Localized Induction of Gene Expression in Embryonic Stem Cell Aggregates Using Holographic Optical Tweezers to Create Biochemical Gradients.

Three-dimensional (3D) cell models that mimic the structure and function of native tissues are enabling more detailed study of physiological and pathological mechanisms in vitro. We have previously demonstrated the ability to build and manipulate 3D multicellular microscopic structures using holographic optical tweezers (HOTs). Here, we show the construction of a precisely patterned 3D microenvironment and biochemical gradient model consisting of mouse embryoid bodies (mEBs) and polymer microparticles loaded with retinoic acid (RA), embedded in a hydrogel. We demonstrate discrete, zonal expression of the RA-inducible protein Stra8 within mEBs in response to release of RA from polymer microparticles, corresponding directly to the defined 3D positioning of the microparticles using HOTs. These results demonstrate the ability of this technology to create chemical microgradients at definable length scales and to elicit, with fidelity and precision, specific biological responses. This technique can be used in the study of in vitro microenvironments to enable new insights on 3D cell models, their cellular assembly, and the delivery of drug or biochemical molecules for engineering and interrogation of functional and morphogenic responses. Graphical abstract.

Diet-induced obesity reduces the production of influenza vaccine-induced antibodies via impaired macrophage function.

Obesity is a metabolic disease characterized by low-level chronic inflammation. Obese individuals are susceptible to infection by viruses, and vaccination against these pathogens is less effective than in nonobese individuals. Here, we sought to explore the immunological environment in a mouse model of obesity induced by a high-fat diet (HFD). HFD treatment increased the body weight and epididymal fat mass. The proportion of activated B cells, T cells, and macrophages was similar between mice in the HFD group and the regular-fat diet (RFD) group. The Th1 cell subpopulation in the HFD group was increased, whereas the proportion of Treg cells was reduced compared with the RFD group. Moreover, T-cell proliferation and cytokine production did not differ between the groups when cells were stimulated with anti-CD3 and anti-CD28 antibodies in vitro. In macrophages, phagocytic activity was higher in mice fed an HFD than in those fed an RFD, but expression levels of CD86 and MHC class II antigens were similar. When macrophages were cultured in vitro, the proportion of CD86-expressing macrophages was lower in those isolated from mice in the HFD group than in those isolated from the RFD group. Furthermore, lipopolysaccharide-induced interleukin 6 (IL-6) and tumor necrosis factor alpha secretions were significantly reduced in macrophages isolated from the HFD group. In addition, influenza vaccine-induced antibodies in the HFD group diminished more rapidly than in the RFD group. These results suggest that poor functionality of macrophages during obesity might contribute to a reduction in vaccine efficacy.

A numerical study on the mechanical characteristics of zinc oxide-based transparent thin film transistors.

Zinc Oxide (ZnO) based Thin Film Transistors (TFTs) have been fabricated and analyzed to investigate mechanical characteristics regarding the stress, strain and deformation of electro circuits using the Finite Element Method (FEM). As the best compromise between the stretching and bending abilities, the coating thickness of SU-8 can be as important for bendability as a neutral mechanical plane. The neutral mechanical plane in electro circuits was designed for obtaining flexibility, e.g., bendability, in a previous numerical study. After that, through experimental validation, we observed what degree of SU-8 thickness was attributable for improved mechanical stability. The results suggest that not only numerical but also experimental measurements of the deformation and SU-8 coating thickness in electro circuits are useful for enhancing structural stability.

Techniques for analysing pattern formation in populations of stem cells and their progeny.

BACKGROUND: To investigate how patterns of cell differentiation are related to underlying intra- and inter-cellular signalling pathways, we use a stochastic individual-based model to simulate pattern formation when stem cells and their progeny are cultured as a monolayer. We assume that the fate of an individual cell is regulated by the signals it receives from neighbouring cells via either diffusive or juxtacrine signalling. We analyse simulated patterns using two different spatial statistical measures that are suited to planar multicellular systems: pair correlation functions (PCFs) and quadrat histograms (QHs). RESULTS: With a diffusive signalling mechanism, pattern size (revealed by PCFs) is determined by both morphogen decay rate and a sensitivity parameter that determines the degree to which morphogen biases differentiation; high sensitivity and slow decay give rise to large-scale patterns. In contrast, with juxtacrine signalling, high sensitivity produces well-defined patterns over shorter lengthscales. QHs are simpler to compute than PCFs and allow us to distinguish between random differentiation at low sensitivities and patterned states generated at higher sensitivities. CONCLUSIONS: PCFs and QHs together provide an effective means of characterising emergent patterns of differentiation in planar multicellular aggregates.

A comparative study of two- versus one-lung ventilation for needlescopic bleb resection.

This prospective study was conducted to evaluate the feasibility of two-lung (TL) ventilation with low tidal volume anaesthesia compared with one-lung (OL) ventilation for needlescopic bleb resection. Patients with spontaneous pneumothorax that underwent bleb resection with a 2-mm thoracoscope were enrolled. During the operation, the tidal volume was set at 4.0 mL·kg⁻¹ in the TL group and 8.0 mL·kg⁻¹ in the OL group; the respiration rate was set at 23 and 12 breaths·min⁻¹, respectively, at the same inspiratory oxygen fraction (50%). A total of 108 patients (55 patients in the TL group and 53 in the OL group) were included in this study. Airway pressure was significantly lower in the TL group (mean ± sd 8.0 ± 3.3 versus 24.0 ± 3.9 mmHg in the OL group; p<0.001). The time from endotracheal intubation to the incision was 17.1 ± 4.0 min in the TL group and 35.3 ± 7.6 min in the OL group, which was significantly different (p<0.001). However, the operation time was not different in comparisons between the two groups. Therefore, the total anaesthesia time was significantly longer in the OL group (77.9 ± 21.6 versus 64.9 ± 14.7 min in the TL group; p = 0.002). Needlescopic bleb resection using TL ventilation anaesthesia with low tidal volume was technically feasible, cost-effective and time-saving compared with OL ventilation anaesthesia.

The usefulness of clinical findings in localising lesions in Bell's palsy: comparison with MRI.

BACKGROUND: Although electrophysiological tests and brain MRI provide information about the site of the lesion in Bell's palsy, clinicians usually depend on clinical data. However, the accuracy of clinical findings in identifying lesions has never been evaluated. METHOD: A total of 57 patients with idiopathic peripheral facial palsy were included in this study. We determined the sites of the lesions based on associated symptoms and by brain MRI. We then compared the two to assess the value of clinical findings in determining lesion sites. RESULTS: Of the 57 patients, 27 were men. The mean age of all patients was 50.6+/-16.7 years. The lesion sites determined from clinical findings were as follows: the infrageniculate-suprastapedial segment, 13 (23%); the infrastapedial-suprachordal segment, 9 (16%); and the mastoid segment, 35 (61%). No sites were classified as involving the suprageniculate segment. On brain MRI, 51 (89%) of the 57 patients showed abnormal enhancement of the facial nerve, with the most common area being the suprageniculate segment, including the distal intrameatal, labyrinthine and geniculate ganglion. CONCLUSIONS: We demonstrate that clinical history is not helpful in determining the site of a lesion in Bell's palsy. The segment most frequently involved in Bell's palsy is the suprageniculate segment.

Inactivation of algal blooms in eutrophic water of drinking water supplies with the photocatalysis of TiO2 thin film on hollow glass beads.

Photocatalytic inactivation of algae, Anabaena, Microcystis, and Melosira, was carried out with TiO2-coated Pyrex hollow glass beads under the illumination of UV light (370 nm wavelength). After being irradiated with UV light in the presence of the TiO2-coated Pyrex glass beads, Anabaena and Microcystis, known as typical cyanobacteria, lost their photosynthetic activity, and the string of Anabaena cells and the colonies of Microcystis cells were completely separated into individual spherical ones. In the case of Melosira, which is a typical diatom, however, somewhat lower photocatalytic inactivation efficiency was obtained, which was believed to be due to the presence of the inorganic siliceous wall surrounding the cells of Melosira. The TiO2-coated hollow glass beads could successfully be employed for the practical application in a eutrophicated river under sunlight. More than 50% of the chlorophyll-a concentration could be reduced by the action of TiO2 photocatalysis.

Development of an In Vitro Screening Assay for Compounds that Increase Bone Formation.

Although parathyroid hormone (PTH) has the ability to stimulate bone growth in both rats and humans, its mechanism of action is not fully understood at the molecular level. An in vitro marker that reflects the in vivo anabolic actions of PTH would facilitate the discovery of small-molecule compounds that stimulate bone growth. We therefore compared the patterns of gene expression in three cell lines treated with PTH. The levels of c-fos, collagenase, interleukin-6 (IL-6), and collagen mRNA were determined by reverse transcription-polymerase chain reaction (RT-PCR) in three osteoblast-like cell lines. The most responsive marker was c-fos, which was induced 5-10-fold after 1 h of PTH treatment in the UMR106-01 cell line. Because it is a critical early response gene in bone growth, we investigated the possibility of using c-fos stimulation as a method to screen for compounds that can stimulate bone formation. A highly sensitive, medium-throughput RT-PCR assay for c-fos mRNA expression was established using the Taqmantrade mark Detection System (Perkin Elmer, Mississauga, Ontario). Cells were treated with a series of compounds to determine the specificity of c-fos stimulation. Of the compounds tested, only PTH, prostaglandin E(2), 8-bromo-cAMP, and forskolin induced c-fos mRNA levels, indicating that this assay was specific for compounds that are known to induce cAMP and stimulate bone growth. These results indicate that a simple in vitro assay for c-fos may be a reliable method for the screening of compounds that stimulate bone growth in vivo.