GPR41 is reportedly expressed in murine adipose tissue and mediates short chain fatty acid (SCFA)-stimulated leptin secretion by activating Galpha(i). Here, we agree with a contradictory report in finding no expression of GPR41 in murine adipose tissue. Nevertheless, in the presence of adenosine deaminase to minimise Galpha(i) signalling via the adenosine A1 receptor, SCFA stimulated leptin secretion by adipocytes from wild-type but not GPR41 knockout mice. Expression of GPR43 was reduced in GPR41 knockout mice. Acetate but not butyrate stimulated leptin secretion in wild-type mesenteric adipocytes, consistent with mediation of the response by GPR43 rather than GPR41. Pertussis toxin prevented stimulation of leptin secretion by propionate in epididymal adipocytes, implicating Galpha(i) signalling mediated by GPR43 in SCFA-stimulated leptin secretion.

The NEFA-responsive G-protein coupled receptor 120 (GPR120) has been implicated in the regulation of inflammation, in the control of incretin secretion and as a predisposing factor influencing the development of type 2 diabetes by regulation of islet cell apoptosis. However, there is still considerable controversy about the tissue distribution of GPR120 and, in particular, it remains unclear which islet cell types express this molecule. In the present study, we have addressed this issue by constructing a Gpr120-knockout/β-galactosidase (LacZ) knock-in (KO/KI) mouse to examine the distribution and functional role of GPR120 in the endocrine pancreas.

Hormone-secreting cells within pancreatic islets of Langerhans play important roles in metabolic homeostasis and disease. However, their transcriptional characterization is still incomplete. Here, we sequenced the transcriptomes of thousands of human islet cells from healthy and type 2 diabetic donors. We could define specific genetic programs for each individual endocrine and exocrine cell type, even for rare δ, γ, ε, and stellate cells, and revealed subpopulations of α, β, and acinar cells. Intriguingly, δ cells expressed several important receptors, indicating an unrecognized importance of these cells in integrating paracrine and systemic metabolic signals. Genes previously associated with obesity or diabetes were found to correlate with BMI. Finally, comparing healthy and T2D transcriptomes in a cell-type resolved manner uncovered candidates for future functional studies. Altogether, our analyses demonstrate the utility of the generated single-cell gene expression resource.

Co-infection with HIV and P. falciparum worsens the prognosis of both infections; however, the mechanisms driving this adverse interaction are not fully delineated. To evaluate this, we studied HIV-1 and P. falciparum interactions in vitro using peripheral blood mononuclear cells (PBMCs) from human malaria naïve volunteers experimentally infected with P. falciparum in a malaria challenge trial. PBMCs collected before the malaria challenge and at several time points post-infection were infected with HIV-1 and co-cultured with either P. falciparum infected (iRBCs) or uninfected (uRBCs) red blood cells. HIV p24Ag and TNF-α, IFN-γ, IL-4, IL-6, IL-10, IL-17, and MIP-1α were quantified in the co-culture supernatants. In general, iRBCs stimulated more HIV p24Ag production by PBMCs than did uRBCs. HIV p24Ag production by PBMCs in the presence of iRBCs (but not uRBCs) further increased during convalescence (days 35, 56, and 90 post-challenge). In parallel, iRBCs induced higher secretion of pro-inflammatory cytokines (TNF-α, IFN-γ, and MIP-1α) than uRBCs, and production increased further during convalescence. Because the increase in p24Ag production occurred after parasitemia and generalized immune activation had resolved, our results suggest that enhanced HIV production is related to the development of anti-malaria immunity and may be mediated by pro-inflammatory cytokines.

Memory retrieval requires coordinated intra- and inter-regional activity in networks of brain structures. Dysfunction of these networks and memory impairment are seen in many psychiatric disorders, but relatively little is known about how memory retrieval and memory failure are represented at the level of local and regional oscillatory activity. To address this question, we measured local field potentials (LFPs) from mice as they explored a novel context, retrieved memories for contextual fear conditioning, and after administration of two amnestic agents: the NMDA receptor antagonist MK-801 and muscarinic acetylcholine receptor antagonist scopolamine (SCOP). LFPs were simultaneously recorded from retrosplenial cortex (RSC), dorsal hippocampus (DH), and anterior cingulate cortex (ACC), which are involved in processing contextual memories, and analyzed for changes in intra-regional power and inter-regional peak coherence of oscillations across multiple frequency bands. Context encoding and memory retrieval sessions yielded similar patterns of changes across all three structures, including decreased delta power and increased theta peak coherence. Baseline effects of MK-801 and SCOP were primarily targeted to gamma oscillations, but in opposite directions. Both drugs also blocked memory retrieval, as indicated by reduced freezing when mice were returned to the conditioning context, but this common behavioral impairment was only associated with power and peak coherence disruptions after MK-801 treatment. These findings point to neural signatures for memory impairment, whose underlying mechanisms may serve as therapeutic targets for related psychiatric disorders.

Protein accumulation and aggregation with a concomitant loss of proteostasis often contribute to neurodegenerative diseases, and the ubiquitin-proteasome system plays a major role in protein degradation and proteostasis. Here, we show that three different proteins from Alzheimer's, Parkinson's, and Huntington's disease that misfold and oligomerize into a shared three-dimensional structure potently impair the proteasome. This study indicates that the shared conformation allows these oligomers to bind and inhibit the proteasome with low nanomolar affinity, impairing ubiquitin-dependent and ubiquitin-independent proteasome function in brain lysates. Detailed mechanistic analysis demonstrates that these oligomers inhibit the 20S proteasome through allosteric impairment of the substrate gate in the 20S core particle, preventing the 19S regulatory particle from injecting substrates into the degradation chamber. These results provide a novel molecular model for oligomer-driven impairment of proteasome function that is relevant to a variety of neurodegenerative diseases, irrespective of the specific misfolded protein that is involved.

The aim of the study was to determine the effects of exercise training on improving the thoracic perivascular adipose tissue (tPVAT) phenotype (inflammation, oxidative stress, and proteasome function) in metabolic syndrome and its subsequent actions on aortic function.

Endothelial barrier (EB) breaching is a frequent event during inflammation, and it is followed by the rapid recovery of microvascular integrity. The molecular mechanisms of EB recovery are poorly understood. Triggering of MHC molecules by migrating T-cells is a minimal signal capable of inducing endothelial contraction and transient microvascular leakage. Using this model, we show that EB recovery requires a CD31 receptor-induced, robust glycolytic response sustaining junction re-annealing. Mechanistically, this response involves src-homology phosphatase activation leading to Akt-mediated nuclear exclusion of FoxO1 and concomitant β-catenin translocation to the nucleus, collectively leading to cMyc transcription. CD31 signals also sustain mitochondrial respiration, however this pathway does not contribute to junction remodeling. We further show that pathologic microvascular leakage in CD31-deficient mice can be corrected by enhancing the glycolytic flux via pharmacological Akt or AMPK activation, thus providing a molecular platform for the therapeutic control of EB response.

Regulated necrosis or necroptosis, mediated by receptor-interacting kinase 1 (RIPK1), RIPK3 and pseudokinase mixed lineage kinase domain-like protein (MLKL), contributes to the pathogenesis of inflammatory, infectious and degenerative diseases. Recently identified necroptosis inhibitors display moderate specificity, suboptimal pharmacokinetics, off-target effects and toxicity, preventing these molecules from reaching the clinic. Here, we developed a cell-based high-throughput screening (HTS) cascade for the identification of small-molecule inhibitors of necroptosis. From the initial library of over 250,000 compounds, the primary screening phase identified 356 compounds that strongly inhibited TNF-α-induced necroptosis, but not apoptosis, in human and murine cell systems, with EC50 < 6.7 μM. From these, 251 compounds were tested for RIPK1 and/or RIPK3 kinase inhibitory activity; some were active and several have novel mechanisms of action. Based on specific chemical descriptors, 110 compounds proceeded into the secondary screening cascade, which then identified seven compounds with maximum ability to reduce MLKL activation, IC50 >100 μM, EC50 2.5-11.5 μM under long-term necroptosis execution in murine fibroblast L929 cells, and full protection from ATP depletion and membrane leakage in human and murine cells. As a proof of concept, compound SN-6109, with binding mode to RIPK1 similar to that of necrostatin-1, confirmed RIPK1 inhibitory activity and appropriate pharmacokinetic properties. SN-6109 was further tested in mice, showing efficacy against TNF-α-induced systemic inflammatory response syndrome. In conclusion, a phenotypic-driven HTS cascade promptly identified robust necroptosis inhibitors with in vivo activity, currently undergoing further medicinal chemistry optimization. Notably, the novel hits highlight the opportunity to identify new molecular mechanisms of action in necroptosis.

We investigate the spatial, temporal, and spectral properties of 10 microflares from AR12721 on 2018 September 9 and 10 observed in X-rays using the Nuclear Spectroscopic Telescope ARray and the Solar Dynamic Observatory's Atmospheric Imaging Assembly and Helioseismic and Magnetic Imager. We find GOES sub-A class equivalent microflare energies of 1026-1028 erg reaching temperatures up to 10 MK with consistent quiescent or hot active region (AR) core plasma temperatures of 3-4 MK. One microflare (SOL2018-09-09T10:33), with an equivalent GOES class of A0.1, has non-thermal hard X-ray emission during its impulsive phase (of non-thermal power ~7 × 1024 erg s-1) making it one of the faintest X-ray microflares to have direct evidence for accelerated electrons. In 4 of the 10 microflares, we find that the X-ray time profile matches fainter and more transient sources in the extreme-ultraviolet, highlighting the need for observations sensitive to only the hottest material that reaches temperatures higher than those of the AR core (>5 MK). Evidence for corresponding photospheric magnetic flux cancellation/emergence present at the footpoints of eight microflares is also observed.

Developmental morphogenesis and tumor progression require a transient or stable breakdown of epithelial junctional complexes to permit programmed migration, invasion, and anoikis resistance, characteristics endowed by the epithelial-mesenchymal transition (EMT). The epithelial master-regulatory transcription factor Grainyhead-like 2 (GRHL2) suppresses and reverses EMT, causing a mesenchymal-epithelial transition to the default epithelial phenotype. Here we investigated the role of GRHL2 in tubulogenesis of Madin-Darby canine kidney cells, a process requiring transient, partial EMT. GRHL2 was required for cystogenesis, but it suppressed tubulogenesis in response to hepatocyte growth factor. Surprisingly, GRHL2 suppressed this process by inhibiting the histone acetyltransferase coactivator p300, preventing the induction of matrix metalloproteases and other p300-dependent genes required for tubulogenesis. A 13-amino acid region of GRHL2 was necessary for inhibition of p300, suppression of tubulogenesis, and interference with EMT. The results demonstrate that p300 is required for partial or complete EMT occurring in tubulogenesis or tumor progression and that GRHL2 suppresses EMT in both contexts through inhibition of p300.

Migration of activated regulatory T (Treg) cells to inflamed tissue is crucial for their immune-modulatory function. While metabolic reprogramming during Treg cell differentiation has been extensively studied, the bioenergetics of Treg cell trafficking remains undefined. We have investigated the metabolic demands of migrating Treg cells in vitro and in vivo. We show that glycolysis was instrumental for their migration and was initiated by pro-migratory stimuli via a PI3K-mTORC2-mediated pathway culminating in induction of the enzyme glucokinase (GCK). Subsequently, GCK promoted cytoskeletal rearrangements by associating with actin. Treg cells lacking this pathway were functionally suppressive but failed to migrate to skin allografts and inhibit rejection. Similarly, human carriers of a loss-of-function GCK regulatory protein gene-leading to increased GCK activity-had reduced numbers of circulating Treg cells. These cells displayed enhanced migratory activity but similar suppressive function, while conventional T cells were unaffected. Thus, GCK-dependent glycolysis regulates Treg cell migration.

The ubiquitin-proteasome system catalyzes the degradation of intracellular proteins. Although ubiquitination of proteins determines their stabilities, there is growing evidence that proteasome function is also regulated. We report the functional characterization of a conserved proteasomal regulatory complex. We identified DmPI31 as a binding partner of the F box protein Nutcracker, a component of an SCF ubiquitin ligase (E3) required for caspase activation during sperm differentiation in Drosophila. DmPI31 binds Nutcracker via a conserved mechanism that is also used by mammalian FBXO7 and PI31. Nutcracker promotes DmPI31 stability, which is necessary for caspase activation, proteasome function, and sperm differentiation. DmPI31 can activate 26S proteasomes in vitro, and increasing DmPI31 levels suppresses defects caused by diminished proteasome activity in vivo. Furthermore, loss of DmPI31 function causes lethality, cell-cycle abnormalities, and defects in protein degradation, demonstrating that DmPI31 is physiologically required for normal proteasome activity.

Binding interactions of the spike proteins of the severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) to a peptide fragment derived from the human angiotensin converting enzyme 2 (hACE2) receptor are investigated. The peptide is employed as capture moiety in enzyme linked immunosorbent assays (ELISA) and quantitative binding interaction measurements that are based on fluorescence proximity sensing (switchSENSE). In both techniques, the peptide is presented on an oligovalent DNA nanostructure, in order to assess the impact of mono- versus trivalent binding modes. As the analyte, the spike protein and several of its subunits are tested as well as inactivated SARS-CoV-2 and pseudo viruses. While binding of the peptide to the full-length spike protein can be observed, the subunits RBD and S1 do not exhibit binding in the employed concentrations. Variations of the amino acid sequence of the recombinant full-length spike proteins furthermore influence binding behavior. The peptide was coupled to DNA nanostructures that form a geometric complement to the trimeric structure of the spike protein binding sites. An increase in binding strength for trimeric peptide presentation compared to single peptide presentation could be generally observed in ELISA and was quantified in switchSENSE measurements. Binding to inactivated wild type viruses could be shown as well as qualitatively different binding behavior of the Alpha and Beta variants compared to the wild type virus strain in pseudo virus models.

Inflammatory signals and increased prostaglandin synthesis play a role during the development of diabetes. The prostaglandin D2 (PGD2) receptor, GPR44/DP2, is highly expressed in human islets and activation of the pathway results in impaired insulin secretion. The role of GPR44 activation on islet function and survival rate during chronic hyperglycaemic conditions is not known. In this study, we investigate GPR44 inhibition by using a selective GPR44 antagonist (AZ8154) in human islets both in vitro and in vivo in diabetic mice transplanted with human islets.

Accurate molecular modeling of the physical and chemical behavior of highly cross-linked epoxy resins at the atomistic scale is important for the design of new property-optimized materials. However, a systematic approach to parametrizing and characterizing these systems in molecular dynamics is missing. We therefore present a unified scheme to derive atomic charges for amine-based epoxy resins, in agreement with the AMBER force field, based on defining reactive fragments─blocks─building the network. The approach is applicable to all stages of curing from pure liquid to gelation to fully cured glass. We utilize this approach to study DGEBA/DDS epoxy systems, incorporating dynamic topology changes into atomistic molecular dynamics simulations of the curing reaction with 127,000 atoms. We study size effects in our simulations and predict the gel point utilizing a rigorous percolation theory to recover accurately the experimental data. Furthermore, we observe excellent agreement between the estimated and the experimentally determined glass transition temperatures as a function of curing rate. Finally, we demonstrate the quality of our model by the prediction of the elastic modulus based on uniaxial tensile tests. The presented scheme paves the way for a broadly consistent approach for modeling and characterizing all amine-based epoxy resins.

The Glucokinase Regulatory Protein GKRP, encoded by GCKR, enables acute regulation of liver glucokinase to support metabolic demand. The common human GCKR rs1260326:Pro446 > Leu variant within a large linkage disequilibrium region associates with pleiotropic traits including lower Type 2 diabetes risk and raised blood triglycerides and cholesterol. Whether the GCKR-P446 > L substitution is causal to the raised lipids is unknown. We determined whether mouse GKRP phenocopies the human GKRP:P446 > L substitution and studied a GKRP:P446L knockin mouse to identify physiological consequences to P446 > L.

Protein degradation in the 20S proteasome is regulated in eukaryotes by the 19S ATPase complex and in archaea by the homologous PAN ATPase ring complex. Subunits of these hexameric ATPases contain on their C-termini a conserved hydrophobic-tyrosine-X (HbYX) motif that docks into pockets in the 20S to stimulate the opening of a gated substrate entry channel. Here, we report the crystal structure of the archaeal 20S proteasome in complex with the C-terminus of the archaeal proteasome regulatory ATPase, PAN. This structure defines the detailed interactions between the critical C-terminal HbYX motif and the 20S alpha-subunits and indicates that the intersubunit pocket in the 20S undergoes an induced-fit conformational change on binding of the HbYX motif. This structure together with related mutagenesis data suggest how in eukaryotes certain proteasomal ATPases bind to specific pockets in an asymmetrical manner to regulate gate opening.

Although targeted in extrapancreatic tissues by several drugs used to treat type 2 diabetes, the role of AMP-activated protein kinase (AMPK) in the control of insulin secretion is still debatable. Previous studies have used pharmacological activators of limited selectivity and specificity, and none has examined in primary pancreatic beta cells the actions of the latest generation of highly potent and specific activators that act via the allosteric drug and metabolite (ADaM) site.

This article contains data on structural characterization of the [C2Mim][NTf2] in bulk and in nano-confined environment obtained using MD simulations. These data supplement those presented in the paper "Insights from Molecular Dynamics Simulations on Structural Organization and Diffusive Dynamics of an Ionic Liquid at Solid and Vacuum Interfaces" [1], where force fields with three different charge methods and three charge scaling factors were used for the analysis of the IL in the bulk, at the interface with the vacuum and the IL film in the contact with a hydroxylated alumina surface. Here, we present details on the construction of the model systems in an extended detailed methods section. Furthermore, for best parametrization, structural and dynamic properties of IL in different environment are studied with certain features presented herein.