Transient Acceleration of Epidermal Growth Factor Receptor Dynamics Produces Higher-Order Signaling Clusters

Cell signaling depends on spatiotemporally regulated molecular interactions. Although the movements of signaling proteins have been analyzed with various technologies, how spatial dynamics influence the molecular interactions that transduce signals is unclear. Here, we developed a single-molecule method to analyze the spatiotemporal coupling between motility, clustering, and signaling. The analysis was performed with the epidermal growth factor receptor (EGFR), which triggers signaling through its dimerization and phosphorylation after association with EGF. Our results show that the few EGFRs isolated in membrane subdomains were released by an EGF-dependent increase in their diffusion area, facilitating molecular associations and producing immobile clusters. Using a two-color single-molecule analysis, we found that the EGF-induced state transition alters the properties of the immobile clusters, allowing them to interact for extended periods with the cytoplasmic protein, GRB2. Our study reveals a novel correlation between this molecular interaction and its mesoscale dynamics, providing the initial signaling node.     

Development of vaccines against pertussis caused by Bordetella holmesii using a mouse intranasal challenge model

Bordetella holmesii is recognized as the third causative agent of pertussis (whooping cough) in addition to Bordetella pertussis and Bordetella parapertussis. Pertussis caused by B. holmesii is not rare around the world. However, to date, there is no effective vaccine against B. holmesii. We examined the protective potency of pertussis vaccines available in Japan and vaccines prepared from B. holmesii. A murine model of respiratory infection was exploited to evaluate protective potency. No Japanese commercial pertussis vaccines were effective against B. holmesii. In contrast, a wBH vaccine and an aBH vaccine prepared from B. holmesii were both protective. Passive immunization with sera from mice immunized with aBH vaccine established protection against B. holmesii, indicating that B. holmesii‐specific serum antibodies might play an important role in protection. Immuno‐proteomic analysis with sera from mice immunized with aBH vaccine revealed that the sera recognized a BipA‐like protein of B. holmesii. An aBH vaccine prepared from a BipA‐like protein‐deficient mutant strain did not have a protective effect against B. holmesii. Taken together, our results suggest that the BipA‐like protein plays an important role in the protective efficacy of aBH vaccine.     

Aquaporin-11 containing a divergent NPA motif has normal water channel activity

Recently, two novel mammalian aquaporins (AQPs), AQPs 11 and 12, have been identified and classified as members of a new AQP subfamily, the "subcellular AQPs". In members of this subfamily one of the two asparagine-proline-alanine (NPA) motifs, which play a crucial role in selective water conduction, are not completely conserved. Mouse AQP11 (mAQP11) was expressed in Sf9 cells and purified using the detergent Fos-choline 10. The protein was reconstituted into liposomes, which were used for water conduction studies with a stopped-flow device. Single water permeability (pf) of AQP11 was measured to be 1.72+/-0.03x10(-13) cm(3)/s, suggesting that other members of the subfamily with incompletely conserved NPA motifs may also function as water channels.     

Direct electrochemical regeneration of NADH from NAD+ using cholesterol-modified gold amalgam electrode

The efficiency of the direct electrochemical regeneration of NADH from NAD+ was enhanced by applying a cholesterol-modified gold amalgam electrode. The modified electrode was prepared by immersing gold plate in mercury and casting few drops of cholesteryl oleate solution over the gold amalgam. Coenzymatically active NADH was formed from NAD+ directly at the cholesterol-modified gold amalgam electrode which is supposed to hinder the dimerization of the NAD radicals on its membrane surface. The direct electrochemical NAD+ reduction process was used favorably to drive an enzymatic reduction of pyruvate to d-lactate. d-Lactate of 18.2 mm was obtained from pyruvate of 25.3 mm at 21 h of total reaction time in the electrolysis of 50 cm3 solution with the electrode of 6 cm2area. The turnover number for NAD+ was estimated as 1400.     

An exception among diatoms: unique organization of genes involved in isoprenoid biosynthesis in Rhizosolenia setigera CCMP 1694

The marine diatom Rhizosolenia setigera is unique among this group of microalgae given that it is only one of a handful of diatom species that can produce highly branched isoprenoid (HBI) hydrocarbons. In our efforts to determine distinguishing molecular characteristics in R. setigera CCMP 1694 that could help elucidate the underlying mechanisms for its ability to biosynthesize HBIs, we discovered the occurrence of independent genes encoding for two isopentenyl diphosphate isomerases (RsIDI1 and RsIDI2) and one squalene synthase (RsSQS), enzymes that catalyze non‐consecutive steps in isoprenoid biosynthesis. These genes are peculiarly fused in all other genome‐sequenced diatoms to date, making their organization in R. setigera CCMP 1694 a clear distinguishing molecular feature. Phylogenetic and sequence analysis of RsIDI1, RsIDI2, and RsSQS revealed that such an arrangement of individually transcribed genes involved in isoprenoid biosynthesis could have arisen through a secondary gene fission event. We further demonstrate that inhibition of squalene synthase (SQS) shifts the flux of exogenous isoprenoid precursors towards HBI biosynthesis suggesting the competition for isoprenoid substrates in the form of farnesyl diphosphate between the sterol and HBI biosynthetic pathways in this diatom.     

Discovery of novel cyclic peptide inhibitors of dengue virus NS2B-NS3 protease with antiviral activity

NS2B-NS3 protease is an essential enzyme for the replication of dengue virus (DENV), which continues to be a serious threat to worldwide public health. We designed and synthesized a series of cyclic peptides mimicking the substrates of this enzyme, and assayed their activity against the DENV-2 NS2B-NS3 protease. The introduction of aromatic residues at the appropriate positions and conformational restriction generated the most promising cyclic peptide with an IC₅₀ of 0.95 μM against NS2B-NS3 protease. Cyclic peptides with proper positioning of additional arginines and aromatic residues exhibited antiviral activity against DENV. Furthermore, replacing the C-terminal amide bond of the polybasic amino acid sequence with an amino methylene moiety stabilized the cyclic peptides against hydrolysis by NS2B-NS3 protease, while maintaining their enzyme inhibitory activity and antiviral activity.     

Discovery of AAT-008, a novel,potent, and selective prostaglandin EP4 receptor antagonist

Starting from acylsufonamide HTS hit 2, a novel series of para-N-acylaminomethylbenzoic acids was identified and developed as selective prostaglandin EP4 receptor antagonists. Structural modifications on lead compound 4a were explored with the aim of improving potency, physicochemical properties, and animal PK predictive of QD (once a day) dosing regimen in human. These efforts led to the discovery of the clinical candidate AAT-008 (4j), which exhibited significantly improved pharmacological profiles over grapiprant (1).     

Microbial resource allocation for phosphatase synthesis reflects the availability of inorganic phosphorus across various soils

According to the resource allocation model for extracellular enzyme synthesis, microorganisms should preferentially allocate their resources to phosphorus (P)-acquiring enzyme synthesis when P availability is low in soils. However, the validity of this model across different soil types and soils differing in their microbial community composition has not been well demonstrated. Here we investigated whether the resource allocation model for phosphatase synthesis is applicable across different soil types (Andosols, Acrisols, Cambisols, and Fluvisols) and land uses (arable and forest), and we examined which soil test P and/or P fraction microorganisms responded to when investing their resources in phosphatase synthesis in the soils. The ratio of alkaline phosphatase (ALP) to β-d-glucosidase (BG) activities in the arable soils and the ratio of acid phosphatase (ACP) to BG activities in the forest soils were significantly negatively related with the available inorganic P concentration. We also observed significant effects of available inorganic P, pH, soil types, and land uses on the (ACP + ALP)/BG ratio when the data for the arable and forest soils were combined and used in a stepwise multiple regression analysis. These results suggest that microbial resource allocation for phosphatase synthesis is primarily controlled by available inorganic P concentration and soil pH, but the effects of soil types and land uses are also significant.     

Synergistic attraction of pine sawyer Monochamus saltuarius (Coleoptera: Cerambycidae) to monochamol and α‐pinene

Monochamus (Coleoptera: Cerambycidae) species are longhorn pine sawyers that serve as insect vectors of the pinewood nematode Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae), which are responsible for debilitating pine wilt disease. An aggregation pheromone, 2‐(1‐undecyloxy)‐1‐ethanol (hereafter referred to as monochamol), was shown to be effective at attracting Monochamus species. However, attraction of the pine sawyers to aggregation pheromones varied depending on semiochemicals, including host plant volatiles and kairomones. In this study, we investigated the abilities of monochamol and the host‐plant volatiles α‐pinene and ethanol to attract M. saltuarius in a pine forest in Cheongsong, Gyeongsangbuk‐do, Korea. A total of 91 M. saltuarius (28 males and 63 females) were captured. The combination of monochamol (700 mg) with α‐pinene and ethanol exhibited a synergistic effect on attracting M. saltuarius (11.0 beetles per trap), whereas monochamol alone and a mixture of α‐pinene and ethanol resulted in the capture of 3.2 beetles and 3.6 beetles per trap, respectively. Our results suggest that multi‐funnel traps baited with a blend of monochamol, α‐pinene and ethanol are highly effective for monitoring M. saltuarius and M. alternatus in pine forests.     

Significant decrease in local butterfly community during the last 15 years in a calcareous hill of the middle Korea

Recently the significant decreases of species richness and abundance among terrestrial animals including butterflies are reported due to habitat change, overexploitation, and global warming. We compared the butterfly species composition and abundance from 1999 and 2014–2015 in a calcareous hill site of the middle part of Korea using a line transect method. There was a significant decrease in the number of individuals (abundance) and the number of species (richness) from 1999 to 2014–2015. This decrease was more prevalent among northern species than southern species, and the local extinct species were more prevalent among northern species, showing the influence of global warming on butterfly assemblages. However, no impact of habitat change was observed because of maintenance of the grasslands, which is caused by the dry soils of the calcareous region.