (i) the identification and isolation of single B cells from rabbit blood expressing IgG antibodies, (ii) an elaborate short term B-cell cultivation to produce sufficient monoclonal antigen specific IgG for comprehensive phenotype screens, (iii) the isolation of VH and VL coding regions via PCR from B-cell clones producing antigen specific and functional antibodies followed by the sequence determination, and (iv) the recombinant expression and purification of IgG antibodies. The fully integrated and to a large degree automated platform (demonstrated in this paper using IL1RL1 immunized rabbits) yielded clonal and very diverse IL1RL1-specific and functional IL1RL1-inhibiting rabbit antibodies. These functional IgGs from individual animals were obtained at a short time range after immunization and could be identified already during primary screening, thus substantially lowering the workload for the subsequent B-cell PCR workflow. Early availability of sequence information permits one to select early-on function- and sequence-diverse antibodies for further characterization. In summary, this powerful technology platform has proven to be an efficient and robust method for the rapid generation of antigen specific and functional monoclonal rabbit antibodies without sacrificing the immunized animal.