Single-molecule dynamic structural biology with vertically arranged DNA on a fluorescence microscope.

The intricate interplay between DNA and proteins is key for biological functions such as DNA replication, transcription and repair. Dynamic nanoscale observations of DNA structural features are necessary for understanding these interactions. Here we introduce graphene energy transfer with vertical nucleic acids (GETvNA), a method to investigate DNA-protein interactions that exploits the vertical orientation adopted by double-stranded DNA on graphene.

Immortalized Human Conjunctival Epithelial Cells Produce Functional Complement C3 and C4 Proteins.

Purpose: The aim of this study was to assess whether complement proteins C3 and C4 are produced by immortalized human conjunctival epithelial (HCjE) cells.

Methods: Supernatants and cell lysates from undifferentiated and differentiated HCjE cells were assayed for C3 and C4 by enzyme-linked immunosorbent assay. To measure complement protein function, supernatants and lysates were treated with heat-aggregated IgG, and soluble C5b-9 was measured.

Exploring the Synergies of Single-Molecule Fluorescence and 2D Materials Coupled by DNA.

The world of 2D materials is steadily growing, with numerous researchers attempting to discover, elucidate, and exploit their properties. Approaches relying on the detection of single fluorescent molecules offer a set of advantages, for instance, high sensitivity and specificity, that allow the drawing of conclusions with unprecedented precision. Herein, it is argued how the study of 2D materials benefits from fluorescence-based single-molecule modalities, and vice versa.

Irritable bowel syndrome-like symptoms before and after bariatric surgery and association with short-chain fermentable carbohydrates consumption: an observational prospective study.

Background And Aims: Irritable Bowel Syndrome (IBS)-like symptoms are frequent following bariatric surgery. This study aims to evaluate the frequency of IBS symptoms severity before and after bariatric surgery and their association with short-chain fermentable carbohydrates (FODMAPs) consumption.

Patients And Methods: IBS symptoms severity in a cohort of obese patients was evaluated prospectively before, 6 and 12 months after bariatric surgery by validated questionnaires and tools (Irritable Bowel Syndrome Severity Scoring System (IBS SSS), Bristol Stool Scale (BSS), Quality of Life Short- Form-12 (SF-12), Hospital Anxiety and Depression scale (HAD)).

Clustering Patients With Gout Based on Comorbidities and Biomarkers: A Cross-Sectional Study.

Objective: This single-center clinical study identifies clusters of different phenotypes and pathophysiology subtypes of patients with gout and associated comorbidities.

Methods: Patients clinically diagnosed with gout were enrolled between January 2018 and December 2019. Hierarchical cluster analyses were performed using clinical data or biological markers, inflammatory markers, and oxidative stress pathway metabolites assayed from serum and plasma samples.

An alternative to MINFLUX that enables nanometer resolution in a confocal microscope.

Localization of single fluorescent emitters is key for physicochemical and biophysical measurements at the nanoscale and beyond ensemble averaging. Examples include single-molecule tracking and super-resolution imaging by single-molecule localization microscopy. Among the numerous localization methods available, MINFLUX outstands for achieving a ~10-fold improvement in resolution over wide-field camera-based approaches, reaching the molecular scale at moderate photon counts.

A Central Role for TRPM4 in Ca-Signal Amplification and Vasoconstriction.

Transient receptor potential melastatin-4 (TRPM4) is activated by an increase in intracellular Ca concentration and is expressed on smooth muscle cells (SMCs). It is implicated in the myogenic constriction of cerebral arteries. We hypothesized that TRPM4 has a general role in intracellular Ca signal amplification in a wide range of blood vessels.

Feasibility of a low FODMAPs diet without initial dietician intervention in the management of patients with irritable bowel syndrome: a prospective study.

Background And Aims: Efficiency of a low FODMAPs diet (LFD) to relieve symptoms in patients with irritable bowel syndrome (IBS) has been proved in several studies. Our study aimed to evaluate the management of IBS-patients when explanations about LFD are given by the physician without dietician intervention.

Patients And Methods: Patients with IBS were evaluated prospectively after explanations about the LFD were given with the help of a leaflet.

Super-resolution FRET measurements.

Super-resolution fluorescence microscopy and Förster Resonance Energy Transfer (FRET) form a well-established family of techniques that has provided unique tools to study the dynamic architecture and functionality of biological systems, as well as to investigate nanomaterials. In the last years, the integration of super-resolution methods with FRET measurements has generated advances in two fronts. On the one hand, FRET-based probes have enhanced super-resolution imaging.

Decreased Levels of STAT1 and Interferon-γ-Induced STAT1 Phosphorylation in Rheumatoid Arthritis CD4 and CD8 T Cells.

Objective: We investigated whether a previously reported association of IFNGR expression with rheumatoid arthritis (RA) and its radiographic severity reflects differences in proximal interferon-γ (IFN-γ) signaling in T cells from patients with RA compared with healthy controls (HC).

Methods: Using phosphoflow cytometry, we compared IFN-γ-stimulated signal transducer and activator of transcription 1 (STAT1) activation in CD4 and CD8 T-cell populations from patients with RA and HC.

Results: Compared with controls, patients with RA had a higher proportion of CD4 T cells, associated with expansion of the CD4 effector memory subset.

Therapeutic Lowering of C-Reactive Protein.

In the blood of healthy individuals C-reactive protein (CRP) is typically quite scarce, whereas its blood concentration can rise robustly and rapidly in response to tissue damage and inflammation associated with trauma and infectious and non-infectious diseases. Consequently, CRP plasma or serum levels are routinely monitored in inpatients to gauge the severity of their initial illness and injury and their subsequent response to therapy and return to health. Its clinical utility as a faithful barometer of inflammation notwithstanding, it is often wrongly concluded that the biological actions of CRP (whatever they may be) are manifested only when blood CRP is elevated.

Super-resolution Imaging of Energy Transfer by Intensity-Based STED-FRET.

Förster resonance energy transfer (FRET) imaging methods provide unique insight into the spatial distribution of energy transfer and (bio)molecular interaction events, though they deliver average information for an ensemble of events included in a diffraction-limited volume. Coupling super-resolution fluorescence microscopy and FRET has been a challenging and elusive task. Here, we present STED-FRET, a method of general applicability to obtain super-resolved energy transfer images.

Three-dimensional total-internal reflection fluorescence nanoscopy with nanometric axial resolution by photometric localization of single molecules.

Single-molecule localization microscopy enables far-field imaging with lateral resolution in the range of 10 to 20 nanometres, exploiting the fact that the centre position of a single-molecule's image can be determined with much higher accuracy than the size of that image itself. However, attaining the same level of resolution in the axial (third) dimension remains challenging. Here, we present Supercritical Illumination Microscopy Photometric z-Localization with Enhanced Resolution (SIMPLER), a photometric method to decode the axial position of single molecules in a total internal reflection fluorescence microscope.

Analysis of sparse molecular distributions in fibrous arrangements based on the distance to the first neighbor in single molecule localization microscopy.

Single Molecule Localization Microscopy (SMLM) currently attains a lateral resolution of around 10 nm approaching molecular size. Together with increasingly specific fluorescent labeling, it opens the possibility to quantitatively analyze molecular organization. When the labeling density is high enough, SMLM provides clear images of the molecular organization.

Quantitative expansion microscopy for the characterization of the spectrin periodic skeleton of axons using fluorescence microscopy.

Fluorescent nanoscopy approaches have been used to characterize the periodic organization of actin, spectrin and associated proteins in neuronal axons and dendrites. This membrane-associated periodic skeleton (MPS) is conserved across animals, suggesting it is a fundamental component of neuronal extensions. The nanoscale architecture of the arrangement (190 nm) is below the resolution limit of conventional fluorescent microscopy.

Mice expressing the variant rs1143679 allele of ITGAM (CD11b) show impaired DC-mediated T cell proliferation.

Genome-wide association studies (GWAS) and functional genomic analyses have implicated several ITGAM (CD11b) single-nucleotide polymorphisms (SNPs) in the development of SLE and other disorders. ITGAM encodes the α chain of the β integrin Mac-1, a receptor that plays important roles in myeloid cell functions. The ITGAM SNP rs1143679, which results in an arginine to histidine change at amino acid position 77 of the CD11b protein, has been shown to reduce binding to several ligands and to alter Mac-1-mediated cellular response in vitro.

C-Reactive Protein Promotes the Expansion of Myeloid Derived Cells With Suppressor Functions.

Previously we established that human C-reactive protein (CRP) exacerbates mouse acute kidney injury and that the effect was associated with heightened renal accumulation of myeloid derived cells with suppressor functions (MDSC). Herein we provide direct evidence that CRP modulates the development and suppressive actions of MDSCs . We demonstrate that CRP dose-dependently increases the generation of MDSC from wild type mouse bone marrow progenitors and enhances MDSC production of intracellular reactive oxygen species (iROS).

Asymmetric bifunctional protein nanoparticles through redesign of self-assembly.

Engineering oligomeric protein self-assembly is an attractive approach to fabricate nanostructures with well-defined geometries, stoichiometry and functions. The homodecamer Lumazine Synthase (BLS) is a highly stable and immunogenic protein nanoparticle (PNP). Here, we engineered the BLS protein scaffold to display two functions in spatially opposite regions of its structure yielding a Janus-like nanoparticle.

A fluorescence nanoscopy marker for corticotropin-releasing hormone type 1 receptor: computer design, synthesis, signaling effects, super-resolved fluorescence imaging, and in situ affinity constant in cells.

Class B G protein-coupled receptors (GPCRs) are involved in a variety of human pathophysiological states. These groups of membrane receptors are less studied than class A GPCRs due to the lack of structural information, delayed small molecule drug discovery, and scarce fluorescence detection tools available. The class B corticotropin-releasing hormone type 1 receptor (CRHR1) is a key player in the stress response whose dysregulation is critically involved in stress-related disorders: psychiatric conditions (i.

C-Reactive Protein Impairs Dendritic Cell Development, Maturation, and Function: Implications for Peripheral Tolerance.

C-reactive protein (CRP) is the prototypical acute phase reactant, increasing in blood concentration rapidly and several-fold in response to inflammation. Recent evidence indicates that CRP has an important physiological role even at low, baseline levels, or in the absence of overt inflammation. For example, we have shown that human CRP inhibits the progression of experimental autoimmune encephalomyelitis (EAE) in CRP transgenic mice by shifting CD4 T cells away from the T1 and toward the T2 subset.

Hyposialylated IgG activates endothelial IgG receptor FcγRIIB to promote obesity-induced insulin resistance.

Type 2 diabetes mellitus (T2DM) is a common complication of obesity. Here, we have shown that activation of the IgG receptor FcγRIIB in endothelium by hyposialylated IgG plays an important role in obesity-induced insulin resistance. Despite becoming obese on a high-fat diet (HFD), mice lacking FcγRIIB globally or selectively in endothelium were protected from insulin resistance as a result of the preservation of insulin delivery to skeletal muscle and resulting maintenance of muscle glucose disposal.