Clinical Isolation of Anaplasma phagocytophilum in South Korea.

We report the first isolation of Anaplasma phagocytophilum in South Korea. A 61-year-old woman presented with a 6-day history of fever, headache, and myalgia. Initial investigation showed neutropenia and thrombocytopenia. We diagnosed human granulocytic anaplasmosis by microscopic examination and serologic testing. The patient recovered fully without antibiotic therapy. The isolate was obtained from the patient's blood by cell culture and mouse inoculation. Its identity was confirmed by an immunofluorescence assay, sequencing of the 16S rRNA gene, msp2 (p44), and ankA genes, and staining and electron microscopy of morulae of A. phagocytophilum in cultured human promyelocytic leukemia HL-60 cells.

Fisetin stimulates autophagic degradation of phosphorylated tau via the activation of TFEB and Nrf2 transcription factors.

The neuronal accumulation of phosphorylated tau plays a critical role in the pathogenesis of Alzheimer's disease (AD). Here, we examined the effect of fisetin, a flavonol, on tau levels. Treatment of cortical cells or primary neurons with fisetin resulted in significant decreases in the levels of phosphorylated tau. In addition, fisetin decreased the levels of sarkosyl-insoluble tau in an active GSK-3ß-induced tau aggregation model. However, there was no difference in activities of tau kinases and phosphatases such as protein phosphatase 2A, irrespective of fisetin treatment. Fisetin activated autophagy together with the activation of transcription factor EB (TFEB) and Nrf2 transcriptional factors. The activation of autophagy including TFEB is likely due to fisetin-mediated mammalian target of rapamycin complex 1 (mTORC1) inhibition, since the phosphorylation levels of p70S6 kinase and 4E-BP1 were decreased in the presence of fisetin. Indeed, fisetin-induced phosphorylated tau degradation was attenuated by chemical inhibitors of the autophagy-lysosome pathway. Together the results indicate that fisetin reduces levels of phosphorylated tau through the autophagy pathway activated by TFEB and Nrf2. Our result suggests fisetin should be evaluated further as a potential preventive and therapeutic drug candidate for AD.

Small interfering (Si) RNA mediated baculovirus replication reduction without affecting target gene expression.

The baculovirus expression vector system (BEVS) is widely used to produce large quantities of recombinant protein with posttranslational modification. Recombinant baculoviruses (such as Autographa californica multiple nuclear polyhedrosis virus) are especially useful in producing recombinant proteins and virus-like particles (VLPs) as biodrugs or candidate vaccines for the prevention of serious infectious diseases. However, during the bioprocessing of recombinant proteins in insect cells, baculovirus replication and viral budding are coincident. In some cases, residual baculovirus contaminants remain in the recombinant protein products, even though various purification processes are applied such as ion-exchange chromatography, ultracentrifugation, or gel filtration. To reduce unexpected contamination caused by replication and budding-out of the baculovirus, we designed short interfering (si) RNAs targeting glycoprotein 64 (GP64) or single-stranded DNA-binding protein (DBP) to inhibit baculovirus replication during overexpression of recombinant foreign genes. GP64 is known to be critical both for the entry of virions into cells and for the assembly of the budded virion at the cell surface. DBP is also essential for virus assembly by regulation of the capsid protein P39 and the polyhedrin protein. This study showed that GP64 expression was suppressed by GP64 siRNAs in Western blot experiments, while the expression of recombinant proteins was unaffected. In addition, transfection of GP64 siRNAs and DBP siRNAs reduced the level of baculovirus replication, compared with the treatment with scrambled siRNAs. However, DBP siRNA also suppressed the expression of recombinant proteins. In conclusion, our GP64 siRNAs showed that an interfering RNA system, such as siRNAs and short hairpin (sh) RNAs, can be applicable to reduce baculovirus contaminants during the bioprocessing of recombinant proteins in insect cells. Further investigation should be carried out to establish transformed insect cell lines with stable expression of corresponding interfering RNAs.

SUMO1 promotes Aß production via the modulation of autophagy.

Autophagy is one of the main mechanisms in the pathophysiology of neurodegenerative disease. The accumulation of autophagic vacuoles (AVs) in affected neurons is responsible for amyloid-ß (Aß) production. Previously, we reported that SUMO1 (small ubiquitin-like modifier 1) increases Aß levels. In this study, we explored the mechanisms underlying this. We investigated whether AV formation is necessary for Aß production by SUMO1. Overexpression of SUMO1 increased autophagic activation, inducing the formation of LC3-II-positive AVs in neuroglioma H4 cells. Consistently, autophagic activation was decreased by the depletion of SUMO1 with small hairpin RNA (shRNA) in H4 cells. The SUMO1-mediated increase in Aß was reduced by the autophagy inhibitors (3-methyladenine or wortmannin) or genetic inhibitors (siRNA targeting ATG5, ATG7, ATG12, or HIF1A), respectively. Accumulation of SUMO1, ATG12, and LC3 was seen in amyloid precursor protein transgenic mice. Our results suggest that SUMO1 accelerates the accumulation of AVs and promotes Aß production, which is a key mechanism for understanding the AV-mediated pathophysiology of Alzheimer disease.

Sulforaphane induces autophagy through ERK activation in neuronal cells.

Sulforaphane (SFN), an activator of nuclear factor E2-related factor 2 (Nrf2), has been reported to induce autophagy in several cells. However, little is known about its signaling mechanism of autophagic induction. Here, we provide evidence that SFN induces autophagy with increased levels of LC3-II through extracellular signal-regulated kinase (ERK) activation in neuronal cells. Pretreatment with NAC (N-acetyl-l-cysteine), a well-known antioxidant, completely blocked the SFN-induced increase in LC3-II levels and activation of ERK. Knockdown or overexpression of Nrf2 did not affect autophagy. Together, the results suggest that SFN-mediated generation of reactive oxygen species (ROS) induces autophagy via ERK activation, independent of Nrf2 activity in neuronal cells.

Three-dimensional analysis of abnormal ultrastructural alteration in mitochondria of hippocampus of APP/PSEN1 transgenic mouse.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder. The deterioration of subcellular organelles, including the mitochondria, is another major ultrastructural characteristic of AD pathogenesis, in addition to amyloid plaque deposition. However, the three-dimensional (3-D) study of mitochondrial structural alteration in AD remains poorly understood. Therefore, ultrastructural analysis, 3-D electron tomography, and immunogold electron microscopy were performed in the present study to clarify the abnormal structural alterations in mitochondria caused by the progression of AD in APP/PSEN1 transgenic mice, expressing human amyloid precursor protein, as a model for AD. Amyloid beta (A beta) plaques accumulated and dystrophic neurites (DN) developed in the hippocampus of transgenic AD mouse brains. We also identified the loss of peroxiredoxin 3, an endogenous cytoprotective antioxidant enzyme and the accumulation of A beta in the hippocampal mitochondria of transgenic mice, which differs from those in age-matched wild-type mice. The mitochondria in A beta plaque-detected regions were severely disrupted, and the patterns of ultrastructural abnormalities were classified into three groups: disappearance of cristae, swelling of cristae, and bulging of the outer membrane. These results demonstrated that morpho-functional alterations of mitochondria and AD progression are closely associated and may be beneficial in investigating the function of mitochondria in AD pathogenesis.

Immunoproteomically identified GBAA_0345, alkyl hydroperoxide reductase subunit C is a potential target for multivalent anthrax vaccine.

Anthrax is caused by the spore-forming bacterium Bacillus anthracis, which has been used as a weapon for bioterrorism. Although current vaccines are effective, they involve prolonged dose regimens and often cause adverse reactions. High rates of mortality associated with anthrax have made the development of an improved vaccine a top priority. To identify novel vaccine candidates, we applied an immunoproteomics approach. Using sera from convalescent guinea pigs or from human patients with anthrax, we identified 34 immunogenic proteins from the virulent B. anthracis H9401. To evaluate vaccine candidates, six were expressed as recombinant proteins and tested in vivo. Two proteins, rGBAA_0345 (alkyl hydroperoxide reductase subunit C) and rGBAA_3990 (malonyl CoA-acyl carrier protein transacylase), have afforded guinea pigs partial protection from a subsequent virulent-spore challenge. Moreover, combined vaccination with rGBAA_0345 and rPA (protective antigen) exhibited an enhanced ability to protect against anthrax mortality. Finally, we demonstrated that GBAA_0345 localizes to anthrax spores and bacilli. Our results indicate that rGBAA_0345 may be a potential component of a multivalent anthrax vaccine, as it enhances the efficacy of rPA vaccination. This is the first time that sera from patients with anthrax have been used to interrogate the proteome of virulent B. anthracis vegetative cells.

Chryseobacterium gwangjuense sp. nov., isolated from soil.

A Gram-stain-negative, strictly aerobic, non-motile, rod-shaped bacterial strain, THG-A18(T), was isolated from soil of Gwangju province in South Korea. Strain THG-A18(T) grew optimally at 25-30 °C, at pH 7.0-8.0 and in the absence of NaCl. Strain THG-A18(T) displayed ß-glucosidase activity, which enabled it to convert ginsenoside Rb1 to Rd. According to 16S rRNA gene sequence analysis, strain THG-A18(T) was shown to belong to the genus Chryseobacterium. The closest phylogenetic neighbours were Chryseobacterium ginsenosidimutans THG 15(T) (97.9 % 16S rRNA gene sequence similariity), C. defluvii B2(T) (97.7 %), C. daeguense K105(T) (97.6 %), C. taiwanense BCRC 17412(T) (97.5 %), C. indoltheticum LMG 4025(T) (97.4 %), C. gregarium P 461/12(T) (97.4 %) and C. lathyri RBA2-6(T) (97.3 %), but DNA-DNA relatedness values between these strains and strain THG-A18(T) were below 41.9 %. The G+C content of the genomic DNA was 36.4 mol%. The major respiratory quinone (MK-6) and fatty acids [iso-C15 : 0, iso-C17 : 0 3-OH, summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c) and summed feature 9 (comprising iso-C17 : 1ω9c and/or 10-methyl C16 : 0)] supported the affiliation of strain THG-A18(T) with the genus Chryseobacterium. The polar lipids of strain THG-A18(T) were phosphatidylethanolamine, four unidentified aminolipids and seven unidentified lipids. A number of physiological and biochemical tests allowed phenotypic differentiation of strain THG-A18(T) from recognized species of the genus Chryseobacterium. The name Chryseobacterium gwangjuense sp. nov. is proposed, with THG-A18(T) ( = KACC 16227(T) = LMG 26579(T)) as the type strain.

Acute modulations in stratum corneum permeability barrier function affect claudin expression and epidermal tight junction function via changes of epidermal calcium gradient.

Tight junction (TJ) is recognized as a second barrier of the skin. Altered expression of TJ proteins in various skin diseases characterized by the abnormal permeability barrier such as psoriasis suggests that TJ could be affected by stratum corneum (SC) barrier status. However, the physiological relationship between SC and TJ barrier remains to be investigated. Therefore, we examined the effect of SC barrier disruption on the expression of TJ proteins, claudin (Cldn)-1 and Cldn-4, and TJ barrier function in hairless mouse skin. We also investigated whether the alterations in epidermal Ca2+ affected TJ proteins expression in vivo. Repeated tape-stripping induced a sequential change of the expression and function of TJ. As early as 15-30 minutes after tape-stripping, downregulation of Cldn-1 and Cldn-4 immunoreactivity and protein level without change in mRNA level was found. This was accompanied by the abnormal leakage of lanthanum. However, by 1 hour Cldn-1 and Cldn-4 immunolocalization recovered along with normalized lanthanum permeation pattern. Moreover, the mRNA and protein levels of Cldn-1 and Cldn-4 were increased by 1 to 6 hours after tape-stripping. Inhibition of calcium loss by immersion of barrier-disrupted skin into a high Ca2+ solution prevented the dislocation of Cldn-1 and Cldn-4. Occlusion of barrier-disrupted skin delayed the restoration of Cldn-1 and Cldn-4. Our results suggest that the alteration of epidermal Ca2+ gradient caused by SC barrier perturbation affects the TJ structure and function and the faster recovery of TJ as compared to the SC barrier may imply the protective homeostatic mechanism of skin barrier.

Biologically inspired LED lens from cuticular nanostructures of firefly lantern.

Cuticular nanostructures found in insects effectively manage light for light polarization, structural color, or optical index matching within an ultrathin natural scale. These nanostructures are mainly dedicated to manage incoming light and recently inspired many imaging and display applications. A bioluminescent organ, such as a firefly lantern, helps to out-couple light from the body in a highly efficient fashion for delivering strong optical signals in sexual communication. However, the cuticular nanostructures, except the light-producing reactions, have not been well investigated for physical principles and engineering biomimetics. Here we report a unique observation of high-transmission nanostructures on a firefly lantern and its biological inspiration for highly efficient LED illumination. Both numerical and experimental results clearly reveal high transmission through the nanostructures inspired from the lantern cuticle. The nanostructures on an LED lens surface were fabricated by using a large-area nanotemplating and reconfigurable nanomolding with heat-induced shear thinning. The biologically inspired LED lens, distinct from a smooth surface lens, substantially increases light transmission over visible ranges, comparable to conventional antireflection coating. This biological inspiration can offer new opportunities for increasing the light extraction efficiency of high-power LED packages.

Overlapping distribution of osteopontin and calcium in the ischemic core of rat brain after transient focal ischemia.

Osteopontin (OPN), an adhesive glycoprotein, has recently been proposed to act as an opsonin that facilitates phagocytosis of neuronal debris by macrophages in the ischemic brain. The present study was designed to elucidate the process whereby OPN binds to neuronal cell debris in a rat model of ischemic stroke. Significant co-localization of the OPN protein and calcium deposits in the ischemic core were observed by combining alizarin red staining and OPN immunohistochemistry. In addition, electron microscopy (EM) using the osmium/potassium dichromate method revealed that electron-dense precipitates, typical of calcium deposits, were localized mainly along the periphery of putative degenerating neurites. This topical pattern of calcium precipitates resembled the distribution of OPN as detected by immunogold-silver EM. Combining immunogold-silver EM and electron probe microanalysis further demonstrated that the OPN protein was localized at the periphery of cell debris or degenerating neurites, corresponding with locally higher concentrations of calcium and phosphorus, and that the relative magnitude of OPN accumulation was comparable to that of calcium and phosphorus. These data suggest that calcium precipitation provides a matrix for the binding of the OPN protein within the debris or degenerating neurites induced by ischemic injury. Therefore, OPN binding to calcium deposits may be involved in phagocytosis of such debris, and may participate in the regulation of ectopic calcification in the ischemic brain.

Penetration pathways induced by low-frequency sonophoresis with physical and chemical enhancers: iron oxide nanoparticles versus lanthanum nitrates.

Low-frequency sonophoresis (LFS) has been shown to disrupt the structure of stratum corneum (SC) lipid bilayers and enhance SC permeability. In this study, we examined the penetration pathway of lanthanum nitrate (LaNO(3)) tracer in viable epidermis after combined treatment of LFS and tape stripping (TS), as a physical enhancer, or oleic acid (OA) application, as a chemical enhancer, using transmission electron microscopy (TEM). As a positive control, we visualized the passive diffusion pathway of LaNO(3) and iron oxide (Fe(3)O(4)) nanoparticles after the incision of hairless mouse skin. Next, we applied LFS immediately after TS or OA application and visualized the penetration pathway of LaNO(3). Each treatment showed restricted penetration to the SC-stratum granulosum (SG) interface or upper SG layer. However, the additional application of LFS induced diffuse intracellular distribution of LaNO(3) throughout the viable epidermis. Quantitative analysis also revealed that combined treatment significantly increases LaNO(3) penetration into viable epidermis when compared with each treatment. Our ultrastructural findings show the synergistic effect of LFS and TS or OA application on transdermal drug delivery. We also found that this combined treatment enhances the penetration of LaNO(3) through the viable epidermis through an intracellular pathway.

Scanning electron microscope study of ancient parasite eggs recovered from Korean mummies of the Joseon Dynasty.

We have previously shown that parasite eggs have been identified in the coprolites of Korean mummies. These eggs have shed light on parasitic infection patterns in Korean populations living several hundred years ago. We conducted a scanning electron microscopy (SEM) study on ancient Trichuris trichiura, Ascaris lumbricoides, Metagonimus yokogawai, Paragonimus westermani, and Gymnophalloides seoi eggs recovered from Korean mummies of the Joseon Dynasty. We anticipated that the taphonomic conditions of mummification would alter the eggs of certain species but not of others. Our SEM data show that each species of ancient egg exhibited different degrees of preservation. Thus, some of them, for example, M. yokogawai, exhibited a better preservation status than others, suggesting that they should be the first candidates considered when choosing subjects for future paleoparasitological studies.

The potential for non-invasive study of mummies: validation of the use of computerized tomography by post factum dissection and histological examination of a 17th century female Korean mummy.

The socio-cultural antipathies of some descendants with regard to invasive examinations of age-old human remains make permission for dissection of Korean mummies of the Joseon Dynasty (1392-1910) difficult to obtain. Overcoming this obstacle necessitated the use of non-invasive techniques, such as multi-detector computerized tomography (MDCT) and endoscopic examination, enabling determination of the preservation status of internal organs of mummies without significantly damaging the mummies themselves. However, MDCT alone cannot clearly differentiate specific mummified organs. Therefore, in much the same way as diagnostic radiologists make their MDCT readings on living patients more reliable by means of comparison with accumulated post-factum data from autopsies or histological studies, examinations of mummies by invasive techniques should not be decried as mere destruction of age-old human remains. Rather, providing that due permission from descendants and/or other relevant authorities can be obtained, dissection and histological examination should be performed whenever opportunities arise. Therefore, in this study, we compared the radiological data acquired from a 17th century mummy with our dissection results for the same subject. As accumulation of this kind of data could be very crucial for correct interpretation of MDCT findings on Korean mummies, we will perform similar trials on other Korean mummies found in forthcoming days if conditions permit.

Tegumental ultrastructure of adult Gynaecotyla squatarolae (Digenea: Microphallidae).

Gynaecotyla squatarolae (Digenea: Microphallidae) adult flukes were recovered from experimental chicks at day 4-6 post-infection and their tegumental ultrastructure was observed with a scanning electron microscopy. They were pyriform in shape, and their anterior halves were concaved ventrally. The whole body surface was covered with tegumental spines, which were wide and 16-17 digitated between oral and ventral suckers. The density of spines and number of digits decreased posteriorly. The oral sucker was subterminal and the excretory pore was at the posterior end of the worm. Two ventral suckers were similar in appearance and protruded near midline of the worm. The genital atrium was dextral to the small ventral sucker. The dorsal surface was covered with tegumental spines, but the spines were sparser than on the ventral surface. On the middle portion of the dorsal surface, a small opening presumed to be the Laurer's canal was seen. From these findings, it has been confirmed that the adult G. squatarolae has unique characteristics in the surface ultrastructure.