Association between Disability,Cognition, Fatigue,EQ-5D-3L Domains,and Utilities Estimated with Different Western European Value Sets in Patients with Multiple Sclerosis

Objectives

To assess the association between fatigue, cognition, domains of the EuroQol five-dimensional questionnaire (EQ-5D-3L), disability, and utilities estimated with several Western European value sets in patients with multiple sclerosis (MS).

An audit on HIV antibody test uptake amongst GUM clinic attendees in the West Midland region

We audited the practice of offering, and the uptake of, an HIV antibody test amongst genitourinary medicine clinic patients in the West Midlands region. There were wide variations in the offering (from 14 to 100%) and uptake (18 to 64%) of the test in the different clinics within the same region.     

Mapping glycan-mediated galectin-3 interactions by live cell proximity labeling

Galectin-3 is a glycan-binding protein (GBP) that binds β-galactoside glycan structures to orchestrate a variety of important biological events, including the activation of hepatic stellate cells and regulation of immune responses. While the requisite glycan epitopes needed to bind galectin-3 have long been elucidated, the cellular glycoproteins that bear these glycan signatures remain unknown. Given the importance of the three-dimensional (3D) arrangement of glycans in dictating GBP interactions, strategies that allow the identification of GBP receptors in live cells, where the native glycan presentation and glycoprotein expression are preserved, have significant advantages over static and artificial systems. Here we describe the integration of a proximity labeling method and quantitative mass spectrometry to map the glycan and glycoprotein interactors for galectin-3 in live human hepatic stellate cells and peripheral blood mononuclear cells. Understanding the identity of the glycoproteins and defining the structures of the glycans will empower efforts to design and develop selective therapeutics to mitigate galectin-3–mediated biological events.

The noncovalent interactions between glycan-binding proteins (GBPs) and glycans dictate many important biological events. Among such GBPs is galectin-3, a 26-kDa β-galactoside GBP that plays key roles in many physiological and pathological events (1). In hepatic fibrosis, a disease that manifests as the excessive buildup of scar tissue, liver-resident macrophages secrete galectin-3 (2, 3), which then binds cell surface glycans on quiescent hepatic stellate cells (HSCs), activating them to transdifferentiate into a muscle-like phenotype. Galectin-3–null mice exhibit attenuated liver fibrosis even after induced injury, highlighting its critical role (3). Galectin-3 is also known to interact with cells of the innate immune system (4, 5) to regulate apoptosis (6) or control dendritic cell differentiation (7). In these cases, as well as in other cases in which galectin-3 is involved, the full complement of interacting glycoprotein receptors remains unknown.Despite significant advances in glycoscience, the study of GBP–glycan interactions and the identification of glycan-mediated counter-receptors remains a recurring challenge. Capturing these binding events often requires some form of artificial reconstitution to amplify individually weak interactions into high-avidity binding. Indeed, glycan microarrays with defined mixtures of homogenous glycans or recombinant GBPs have significantly propelled our understanding of glycan-mediated function (8). Conventional immunoprecipitation and lectin affinity techniques using cell lysates have similarly been used to reveal an initial catalog of 100 to 185 galectin-3–associated proteins (9–14). However, these manipulations alter the cell’s native and three-dimensional (3D) configuration and multivalent arrangement, both of which are critically important in the study of GBP–glycan interactions (15, 16).Another key issue involves the underlying glycoprotein ligand. Although many glycoproteins carry the glycan epitope for binding a GBP, only a limited set should be recognized as physiologically relevant receptors, owing to the physical constraints imposed by the living cell (17). While often overlooked, the glycoprotein carrying the glycan can impart specific biological functions to a GBP–glycan binding event (17). Recent work has put forth the concept of “professional glycoprotein ligands,” in which a specific set of glycoproteins (instead of a broadly defined glycome) can exhibit exquisite binding and functional roles (18). Thus, determining the identity of the underlying core protein that anchors the glycan can be greatly empowering. Not only can it provide an understanding of the 3D arrangement of the glycan (if the 3D structure of the core protein is known), but it can also provide additional insight into its expression levels in different cell types and tissues, further informing strategies for selective drug development.Thus, comprehensive approaches that permit the study of GBP–glycan interactions in live cells while simultaneously facilitating identification of the physiological glycoprotein receptors have great potential to impact glycoscience. We hypothesize that proximity labeling strategies (19) using an engineered ascorbate peroxidase, APEX2 (20), could be compatible for elucidating glycan-mediated GBP–glycoprotein interactions. In this approach (Fig. 1), APEX2 is fused to a protein of interest, followed by the treatment of cells with biotin-phenol and subsequently with hydrogen peroxide (H₂O₂). Under these conditions, APEX2 catalyzes the formation of highly reactive, short-lived (<1 ms), and proximally restricted (<20 nm) biotin-phenoxyl radicals that covalently tag nearby electron-rich residues. The biotinylated proteins can then be enriched and identified using quantitative mass spectrometry (MS)-based proteomics. Because the (glyco)proteins adjacent to the APEX2 fusion protein are preferentially biotinylated, the resulting MS data provide a readout of its immediate environment.Open in a separate windowFig. 1.Schematic illustration of the identification of galectin-3 (Gal-3) interacting proteins by in situ proximity labeling. Recombinant APEX2 and galectin-3 fusion proteins are applied to living cells where galectin-3 can freely diffuse to bind its cognate ligands. On addition of biotin phenol (yellow circle with “B”; 30 min) and hydrogen peroxide (H₂O₂; 1 min), APEX2 catalyzes the formation of highly-reactive biotin-phenoxyl radicals that react within a short range (<20 nm) of the galectin-3 complex within a short time frame (<1 ms). The biotin-tagged protein interactors can then be identified using MS-based proteomics.We reasoned that proximity labeling could offer significant advantages over other approaches to determining GBP–glycan interactions, including the opportunity to perform the labeling in live cells and the ability to tag weakly bound glycan-mediated interactors, as the covalent biotinylation reaction ensures that the enrichment step no longer relies on weak GBP–glycan interactions alone. When coupled with inhibitors, the proximity labeling strategy can also distinguish between glycan-mediated and non–glycan-mediated interactors. Integration of this approach with quantitative MS-based proteomics would also expedite the assignment of the interacting proteins and provide calculable measures to distinguish interactors from nonspecific binders.Here we report that the use of an APEX2 and galectin-3 fusion protein (PX-Gal3) provides a sensitive and comprehensive approach to mapping the proteome-wide glycan-mediated galectin-3 interactome in live human HSCs and peripheral blood mononuclear cells (PBMCs). We found that the exogenous incubation of cells with PX-Gal3 in HSCs leads to glycan-dependent interactions, whereas its cellular overexpression does not. We further validated the interactions between galectin-3 and candidate proteins in vitro and discovered that some proteins are direct glycan-mediated receptors. Using MS-based glycomics, we also examined the glycomes of HSC surfaces, PX-Gal3 tagged glycoproteins, and an individual glycoprotein receptor for galectin-3. Our results highlight the utility of the in situ proximity labeling approach in discovering physiologically relevant GBP interactors in living cells.     

In vitro generation of HIV type 1 subtype C isolates resistant to enfuvirtide

Enfuvirtide (ENF) is the first in a new class of antiretroviral agents targeting the fusion process of the viral life cycle. ENF is a synthetic 36-amino acid peptide that binds to the HR-1 region of gp41 preventing fusion of viral and cellular membranes. With the introduction of ENF there are now four classes of antiretrovirals each with distinct and different resistance pathways. Resistance to ENF among subtype B HIV-1 isolates is associated with amino acid changes mainly in the HR-1 region, although other regions of envelope have also been implicated. To determine whether subtype C viruses developed resistance mutations similar to subtype B viruses, 11 subtype C and 4 subtype B viruses were passaged in the presence of increasing concentrations of ENF. The subtype C isolates showed varying levels of replication at 1 microg/ml ENF by day 18, but by day 29 all replicated efficiently at 10 microg/ml ENF. All subtype C isolates showed evidence of genotypic changes in gp41 HR-1 following exposure to ENF that included G36S/E/D, I37T, V38M/A/L/E, N/S42D, N43K, L45R/M, and A50T/V. Three subtype C viruses had compensatory changes in the HR-2 region, which corresponds to the ENF sequence, and two isolates had changes in the V3 region. Mutational patterns among the four subtype B viruses were similar to those for subtype C and those previously published in the literature. These data indicate that in vitro resistance to ENF develops rapidly among HIV-1 subtype C isolates. In general, mutational patterns for subtype C were similar to those described for subtype B, suggesting that the mechanism of action for ENF is similar for HIV-1 subtype B and C isolates.     

Resting-State Functional Connectivity of Antero-Medial Prefrontal Cortex Sub-Regions in Major Depression and Relationship to Emotional Intelligence

Background:

Major depressive disorder has been associated with abnormal resting-state functional connectivity (FC), especially in cognitive processing and emotional regulation networks. Although studies have found abnormal FC in regions of the default mode network (DMN), no study has investigated the FC of specific regions within the anterior DMN based on cytoarchitectonic subdivisions of the antero-medial pre-frontal cortex (PFC). Studies from different areas in the field have shown regions within the anterior DMN to be involved in emotional intelligence. Although abnormalities in this region have been observed in depression, the relationship between the ventromedial PFC (vmPFC) function and emotional intelligence has yet to be investigated in depressed individuals.

Methods:

Twenty-one medication-free, non–treatment resistant, depressed patients and 21 healthy controls underwent a resting state functional magnetic resonance imaging session. The participants also completed an ability-based measure of emotional intelligence: the Mayer-Salovey-Caruso Emotional Intelligence Test. FC maps of Brodmann areas (BA) 25, 10m, 10r, and 10p were created and compared between the two groups.

Results:

Mixed-effects analyses showed that the more anterior seeds encompassed larger areas of the DMN. Compared to healthy controls, depressed patients had significantly lower connectivity between BA10p and the right insula and between BA25 and the perigenual anterior cingulate cortex. Exploratory analyses showed an association between vmPFC connectivity and emotional intelligence.

Conclusions:

These results suggest that individuals with depression have reduced FC between antero-medial PFC regions and regions involved in emotional regulation compared to control subjects. Moreover, vmPFC functional connectivity appears linked to emotional intelligence.