Pressure Cycling Technology Assisted Mass Spectrometric Quantification of Gingival Tissue Reveals Proteome Dynamics during the Initiation and Progression of Inflammatory Periodontal Disease

Understanding the progression of periodontal tissue destruction is at the forefront of periodontal research. The authors aimed to capture the dynamics of gingival tissue proteome during the initiation and progression of experimental (ligature‐induced) periodontitis in mice. Pressure cycling technology (PCT), a recently developed platform that uses ultra‐high pressure to disrupt tissues, is utilized to achieve efficient and reproducible protein extraction from ultra‐small amounts of gingival tissues in combination with liquid chromatography‐tandem mass spectrometry (MS). The MS data are processed using Progenesis QI and the regulated proteins are subjected to METACORE, STRING, and WebGestalt for functional enrichment analysis. A total of 1614 proteins with ≥2 peptides are quantified with an estimated protein false discovery rate of 0.06%. Unsupervised clustering analysis shows that the gingival tissue protein abundance is mainly dependent on the periodontitis progression stage. Gene ontology enrichment analysis reveals an overrepresentation in innate immune regulation (e.g., neutrophil‐mediated immunity and antimicrobial peptides), signal transduction (e.g., integrin signaling), and homeostasis processes (e.g., platelet activation and aggregation). In conclusion, a PCT‐assisted label‐free quantitative proteomics workflow that allowed cataloging the deepest gingival tissue proteome on a rapid timescale and provided novel mechanistic insights into host perturbation during periodontitis progression is applied.     

Membrane Protein Location-Dependent Regulation by PI3K (III) and Rabenosyn-5 in Drosophila Wing Cells

The class III phosphatidylinositol-3 kinase (PI3K (III)) regulates intracellular vesicular transport at multiple steps through the production of phosphatidylinositol-3-phosphate (PI(3)P). While the localization of proteins at distinct membrane domains are likely regulated in different ways, the roles of PI3K (III) and its effectors have not been extensively investigated in a polarized cell during tissue development. In this study, we examined in vivo functions of PI3K (III) and its effector candidate Rabenosyn-5 (Rbsn-5) in Drosophila wing primordial cells, which are polarized along the apical-basal axis. Knockdown of the PI3K (III) subunit Vps15 resulted in an accumulation of the apical junctional proteins DE-cadherin and Flamingo and also the basal membrane protein β-integrin in intracellular vesicles. By contrast, knockdown of PI3K (III) increased lateral membrane-localized Fasciclin III (Fas III). Importantly, loss-of-function mutation of Rbsn-5 recapitulated the aberrant localization phenotypes of β-integrin and Fas III, but not those of DE-cadherin and Flamingo. These results suggest that PI3K (III) differentially regulates localization of proteins at distinct membrane domains and that Rbsn-5 mediates only a part of the PI3K (III)-dependent processes.     

Descriptions of two new endoparasitic cecidomyiids (Diptera: Cecidomyiidae) from Japan

Two endoparasitic species of Cecidomyiidae (Diptera) new to science are reported from Japan. Females of Endaphis psyllophaga sp. nov. lay their eggs on the wing of Calophya nigridorsalis (Hemiptera: Psylloidea: Calophyidae) on Rhus succedanea (Anacardiaceae), and newly hatched larvae bore into the adult body. The six nominal species of the genus Endaphis are endoparasitoids of aphids. The genus Endopsylla, which is morphologically similar to the genus Endaphis, consists of two species whose larvae attack psyllids or tingids. Females of Endaphis muraii sp. nov. lay their eggs near colonies of host aphids and newly hatched larvae bore into the body of aphids such as Macrosiphum euphorbiae and Aphis glycines (Hemiptera: Aphididae). The two new species are described, illustrated, and compared to known congeners, and information is given for the two species on their distribution, host range and ecological traits. Now, E. muraii is considered to be a potential biological control agent against aphids.     

Functional Interactions between Sphingolipids and Sterols in Biological Membranes Regulating Cell Physiology

Sterols and sphingolipids are limited to eukaryotic cells, and their interaction has been proposed to favor formation of lipid microdomains. Although there is abundant biophysical evidence demonstrating their interaction in simple systems, convincing evidence is lacking to show that they function together in cells. Using lipid analysis by mass spectrometry and a genetic approach on mutants in sterol metabolism, we show that cells adjust their membrane composition in response to mutant sterol structures preferentially by changing their sphingolipid composition. Systematic combination of mutations in sterol biosynthesis with mutants in sphingolipid hydroxylation and head group turnover give a large number of synthetic and suppression phenotypes. Our unbiased approach provides compelling evidence that sterols and sphingolipids function together in cells. We were not able to correlate any cellular phenotype we measured with plasma membrane fluidity as measured using fluorescence anisotropy. This questions whether the increase in liquid order phases that can be induced by sterol–sphingolipid interactions plays an important role in cells. Our data revealing that cells have a mechanism to sense the quality of their membrane sterol composition has led us to suggest that proteins might recognize sterol–sphingolipid complexes and to hypothesize the coevolution of sterols and sphingolipids.     

How is autonomic nervous system activity in subjects who are sleepy but are unable to sleep in the daytime?

We investigated changes in the activity of the autonomic nervous system (ANS) in the relaxed condition in subjects who felt sleepy, but were unable to sleep. A total of 1021 subjects underwent daytime polysomnography. The sleep latency (SL) and the visual analog scale (VAS) were used to assess “immediate” objective and subjective sleepiness, respectively. The subjects were assigned to an “Alert-Alert” group (VAS ≤ 25 mm, SL ≥ 8 min), a “Sleepy-Alert” group (VAS ≥ 75 mm, SL ≥ 8 min), or a “Sleepy-Sleepy” group (VAS ≥ 75 mm, SL ≤ 4 min). In order to assess the ANS, the spectral analysis and the geometric method were used. The ANS data collected during the relaxed condition (after lights off, post-LO) was compared to that obtained during the control condition (before lights off, pre-LO). From the spectral analysis, a significant decrease of sympathetic function and an increase of parasympathetic function at post-LO in the Sleepy-Sleepy group, a tendency for sympathetic function decrease at post-LO in the Alert-Alert group, and no significant changes to sympathetic and parasympathetic function in the Sleepy-Alert group were observed. The results from the geometric method supported the results of the spectral analysis in the Alert-Alert group and the Sleepy-Sleepy group. The results of this study suggest that the ANS plays a role in individuals who are unable to sleep even though they feel sleepy and are given the opportunity to sleep.

     

Development of ultra‐high sensitivity bioluminescent enzyme immunoassay for hepatitis B virus surface antigen using firefly luciferase

Hepatitis B virus (HBV) infection continues to be a global public health concern. Efficient diagnosis of HBV surface antigen (HBsAg) is useful for identification of infection, treatment and prevention of transfusion‐transmitted viral infections. Seronegative window reduction afforded by a highly sensitive measurement methodology is necessary as a small quantity of virus with infection risk exists for the period characterized by undetectable HBsAg following HBV infection. In this study, a bioluminescent enzyme immunoassay (BLEIA) for HBsAg was developed employing firefly luciferase as a labeling enzyme and a two‐step sandwich immunoassay method. The cut‐off value (10 mIU/mL) was 50‐fold more sensitive relative to conventional chemiluminescent enzyme immunoassay based on luminol luminescence involving peroxidase as the labeling enzyme and the identical antibodies. Preliminary clinical data for this BLEIA revealed that the HBV seroconversion panel derived sequentially from HBV‐infected human blood was detected 11 days following window closure from the first bleed, whereas detection occurred 14–25 days following window closure with the three conventional commercial kits. Copyright © 2009 John Wiley & Sons, Ltd.     

A simple method for isolation of nuclei from <Emphasis Type="Italic">Basidiobolus ranarum</Emphasis> (Zygomycota)

A simple method for isolating the nuclei from Basidiobolus ranarum was established. To improve the yield and purity of nuclei, we investigated maceration methods, buffer composition, and centrifugation conditions to establish an optimal procedure. Basidiobolus ranarum cultured for 5 days was enzymatically macerated and then homogenized and filtrated through stainless steel sieves. The crude cell homogenate was loaded on a layer of buffer containing 50% glycerol and centrifuged at 1500 g. The resultant pellet contained pure nuclei.     

Induction and isolation of pigmentation mutants of Porphyra yezoensis (Bangiales, Rhodophyta) by heavy-ion beam irradiation

The present study describes the isolation of pigmentation mutants of Porphyra yezoensis Ueda induced by heavy-ion beam irradiation for the first time. The gametophytic blades were irradiated with ¹²C⁺⁶ ion beams within a dose range of 25–400 Gy. From the survival rate and cell growth of the irradiated blades, it is suggested that a dose of 150 Gy or less is suitable to induce mutation for the isolation of mutants of P. yezoensis . After irradiation, red, green and deep reddish brown-colored gametophytic blades developed from archeospores that were released from each of the mutated cell clusters of the respective different colors, and the red mutant strain (IBY-R1) and green mutant strain (IBY-G1) were established as a conchocelis colony in culture. Blades of the mutants were characterized by their growth and photosynthetic pigment contents compared with those of the wild-type. From these results, it is clear that heavy-ion beam mutagenesis will be an effective tool for genetic and breeding studies of Porphyra , and also for other algal research.