Semi-automated approaches for interrogating spatial heterogeneity of tissue samples.

Tissues are spatially orchestrated ecosystems composed of heterogeneous cell populations and non-cellular elements. Tissue components' interactions shape the biological processes that govern homeostasis and disease, thus comprehensive insights into tissues' composition are crucial for understanding their biology. Recently, advancements in the spatial biology field enabled the in-depth analyses of tissue architecture at single-cell resolution, while preserving the structural context.

Fully automated sequential immunofluorescence (seqIF) for hyperplex spatial proteomics.

Tissues are complex environments where different cell types are in constant interaction with each other and with non-cellular components. Preserving the spatial context during proteomics analyses of tissue samples has become an important objective for different applications, one of the most important being the investigation of the tumor microenvironment. Here, we describe a multiplexed protein biomarker detection method on the COMET instrument, coined sequential ImmunoFluorescence (seqIF).

Spatially multiplexed single-molecule translocations through a nanopore at controlled speeds.

In current nanopore-based label-free single-molecule sensing technologies, stochastic processes influence the selection of translocating molecule, translocation rate and translocation velocity. As a result, single-molecule translocations are challenging to control both spatially and temporally. Here we present a method using a glass nanopore mounted on a three-dimensional nanopositioner to spatially select molecules, deterministically tethered on a glass surface, for controlled translocations.

Engineering Optically Active Defects in Hexagonal Boron Nitride Using Focused Ion Beam and Water.

Hexagonal boron nitride (hBN) has emerged as a promising material platform for nanophotonics and quantum sensing, hosting optically active defects with exceptional properties such as high brightness and large spectral tuning. However, precise control over deterministic spatial positioning of emitters in hBN remained elusive for a long time, limiting their proper correlative characterization and applications in hybrid devices. Recently, focused ion beam (FIB) systems proved to be useful to engineer several types of spatially defined emitters with various structural and photophysical properties.

Time-Resolved Scanning Ion Conductance Microscopy for Three-Dimensional Tracking of Nanoscale Cell Surface Dynamics.

Nanocharacterization plays a vital role in understanding the complex nanoscale organization of cells and organelles. Understanding cellular function requires high-resolution information about how the cellular structures evolve over time. A number of techniques exist to resolve static nanoscale structure of cells in great detail (super-resolution optical microscopy, EM, AFM).

Bio-orthogonal Red and Far-Red Fluorogenic Probes for Wash-Free Live-Cell and Super-resolution Microscopy.

Small-molecule fluorophores enable the observation of biomolecules in their native context with fluorescence microscopy. Specific labeling via bio-orthogonal tetrazine chemistry combines minimal label size with rapid labeling kinetics. At the same time, fluorogenic tetrazine-dye conjugates exhibit efficient quenching of dyes prior to target binding.

Correlative 3D microscopy of single cells using super-resolution and scanning ion-conductance microscopy.

High-resolution live-cell imaging is necessary to study complex biological phenomena. Modern fluorescence microscopy methods are increasingly combined with complementary, label-free techniques to put the fluorescence information into the cellular context. The most common high-resolution imaging approaches used in combination with fluorescence imaging are electron microscopy and atomic-force microscopy (AFM), originally developed for solid-state material characterization.

High-Throughput Nanocapillary Filling Enabled by Microwave Radiation for Scanning Ion Conductance Microscopy Imaging.

Solid-state nanopores provide a highly sensitive tool for single-molecule sensing and probing nanofluidic effects in solutions. Glass nanopipettes are a cheap and robust type of solid-state nanopore produced from pulling glass capillaries with opening orifice diameters down to below tens of nanometers. Sub-50 nm nanocapillaries allow an unprecedented resolution for translocating single molecules or for scanning ion conductance microscopy imaging.

US, EU, and Japanese Regulatory Guidelines for Development of Drugs for Treatment of Alzheimer's Disease: Implications for Global Drug Development.

Drug development guidelines from regulatory authorities provide important information to sponsors on requirements for clinical evidence needed to support approval of new drugs. In the field of Alzheimer's disease (AD), recently published guidelines are available from EU, US, and Japanese regulatory authorities. In this review, these three guidelines are compared and discussed with emphasis on the recommendations provided for demonstration of efficacy in pivotal clinical trials conducted in predementia stages of AD.

Scanning Probe-Directed Assembly and Rapid Chemical Writing Using Nanoscopic Flow of Phospholipids.

Nanofluidic systems offer a huge potential for discovery of new molecular transport and chemical phenomena that can be employed for future technologies. Herein, we report on the transport behavior of surface-reactive compounds in a nanometer-scale flow of phospholipids from a scanning probe. We have investigated microscopic deposit formation on polycrystalline gold by lithographic printing and writing of 1,2-dioleoyl--glycero-3-phosphocholine and eicosanethiol mixtures, with the latter compound being a model case for self-assembled monolayers (SAMs).

Facile Production of Hexagonal Boron Nitride Nanoparticles by Cryogenic Exfoliation.

Fluorescent nanoparticles with optically robust luminescence are imperative to applications in imaging and labeling. Here we demonstrate that hexagonal boron nitride (hBN) nanoparticles can be reliably produced using a scalable cryogenic exfoliation technique with sizes below 10 nm. The particles exhibit bright fluorescence generated by color centers that act as atomic-size quantum emitters.

Fixed DNA Molecule Arrays for High-Throughput Single DNA-Protein Interaction Studies.

The DNA Curtains assay is a recently developed experimental platform for protein-DNA interaction studies at the single-molecule level that is based on anchoring and alignment of DNA fragments. The DNA Curtains so far have been made by using chromium barriers and fluid lipid bilayer membranes, which makes such a specialized assay technically challenging and relatively unstable. Herein, we report on an alternative strategy for DNA arraying for analysis of individual DNA-protein interactions.

Wide-Field Spectral Super-Resolution Mapping of Optically Active Defects in Hexagonal Boron Nitride.

Point defects can have significant impact on the mechanical, electronic, and optical properties of materials. The development of robust, multidimensional, high-throughput, and large-scale characterization techniques of defects is thus crucial for the establishment of integrated nanophotonic technologies and material growth optimization. Here, we demonstrate the potential of wide-field spectral single-molecule localization microscopy (SMLM) for the determination of ensemble spectral properties as well as the characterization of spatial, spectral, and temporal dynamics of single defects in chemical vapor deposition (CVD)-grown and irradiated exfoliated hexagonal boron-nitride materials.

Fluorescent Nanodiamonds as Versatile Intracellular Temperature Sensors.

Temperature is a widely known phenomenon, which plays an extremely important role in biological systems. Its behavior on the macro-scale has been quite well investigated and understood, thanks to the availability of reliable and precise thermometers such as thermocouples and infrared cameras. However, temperature measurements on the subcellular scale present an ongoing challenge due to the absence of universal nanoscale temperature sensors.

Suppression of EAE by oral tolerance is independent of endogenous IFN-beta whereas treatment with recombinant IFN-beta ameliorates EAE.

IFN-beta is anticipated to have an important function in mucosal tolerance, as it is one of the major cytokines produced by plasmacytoid dendritic cells, and has recently been suggested as central to the maintenance of mucosal homeostasis. Here, we have investigated whether oral tolerance is dependent on endogenous IFN-beta by feeding low-dose self-antigen myelin basic protein to IFN-beta(-/-) mice with subsequent induction of experimental autoimmune encephalomyelitis (EAE). Our study shows that oral tolerance was readily induced in IFN-beta(-/-) mice compared with their wild-type littermates (IFN-beta(+/+)).

Similar response in male and female B10.RIII mice in a murine model of allergic airway inflammation.

Background: Several reports have been published on the gender differences associated with allergies in mice.

Goal: In the present study we investigate the influence of gender on allergy response using a strain of mice, B10.RIII, which is commonly used in the collagen-induced arthritis murine model.

Local therapy with CpG motifs in a murine model of allergic airway inflammation in IFN-beta knock-out mice.

Background: CpG oligodeoxynucleotides (CpG-ODN) are capable of inducing high amounts of type I IFNs with many immunomodulatory properties. Furthermore, type-I IFNs have been proposed to play a key role in mediating effects of CpG-ODN. The precise role of IFN-beta in the immunomodulatory effects of CpG-ODN is not known.

IFN-beta gene deletion leads to augmented and chronic demyelinating experimental autoimmune encephalomyelitis.

Since the basic mechanisms behind the beneficial effects of IFN-beta in multiple sclerosis (MS) patients are still obscure, here we have investigated the effects of IFN-beta gene disruption on the commonly used animal model for MS, experimental autoimmune encephalomyelitis (EAE). We show that IFN-beta knockout (KO) mice are more susceptible to EAE than their wild-type (wt) littermates; they develop more severe and chronic neurological symptoms with more extensive CNS inflammation and demyelination. However, there was no discrepancy observed between wt and KO mice regarding the capacity of T cells to proliferate or produce IFN-gamma in response to recall Ag.

Upregulation of B7 molecules (CD80 and CD86) and exacerbated eosinophilic pulmonary inflammatory response in mice lacking the IFN-beta gene.

Background: IFN-beta has been shown to be effective as therapy for multiple sclerosis. Some reports attributed its beneficial effects to the capacity to induce a T(H)2 response. However, other studies have suggested that endogenous type I IFN might downregulate the allergic response in mice.

Influence of IFN-beta1b (Betaferon) on cytokine mRNA profiles in blood mononuclear cells and plasma levels of soluble VCAM-1 in multiple sclerosis.

Inflammatory cell infiltration within the central nervous system (CNS) and upregulation of both pro- and anti-inflammatory cytokines are characteristic for multiple sclerosis (MS). Treatment with interferon-beta 1b (IFN-beta1b) reduces the number and severity of MS relapses. To examine whether treatment with IFN-beta1b affects levels of cytokine mRNA expressing blood mononuclear cells (MNC) we employed in-situ hybridization with synthetic oligonucleotide probes to detect and enumerate IFN-gamma, TNF-alpha, IL-10, TGF-beta and perforin mRNA expressing cells in MS patients before treatment with IFN-beta1b and during treatment for 3-6 weeks and for 3-6 months.

Linomide (roquinimex) affects the balance between pro- and anti-inflammatory cytokines in vitro in multiple sclerosis.

Objectives: Multiple sclerosis (MS) is characterized by high levels of circulating mononuclear cells (MNC) that respond to myelin proteins like myelin basic protein (MBP) in vitro by expressing mRNA of both pro-inflammatory cytokines, e.g. interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and lymphotoxin (LT) that may make MS worse, and anti-inflammatory cytokines like IL-4, IL-10 and transforming growth factor-beta (TGF-beta) that may act beneficially.

The phosphodiesterase i.v. inhibitor rolipram in vitro reduces the numbers of MBP-reactive IFN-gamma and TNF-alpha mRNA expressing blood mononuclear cells in patients with multiple sclerosis.

The inflammatory nature of multiple sclerosis (MS) implicates the participation of cytokines as immune response mediators. Targeting the cytokine balance by downregulating proinflammatory cytokines and/or upregulating immunosuppressive cytokines could benefit patients with MS. This article reports on the in vitro effects of the phosphodiesterase i.

Kinetics of expression of costimulatory molecules and their ligands in murine relapsing experimental autoimmune encephalomyelitis in vivo.

We studied the kinetics of expression of costimulatory molecules and cytokines in the central nervous system (CNS) in murine relapsing experimental autoimmune encephalomyelitis (EAE). During the natural course of EAE, B7-2 expression in the CNS correlated with clinical signs, while B7-1 was exclusively expressed during remissions. Interestingly, B7-1 was expressed on infiltrating mononuclear cells as well as neuronal cells in the CNS.

Interleukin-12 and perforin mRNA expression is augmented in blood mononuclear cells in multiple sclerosis.

Cytokines are suggested to orchestrate an abnormal immune response in multiple sclerosis (MS). The regulatory cytokine interleukin (IL)-12 induces T-helper (Th) cell switch to the Th1 type and the production by cytotoxic T cells of perforin, a cell lysis-inducing factor. It has been suggested that Th1-like cytokines may promote the development of MS, and the production of perforin to induce oligodendrocyte damage.

Cytokines and the pathogenesis of myasthenia gravis.

Myasthenia gravis (MG) and its animal model experimental autoimmune myasthenia gravis (EAMG) are caused by autoantibodies against nicotinic acetylcholine receptor (AChR) in skeletal muscle. The production of anti-AChR antibodies is mediated by cytokines produced by CD4+ and CD8+ T helper (Th) cells. Emerging investigations of the roles of cytokines in MG and EAMG have revealed that the Th2 cell related cytokine interleukin 4 (IL-4), an efficient growth promoter for B-cell proliferation and differentiation, is important for anti-AChR antibody production.