Separation of extracellular nanovesicles and apoptotic bodies from cancer cell culture broth using tunable microfluidic systems.

Extracellular vesicles (EVs) are the cell-secreted nano- and micro-sized particles consisted of lipid bilayer containing nucleic acids and proteins for diagnosis and therapeutic applications. The inherent complexity of EVs is a source of heterogeneity in various potential applications of the biological nanovesicles including analysis. To diminish heterogeneity, EV should be isolated and separated according to their sizes and cargos. However, current technologies do not meet the requirements. We showed noninvasive and precise separation of EVs based on their sizes without any recognizable damages. We separated atto-liter volumes of biological nanoparticles through operation of the present system showing relatively large volume of sample treatment to milliliters within an hour. We observed distinct size and morphological differences of 30 to 100 nm of exosomes and apoptotic bodies through TEM analysis. Indeed, we confirmed the biological moiety variations through immunoblotting with noninvasively separated EVs opening new windows in study and application of the biological nanoparticles.

Imitating Musical Scores on a Chip Through Droplet Generation.

We have demonstrated fine control of droplet volumes on a chip using pneumatically actuated micro valve systems resulting in confined nanoliter or picoliter reagent generation. Using the micro valves, we are able to align multi-inlet droplet generating chips for systematic generation of combinatorial mixtures of chemical constituents. By following the change of chemical conditions such as pH we converted musical note information into sequential droplets representing musical scores of 'Twinkle little star.' It would just one of the interesting application of micro-droplet generation systems based on accurate droplet generation, mixing, and incubation. It, thus, would be applied to combinatorial chemical and biological reactions increasing throughput and efficiency with various kinds of reagents.

Evaluation of peristaltic micromixers for highly integrated microfluidic systems.

Microfluidic devices based on the multilayer soft lithography allow accurate manipulation of liquids, handling reagents at the sub-nanoliter level, and performing multiple reactions in parallel processors by adapting micromixers. Here, we have experimentally evaluated and compared several designs of micromixers and operating conditions to find design guidelines for the micromixers. We tested circular, triangular, and rectangular mixing loops and measured mixing performance according to the position and the width of the valves that drive nanoliters of fluids in the micrometer scale mixing loop. We found that the rectangular mixer is best for the applications of highly integrated microfluidic platforms in terms of the mixing performance and the space utilization. This study provides an improved understanding of the flow behaviors inside micromixers and design guidelines for micromixers that are critical to build higher order fluidic systems for the complicated parallel bio/chemical processes on a chip.

A Microfluidic Approach to Investigating a Synergistic Effect of Tobramycin and Sodium Dodecyl Sulfate on Pseudomonas aeruginosa Biofilms.

In recent years, a microfluidic technology has contributed a significant role in biological research, specifically for the study of biofilms. Bacterial biofilms are a source of infections and contamination in the environment due to an extra polymeric matrix. Inadequate uses of antibiotics make the bacterial biofilms antibiotic resistant. Therefore, it is important to determine the effective concentration of antibiotics in order to eliminate bacterial biofilms. The present microfluidic study was carried out to analyze the activities of tobramycin and sodium dodecyl sulfate (SDS) against Pseudomonas aeruginosa biofilms with a continuous flow in order to achieve a greater delivery of the agents. The results show that a co-treatment of tobramycin and SDS significantly reduced the biomass of biofilms (by more than 99%) after 24 h. Tobramycin and SDS killed and detached bacteria in the cores of biofilms. Evidently, our data suggest that a microchannel would be effective for both quantitative and qualitative evaluations in order to test combinatorial effect of drugs and chemicals on a complexed biological system including biofilm.

Oral toxicity of isotretinoin, misoprostol, methotrexate, mifepristone and levonorgestrel as pregnancy category X medications in female mice.

An oral toxicity study of several pregnancy category X drugs was performed in female ICR mice. The drugs were administered orally once daily for 3 days at doses of 1, 10 and 100 µg/kg for isotretinoin; 6.7, 67 and 670 µg/kg for misoprostol; 83, 830 and 8,300 µg/kg for methotrexate; 3.3, 33 and 330 µg/kg for mifepristone; and 25, 250 and 2,500 µg/kg for levonorgestrel. During the test period, clinical signs, mortality, body weight, hematology, serum biochemistry and necropsy findings were examined. Following administration of methotrexate at 8,300 µg/kg, a number of animals exhibited decreased spontaneous activity, and one animal died. In the hematological analysis, compared with those treated with the control, the animals treated with the drugs exhibited similar significant decreases in the number of granulocytes and granulocyte differentiation, and increases in lymphocyte differentiation. In the serum biochemical analysis, animals receiving high doses of the five drugs demonstrated significant changes in uric acid, glucose, alkaline phosphatase, total bilirubin, lipase, total cholesterol and calcium. At necropsy, intestinal redness was frequently observed in animals that received the high dose of methotrexate. Uterus enlargement and ovary dropsy were also detected in the groups receiving mifepristone and levonorgestrel. Despite the short-term exposure, these drugs exhibited significant side effects, including white blood cell toxicity, in the mouse model. Category X drugs can be traded illegally via the internet for the purpose of early pregnancy termination. Thus, illegal abuse of the drugs should be further discouraged to protect mothers.

Microgravity Separation of Alginate Empty Capsules from Encapsulated Pancreatic Islets Using a Microfluidic System.

Although microencapsulated pancreatic islets have merits, such as ease of transplantation, viability and functionality improvement, and immune protection in vivo, the co-production of alginate empty capsules during the encapsulation of islets with alginate makes them unusable for biomedical application. In previous research, the removal of empty alginate capsules with high yield was achieved using density-gradient centrifugation. Here, we report advanced microgravity-based separation techniques in a microfluidic format for alginate empty capsules. The optimal separation conditions were mathematically evaluated using Stokes' law and the separation of the encapsulation product was accomplished. A microfluidic chip was designed with two inlets and two outlets at different elevations to mimic the vertical percoll gradient in density-gradient centrifugation. The separation of alginate empty capsules using microgravitational force resulted in effective separation of encapsulated islets from alginate empty capsules with more than 70% efficiency. Moreover, no loss of encapsulated islets was expected because the process is a one-pot separation, unlike the previous method. This type of microgravitational particle separation could be used both for the fractionization of heterogeneous encapsulated cells and to remove empty capsules.

Mild cognitive impairment due to alzheimer disease is less likely under the age of 65.

Patients with amnestic mild cognitive impairment (aMCI) are considered to have a high risk for Alzheimer dementia (AD). Even high positive predictive values, however, cannot be guaranteed even by tests with high sensitivity and specificity when disease prevalence is low. If we regard the clinical criteria for aMCI as a test for predicting aMCI due to AD, the positive predictive value of the criteria will be low by definition in young patients with aMCI (age below 65 years) because of the low prevalence of AD in this age group. To test this hypothesis, we compared CSF biomarkers for AD between young (age below 65 years) and old (age 65 years or older) age groups of normal cognition, aMCI, and AD of the Alzheimer's Disease Neuroimaging Initiative database. Using these biomarkers, we observed that the prevalence of aMCI due to AD differed significantly between the young and the old. For example, only 28.2% young aMCI, but 63.2% old aMCI, had abnormal CSF amyloid measures consistent with AD pathology. As posited, the presence of aMCI due to AD was lower in young aMCI than in old aMCI. Given that the likelihood of aMCI due to AD is reduced in younger subjects, more attention to and evaluation of alternative diagnoses need to be considered in this group.

Exogenously applied 24-epi brassinolide reduces lignification and alters cell wall carbohydrate biosynthesis in the secondary xylem of Liriodendron tulipifera.

The roles of brassinosteroids (BRs) in vasculature development have been implicated based on an analysis of Arabidopsis BR mutants and suspension cells of Zinnia elegans. However, the effects of BRs in vascular development of a woody species have not been demonstrated. In this study, 24-epi brassinolide (BL) was applied to the vascular cambium of a vertical stem of a 2-year-old Liriodendron, and the resulting chemical and anatomical phenotypes were characterized to uncover the roles of BRs in secondary xylem formation of a woody species. The growth in xylary cells was clearly promoted when treated with BL. Statistical analysis indicated that the length of both types of xylary cells (fiber and vessel elements) increased significantly after BL application. Histochemical analysis demonstrated that BL-induced growth promotion involved the acceleration of cell division and cell elongation. Histochemical and expression analysis of several lignin biosynthetic genes indicated that most genes in the phenylpropanoid pathway were significantly down-regulated in BL-treated stems compared to that in control stems. Chemical analysis of secondary xylem demonstrated that BL treatment induced significant modification in the cell wall carbohydrates, including biosynthesis of hemicellulose and cellulose. Lignocellulose crystallinity decreased significantly, and the hemicellulose composition changed with significant increases in galactan and arabinan. Thus, BL has regulatory roles in the biosynthesis and modification of secondary cell wall components and cell wall assembly during secondary xylem development in woody plants.

Separation of empty microcapsules after microencapsulation of porcine neonatal islets.

Pancreatic islet transplantation is used to treat diabetes mellitus that has minimal complications and avoids hypoglycemic shock. Conformal microencapsulation of pancreatic islets improves their function by blocking immunogenic molecules while protecting fragile islets. However, production of empty alginate capsules during microencapsulation causes enlargement of the transplantation volume of the encapsulated islets and interferes with efficient transfer of nutrients and insulin. In this study, empty alginate capsules were separated after microencapsulation of neonatal porcine islet-like cell clusters (NPCC) using density-gradient centrifugation. Densities of NPCC and alginate capsules were determined using Percoll. Encapsulation products following alginate removal were 97 % of products, with less than 10 % of the capsules remaining empty. The viability of this process compared with manually-selected encapsulated islets indicates the separation process does not harm islets.

Attachment of alginate microcapsules onto plasma-treated PDMS sheet for retrieval after transplantation.

Although transplantation of microencapsulated islets has been proposed as a therapy for the treatment of diabetes mellitus, limited retrievability of the cells has impeded its medical usage. To achieve retrieval of microencapsulated islets, capsules were attached to polydimethylsiloxane (PDMS) with a biocompatible adhesive. Because the hydrophobic nature of the PDMS surface prevents attachment, surface modification is essential. Alginate microcapsules were attached to modified PDMS sheets, and the mechanical stability of the resulting constructs was determined. Acrylic acid (AA) and acrylamide (AM) mixtures were grafted on the surfaces of PDMS sheets using a two-step oxygen plasma treatment (TSPT). TSPT-PDMS was characterized according to water contact angle and zeta-potential measurements. The contact angle was altered by changing the ratio of AM to AA to generate hydrophilic surface. Evaluation of the surface charge at pH 2, 7, and 12 confirmed the presence of polar groups on the modified surface. Microcapsules were attached to TSPT-PDMS using Histoacryl® and shown to be in a monolayered and half-exposed state. The shear stress resistance of alginate capsules attached to the PDMS sheet indicates the possibility of transplantation of encapsulated cells without scattering in vivo. This method is applicable to retrieve microencapsulated porcine islets when required.

Neuroanatomical substrates of visual hallucinations in patients with non-demented Parkinson's disease.

BACKGROUND: Visual hallucinations (VH), which are common in patients with Parkinson's disease (PD), lead to increased disability and are a significant predictor of the development of dementia. However, the neuroanatomical basis for VH in non-demented PD patients remains controversial. METHODS: A total of 110 patients with PD were classified into PD with VH (n=46) and PD without VH (n=64) groups, depending on the presence of VH assessed by the caregiver-based structured interview of the Neuropsychiatric Inventory. We performed voxel-based morphometry (VBM) for grey matter (GM) volume and a region-of-interest-based volumetric analysis of the substantia innominata (SI) between two groups. RESULTS: The comprehensive neuropsychological assessment showed that PD patients with VH showed more severe cognitive deficits in delayed visual memory and frontal executive functions compared with those without VH. A VBM analysis revealed that PD patients with VH had significantly lower GM volume in the right orbitofrontal, left temporal and left thalamic areas compared with those without VH. The normalised SI volume was significantly reduced in PD patients with VH compared with those without VH (1.28 ± 0.22 vs 1.41 ± 0.25, p=0.005). CONCLUSIONS: The present study demonstrates that non-demented PD patients with VH exhibited a smaller volume in the frontal, temporal and thalamic areas as well as the SI, suggesting that PD hallucinators may have distinctive neuroanatomical bases relative to PD non-hallucinators.

An objective pronator drift test application (iPronator) using handheld device.

BACKGROUND: The pronator drift test is widely used to detect mild arm weakness. We developed an application that runs on a handheld device to objectify the pronator drift test and investigated its feasibility in stroke patients. METHODS: The iPronator application, which uses the built-in accelerometer in handheld devices, was developed. We enrolled acute ischemic stroke patients (n = 10) with mild arm weakness and healthy controls (n = 10) to validate the iPronator. In addition to conventional neurological examinations, the degree of average, maximum, and oscillation in drift and pronation were measured and compared using the iPronator. Follow-up tests using the iPronator were also conducted in the patient group one week later. RESULTS: There was a strong correlation between the average degree of pronation and drift measured by the iPronator (r = 0.741, p<0.001). The degrees of average and maximum in pronation were greater in the patient group than in the control group [in average, 28.9°, interquartile range (IQR) 18.7-40.3 vs. 3.8° (IQR 0.3-7.5), p<0.001], in maximum, 33.0° (IQR 24.0-52.1) vs. 6.2° (IQR 1.4-9.4), p<0.001]. The degree of oscillation in pronation was not different between the groups (p = 0.166). In drift, the degrees of average, maximum, and oscillation were greater in the patient group. In stroke patients, a follow-up study at one week revealed improvements in the degrees of pronation and drift compared with baseline parameters. CONCLUSIONS: The iPronator can reliably detect mild arm weakness of stroke patients and was also useful in detecting functional recovery for one week in patients with acute stroke.

Functional improvement of porcine neonatal pancreatic cell clusters via conformal encapsulation using an air-driven encapsulator.

Transplantation of islet cells into diabetic patients is a promising therapy, provided that the islet cells are able to evade host immune rejection. With improved islet viability, this strategy may effectively reverse diabetes. We applied 2% calcium alginate to generate small and large capsules to encapsulate porcine neonatal pancreatic cell clusters (NPCCs) using an air-driven encapsulator. After encapsulation, the viability was assessed at 1, 4, 7, 14 and 28 days and secretion of functional insulin in response to glucose stimulation were tested at days 14 and 28. Selective permeability of the small alginate capsules was confirmed using various sizes of isothiocyanate-labeled dextran (FITC-dextran). Encapsulation of NPCCs was performed without islet protrusion in the small and large capsules. The viability of NPCCs in all experimental groups was greater than 90% at day 1 and then gradually decreased after day 7. The NPCCs encapsulated in large capsules showed significantly lower viability (79.50 ± 2.88%) than that of naïve NPCCs and NPCCs in small capsule (86.83 ± 2.32%, 87.67 ± 2.07%, respectively) at day 7. The viability of naïve NPCCs decreased rapidly at day 14 (75.67 ± 1.75%), whereas the NPCCs encapsulated in small capsules maintained (82.0 ± 2.19%). After 14 and 28 days NPCCs' function in small capsules (2.67 ± 0.09 and 2.13 ± 0.09) was conserved better compared to that of naïve NPCCs (2.04 ± 0.25 and 1.53 ± 0.32, respectively) and NPCCs in large capsules (2.04 ± 0.34 and 1.13 ± 0.10, respectively), as assessed by a stimulation index. The small capsules also demonstrated selective permeability. With this encapsulation technique, small capsules improved the viability and insulin secretion of NPCCs without islet protrusion.

Overexpression of constitutively active Arabidopsis RabG3b promotes xylem development in transgenic poplars.

An Arabidopsis small GTPase, RabG3b, was previously characterized as a component of autophagy and as a positive regulator for xylem development in Arabidopsis. In this work, we assessed whether RabG3b modulates xylem-associated traits in poplar in a similar way as in Arabidopsis. We generated transgenic poplars (Populus alba × Populus tremula var. glandulosa) overexpressing a constitutively active form of RabG3b (RabG3bCA) and performed a range of morphological, histochemical and molecular analyses to examine xylogenesis. RabG3bCA transgenic poplars showed increased stem growth due to enhanced xylem development. Autophagic structures were observed in differentiating xyelm cells undergoing programmed cell death (PCD) in wild-type poplar, and were more abundant in RabG3bCA transgenic poplar plants and cultured cells. Xylogenic activation was also accompanied by the expression of secondary wall-, PCD- and autophagy-related genes. Collectively, our results suggest that Arabidopsis RabG3b functions to regulate xylem growth through the activation of autophagy during wood formation in Populus, as does the same in Arabidopsis.

Partial nitrification using an electrolytic aerating bioreactor with ammonia-oxidizing bacteria-dominant activated sludge.

An electrolytic aerating bioreactor was used to partially nitrify ammonia from wastewater. Activated sludge was cultured for 8 months to increase the population of ammonia-oxidizing bacteria (AOB) and then used in the bioreactor. The maximum ammonia removal rate was 0.64 mM NH(3)/l h in a 50 ml reactor using 5.4 g mixed liquor suspended solids per litre of AOB-dominant activated sludge.

Ethanol production from acid hydrolysates based on the construction and demolition wood waste using Pichia stipitis.

The feasibility of ethanol production from the construction and demolition (C&D) wood waste acid hydrolysates was investigated. The chemical compositions of the classified C&D wood waste were analyzed. Concentrated sulfuric acid hydrolysis was used to obtain the saccharide hydrolysates and the inhibitors in the hydrolysates were also analyzed. The C&D wood waste composed of lumber, plywood, particleboard, and medium density fiberboard (MDF) had polysaccharide (cellulose, xylan, and glucomannan) fractions of 60.7-67.9%. The sugar composition (glucose, xylose, and mannose) of the C&D wood wastes varied according to the type of wood. The additives used in the wood processing did not appear to be released into the saccharide solution under acid hydrolysis. Although some fermentation inhibitors were detected in the hydrolysates, they did not affect the ethanol production by Pichia stipitis. The hexose sugar-based ethanol yield and ethanol yield efficiency were 0.42-0.46 g ethanol/g substrate and 84.7-90.7%, respectively. Therefore, the C&D wood wastes dumped in landfill sites could be used as a raw material feedstock for the production of bioethanol.

Enhanced ethanol production from deacetylated yellow poplar acid hydrolysate by Pichia stipitis.

In this study, alkaline-pretreatment for the extraction of acetic acid from xylan of hemicellulose was introduced prior to concentrated acid hydrolysis of yellow poplar wood meal. Ethanol fermentability in deacetylated yellow poplar hydrolysate (DYPH) by Pichia stipitis was also investigated. The alkali-pretreatment conditions were evaluated in terms of temperature, reaction time, and alkalinity. 94% of the acetyl group in xylan of the yellow poplar hemicellulose fraction was extracted using 0.5% sodium hydroxide solution at 60 degrees C for 60 min. The cell growth and ethanol production of P. stipitis was strongly affected by acetic acid, either in synthetic medium with 7.1g/l of acetic acid added or in yellow poplar hydrolysate (YPH) containing 7.1g/l of acetic acid. On the other hand, ethanol production in DYPH was slightly higher than that of the control although cell growth decreased by 34%. In the case of DYPH, the ethanol yield, volumetric ethanol productivity, and theoretical yield percentage was 0.48 g/g, 0.40 g/lh, and 93.2%, respectively. Thus, the alkaline-pretreatment method greatly enhanced the ethanol fermentability of yellow poplar hydrolysate.