KIN‐4/MAST kinase promotes PTEN‐mediated longevity of Caenorhabditis elegans via binding through a PDZ domain

PDZ domain‐containing proteins (PDZ proteins) act as scaffolds for protein–protein interactions and are crucial for a variety of signal transduction processes. However, the role of PDZ proteins in organismal lifespan and aging remains poorly understood. Here, we demonstrate that KIN‐4, a PDZ domain‐containing microtubule‐associated serine‐threonine (MAST) protein kinase, is a key longevity factor acting through binding PTEN phosphatase in Caenorhabditis elegans. Through a targeted genetic screen for PDZ proteins, we find that kin‐4 is required for the long lifespan of daf‐2/insulin/IGF‐1 receptor mutants. We then show that neurons are crucial tissues for the longevity‐promoting role of kin‐4. We find that the PDZ domain of KIN‐4 binds PTEN, a key factor for the longevity of daf‐2 mutants. Moreover, the interaction between KIN‐4 and PTEN is essential for the extended lifespan of daf‐2 mutants. As many aspects of lifespan regulation in C. elegans are evolutionarily conserved, MAST family kinases may regulate aging and/or age‐related diseases in mammals through their interaction with PTEN.     

Preliminary report of knockdown resistance in Culex pipiens pallens and Aedes koreicus from Korea

Pyrethroid insecticides have been effective and powerful for controlling mosquitoes. However, abuse of these insecticides increases the number of resistant mosquitoes. In this study, Culex pipiens pallens and Aedes koreicus were collected from an artificial reservoir in the vicinity of a populated area in Korea, which is also a migratory bird catchment area. To monitor resistance to pyrethroid insecticides in mosquitoes, genomic DNA from the collected mosquitoes was sequenced for the kdr mutation in the voltage‐gated sodium channel (VGSC) gene. As a result, three samples with homozygous resistance (17.6%) and one with heterozygous resistance (5.9%) were found among 17 Cx. pipiens pallens specimens. One of the samples had a unique sequence at the amplified VGSC region. Of the 15 Ae. koreicus, no insecticide resistant individuals were found. In Korea, this is the first report of kdr genetic traits in Ae. koreicus and Cx. pipiens pallens and of a unique VGSC allele in Cx. pipiens pallens. Further investigation is needed to monitor the kdr resistance of these species in Korea and to determine how the unique sequence found in Cx. pipiens pallens is related to insecticide resistance.     

Soil depth- and root diameter-related variations affect root decomposition in temperate pine and oak forests

Aims Assessment of factors regulating root decomposition is needed to understand carbon and nutrient cycling in forest ecosystems. The objective of this study is to examine the effects of soil depth and root diameter on root decomposition and to analyze the relationship of root decomposition with factors such as soil environmental conditions and initial litter quality.     

Structural hybridization of pyrrolidine-based T-type calcium channel inhibitors and exploration of their analgesic effects in a neuropathic pain model

Highly effective and safe drugs for the treatment of neuropathic pain are urgently required and it was shown that blocking T-type calcium channels can be a promising strategy for drug development for neuropathic pain. We have developed pyrrolidine-based T-type calcium channel inhibitors by structural hybridization and subsequent assessment of in vitro activities against Ca_v3.1 and Ca_v3.2 channels. Profiling of in vitro ADME properties of compounds was also carried out. The representative compound 17h showed comparable in vivo efficacy to gabapentin in the SNL model, which indicates T-type calcium channel inhibitors can be developed as effective therapeutics for neuropathic pain.     

Predicting and validating a model of suppressor of IKKepsilon through biophysical characterization

Suppressor of IKKepsilon (SIKE) is a 207 residue protein that is implicated in the TLR3‐TANK‐binding kinase‐1‐mediated response to viral infection. SIKE's function in this pathway is unknown, but SIKE forms interactions with two distinct cytoskeletal proteins, α‐actinin and tubulin, and SIKE knockout reduces cell migration. As structure informs function and in the absence of solved structural homologs, our studies were directed toward creating a structural model of SIKE through biochemical and biophysical characterization to probe and interrogate SIKE function. Circular dichroism revealed a primarily (73%) helical structure of minimal stability (<T_m > =32°C) but reversibly denatured. Limited proteolysis (LP) and chemical modification identified the N‐terminal 2/3 of the protein as dynamic and accessible, whereas size exclusion chromatography (SEC) confirmed three homo‐oligomeric species. SEC coupled to chemical crosslinking characterized the primary species as dimeric, a secondary hexameric species, and a higher order aggregate/polymer. Fluorescence polarization using intrinsic tryptophan fluorescence contextualized the anisotropy value for the SIKE dimer (molecular weight 51.8 kDa) among proteins of known structure, bovine serum albumin (BSA; 66 kDa), and glutamate dehydrogenase (GDH; 332 kDa). Radii of gyration for BSA and GDH provided exclusionary values for SIKE tertiary and dimeric quaternary models that otherwise conformed to secondary structure, LP, and modification data. Dimeric quaternary models were further culled using acrylamide quenching data of SIKE's single tryptophan that showed a single, protected environment. The low cooperativity of folding and regions of dynamic and potentially disordered structure advance the hypothesis that SIKE forms a conformational ensemble of native states that accommodate SIKE's interactions with multiple, distinct protein‐binding partners.     

Detection of dental plaque and its potential pathogenicity using quantitative light‐induced fluorescence

Quantitative light‐induced fluorescence (QLF) technology can detect some dental plaque as red fluorescence. This in vivo study aimed to identify the microbial characteristics of red fluorescent (RF) dental plaque using 16S rRNA gene sequencing and evaluate the correlations between RF plaque and the clinical symptoms of dental diseases. Paired supragingival plaque samples collected from each 10 subjects and consisted of RF and non‐RF dental plaques as observed by QLF technology using a 405 nm blue light source for excitation. The characteristics of the bacterial communities in the RF and non‐RF plaque samples were compared by sequencing analysis. An increase in microbial diversity was observed in RF plaque compared with the non‐RF plaque. There were significant differences in the community compositions between the 2 types of dental plaque. Periodontopathic bacteria were significantly more abundant in the RF plaque than non‐RF plaque. The fluorescence intensity of RF plaque was significantly related to the proportion of the periodontopathic bacterial community and the presence of gingival inflammation. In conclusion, the plaque red fluorescence is associated with changes in the microbial composition and enrichment of periodontopathic pathogens, which suggests that RF plaque detected by QLF technology could be used as a risk indicator for gingival inflammation.      

Mass Balance Model with Donnan Equilibrium Accurately Describes Unusual pH and Excipient Profiles during Diafiltration of Monoclonal Antibodies

There is extensive experimental data showing that the final pH and buffer composition after protein diafiltration (DF), particularly with monoclonal antibodies, can be considerably different than that in the DF buffer due to electrostatic interactions between the charged protein and the charged ions. Previous models for this behavior have focused on the final (equilibrium) partitioning and are unable to explain the complex pH and concentration profiles during the DF process. The objective of this study is to develop a new model for antibody DF based on solution of the transient mass balance equations, with the permeate concentrations of the charged species evaluated assuming Donnan equilibrium across the semipermeable membrane in combination with electroneutrality constraints. Model predictions are in excellent agreement with experimental data obtained during DF of both acidic and basic monoclonal antibodies, with the protein charge determined from independent electrophoretic mobility measurements. The model is able to predict the entire pH/histidine concentration profiles during DF, providing a framework for the development of DF processes that yield the desired antibody formulation.     

dXNP, a Drosophila homolog of XNP/ATRX, induces apoptosis via Jun-N-terminal kinase activation

XNP/ATRX, a causative gene of X-linked alpha-thalassemia/mental retardation syndrome, encodes an SNF2 family ATPase/helicase protein. To better understand the role of XNP/ATRX in development, we isolated and characterized a Drosophila XNP/ATRX homolog, dXNP, which contains highly conserved SNF2 and helicase domains. Ectopically expressed dXNP induced strong apoptosis in the developing eye and wing, but did not affect cell cycle progression or the expression of wingless and engrailed, essential regulators of development. The dXNP-induced apoptosis was strongly suppressed by DJNKK/hemipterous mutation, and dXNP increased JNK activity. Taken together, these results suggest that dXNP regulates apoptosis via JNK activation.     

Role of Scarf and its binding target proteins in epidermal calcium homeostasis

The novel Ca2+-binding protein, Scarf (skin calmodulin-related factor) belongs to the calmodulin-like protein family and is expressed in the differentiated layers of the epidermis. To determine the roles of Scarf during stratification, we set out to identify the binding target proteins by affinity chromatography and subsequent analysis by mass spectrometry. Several binding factors, including 14-3-3s, annexins, calreticulin, ERp72 (endoplasmic reticulum protein 72), and nucleolin, were identified, and their interactions with Scarf were corroborated by co-immunoprecipitation and co-localization analyses. To further understand the functions of Scarf in epidermis in vivo, we altered the epidermal Ca2+ gradient by acute barrier disruption. The change in the expression levels of Scarf and its binding target proteins were determined by immunohistochemistry and Western blot analysis. The expression of Scarf, annexins, calreticulin, and ERp72 were up-regulated by Ca2+ gradient disruption, whereas the expression of 14-3-3s and nucleolin was reduced. Because annexins, calreticulin, and ERp72 have been implicated in Ca2+-induced cellular trafficking, including the secretion of lamellar bodies and Ca2+ homeostasis, we propose that the interaction of Scarf with these proteins might be crucial in the process of barrier restoration. On the other hand, down-regulation of 14-3-3s and nucleolin is potentially involved in the process of keratinocyte differentiation and growth inhibition. The calcium-dependent localization and up-regulation of Scarf and its binding target proteins were studied in mouse keratinocytes treated with ionomycin and during the wound-healing process. We found increased expression and nuclear presence of Scarf in the epidermis of the wound edge 4 and 7 days post-wounding, entailing the role of Scarf in barrier restoration. Our results suggest that Scarf plays a critical role as a Ca2+ sensor, potentially regulating the function of its binding target proteins in a Ca2+-dependent manner in the process of restoration of epidermal Ca2+ gradient as well as during epidermal barrier formation.