ß-Arrestins as Important Regulators of Glucose and Energy Homeostasis.

ß-Arrestin-1 and -2 (also known as arrestin-2 and -3, respectively) are ubiquitously expressed cytoplasmic proteins that dampen signaling through G protein-coupled receptors. However, ß-arrestins can also act as signaling molecules in their own right. To investigate the potential metabolic roles of the two ß-arrestins in modulating glucose and energy homeostasis, recent studies analyzed mutant mice that lacked or overexpressed ß-arrestin-1 and/or -2 in distinct, metabolically important cell types. Metabolic analysis of these mutant mice clearly demonstrated that both ß-arrestins play key roles in regulating the function of most of these cell types, resulting in striking changes in whole-body glucose and/or energy homeostasis. These studies also revealed that ß-arrestin-1 and -2, though structurally closely related, clearly differ in their metabolic roles under physiological and pathophysiological conditions. These new findings should guide the development of novel drugs for the treatment of various metabolic disorders, including type 2 diabetes and obesity.

Yb-to-Eu Cooperative Sensitization Upconversion in a Multifunctional Molecular Nonanuclear Lanthanide Cluster in Solution.

Lanthanide metal clusters excel in combining molecular and material chemistry properties. Here, we report an efficient cooperative sensitization UC phenomenon of a Eu3+/Yb3+ nonanuclear lanthanide cluster in CD3OD. The synthesis and characterization of the heteronuclear cluster in the solid state and solution are described together with the UC phenomenon showing Eu3+ luminescence in the visible region upon 980 nm NIR excitation of Yb3+ at concentrations as low as 100 nM. Alongside being the Eu/Yb cluster to display UC (with a quantum yield value of 4.88 × 10-8 upon 1.13 W cm-2 excitation at 980 nm), the cluster exhibits downshifted light emission of Yb3+ in the NIR region upon 578 nm visible excitation of Eu3+, which is ascribed to sensitization pathways for Yb through the 5D0 energy levels of Eu3+. Additionally, a faint emission is also observed at ca. 500 nm upon 980 nm excitation, originating from the cooperative luminescence of Yb3+. The [Eu8Yb(BA)16(OH)10]Cl cluster (BA = benzoylacetonate) is also a field-induced single-molecular magnet (SMM) under 4K with a modest Ueff/kB of 8.48 K, thereby joining the coveted list of Yb-SMMs and emerging as a prototype system for next-generation devices, combining luminescence with single-molecular magnetism in a molecular cluster.

Use of DREADD Technology to Identify Novel Targets for Antidiabetic Drugs.

G protein-coupled receptors (GPCRs) form a superfamily of plasma membrane receptors that couple to four major families of heterotrimeric G proteins, Gs, Gi, Gq, and G12. GPCRs represent excellent targets for drug therapy. Since the individual GPCRs are expressed by many different cell types, the in vivo metabolic roles of a specific GPCR expressed by a distinct cell type are not well understood. The development of designer GPCRs known as DREADDs (designer receptors exclusively activated by a designer drug) that selectively couple to distinct classes of heterotrimeric G proteins has greatly facilitated studies in this area. This review focuses on the use of DREADD technology to explore the physiological and pathophysiological roles of distinct GPCR/G protein cascades in several metabolically important cell types. The novel insights gained from these studies should stimulate the development of GPCR-based treatments for major metabolic diseases such as type 2 diabetes and obesity.

Thrombospondin 2 is a key determinant of fibrogenesis in non-alcoholic fatty liver disease.

OBJECTIVE: Hepatic overexpression of the thrombospondin 2 gene (THBS2) and elevated levels of circulating thrombospondin 2 (TSP2) have been observed in patients with chronic liver disease. This study aimed to identify the specific cells expressing THBS2/TSP2 in non-alcoholic fatty liver disease (NAFLD) and investigate the underlying mechanism behind THBS2/TSP2 upregulation. DESIGN: Comprehensive NAFLD liver gene datasets, including single-cell RNA sequencing (scRNA-seq), in-house NAFLD liver tissue, and LX-2 cells derived from human hepatic stellate cells (HSCs), were analysed using a combination of computational biology, genetic, immunological, and pharmacological approaches. RESULTS: Analysis of the genetic dataset revealed the presence of 1433 variable genes in patients with advanced fibrosis NAFLD, with THBS2 ranked among the top 2 genes. Quantitative polymerase chain reaction (qPCR) examination of NAFLD livers showed a significant correlation between THBS2 expression and fibrosis stage (r = .349, p < .001). In support of this, scRNA-seq data and in situ hybridization demonstrated that the THBS2 gene was highly expressed in HSCs of NAFLD patients with advanced fibrosis. Pathway analysis of the gene dataset revealed THBS2 expression to be associated with the transforming growth factor beta (TGFß) pathway and collagen gene activation. Moreover, the activation of LX-2 cells with TGFß increased THBS2/TSP2 and collagen expression independently of the TGFß-SMAD2/3 pathway. THBS2 gene knockdown significantly decreased collagen expression in LX-2 cells. CONCLUSIONS: THBS2/TSP2 is highly expressed in HSCs and plays a role in regulating fibrogenesis in NAFLD patients. THBS2/TSP2 may therefore represent a potential target for anti-fibrotic therapy in NAFLD.

Lack of adipocyte purinergic P2Y6 receptor greatly improves whole body glucose homeostasis.

Uridine diphosphate (UDP)-activated purinergic receptor P2Y6 (P2Y6R) plays a crucial role in controlling energy balance through central mechanisms. However, P2Y6R's roles in peripheral tissues regulating energy and glucose homeostasis remain unexplored. Here, we report the surprising finding that adipocyte-specific deletion of P2Y6R protects mice from diet-induced obesity, improving glucose tolerance and insulin sensitivity with reduced systemic inflammation. These changes were associated with reduced JNK signaling and enhanced expression and activity of PPARα affecting downstream PGC1α levels leading to beiging of white fat. In contrast, P2Y6R deletion in skeletal muscle reduced glucose uptake, resulting in impaired glucose homeostasis. Interestingly, whole body P2Y6R knockout mice showed metabolic improvements similar to those observed with mice lacking P2Y6R only in adipocytes. Our findings provide compelling evidence that P2Y6R antagonists may prove useful for the treatment of obesity and type 2 diabetes.

The trimeric coiled-coil HSBP1 protein promotes WASH complex assembly at centrosomes.

The Arp2/3 complex generates branched actin networks that exert pushing forces onto different cellular membranes. WASH complexes activate Arp2/3 complexes at the surface of endosomes and thereby fission transport intermediates containing endocytosed receptors, such as α5ß1 integrins. How WASH complexes are assembled in the cell is unknown. Here, we identify the small coiled-coil protein HSBP1 as a factor that specifically promotes the assembly of a ternary complex composed of CCDC53, WASH, and FAM21 by dissociating the CCDC53 homotrimeric precursor. HSBP1 operates at the centrosome, which concentrates the building blocks. HSBP1 depletion in human cancer cell lines and in Dictyostelium amoebae phenocopies WASH depletion, suggesting a critical role of the ternary WASH complex for WASH functions. HSBP1 is required for the development of focal adhesions and of cell polarity. These defects impair the migration and invasion of tumor cells. Overexpression of HSBP1 in breast tumors is associated with increased levels of WASH complexes and with poor prognosis for patients.

2-Step PLT16-AST44 method: Simplified liver fibrosis detection system in patients with non-alcoholic fatty liver disease.

AIM: Accurate detection of the hepatic fibrosis stage is essential to estimate the outcome of patients with non-alcoholic fatty liver disease (NAFLD). Many formulas, biomarkers, and imaging tests are being developed to predict advanced liver fibrosis without performing a liver biopsy. However, these tests do not have high efficiency in detecting early-stage hepatic fibrosis. Therefore, we aimed to detect the presence of hepatic fibrosis (≥F1) merely by using only standard clinical markers. METHODS: A total of 436 patients with NAFLD who underwent liver biopsy were retrospectively enrolled as the discovery cohort (316 patients) and the validation cohort (120 patients). Liver biopsy and laboratory data were matched to extract simple parameters for identifying ≥F1. RESULTS: We developed a novel simplified ≥F1 detecting system, designated as 2-Step PLT16-AST44 method, where (1) PLT of 16 × 104 /µl or less, or (2) PLT greater than 16 × 104 /µl and AST greater than 44 U/L is determined as having ≥F1 fibrosis. The 2-Step PLT16-AST44 method had a sensitivity of 68%, a specificity of 90%, a positive predictive value (PPV) of 97%, a negative predictive value (NPV) of 40%, and an accuracy of 72% to detect ≥F1 fibrosis in the discovery cohort. Validation studies further supported these results. Despite its simplicity, the 2-Step PLT16-AST44 method's power to detect ≥F1 fibrosis in total NAFLD patients was comparable to hyaluronic acid, type 4 collagen 7S, FIB-4, and APRI. CONCLUSIONS: We propose the 2-Step PLT16-AST44 method as a simple and beneficial early-stage hepatic fibrosis detection system.

Chemogenetic approaches to identify metabolically important GPCR signaling pathways: Therapeutic implications.

DREADDs (Designer Receptors Exclusively Activated by a Designer Drug) are designer G protein-coupled receptors (GPCRs) that are widely used in the neuroscience field to modulate neuronal activity. In this review, we will focus on DREADD studies carried out with genetically engineered mice aimed at elucidating signaling pathways important for maintaining proper glucose and energy homeostasis. The availability of muscarinic receptor-based DREADDs endowed with selectivity for one of the four major classes of heterotrimeric G proteins (Gs , Gi , Gq , and G12 ) has been instrumental in dissecting the physiological and pathophysiological roles of distinct G protein signaling pathways in metabolically important cell types. The novel insights gained from this work should inform the development of novel classes of drugs useful for the treatment of several metabolic disorders including type 2 diabetes and obesity.

Synthesis and Photophysical Properties of Soluble N-Doped Rubicenes via Ruthenium-Catalyzed Transfer Hydrogenative Benzannulation.

Ruthenium-catalyzed butadiene-mediated benzannulation enabled the first synthesis of 3,10-(di-tert-butyl)rubicene and its N-doped derivatives as well as preliminary studies on their photophysical properties. Unlike the parent rubicene and 3,10-(di-tert-butyl)rubicene, which adopt classical herringbone-type packing motifs in the solid state, the N-doped congener 7 b displayed columnar packing with an alternating co-facial arrangement of aromatic and heteroaromatic substructures.

Serum thrombospondin 2 is a novel predictor for the severity in the patients with NAFLD.

AIM: Thrombospondins are a family of multidomain and secretory glycoproteins. Among them, thrombospondin 2 (TSP2) encoded by TSP2 gene has been reported to be involved in various functions such as collagen/fibrin formation, maintenance of normal blood vessel density and cell adhesion properties. Microarray analyses ranked TSP2 as one of the most highly up-regulated genes in the fibrotic liver in patients with non-alcoholic fatty liver disease (NAFLD). Since TSP2 possesses unique properties as a secretory protein, we hypothesized that hepatic TSP2 gene expression levels would be reflected in serum TSP2 levels. In this study, we examined the relationship between serum TSP2 concentrations and clinicopathological findings in NAFLD patients. METHODS: One hundred and thirty NAFLD patients who had undergone liver biopsy between 2009 and 2015 were retrospectively enrolled. Serum samples were collected at the time of biopsy, and TSP2 was measured by enzyme immunoassays. RESULTS: Serum TSP2 levels moderately correlated with ballooning (r = 0.56, P < .001) and fibrosis stage (r = 0.53, P < .001). The AUC values of TSP2 for predicting mild fibrosis (≧F1), moderate fibrosis (≧F2) and severe fibrosis (≧F3) were 0.73, 0.76 and 0.82 respectively. Additionally, NAFLD activity score (NAS) correlated best with TSP2 (r = 0.52, P < .001) compared to conventional NAFLD-related biomarkers, such as cytokeratin 18 M30, hyaluronic acid, type IV collagen 7S, APRI and FIB-4 index. CONCLUSION: Serum TSP2 levels reflected hepatocyte ballooning, fibrosis and NAS in NAFLD patients. For clinical application of serum TSP2 as a predictor of NAFLD histological activity, additional validation and mechanistic investigations are required.

Core outcomes sets for studies evaluating critical illness and patient recovery.

PURPOSE OF REVIEW: Given the growing body of critical care clinical research publications, core outcome sets (COSs) are important to help mitigate heterogeneity in outcomes assessed and measurement instruments used, and have potential to reduce research waste. This article provides an update on COS projects in critical care medicine, and related resources and tools for COS developers. RECENT FINDINGS: We identified 28 unique COS projects, of which 15 have published results as of May 2020. COS topics relevant to critical care medicine include mechanical ventilation, cardiology, stroke, rehabilitation, and long-term outcomes (LTOs) after critical illness. There are four COS projects for coronavirus disease 2019 (COVID-19), with a 'meta-COS' summarizing common outcomes across these projects. To help facilitate COS development, there are existing resources, standards, guidelines, and tools available from the Core Outcome Measures in Effectiveness Trials Initiative (www.comet-initiative.org/) and the National Institutes of Health-funded Improve LTO project (www.improvelto.com/). SUMMARY: Many COS projects have been completed in critical care, with more on-going COS projects, including foci from across the spectrum of acute critical care, COVID-19, critical care rehabilitation, and patient recovery and LTOs. Extensive resources are accessible to help facilitate rigorous COS development.

Comparative Pharmacokinetics of Mitragynine after Oral Administration of Mitragyna speciosa (Kratom) Leaf Extracts in Rats.

Kratom (Mitragyna speciosa) has been examined for its opioid activity, especially for the treatment of opioid withdrawal and pain. Mitragynine, the most abundant alkaloid in kratom, is thought to be the major psychoactive alkaloid. An HPLC method was developed for the quantification of mitragynine in kratom leaf extracts. In addition, a multiple reaction mode based UPLC-MS/MS method was developed and validated for the quantification of mitragynine in rat plasma. Pharmacokinetic studies were performed by comparing a single intravenous dose of mitragynine (5 mg/kg, mitragynine hydrochloride) to a single oral dose of mitragynine (20 mg/kg, mitragynine hydrochloride), lyophilized kratom tea, and the organic fraction of the lyophilized kratom tea at an equivalent mitragynine dose of 20 mg/kg in rats. After intravenous administration, mitragynine exhibited a biexponential decrease in the concentration-time profile, indicating the fast distribution of mitragynine from the systemic circulation or central compartment to the peripheral compartments. Mitragynine hydrochloride, lyophilized kratom tea, and the lyophilized kratom tea organic fraction were dosed orally and the absolute oral bioavailability of mitragynine in rats was found to be 1.5- and 1.8-fold higher than that of mitragynine dosed alone. The results provide evidence that an equivalent oral dose of the traditional preparation (lyophilized kratom tea) and formulated/manufactured products (organic fraction) of kratom leaves provide better systemic exposure of mitragynine than that of mitragynine dosed alone.

Formation of Small Gold Nanoparticle Chains with High NIR Extinction through Bridging with Calcium Ions.

The self-assembly of citrate-capped Au nanoparticles (5 nm) resulted in branched nanochains by adding CaCl2 versus spherical nanoclusters for NaCl. These assemblies were formed between 1 s to 30 min by tuning the electrostatic repulsion and the interparticle bridging attraction between the cations and citrate ligands as a function of electrolyte concentration. For dilute Ca(2+), strong interparticle bridging favored particle attachment at chain ends. This resulted in the formation of small, branched chains with lengths as short as 20 nm, due to the large Debye length for the diffuse counterions. Furthermore, the bridging produced very small interparticle spacings and sintering, as evident in high-resolution TEM despite the low temperature. This morphology produced a large red shift in the surface plasmon resonance, as characterized by a broad extinction peak with NIR absorption out to 1000 nm, which is unusual for such small particles. Whereas these properties were seen for primary particles with partial citrate monolayers, the degrees of sintering and NIR extinction were small in the case of citrate multilayers. The ability to design the size and shape of nanoparticle clusters as well as the interparticle spacing by tuning bridging and electrostatic interactions may be expected to be quite general and of broad applicability in materials synthesis.

Abscisic Acid Acts as a Blocker of the Bitter Taste G Protein-Coupled Receptor T2R4.

Bitter taste receptors (T2Rs) belong to the G protein-coupled receptor superfamily. In humans, 25 T2Rs mediate bitter taste sensation. In addition to the oral cavity, T2Rs are expressed in many extraoral tissues, including the central nervous system, respiratory system, and reproductive system. To understand the mechanistic roles of the T2Rs in oral and extraoral tissues, novel blockers or antagonists are urgently needed. Recently, we elucidated the binding pocket of T2R4 for its agonist quinine, and an antagonist and inhibitory neurotransmitter, γ-aminobutyric acid. This structure-function information about T2R4 led us to screen the plant hormone abscisic acid (ABA), its precursor (xanthoxin), and catabolite phaseic acid for their ability to bind and activate or inhibit T2R4. Molecular docking studies followed by functional assays involving calcium imaging confirmed that ABA is an antagonist with an IC50 value of 34.4 ± 1.1 µM. However, ABA precursor xanthoxin acts as an agonist on T2R4. Interestingly, molecular model-guided site-directed mutagenesis suggests that the T2R4 residues involved in quinine binding are also predominantly involved in binding to the novel antagonist, ABA. The antagonist ability of ABA was tested using another T2R4 agonist, yohimbine. Our results suggest that ABA does not inhibit yohimbine-induced T2R4 activity. The discovery of natural bitter blockers has immense nutraceutical and physiological significance and will help in dissecting the T2R molecular pathways in various tissues.

Amino acid derivatives as bitter taste receptor (T2R) blockers.

In humans, the 25 bitter taste receptors (T2Rs) are activated by hundreds of structurally diverse bitter compounds. However, only five antagonists or bitter blockers are known. In this study, using molecular modeling guided site-directed mutagenesis, we elucidated the ligand-binding pocket of T2R4. We found seven amino acids located in the extracellular side of transmembrane 3 (TM3), TM4, extracellular loop 2 (ECL2), and ECL3 to be involved in T2R4 binding to its agonist quinine. ECL2 residues Asn-173 and Thr-174 are essential for quinine binding. Guided by a molecular model of T2R4, a number of amino acid derivatives were screened for their ability to bind to T2R4. These predictions were tested by calcium imaging assays that led to identification of γ-aminobutryic acid (GABA) and Nα,Nα-bis(carboxymethyl)-L-lysine (BCML) as competitive inhibitors of quinine-activated T2R4 with an IC50 of 3.2 ± 0.3 µM and 59 ± 18 nM, respectively. Interestingly, pharmacological characterization using a constitutively active mutant of T2R4 reveals that GABA acts as an antagonist, whereas BCML acts as an inverse agonist on T2R4. Site-directed mutagenesis confirms that the two novel bitter blockers share the same orthosteric site as the agonist quinine. The signature residues Ala-90 and Lys-270 play important roles in interacting with BCML and GABA, respectively. This is the first report to characterize a T2R endogenous antagonist and an inverse agonist. The novel bitter blockers will facilitate physiological studies focused on understanding the roles of T2Rs in extraoral tissues.

Nod2 deficiency protects mice from cholestatic liver disease by increasing renal excretion of bile acids.

BACKGROUND & AIMS: Chronic liver disease is characterized by fibrosis that may progress to cirrhosis. Nucleotide oligomerization domain 2 (Nod2), a member of the Nod-like receptor (NLR) family of intracellular immune receptors, plays an important role in the defense against bacterial infection through binding to the ligand muramyl dipeptide (MDP). Here, we investigated the role of Nod2 in the development of liver fibrosis. METHODS: We studied experimental cholestatic liver disease induced by bile duct ligation or toxic liver disease induced by carbon tetrachloride in wild type and Nod2(-/-) mice. RESULTS: Nod2 deficiency protected mice from cholestatic but not toxin-induced liver injury and fibrosis. Most notably, the hepatic bile acid concentration was lower in Nod2(-/-) mice than wild type mice following bile duct ligation for 3 weeks. In contrast to wild type mice, Nod2(-/-) mice had increased urinary excretion of bile acids, including sulfated bile acids, and an upregulation of the bile acid efflux transporters MRP2 and MRP4 in tubular epithelial cells of the kidney. MRP2 and MRP4 were downregulated by IL-1ß in a Nod2 dependent fashion. CONCLUSIONS: Our findings indicate that Nod2 deficiency protects mice from cholestatic liver injury and fibrosis through enhancing renal excretion of bile acids that in turn contributes to decreased concentration of bile acids in the hepatocyte.

Enforced hematopoietic cell E- and L-selectin ligand (HCELL) expression primes transendothelial migration of human mesenchymal stem cells.

According to the multistep model of cell migration, chemokine receptor engagement (step 2) triggers conversion of rolling interactions (step 1) into firm adhesion (step 3), yielding transendothelial migration. We recently reported that glycosyltransferase-programmed stereosubstitution (GPS) of CD44 on human mesenchymal stem cells (hMSCs) creates the E-selectin ligand HCELL (hematopoietic cell E-selectin/L-selectin ligand) and, despite absence of CXCR4, systemically administered HCELL(+)hMSCs display robust osteotropism visualized by intravital microscopy. Here we performed studies to define the molecular effectors of this process. We observed that engagement of hMSC HCELL with E-selectin triggers VLA-4 adhesiveness, resulting in shear-resistant adhesion to ligand VCAM-1. This VLA-4 activation is mediated via a Rac1/Rap1 GTPase signaling pathway, resulting in transendothelial migration on stimulated human umbilical vein endothelial cells without chemokine input. These findings indicate that hMSCs coordinately integrate CD44 ligation and integrin activation, circumventing chemokine-mediated signaling, yielding a step 2-bypass pathway of the canonical multistep paradigm of cell migration.

Mixed neuroendocrine-nonneuroendocrine epithelial neoplasm of muscle invasive bladder cancer: a clinicopathologic case study.

Mixed neuroendocrine-nonneuroendocrine epithelial neoplasms are rare malignant neoplasms that may occur in the bladder with highly aggressive behavior. Because of its worse prognosis, when compared to the pure urothelial carcinoma without the neuroendocrine component, the bladder mixed neuroendocrine-nonneuroendocrine epithelial neoplasm may be considered a distinct clinicopathologic entity. We present a case of mixed neuroendocrine-nonneuroendocrine epithelial neoplasm occurring in the urinary bladder of an elderly female with a personal history of chronic kidney disease, drug-resistant urinary tract infections, and neurogenic bladder. Her presenting symptoms included complaints of abdominal pain, urinary urgency, oliguria, dysuria, and occasional hematuria. Recognition of the clinicopathologic features of these rare aggressive neoplasms is important for accurate early diagnosis, necessitating appropriate therapeutic management.

Localized pseudomembranous gastritis associated with hepatic cirrhosis and portal hypertensive gastropathy: a case report.

Pseudomembranes in the large and small intestines are common in hospitalized patients that are immunosuppressed or on certain oral antibiotics. Pseudomembranous enterocolitis, histologically characterized by volcanic-like eruption of inflammatory cellular exudate from the mucosal surface, is mainly attributed to Clostridium difficile toxins and often presents with symptomatic diarrhea. Rarely, there are case reports of similar pseudomembranous lesions limited to the stomach in the absence of intestinal involvement. In this paper, we present a case of localized pseudomembranous gastritis in a 76-year-old patient with personal history limited to prior gastrointestinal bleed, liver cirrhosis, alcohol dependence, diabetes mellitus, and hypertension who was referred to the emergency department from his primary care physician's office due to low hemoglobin.