Evaluation of a Series of Turnip Mosaic Virus Chimeric Clones Reveals Two Amino Acid Sites Critical for Systemic Infection in Chinese Cabbage.

Two infectious clones of turnip mosaic virus (TuMV), pKBC-1 and pKBC-8, with differential infectivity in Chinese cabbage (Brassica rapa subsp. pekinensis), were obtained. Both infected Nicotiana benthamiana systemically, inducing similar symptoms, whereas only virus KBC-8 infected Chinese cabbage systemically. To identify the determinants affecting infectivity on Chinese cabbage, chimeric clones were constructed by restriction fragment exchange between the parental clones and tested on several Chinese cabbage cultivars. Chimeric clones p1N8C and p8N1C demonstrated that the C-terminal portion of the polyprotein determines systemic infection of Chinese cabbage despite only three amino acid differences in this region, in the cylindrical inclusion (CI), viral protein genome-linked (VPg), and coat protein (CP). A second pair of hybrid constructs, pHindIII-1N8C and pHindIII-8N1C, failed to infect cultivars CR Victory and Jinseonnorang systemically, yet pHindIII-1N8C caused hypersensitive response-like lesions on inoculated leaves of these cultivars, and could systemically infect cultivars CR Chusarang and Jeongsang; this suggests that R genes effective against TuMV may exist in the first two cultivars but not the latter two. Constructs with single amino acid changes in both VPg (K2045E) and CP (Y3095H) failed to infect Chinese cabbage, implying that at least one of these two amino acid substitutions is essential for successful infection on Chinese cabbage. Successful infection by mutant KBC-8-CP-H and delayed infection with mutant HJY1-VPg-E following mutation or reversion suggested that VPg (2045K) is the residue required for infection of Chinese cabbage and involved in the interaction between VPg and eukaryotic initiation factor eIF(iso)4E, confirmed by yeast two-hybrid assay.

Reassortment of Infectious Clones of Radish Mosaic Virus Shows that Systemic Necrosis in Nicotiana benthamiana Is Determined by RNA1.

Three infectious clones of radish mosaic virus (RaMV) were generated from isolates collected in mainland Korea (RaMV-Gg) and Jeju Island (RaMV-Aa and RaMV-Bb). These isolates differed in sequences and pathogenicity. Examination of the wild-type isolates and reassortants between the genomic RNA1 and RNA2 of these three isolates revealed that severe symptoms were associated with RNA1 of isolates Aa or Gg causing systemic necrosis in Nicotiana benthamiana, or with RNA1 of isolate Bb for induction of veinal necrosis and severe mosaic symptoms in radish. Reverse transcription, followed by quantitative real-time PCR (Q-RT-PCR), results from infected N. benthamiana confirmed that viral RNA2 accumulation level was correlated to RaMV necrosis-inducing ability, and that the RNA2 accumulation level was mostly dependent on the origin of RNA1. However, in radish, Q-RT-PCR results showed more similar viral RNA2 accumulation levels regardless of the ability of the isolate to induce necrosis. Phylogenetic analysis of genomic RNAs sequence including previously characterized isolates from North America, Europe, and Asia suggest possible recombination within RNA1, while analysis of concatenated RNA1+RNA2 sequences indicates that reassortment of RNA1 and RNA2 has been more important in the evolution of RaMV isolates than recombination. Korean isolate Aa is a potential reassortant between isolates RaMV-J and RaMV-TW, while isolate Bb might have evolved from reassortment between isolates RaMV-CA and RaMV-J. The Korean isolates were shown to also be able to infect Chinese cabbage, raising concerns that RaMV may spread from radish fields to the Chinese cabbage crop in Korea, causing further economic losses.

A microfluidic microparticle-labeled impedance sensor array for enhancing immunoassay sensitivity.

An impedimetric biosensor is used to measure electrical impedance changes in the presence of biomolecules from sinusoidal input voltages. In this paper, we present a new portable impedance-based biosensor platform to improve the sensitivity of immunoassays with microparticles as a label. Using a 2 × 4 interdigitated electrode array with a 10/10 µm electrode/gap and a miniaturized impedance analyzer, we performed immunoassays with microparticles by integrating a microfluidic channel to evaluate signal enhancement. First, to understand the material dependency of microparticles on the sensor array, magnetic, silica, and polystyrene microparticles were tested. Among these microparticles, magnetic microparticles presented a high signal enhancement with relevant stability from the sensor array. With the magnetic microparticles, we demonstrate a series of immunoassays to detect human tumor necrosis factor (TNF-α) and compare the level of signal enhancement by measuring the limit of detection (LOD). With the microparticles, we achieved over ten times improvement of LOD from sandwich immunoassays. By incorporating with sample preparation and flow manipulation systems, this impedance sensor array can be utilized for digital diagnostics for a real sample-in answer-out system.

A single nucleotide change in the overlapping MP and CP reading frames results in differences in symptoms caused by two isolates of Youcai mosaic virus.

Two isolates of Youcai mosaic virus (YoMV) were obtained, and their full-length genomic sequences were determined. Full-length infectious cDNA clones of each isolate were generated in which the viral sequence was under the control of dual T7 and 35S promoters for both in vitro transcript production and agro-infiltration. Comparison of the predicted amino acid sequences of the encoded proteins revealed only four differences between the isolates: three in the RNA-dependent RNA polymerase (RdRp) (V383I and M492I in the 125-kDa protein and T1245M in the 182-kDa protein); and one in the overlapping region of the movement protein (MP) and coat protein (CP) genes, affecting only the N-terminal domain of CP (CP M17T). One of the isolates caused severe symptoms in Nicotiana benthamiana plants, while the other caused only mild symptoms. In order to identify the amino acid residues associated with symptom severity, chimeric constructs were generated by combining parts of the two infectious YoMV clones, and the symptoms in infected plants were compared to those induced by the parental isolates. This allowed us to conclude that the M17T substitution in the N-terminal domain of CP was responsible for the difference in symptom severity. The M17T variation was found to be unique among characterized YoMV isolates. A difference in potential post-translational modification resulting from the presence of a predicted casein kinase II phosphorylation site only in the CP of isolate HK2 may be responsible for the symptom differences.

Sequence Variations Among 17 New Radish Isolates of Turnip mosaic virus Showing Differential Pathogenicity and Infectivity in Nicotiana benthamiana, Brassica rapa, and Raphanus sativus.

Infectious clones were generated from 17 new Korean radish isolates of Turnip mosaic virus (TuMV). Phylogenetic analysis indicated that all new isolates, and three previously characterized Korean radish isolates, belong to the basal-BR group (indicating that the pathotype can infect both Brassica and Raphanus spp.). Pairwise analysis revealed genomic nucleotide and polyprotein amino acid identities of >87.9 and >95.7%, respectively. Five clones (HJY1, HJY2, KIH2, BE, and prior isolate R007) had lower sequence identities than other isolates and produced mild symptoms in Nicotiana benthamiana. These isolates formed three distinct sequence classes (HJY1/HJY2/R007, KIH2, and BE), and several differential amino acid residues (in P1, P3, 6K2, and VPg) were present only in mild isolates HJY1, HJY2, and R007. The remaining isolates all induced systemic necrosis in N. benthamiana. Four mild isolates formed a phylogenetic subclade separate from another subclade including all of the necrosis-inducing isolates plus mild isolate KIH2. Symptom severity in radish and Chinese cabbage genotypes was not correlated with pathogenicity in N. benthamiana; indeed, Chinese cabbage cultivar Norang was not infected by any isolate, whereas Chinese cabbage cultivar Chusarang was uniformly susceptible. Four isolates were unable to infect radish cultivar Iljin, but no specific amino acid residues were correlated with avirulence. These results may lead to the identification of new resistance genes against TuMV.

A capillary flow-driven microfluidic system for microparticle-labeled immunoassays.

A simple, reliable, and self-powered capillary flow-driven microfluidic platform is developed for conducting microparticle-labeled immunoassays. To obtain the washing forces and binding kinetics appropriate for microparticle-labeled immunoassays, both microchannel networks and sample access holes are designed and characterized to confirm the fluidic routes. To demonstrate two different types of immunoassays, serial and parallel capillary-driven microfluidic platforms were developed for mouse immunoglobulin G (IgG) and cardiac troponin I (cTnI) using detection antibody-conjugated microparticles, respectively. With the serial capillary-driven microfluidic platform, we successfully demonstrated IgG quantification using direct immunoassay and achieved a limit of detection (LOD) of 30 pM by using pre-immobilized mouse IgG. In the parallel capillary-driven microfluidic platform, a sandwich immunoassay for detecting cTnI was demonstrated and a clinically relevant LOD as low as 4.2 pM was achieved with minimal human intervention. In both assays, the association rate constants (Ka) were measured to estimate the overall assay time. According to these estimations, microparticle-labeled immunoassays could be conducted in a few minutes using the proposed capillary-driven microfluidic devices. By coupling with various magnetic sensors, these simple immunoassay platforms enable us to achieve a true sample-in-answer-out device that can screen for a variety of targets without relying on external power sources for fluidic manipulation.

Complete nucleotide sequences and construction of full-length infectious cDNA clones of cucumber green mottle mosaic virus (CGMMV) in a versatile newly developed binary vector including both 35S and T7 promoters.

Seed-transmitted viruses have caused significant damage to watermelon crops in Korea in recent years, with cucumber green mottle mosaic virus (CGMMV) infection widespread as a result of infected seed lots. To determine the likely origin of CGMMV infection, we collected CGMMV isolates from watermelon and melon fields and generated full-length infectious cDNA clones. The full-length cDNAs were cloned into newly constructed binary vector pJY, which includes both the 35S and T7 promoters for versatile usage (agroinfiltration and in vitro RNA transcription) and a modified hepatitis delta virus ribozyme sequence to precisely cleave RNA transcripts at the 3' end of the tobamovirus genome. Three CGMMV isolates (OMpj, Wpj, and Mpj) were separately evaluated for infectivity in Nicotiana benthamiana, demonstrated by either Agroinfiltration or inoculation with in vitro RNA transcripts. CGMMV nucleotide identities to other tobamoviruses were calculated from pairwise alignments using DNAMAN. CGMMV identities were 49.89% to tobacco mosaic virus; 49.85% to pepper mild mottle virus; 50.47% to tomato mosaic virus; 60.9% to zucchini green mottle mosaic virus; and 60.96% to kyuri green mottle mosaic virus, confirming that CGMMV is a distinct species most similar to other cucurbit-infecting tobamoviruses. We further performed phylogenetic analysis to determine relationships of our new Korean CGMMV isolates to previously characterized isolates from Canada, China, India, Israel, Japan, Korea, Russia, Spain, and Taiwan available from NCBI. Analysis of CGMMV amino acid sequences showed three major clades, broadly typified as 'Russian,' 'Israeli,' and 'Asian' groups. All of our new Korean isolates fell within the 'Asian' clade. Neither the 128 nor 186 kDa RdRps of the three new isolates showed any detectable gene silencing suppressor function.

New Korean isolates of Pepper mild mottle virus (PMMoV) differ in symptom severity and subcellular localization of the 126 kDa protein.

Two isolates of Pepper mild mottle virus (PMMoV) were selected from a nationwide survey of pepper fields in South Korea in 2014 and 2015, in which Cucumber mosaic virus was also detected; the two PMMoV isolates, Sangcheong 47 (S-47, KX399390) and Jeongsong 76 (J-76, KX399389), share ~99% nucleotide and amino acid identity and are closely related to Japanese and Chinese isolates at the nucleotide level. Amino acid sequence comparisons revealed 99.73, 99.81, 98.44, and 100% identity in the ORF1, ORF2, MP, and CP, respectively, between S-47 and J-76. In addition, we generated infectious clones of S-47 and J-76, and T7 promoter driven transcripts of each inoculated to Nicotiana benthamiana produced very severe symptoms, whereas only mild symptoms developed in Capsicum annuum. Gene silencing suppressor function of 126 kDa and cytoskeleton-connected plasmodesmata localization of movement protein of S-47 and J-76 showed no difference between isolates, whereas 126 kDa of J-76 clearly formed intracellular aggregates not observed with S-47 126 kDa protein. Differences between these isolates in 126/183 kDa-related functions including subcellular localization suggest that differential interactions with host proteins may affect symptom development in C. annuum.

Utilizing intentional internal resonance to achieve multi-harmonic atomic force microscopy.

During dynamic atomic force microscopy (AFM), the deflection of a scanning cantilever generates multiple frequency terms due to the nonlinear nature of AFM tip-sample interactions. Even though each frequency term is reasonably expected to encode information about the sample, only the fundamental frequency term is typically decoded to provide topographic mapping of the measured surface. One of main reasons for discarding higher harmonic signals is their low signal-to-noise ratio. Here, we introduce a new design concept for multi-harmonic AFM, exploiting intentional nonlinear internal resonance for the enhancement of higher harmonics. The nonlinear internal resonance, triggered by the non-smooth tip-sample dynamic interactions, results in nonlinear energy transfers from the directly excited fundamental bending mode to the higher-frequency mode and, hence, enhancement of the higher harmonic of the measured response. It is verified through detailed theoretical and experimental study that this AFM design can robustly incorporate the required internal resonance and enable high-frequency AFM measurements. Measurements on an inhomogeneous polymer specimen demonstrate the efficacy of the proposed design, namely that the higher harmonic of the measured response is capable of enhanced simultaneous topography imaging and compositional mapping, exhibiting less crosstalk with an abrupt height change.

Comparison of helper component-protease RNA silencing suppression activity, subcellular localization, and aggregation of three Korean isolates of Turnip mosaic virus.

In 2014, we performed a nationwide survey in Korean radish fields to investigate the distribution and variability of Turnip mosaic virus (TuMV). Brassica rapa ssp. pekinensis sap-inoculated with three isolates of TuMV from infected radish tissue showed different symptom severities, whereas symptoms in Raphanus sativus were similar for each isolate. The helper component-protease (HC-Pro) genes of each isolate were sequenced, and phylogenetic analysis showed that the three Korean isolates were clustered into the basal-BR group. The HC-Pro proteins of these isolates were tested for their RNA silencing suppressor (VSR) activity and subcellular localization in Nicotiana benthamiana. A VSR assay by co-agroinfiltration of HC-Pro with soluble-modified GFP (smGFP) showed that HC-Pro of isolate R007 and R041 showed stronger VSR activity than R065. The HC-Pros showed 98.25 % amino acid identity, and weak VSR isolate (R065) has a single variant residue in the C-terminal domain associated with protease activity and self-interaction compared to isolates with strong VSR activity. Formation of large subcellular aggregates of GFP:HC-Pro fusion proteins in N. benthamiana was only observed for HC-Pro from isolates with strong VSR activity, suggesting that R065 'weak' HC-Pro may have diminished self-association; substitution of the variant C-terminal residue largely reversed the HC-Pro aggregation and silencing suppressor characteristics. The lack of correlation between VSR efficiency and induction of systemic necrosis (SN) suggests that differences in viral accumulation due to HC-Pro are not responsible for SN.

Microfluidic linear hydrogel array for multiplexed single nucleotide polymorphism (SNP) detection.

A PDMS-based microfluidic linear hydrogel array is developed for multiplexed single nucleotide polymorphism (SNP) detection. A sequence of three-dimensional (3D) hydrogel plugs containing the desired DNA probes is prepared by UV polymerization within a PDMS microchannel system. The fluorescently labeled target DNA is then electrophoresed through the sequence of hydrogel plugs for hybridization. Continued electrophoresis provides an electrophoretic wash that removes nonspecific binders. The capture gel array is imaged after washing at various temperatures (temperature gradient electrophoresis) to further distinguish perfect matches from mismatches. The ability of this microdevice to perform multiplex SNP genotyping is demonstrated by analyzing a mixture of model E. coli bacterial targets. This microfluidic hydrogel array is ∼1000 times more sensitive than planar microarrays due to the 3D gel capture, the hybridization time is much shorter due to electrophoretic control of the transport properties, and the stringent wash with temperature gradient electrophoresis enables analysis of single nucleotide mismatches with high specificity.

Sequence variability in the HC-Pro coding regions of Korean soybean mosaic virus isolates is associated with differences in RNA silencing suppression.

Soybean mosaic virus (SMV), a member of the family Potyviridae, is an important viral pathogen affecting soybean production in Korea. Variations in helper component proteinase (HC-Pro) sequences and the pathogenicity of SMV samples from seven Korean provinces were compared with those of previously characterized SMV isolates from China, Korea and the United States. Phylogenetic analysis separated 16 new Korean SMV isolates into two groups. Fourteen of the new Korean SMV samples belonged to group II and were very similar to U.S. strain SMV G7 and Chinese isolate C14. One isolate in group II, A297-13, differed at three amino acid positions (L54F, N286D, D369N) in the HC-Pro coding sequence from severe isolates and SMV 413, showed very weak silencing suppressor activity, and produced only mild symptoms in soybean. To test the role of each amino acid substitution in RNA silencing and viral RNA accumulation, a series of point mutations was constructed. Substitution of N for D at position 286 in HC-Pro of SMV A297-12 significantly reduced silencing suppression activity. When the mutant HC-Pro of A297-13 was introduced into an infectious clone of SMV 413, accumulation of viral RNA was reduced to less than 3 % of the level of SMV 413 containing HC-Pro of A297-12 at 10 days post-inoculation (dpi) but increased to 40 % of SMV 413(HC-Pro A297-12) at 40 dpi. At 50 dpi RNA accumulation of SMV 413(HC-Pro A297-13) was similar to that of SMV 413(HC-Pro A297-12). However, at 50 dpi, the D at position 286 of HC-Pro in SMV 413(HC-Pro A297-13) was found to have reverted to N. The results showed that 1) a naturally occurring mutation in HC-Pro significantly reduced silencing suppression activity and accumulation of transgene and viral RNAs, and 2) that there was strong selection for revision to wild type when the mutation was introduced into an infectious clone of SMV.

Regulation of Corneal Stromal Cell Behavior by Modulating Curvature Using a Hydraulically Controlled Organ Chip Array.

Curvature is a critical factor in cornea mechanobiology, but its impact on phenotypic alterations and extracellular matrix remodeling of cornea stroma remains unclear. In this work, we investigated how curvature influences the corneal stroma using a hydraulically controlled curvature array chip. The responses of stromal cells to low, medium, and high curvatures were observed by preparing three phenotypes of corneal stromal cells: corneal keratocytes, fibroblasts, and myofibroblasts. Keratocytes exhibited phenotypic alterations in response to curvature changes, notably including a decrease in ALDH3 expression and an increase in α-SMA expression. For focal adhesion, corneal fibroblast and myofibroblasts showed enhanced vinculin localization in response to curvature, while corneal keratocytes presented reduced vinculin expression. For cell alignment and ECM expression, most stromal cells under all curvatures showed a radially organized f-actin and collagen fibrils. Interestingly, for corneal fibroblast under medium curvature, we observed orthogonal cell alignment, which is linked to the unique hoop and meridional stress profiles of the curved surface. Furthermore, lumican expression was upregulated in corneal keratocytes, and keratocan expression was increased in corneal fibroblasts and myofibroblasts due to curvature. These results demonstrate that curvature influences both the phenotype of corneal stromal cells and the structural organization of corneal stroma tissue without any external stimuli. This curvature-dependent behavior of corneal stromal cells presents potential opportunities for creating therapeutic strategies for corneal shape dysfunctions.

Analysis of Early Iron Sulfide, Carbonate, and Phosphate Mineral Analogues Produced by Flow-Driven Precipitation in a Microchannel.

Most of the chemical and physical interactions of interest to the astrobiology community are influenced by the mineralogy of the systems under consideration. Often, this mineralogy occurs in sediment or sediment-like aqueous microenvironments in which the early minerals differ dramatically from the mature version that results from a long diagenesis, which are tied to complex interactions of pH, redox state, concentration, and temperature. This interconnectedness is difficult to reproduce in a laboratory setting yet is essential to understanding how the physical and chemical demands of living systems alter and are altered by their geological context. We present a facile means for producing precipitated mineral analogues within a microchannel and demonstrate its analytical efficacy through instrumental and modeling techniques. We show that amorphous, early-stage analogues of iron sulfide, iron carbonate, and iron phosphate can be formed at the boundary between flowing solutions, modeled on the microscale, and analyzed by standard instrumental techniques such as scanning electron microscopy/energy-dispersive spectroscopy, X-ray photoelectron spectroscopy, and Raman spectroscopy.

Effect of hemicellulose hydrolysate addition on the dehydration and redispersion characteristic of cellulose nanofibrils.

Nanocellulose, owing to its environmentally friendly and unique attributes, is gaining traction in various industries. However, commercialization of nanocellulose faces challenges due to structural alterations during drying process, leading to irreversible aggregation. This study, inspired by wood's natural structure, introduces a cellulose nanofibril (CNF) drying system using hemicellulose hydrolysate (HH) as a capping agent. The addition of only 1 wt% of HH to the CNF suspension not only prevents aggregation among CNFs during dehydration and drying but also dramatically enhances the redispersion rate and dispersion stability of the dried CNFs. The redispersed CNF/HH suspension exhibits physicochemical properties comparable to the original CNF suspension before drying. This confirms that HH inhibits irreversible hydrogen bonding among CNFs, leading to the restoration of the nanostructure during redispersion. Moreover, HH in the CNF suspension after redispersion can be easily removed through a simple water rinsing process, highlighting HH as a highly suitable candidate for preventing aggregation of CNFs.

Lignin/PVA hydrogel with enhanced structural stability for cationic dye removal.

In this study, a lignin-based hydrogel for wastewater treatment was prepared by incorporating kraft lignin (KL) into a poly (vinyl alcohol) (PVA) matrix. The underwater structural stability of the KL-PVA hydrogel was guaranteed through physicochemical crosslinking, involving freeze-thaw process and chemical crosslinking reaction. The KL-PVA hydrogel displayed superior compressive characteristics compared to the original PVA hydrogel. This improvement was attributed to the chemical crosslinking and the reinforcing effect of the incorporated KL microparticles. The incorporation of anionic KL microparticles into the PVA three-dimensional network structure enhanced the cationic methylene blue (MB) and crystal violet (CV) adsorption efficiency of the prepared KL-PVA hydrogel. The MB adsorption results were well explained by pseudo-2nd order kinetics model and Langmuir isotherm model. Electrostatic forces, hydrogen bonding and π-π stacking interactions were the main adsorption mechanisms between cationic dyes and KL surfaces, indicating the potential of KL-PVA hydrogel as an effective adsorption material. Moreover, regulating the molecular weight of PVA not only prevented lignin leakage from the KL-PVA hydrogel but also elevated the KL content within the hydrogel, consequently improving its dye removal performance. For KL-PVA hydrogel with high molecular weight PVA, the MB and CV adsorption capacities were 193.8 mg/g and 190.0 mg/g, respectively.

Integrated high performance microfluidic organic analysis instrument for planetary and space exploration.

The exploration of our solar system to characterize the molecular organic inventory will enable the identification of potentially habitable regions and initiate the search for biosignatures of extraterrestrial life. However, it is challenging to perform the required high-resolution, high-sensitivity chemical analyses in space and in planetary environments. To address this challenge, we have developed a microfluidic organic analyzer (MOA) instrument that consists of a multilayer programmable microfluidic analyzer (PMA) for fluidic processing at the microliter scale coupled with a microfabricated glass capillary electrophoresis (CE) wafer for separation and analysis of the sample components. Organic analytes are labeled with a functional group-specific (e.g. amine, organic acid, aldehyde) fluorescent dye, separated according to charge and hydrodynamic size by capillary electrophoresis (CE), and detected with picomolar limit of detection (LOD) using laser-induced fluorescence (LIF). Our goal is a sensitive automated instrument and autonomous process that enables sample-in to data-out performance in a flight capable format. We present here the design, fabrication, and operation of a technology development unit (TDU) that meets these design goals with a core mass of 3 kg and a volume of <5 L. MOA has a demonstrated resolution of 2 × 105 theoretical plates for relevant amino acids using a 15 cm long CE channel and 467 V cm-1. The LOD of LIF surpasses 100 pM (0.01 ppb), enabling biosignature detection in harsh environments on Earth. MOA is ideally suited for probing biosignatures in potentially habitable destinations on icy moons such as Europa and Enceladus, and on Mars.

Universal microfluidic automaton for autonomous sample processing: application to the Mars Organic Analyzer.

A fully integrated multilayer microfluidic chemical analyzer for automated sample processing and labeling, as well as analysis using capillary zone electrophoresis is developed and characterized. Using lifting gate microfluidic control valve technology, a microfluidic automaton consisting of a two-dimensional microvalve cellular array is fabricated with soft lithography in a format that enables facile integration with a microfluidic capillary electrophoresis device. The programmable sample processor performs precise mixing, metering, and routing operations that can be combined to achieve automation of complex and diverse assay protocols. Sample labeling protocols for amino acid, aldehyde/ketone and carboxylic acid analysis are performed automatically followed by automated transfer and analysis by the integrated microfluidic capillary electrophoresis chip. Equivalent performance to off-chip sample processing is demonstrated for each compound class; the automated analysis resulted in a limit of detection of ~16 nM for amino acids. Our microfluidic automaton provides a fully automated, portable microfluidic analysis system capable of autonomous analysis of diverse compound classes in challenging environments.

Bee venom at different concentrations modulates the aeroallergen-induced activation of nasal polyp epithelial cells.

Bee venom (BV) has long been used as an oriental traditional medicine for the control of pain and inflammation. However, BV's anti-inflammatory mechanisms remain unclear. This study aimed to clarify the potential clinical efficacy of BV concerning the anti-inflammatory effect on nasal epithelial cell inflammation. Nasal polyp epithelial cells were obtained from patients. Cells were exposed to Alternaria alternata, Aspergillus nigra, Dermatophagoides pteronyssinus, Dermatophagoides farina and lipopolysaccharide with or without various concentrations of BV. Interleukin (IL)-6, IL-8, and granulocyte macrophage colony-stimulating factor were measured to determine the activation of epithelial cells. Nuclear factor-ĸB (NF-ĸB) and activator protein 1 expression and activity were determined with Western blot analysis and ELISA. Cytotoxicity of BV was measured using a CellTiter-96® aqueous cell proliferation assay kit. Cell survival was significantly decreased at BV concentrations exceeding 5 µg/ml. Fungi-induced cytokine production was more effectively inhibited by BV than house dust mite. Alternaria enhanced NF-ĸB expression, which was strongly inhibited by BV. BV appears to be relatively safe, and is of potential value for the treatment of airway inflammation and/or immunologic diseases.