Photolithographic fabrication of poly(ethylene glycol) microstructures for hydrogel-based microreactors and spatially addressed microarrays

We describe the fabrication of poly(ethylene glycol) diacrylate (PEG-DA) hydrogel microstructures with a high aspect ratio and the use of hydrogel microstructures containing the enzyme beta-galactosidase (beta-Gal) or glucose oxidase (GOx)/horseradish peroxidase (HRP) as biosensing components for the simultaneous detection of multiple analytes. The diameters of the hydrogel microstructures were almost the same at the top and at the bottom, indicating that no differential curing occurred through the thickness of the hydrogel microstructure. Using the hydrogel microstructures as microreactors, beta-Gal or GOx/HRP was trapped in the hydrogel array, and the time-dependent fluorescence intensities of the hydrogel array were investigated to determine the dynamic uptake of substrates into the PEG-DA hydrogel. The time required to reach steady-state fluorescence by glucose diffusing into the hydrogel and its enzymatic reactions with GOx and HRP was half the time required for resorufin beta-D-galactopyranoside (RGB) when used as the substrate for beta-Gal. Spatially addressed hydrogel microarrays containing different enzymes were micropatterned for the simultaneous detection of multiple analytes, and glucose and RGB solutions were incubated as substrates. These results indicate that there was no cross-talk between the beta-Gal-immobilizing hydrogel micropatches and the GOx/HRP-immobilizing micropatches.     

PUGNAc induces protein ubiquitination in C2C12 myotube cells

O‐linked β‐N‐acetylglucosaminylation (O‐GlcNAcylation) regulates many cellular processes including the cell cycle, cell signaling, and protein trafficking. Dysregulation of O‐GlcNAcylation may be involved in the development of insulin resistance and type 2 diabetes. Therefore, it is necessary to identify cellular proteins that are induced by elevated O‐GlcNAcylation. Here, using adenosine 5′‐triphosphate affinity chromatography, we employed a proteomic approach in order to identify differentially expressed proteins in response to treatment with the O‐GlcNAcase inhibitor, O‐(2‐acetamido‐2‐deoxy‐d ‐glucopyranosylidene)amino‐N‐phenylcarbamate (PUGNAc), in mouse C2C12 myotube cells. Among 205 selected genes, we identified 68 nucleotide‐binding proteins, 14 proteins that have adenosinetriphosphatase activity, and 10 proteins with ligase activity. Upregulation of proteins, including ubiquitin‐activating enzyme E1, proteasome subunit 20S, cullin‐associated NEDD8‐dissociated protein 1, ezrin, and downregulation of the protein nucleoside diphosphate kinase B, were confirmed by western blot analysis. In particular, we found that the protein ubiquitination level in C2C12 cells was increased by PUGNAc treatment. This is the first report of quantitative proteomic profiles of myotube cells after treatment with PUGNAc, and our results demonstrate the potential to enhance understanding of the relationship between insulin resistance, O‐GlcNAc, and PUGNAc in the future. Copyright © 2015 John Wiley & Sons, Ltd.     

Glial proteome changes in response to moderate hypothermia

Reactive glia plays a central role in neuroinflammation associated with secondary damage after brain injury. In order to understand the global effects of therapeutic hypothermia on glial activation and neuroinflammation, we performed proteomic profiling of glial cultures following inflammatory stimulation and hypothermic exposure. Primary mixed glial cultures prepared from mouse brains were stimulated with lipopolysaccharide and interferon-γ under normothermic (37°C) or moderate hypothermic (29°C) conditions, and their proteome profiles were compared by LC-ESI-MS/MS. Differentially expressed proteins were determined by high-throughput label-free quantification. Under hypothermic conditions, 64 and 16 proteins were upregulated (≥1.5-fold) and downregulated (≤ 0.7-fold), respectively, compared to normothermic conditions. More importantly, hypothermia altered the abundance of 143 proteins that were either increased or decreased by inflammatory stimulation. The results were validated for several proteins (ICAM-1, STAT-1, YWHAB, and IFIT-3) by Western blot analysis. Pathway and network analysis indicate that hypothermia influences various biological functions of glia such as molecular transport, cell movement, immune response, cell death, and stress response. In conclusion, moderate hypothermia seems to have a significant effect on the protein expression profiles of brain glia and possibly ensuing neuroinflammation. These proteins may be involved in the protective mechanism of hypothermia against brain injuries.     

Involvement of mTOR signaling in sphingosylphosphorylcholine-induced hypopigmentation effects

Background  

Sphingosylphosphorylcholine (SPC) acts as a potent lipid mediator and signaling molecule in various cell types. In the present study, we investigated the effects of SPC on melanogenesis and SPC-modulated signaling pathways related to melanin synthesis.     

Effects of nicotine on the growth and protein expression of <Emphasis Type="Italic">Porphyromonas gingivalis</Emphasis>

Tobacco smoking is considered one of the most significant environmental risk factors for destructive periodontal disease. The effect of smoking on periodontopathic microbiota has not yet been elucidated, as previous studies failed to identify a concrete relationship between periodontopathic microorganisms and smoking. However, it is likely that smoking, as an environmental stress factor, may affect the behavior of dental plaque microorganisms, ultimately leading to alteration of the host-parasite interaction. The goal of this study was to examine the effect of nicotine, a major component of tobacco, on the growth and protein expression of the crucial periodontal pathogen Porphyromonas gingivalis. The growth of P. gingivalis 381 was measured after bacterial cells were cultivated in liquid broth containing various nicotine concentrations. First, P. gingivalis cells were allowed to grow in the presence of a single dose of nicotine (the single exposure protocol) at 0, 1, 2, 4, and 8 mg/L, respectively. Second, P. gingivalis cells were exposed to five consecutive doses of nicotine (the multiple exposure protocol) at 0, 1, 2, and 4 mg/L, respectively. Bacterial growth was measured by optical density and protein expression was analyzed by SDS-PAGE and 2-D gel electrophoresis. In the single nicotine exposure protocol, it was observed that the growth of P. gingivalis 381 was inhibited by nicotine in a dose-dependent manner. In the multiple nicotine exposure protocol, the growth rate of P. gingivalis increased with each subsequent nicotine exposure, even though bacterial growth was also inhibited in a dose dependent fashion. SDS-PAGE and 2-D gel electrophoresis analyses revealed a minor change in the pattern of protein expression, showing differences in proteins with low molecular weights (around 20 kDa) on exposure to nicotine. The results of this study suggest that nicotine exerts an inhibitory effect on the growth of P. gingivalis, and has a potential to modulate protein expression in P. gingivalis.     

Tick-Borne Rickettsial Pathogens in Ticks and Small Mammals in Korea

In order to investigate the prevalence of tick-borne infectious agents among ticks, ticks comprising five species from two genera (Hemaphysalis spp. and Ixodes spp.) were screened using molecular techniques. Ticks (3,135) were collected from small wild-caught mammals or by dragging/flagging in the Republic of Korea (ROK) and were pooled into a total of 1,638 samples (1 to 27 ticks per pool). From the 1,638 tick samples, species-specific fragments of Anaplasma phagocytophilum (1 sample), Anaplasma platys (52 samples), Ehrlichia chaffeensis (29 samples), Ehrlichia ewingii (2 samples), Ehrlichia canis (18 samples), and Rickettsia rickettsii (28 samples) were amplified by PCR assay. Twenty-one pooled and individual tick samples had mixed infections of two (15 samples) or three (6 samples) pathogens. In addition, 424 spleen samples from small captured mammals (389 rodents, 33 insectivores, and 2 weasels) were screened for selected zoonotic pathogens. Species-specific DNA fragments of A. phagocytophilum (110 samples), A. platys (68 samples), E. chaffeensis (8 samples), E. ewingii (26 samples), E. canis (51 samples), and Rickettsia sp. (22 samples) were amplified by PCR assay. One hundred thirty small mammals had single infections, while 4, 14, and 21 striped field mice (Apodemus agrarius) had mixed infections of four, three, and two pathogens, respectively. Phylogenetic analysis based on nucleotide sequence comparison also revealed that Korean strains of E. chaffeensis clustered closely with those from China and the United States, while the Rickettsia (rOmpA) sequences clustered within a clade together with a Chinese strain. These results suggest that these agents should be considered in differential diagnosis while examining cases of acute febrile illnesses in humans as well as animals in the ROK.     

Remodeling of nuclear architecture during the cell cycle in Drosophila embryos

Little is known about what determines the nuclear matrix or how its reorganization is regulated during mitosis. In this study we report on a monoclonal antibody, mAb2A, which identifies a novel nuclear structure in Drosophila embryos which forms a diffuse meshwork at interphase but which undergoes a striking reorganization into a spindle-like structure during pro- and metaphase. Double labelings with α-tubulin and mAb2A antibodies demonstrate that the microtubules of the mitotic apparatus co-localize with this mAb2A labeled structure during metaphase, suggesting it may serve a role in microtubule spindle assembly and/or function during nuclear division. That the mAb2A-labeled nuclear structure is essential for cell division and/or maintenance of nuclear integrity was directly demonstrated by microinjection of mAb2A into early syncytial embryos which resulted in a disintegration of nuclear morphology and perturbation of mitosis. © 1996 Wiley-Liss, Inc.