Pathologically Activated Neuroprotection via Uncompetitive Blockade of N-Methyl-d-aspartate Receptors with Fast Off-rate by Novel Multifunctional Dimer Bis(propyl)-cognitin

Uncompetitive N-methyl-d-aspartate (NMDA) receptor antagonists with fast off-rate (UFO) may represent promising drug candidates for various neurodegenerative disorders. In this study, we report that bis(propyl)-cognitin, a novel dimeric acetylcholinesterase inhibitor and γ-aminobutyric acid subtype A receptor antagonist, is such an antagonist of NMDA receptors. In cultured rat hippocampal neurons, we demonstrated that bis(propyl)-cognitin voltage-dependently, selectively, and moderately inhibited NMDA-activated currents. The inhibitory effects of bis(propyl)-cognitin increased with the rise in NMDA and glycine concentrations. Kinetics analysis showed that the inhibition was of fast onset and offset with an off-rate time constant of 1.9 s. Molecular docking simulations showed moderate hydrophobic interaction between bis(propyl)-cognitin and the MK-801 binding region in the ion channel pore of the NMDA receptor. Bis(propyl)-cognitin was further found to compete with [³H]MK-801 with a K_i value of 0.27 μm, and the mutation of NR1(N616R) significantly reduced its inhibitory potency. Under glutamate-mediated pathological conditions, bis(propyl)-cognitin, in contrast to bis(heptyl)-cognitin, prevented excitotoxicity with increasing effectiveness against escalating levels of glutamate and much more effectively protected against middle cerebral artery occlusion-induced brain damage than did memantine. More interestingly, under NMDA receptor-mediated physiological conditions, bis(propyl)-cognitin enhanced long-term potentiation in hippocampal slices, whereas MK-801 reduced and memantine did not alter this process. These results suggest that bis(propyl)-cognitin is a UFO antagonist of NMDA receptors with moderate affinity, which may provide a pathologically activated therapy for various neurodegenerative disorders associated with NMDA receptor dysregulation.     

Double stranded RNA- relative to other TLR ligand-activated dendritic cells induce extremely polarized human Th1 responses

To better understand the relative efficiencies of using different TLR ligand-activated DCs to induce human CD4⁺ T lymphocyte responses, human DCs were activated with two viral and two bacterial TLR ligands, and their production of IL12, TNFα, and IL10 was examined. While the two viral TLR ligands (ssRNA and dsRNA) induced DC production of detectable levels of IL12p70, DCs activated by the two bacterial TLR ligands (LPS and flagellin) induced increased proliferation of human allogeneic naïve CD4⁺ T cells. dsRNA-activated DCs induced increased Th1 and decreased Th2 differentiation, resulting in extremely polarized responses relative to those induced by unstimulated and other TLR ligand-activated DCs. Neutralization of IL12p70 abrogated most of the Th1 skewing induced by all TLR ligand-activated moDCs. Collectively, these results demonstrate that dsRNA-activated DCs induce more highly polarized human Th1 responses than the other TLR ligand-activated DCs tested here. These results have implications for TLR ligands in immunotherapy.     

Interleukin-15 Is Critical in the Pathogenesis of Influenza A Virus-Induced Acute Lung Injury

Highly pathogenic influenza A viruses cause acute severe pneumonia to which the occurrence of “cytokine storm” has been proposed to contribute. Here we show that interleukin-15 (IL-15) knockout (KO) mice exhibited reduced mortality after infection with influenza virus A/FM/1/47 (H1N1, a mouse-adapted strain) albeit the viral titers of these mice showed no difference from those of control mice. There were significantly fewer antigen-specific CD44⁺ CD8⁺ T cells in the lungs of infected IL-15 KO mice, and adoptive transfer of the CD8⁺ T cells caused reduced survival of IL-15 KO mice following influenza virus infection. Mice deficient in β₂-microglobulin by gene targeting and those depleted of CD8⁺ T cells by in vivo administration of anti-CD8 monoclonal antibody displayed a reduced mortality rate after infection. These results indicate that IL-15-dependent CD8⁺ T cells are at least partly responsible for the pathogenesis of acute pneumonia caused by influenza A virus.Highly pathogenic influenza A viruses cause acute severe pneumonia that results in high morbidity and significant mortality (11, 12, 24, 26). Elevated levels of serum cytokines and chemokines accompany these clinical manifestations, and the possibility that this “cytokine storm” contributes to increased severity of the disease caused by avian H5N1 virus and by other strains of influenza A virus has been proposed (10, 21, 33). In fact, CCR2-deficient mice [CCR2 is chemokine (C-C motif) receptor 2] were protected from early pathological manifestations despite higher pulmonary titers of the influenza virus A/PR/8/34 (H1N1) strain (7). Tumor necrosis factor receptor 1 (TNFR-1)-deficient mice exhibited significantly reduced morbidity following challenge with H5N1 virus (31). Other cytokines or chemokines have also been investigated (8, 28, 34, 35, 38). Thus, at least some of the elevated proinflammatory cytokines may contribute to the pathogenesis of influenza A virus.Interleukin-15 (IL-15) is a pleiotropic cytokine involved in both innate and adaptive immune responses (20, 36). IL-15 utilizes the β-chain of the IL-2 receptor (IL-2R) (CD122) and the common cytokine receptor γ-chain (CD132) for signal transduction in lymphocytes and therefore shares many biological properties with IL-2 (3). Memory CD8⁺ T cells, natural killer (NK) cells, NKT cells, and intraepithelial lymphocyte (IEL) T cells (15, 23, 42) decrease in mice with defective IL-15 signaling, indicating the importance of IL-15 in their development and/or maintenance. IL-15 regulates not only the number of memory CD8⁺ T cells but also activation of their functions, including gamma interferon (IFN-γ) production and cytotoxic activity (40), which are important to target the virus (9). Therefore, it is possible that we may be able to use IL-15 as an immune-enhancing molecular adjuvant in vaccines for protection against various pathogens, including influenza A virus (37).In the present study, we demonstrate that IL-15 knockout (KO) mice exhibited high resistance against infection with mouse-adapted influenza virus A/FM/1/47 (H1N1) strain. We show for the first time that IL-15-dependent CD8⁺ T cells are at least partly responsible for the pathogenesis of acute pneumonia caused by influenza A virus. In addition, our observations are important in the light of recent research into the use of IL-15 as an adjuvant for vaccination.     

A Novel Antidiabetic Drug,Fasiglifam/TAK-875, Acts as an Ago-Allosteric Modulator of FFAR1

Selective free fatty acid receptor 1 (FFAR1)/GPR40 agonist fasiglifam (TAK-875), an antidiabetic drug under phase 3 development, potentiates insulin secretion in a glucose-dependent manner by activating FFAR1 expressed in pancreatic β cells. Although fasiglifam significantly improved glycemic control in type 2 diabetes patients with a minimum risk of hypoglycemia in a phase 2 study, the precise mechanisms of its potent pharmacological effects are not fully understood. Here we demonstrate that fasiglifam acts as an ago-allosteric modulator with a partial agonistic activity for FFAR1. In both Ca²⁺ influx and insulin secretion assays using cell lines and mouse islets, fasiglifam showed positive cooperativity with the FFAR1 ligand γ-linolenic acid (γ-LA). Augmentation of glucose-induced insulin secretion by fasiglifam, γ-LA, or their combination was completely abolished in pancreatic islets of FFAR1-knockout mice. In diabetic rats, the insulinotropic effect of fasiglifam was suppressed by pharmacological reduction of plasma free fatty acid (FFA) levels using a lipolysis inhibitor, suggesting that fasiglifam potentiates insulin release in conjunction with plasma FFAs in vivo. Point mutations of FFAR1 differentially affected Ca²⁺ influx activities of fasiglifam and γ-LA, further indicating that these agonists may bind to distinct binding sites. Our results strongly suggest that fasiglifam is an ago-allosteric modulator of FFAR1 that exerts its effects by acting cooperatively with endogenous plasma FFAs in human patients as well as diabetic animals. These findings contribute to our understanding of fasiglifam as an attractive antidiabetic drug with a novel mechanism of action.     

2-(Benzothiazol-2-yl)-phenyl-β-d-galactopyranoside derivatives as fluorescent pigment dyeing substrates and their application for the assay of β-d-galactosidase activities

2-(Benzothiazol-2-yl)-phenyl-β-d-galactopyranoside derivatives were synthesized as novel artificial fluorescent pigment dyeing substrates for β-d-galactosidase. The substrates, which exhibited non-fluorescence or weak fluorescence in solution phase, were smoothly hydrolyzed by β-d-galactosidase from Aspergillus oryzae and yielded a water-insoluble strong fluorescent pigment. The difference of fluorescent intensity exhibited a linear relationship with the amount of enzyme.     

Analysis of inbreeding depression in mixed-mating plants provides evidence for selective interference and stable mixed mating

Hermaphroditic individuals can produce both selfed and outcrossed progeny, termed mixed mating. General theory predicts that mixed-mating populations should evolve quickly toward high rates of selfing, driven by rapid purging of genetic load and loss of inbreeding depression (ID), but the substantial number of mixed-mating species observed in nature calls this prediction into question. Lower average ID reported for selfing than for outcrossing populations is consistent with purging and suggests that mixed-mating taxa in evolutionary transition will have intermediate ID. We compared the magnitude of ID from published estimates for highly selfing (r > 0.8), mixed-mating (0.2 ≤ r ≥ 0.8), and highly outcrossing (r < 0.2) plant populations across 58 species. We found that mixed-mating and outcrossing taxa have equally high average lifetime ID (δ= 0.58 and 0.54, respectively) and similar ID at each of four life-cycle stages. These results are not consistent with evolution toward selfing in most mixed-mating taxa. We suggest that prevention of purging by selective interference could explain stable mixed mating in many natural populations. We identify critical gaps in the empirical data on ID and outline key approaches to filling them.     

A calcium-dependent xylan-binding domain of alkaline xylanase from alkaliphilic Bacillus sp. strain 41M-1

Xylanase J of alkaliphilic Bacillus sp. strain 41M-1 contains a carbohydrate-binding module family 36 xylan-binding domain (XBD). Mutational analysis of the XBD revealed that Tyr237, Asp313, Trp317, and Asp318 were involved in Ca(2+)-dependent xylan-binding, and that Asp313 and Asp318 were especially important.     

Acceptability of Using Electronic Vending Machines to Deliver Oral Rapid HIV Self-Testing Kits: A Qualitative Study

Introduction

Rates of unrecognized HIV infection are significantly higher among Latino and Black men who have sex with men (MSM). Policy makers have proposed that HIV self-testing kits and new methods for delivering self-testing could improve testing uptake among minority MSM. This study sought to conduct qualitative assessments with MSM of color to determine the acceptability of using electronic vending machines to dispense HIV self-testing kits.

Materials and Methods

African American and Latino MSM were recruited using a participant pool from an existing HIV prevention trial on Facebook. If participants expressed interest in using a vending machine to receive an HIV self-testing kit, they were emailed a 4-digit personal identification number (PIN) code to retrieve the test from the machine. We followed up with those who had tested to assess their willingness to participate in an interview about their experience.

Results

Twelve kits were dispensed and 8 interviews were conducted. In general, participants expressed that the vending machine was an acceptable HIV test delivery method due to its novelty and convenience.

Discussion

Acceptability of this delivery model for HIV testing kits was closely associated with three main factors: credibility, confidentiality, and convenience. Future research is needed to address issues, such as user-induced errors and costs, before scaling up the dispensing method.