Structural basis for ion selectivity in potassium-selective channelrhodopsins.

KCR channelrhodopsins (K+-selective light-gated ion channels) have received attention as potential inhibitory optogenetic tools but more broadly pose a fundamental mystery regarding how their K+ selectivity is achieved. Here, we present 2.5-2.7 Å cryo-electron microscopy structures of HcKCR1 and HcKCR2 and of a structure-guided mutant with enhanced K+ selectivity. Structural, electrophysiological, computational, spectroscopic, and biochemical analyses reveal a distinctive mechanism for K+ selectivity; rather than forming the symmetrical filter of canonical K+ channels achieving both selectivity and dehydration, instead, three extracellular-vestibule residues within each monomer form a flexible asymmetric selectivity gate, while a distinct dehydration pathway extends intracellularly. Structural comparisons reveal a retinal-binding pocket that induces retinal rotation (accounting for HcKCR1/HcKCR2 spectral differences), and design of corresponding KCR variants with increased K+ selectivity (KALI-1/KALI-2) provides key advantages for optogenetic inhibition in vitro and in vivo. Thus, discovery of a mechanism for ion-channel K+ selectivity also provides a framework for next-generation optogenetics.

Establishment of an inspection standard for decomposition and methane production rates of organic waste co-digestion facilities in Korea.

Domestic organic waste resources have increased over the past decade and treatment of this waste via co-digested biogasification facilities is increasing annually. However, inspection standards for such facilities are not well-established. Herein, we aimed to derive calculation formulas and factors related to organic matter decomposition efficiency and methane production rate in accordance with waste treatment facility inspection standards. We also aimed to determine the optimum waste mixing ratio. Sample (field) surveys of 18 treatment facilities and complete enumeration of 110 facilities were conducted. Calculation formulas and factors were derived using the survey data and biochemical methane potential (BMP) test. The calculated coefficients derived through the BMP test were 0.512 m3 CH4/kgVSin for food waste, 0.601 m3 CH4/kgVSin for livestock manure, and 0.382 m3 CH4/kgVSin for sewage sludge. The final derived calculation factors were 65.0% for food waste, 36.0% for livestock manure, and 20.0% for sewage sludge for organic matter decomposition efficiency, and 0.380 m3 CH4/kgVSin for food waste, 0.27 m3 CH4/kgVSin for livestock manure, and 0.140 m3 CH4/kgVSin for sewage sludge for methane production rates. The derived effective capacity calculation factors can be utilized in future waste treatment facility inspection methods by aiding in the establishment of appropriate inspection standards for co-digested biogasification facilities other than single food waste treatment facilities. In addition, the optimum mixing ratio can be used as design data for co-digested biogasification facilities.

A unique photochromic UV-A sensor protein, Rc-PYP, interacting with the PYP-binding protein.

Photoactive yellow protein (PYP) is one of the typical light sensor proteins. Although its photoreaction has been extensively studied, no downstream partner protein has been identified to date. In this study, the intermolecular interaction dynamics observed between PYP from Rhodobacter capsulatus (Rc-PYP) and a possible downstream protein, PYP-binding protein (PBP), were investigated. It was found that UV light induced a long-lived product (pUV*), which interacts with PBP to form a stable hetero-hexamer (Complex-2). The reaction scheme for this interaction was revealed using transient absorption and transient grating methods. Time-resolved diffusion detection showed that a hetero-trimer (Complex-1) is formed transiently, which produced Complex-2 via a second-order reaction. Any other intermediates, including those from pBL, do not interact with PBP. The reaction scheme and kinetics are determined. Interestingly, long-lived Complex-2 dissociates upon excitation with blue light. These results demonstrate that Rc-PYP is a photochromic and new type of UV sensor to sense the relative intensities of UV-A and blue light.

Effect of Hydrated Ionic Liquid on Photocycle and Dynamics of Photoactive Yellow Protein.

The mechanism by which proteins are solvated in hydrated ionic liquids remains an open question. Herein, the photoexcitation dynamics of photoactive yellow protein dissolved in hydrated choline dihydrogen phosphate (Hy[ch][dhp]) were studied by transient absorption and transient grating spectroscopy. The photocyclic reaction of the protein in Hy[ch][dhp] was similar to that observed in the buffer solution, as confirmed by transient absorption spectroscopy. However, the structural change of the protein during the photocycle in Hy[ch][dhp] was found to be different from that observed in the buffer solution. The known change in the diffusion coefficient of the protein was apparently suppressed in high concentrations of [ch][dhp], plausibly due to stabilization of the secondary structure.

Wavelength-Dependent Photoreaction of PYP from Rhodobacter capsulatus.

PYPs (photoactive yellow proteins) are blue light sensor proteins found in more than 100 species. Compared with the extensive and intensive studies of the reactions of PYP from Halorhodospira halophila (Hh-PYP), studies of the reactions of other PYPs are scarce. Here, the photoreaction of PYP from Rhodobacter capsulatus (Rc-PYP) was studied in detail using ultraviolet-visible absorption and transient grating methods. Rc-PYP exhibits two absorption peaks at 375 and 438 nm. By using the transient absorption and the temperature-dependent absorption spectrum, the absorption spectra of two forms, pUV and pBL, were determined. Upon photoexcitation of pBL, two intermediates are observed before returning back to the dark state, with a time constant of 1.2 ms, which is 3 orders of magnitude faster than the dark recovery of Hh-PYP. Upon photoexcitation of pUV, two intermediates are observed to produce a long-lived final product, although one of the processes is spectrally silent. The diffusion coefficients decreased transiently for both pBL and pUV reactions, suggesting a relatively large conformational change during the reactions. It is particularly interesting to observe that the blue light irradiation of the long-lived product of pUV returns the product to the dark state. This result suggests different opposing responses of the biological function due to photoexcitation by ultraviolet and blue lights.

Atomistic insight into the promising thermoelectric performance of the copper-based ternary phosphide CaCuP.

A first-principles study on the thermoelectric properties of the copper-based ternary phosphide CaCuP is presented. Self-energy relaxation time approximation and unified theory for lattice transport provide an accurate description of electron-phonon and phonon-phonon scattering. Our work provides an atomistic insight into its high thermoelectric performance, highlighting that nano-structuring can increase the thermoelectric figure of merit ZT by reducing the lattice thermal conductivity.

Combined effects of surface morphology and mechanical straining magnitudes on the differentiation of mesenchymal stem cells without using biochemical reagents.

Existing studies examining the control of mesenchymal stem cell (MSC) differentiation into desired cell types have used a variety of biochemical reagents such as growth factors despite possible side effects. Recently, the roles of biomimetic microphysical environments have drawn much attention in this field. We studied MSC differentiation and changes in gene expression in relation to osteoblast-like cell and smooth muscle-like cell type resulting from various microphysical environments, including differing magnitudes of tensile strain and substrate geometries for 8 days. In addition, we also investigated the residual effects of those selected microphysical environment factors on the differentiation by ceasing those factors for 3 days. The results of this study showed the effects of the strain magnitudes and surface geometries. However, the genes which are related to the same cell type showed different responses depending on the changes in strain magnitude and surface geometry. Also, different responses were observed three days after the straining was stopped. These data confirm that controlling microenvironments so that they mimic those in vivo contributes to the differentiation of MSCs into specific cell types. And duration of straining engagement was also found to play important roles along with surface geometry.

Shear stress magnitude is critical in regulating the differentiation of mesenchymal stem cells even with endothelial growth medium.

Human mesenchymal stem cells (MSC) were seeded onto the inner surface of a tubular silicon construct and, after 24 h, were exposed to a shearing stress of either 2.5 or 10 dyne/cm(2) for 1 day. The fluid contained endothelial growth factors in both cases. Morphological changes and cytoskeletal rearrangements were observed in the stimulated cells. Immunofluorescence staining showed that low (2.5 dyne/cm(2)) and high shear stress (10 dyne/cm(2)) resulted in the expression of von Willebrand factor (vWF) and calponin, respectively. At low shear stress, CD31 (PECAM-1) was significantly expressed whereas vWF and KDR expression was only slightly higher than those under 10 dyne/cm(2). All three markers related to smooth muscle cells (myocardin, myosin heavy chain, and SM-22α) had significantly higher expression under shear stress of 10 dyne/cm(2) compared with a 2.5 dyne/cm(2), even in endothelial growth medium. Shear stress plays a critical role in regulating MSC differentiation and must be considered for bioengineered blood vessels.

ERK 1/2 activation in enhanced osteogenesis of human mesenchymal stem cells in poly(lactic-glycolic acid) by cyclic hydrostatic pressure.

The aim of this study was to identify the signal transduction pathways and mechano-transducers that play critical roles in the processes induced by changes in cyclic hydrostatic pressure and fluid shear in 3-dimensional (3D) culture systems. Mesenchymal stem cells were loaded into a polymeric scaffold and divided into three groups according to the stress treatment: static, fluid shear, and hydrostatic pressure with fluid shear. Cells were exposed daily to a hydrostatic pressure of 0.2 MPa for 1 min followed by 14 min rest with fluid flow at 30 rpm. Protein extracts were analyzed by Western blot for extracellular signal-regulated kinase 1/2 (ERK1/2). The complexes were cultured under the mechanical stimuli for 21 days with or without phospho-ERK1/2 inhibitor (U0126) and evaluated by RT-PCR, calcium contents, and immunohistochemistry. Under conditions of mechanical stimulation, the activation of ERK1/2 was sustained or increased with time. U0126 suppressed mechanical stimuli-induced expression of osteocalcin. In addition, calcium contents and the degrees of osteocalcin and osteopontin staining were decreased by this inhibitor. These results demonstrate that mechanical stimuli, particularly hydrostatic pressure with fluid shear, enhance osteogenesis in 3D culture systems via ERK1/2 activation.

Effects of mechanical stimuli and microfiber-based substrate on neurite outgrowth and guidance.

We introduced mechanical stimuli and micropatterned substrate with microfibers to investigate their effects on neurite outgrowth along with nerve growth factor in vitro. Two types of surface morphology were used: a surface that was coated by laminin alone and a surface where in microfibers was added on the laminin surface. PC-12 cells were seeded on both surface types and cultured for 2 d. The magnitudes of shear stresses ranged from 0.10 to 1.50 Pa. Two days after stimulation by shear stress, neurite outgrowth and its direction were measured by F-actin staining and digital image processing. When a shear stress of 0.50 Pa was applied, neurons were most highly aligned with microfibers. The average length of neurite outgrowth with microfibers was largest at a shear stress of 0.25 Pa. The results suggest that micropatterned fibers and fluid-induced shear stress are promising for stimulating neurite outgrowth in a desired direction.

Biomechanical comparisons of three different tibial tunnel directions in posterior cruciate ligament reconstruction.

PURPOSE: To investigate the effects of 3 different tunnel directions on the outcomes of posterior cruciate ligament (PCL) reconstruction surgery based on the forces exerted on the replacement ligament from a biomechanical point of view. The 3 tunnel directions in the proximal tibia are medial, central, and lateral. TYPE OF STUDY: Biomechanical study. METHODS: The forces exerted on the replaced PCL were calculated using finite element analyses as well as measurements from 6 cadavers. The results of the 3 surgical approaches were then compared. In the finite element analyses, the replaced ligament was assumed to have nonlinear elastic as well as viscoelastic properties. To simulate the overload in exercise, the femur was forced to move in the anterior direction abruptly while the tibia was held. From numerical analyses, the resultant forces, von Mises stresses, and maximum shear stresses on the replacement PCLs were calculated and compared. In the cadaveric study, a pressure-sensitive thin film was inserted between the replacement PCL and the killer turn area of the tibia. The color changes in films were evaluated using digital image processing in each case. RESULTS: The medial approach showed remarkably higher stresses and forces on the interface between the replaced PCL and the killer turn in both the numerical and cadaveric study. In contrast, the lateral approach showed the lowest stresses. CONCLUSIONS: The numerical and cadaveric studies indicate that the lateral approach is highly promising compared with the other approaches. CLINICAL RELEVANCE: The lateral approach has been shown to minimize stress concentration around the killer turn during in vitro experiments and a computer simulation of PCL reconstruction for long-term stability. The lateral approach technique appears to provide a promising clinical outcome in patients undergoing PCL reconstruction.

Chondrogenic differentiation of human mesenchymal stem cells using a thermosensitive poly(N-isopropylacrylamide) and water-soluble chitosan copolymer.

Poly(N-isopropylacrylamide) (PNIPAAm) is known to be thermally responsive material and has a lower critical solution temperature (LCST, 32 degrees C) at which a macromolecular transition from a hydrophilic to a hydrophobic structure occurs. Chitosan is a useful natural polymeric biomaterial due to its biocompatibility and biodegradable properties. It has good characteristics for cell attachment, proliferation and viability. The aim of this study was to assess the ability to differentiate from mesenchymal stem cells (MSCs) to chondrocytes and mass formation using a newly developed injectable material, a thermosensitive (water-soluble chitosan-g-PNIPAAm) gel, and evaluate cartilage formation in vivo after injecting a cell-thermosensitive gel complex. The MSCs were cultured in the chitosan-PNIPAAm in vitro. Fluorescence-activated cell sort analysis, viability test, collagen type I, II, X formation and the aggrecan levels were examined. These cultured cells can be easily recovered from a copolymer gel by simply lowering the temperature. An animal study was performed to assess cartilage formation in the submucosal layer of the bladder of rabbits. The cartilage formation could be detected. This can be used to treat vesicoureteral reflux or reflux esophagitis by the effective mass effect. This is a simple method (sol-gel technique in LCST), and good cartilage formation occurs in the bladder tissue.

Chemical, structural properties, and osteoconductive effectiveness of bone block derived from porcine cancellous bone.

The purpose of this study is to determine the efficacy of bioactive calcium phosphate obtained from porcine cancellous bone for the treatment of bone defects and nonunion. Porcine cancellous bone blocks were heat treated at 1300 degrees C for 2 h. The chemical composition, calcium-to-phosphate ratio, and microstructure of the porcine bone blocks were examined. For in vivo implantation, bone defects were created on the anteromedial aspect of the proximal tibia in seven beagle dogs and the xenograft bone blocks were placed into these defects. Plain radiographs were taken at 2-week intervals for roentgenographic evaluation. At 12 weeks, the specimens were stained with hematoxylin and eosin (H&E). The composition and morphology of heat-treated porcine cancellous bone were found to be similar to heat-treated human cancellous bone. Radiographs showed union between the host bone/bone-block interfaces. At 12 weeks, uniform and substantial new bone formation was observed. It is concluded that heat-treated porcine cancellous bone demonstrated effective osteoconductivity. This high-temperature heat-treatment technique has several advantages, including decreased risk of disease transmission and immunoreactivity, while also offering excellent biocompatibility.

Chondrogenic differentiation of mesenchymal stem cells and its clinical applications.

Tissue engineering has the potential to provide cartilaginous constructs capable of restoring the normal function of native articular cartilage following joint injury or degradation. One approach to functional tissue engineering of cartilage involves the in vitro cultivation of tissue constructs by using: (i) chondrogenic cells that can be selected, expanded, and transfected to overexpress the genes of interest, (ii) scaffolds that provide a defined three-dimensional structure for tissue development and biodegrade at a controlled rate. Understanding the functional potential of the cells and the signaling mechanisms underlying their differentiation should lead to innovative protocols for clinical orthopaedic interventions. A large number of growth factors and hormones have been implicated in the regulation of chondrocyte biology, relatively little is known about the intracellular signaling pathways involved. We have tried to define the roles of specific TGF- dependent signaling pathways involved in the regulation of chondrogenesis from human mesenchymal stem cells. Chondrogenesis induced by TGF-beta3 in alginate bead system was confirmed by examining cartilage specific type II collagen expression and aggrecan, whereas type I collagen expression was not affected by TGF-beta3. Type II collagen mRNA expression was expressed strongly during chondrogenesis and MEK inhibition (U0126) resulted in complete down-regulation of type II collagen. In contrast, aggrecan expression was detected in same level by treatment of U0126. These results strongly suggest that the ERK signaling cascade is involved in TGF-beta3 induced-chondrogenesis signaling pathways and a role of its pathway is necessary over a longer period to promote type II collagen expression. However, their end product properties in vivo have not been well known. In this study, an articular cartilage from chondrogenic MSCs with PLGA scaffolds (75:25 and 65:35) were made and analyzed its biochemical, histological and mechanical properties in vitro and in vivo. And also, we evaluated the cartilage formation in vivo through the injection of cell-thermosensitive gel complex, a newly developed injectable material. At 12 weeks after PLGA scaffolds containing chondrogenic MSCs transplantation, the separated rabbit distal femur showed a good gross articular cartilage appearance in the transplanted site. In indentation test, compare to the native articular cartilage, the engineered cartilage from two types of (75:25 and 65:35) achieved up to 30-60% in mechanical stiffness. And also, a new model for cartilage formation in bladder, at 14 weeks after injection, we could find out mass formation in the submucosal area grossly. H&E staining, alcian blue staining and other special staining confirmed the chondrogenic differentiation in the mass. These cell therapy technologies can provide the possibility of clinical applications for vesicoureteral reflux and reflux esophagitis, and urinary incontinence as well as articular cartilage regeneration.

Effects of flow-induced shear stress on limbal epithelial stem cell growth and enrichment.

The roles of limbal epithelial stem cells (LESCs) are widely recognized, but for these cells to be utilized in basic research and potential clinical applications, researchers must be able to efficiently isolate them and subsequently maintain their stemness in vitro. We aimed to develop a biomimetic environment for LESCs involving cells from their in vivo niche and the principle of flow-induced shear stress, and to subsequently demonstrate the potential of this novel paradigm. LESCs, together with neighboring cells, were isolated from the minced limbal tissues of rabbits. At days 8 and 9 of culture, the cells were exposed to a steady flow or intermittent flow for 2 h per day in a custom-designed bioreactor. The responses of LESCs and epithelial cells were assessed at days 12 and 14. LESCs and epithelial cells responded to both types of flow. Proliferation of LESCs, as assessed using a BrdU assay, was increased to a greater extent under steady flow conditions. Holoclones were found under intermittent flow, indicating that differentiation into transient amplifying cells had occurred. Immunofluorescent staining of Bmi-1 suggested that steady flow has a positive effect on the maintenance of stemness. This finding was confirmed by real-time PCR. Notch-1 and p63 were more sensitive to intermittent flow, but this effect was transient. K3 and K12 expression, indicative of differentiation of LESCs into epithelial cells, was induced by flow and lasted longer under intermittent flow conditions. In summary, culture of LESCs in a bioreactor under a steady flow paradigm, rather than one of intermittent flow, is beneficial for both increasing proliferation and maintaining stemness. Conversely, intermittent flow appears to induce differentiation of LESCs. This novel experimental method introduces micro-mechanical stimuli to traditional culture techniques, and has potential for regulating the proliferation and differentiation of LESCs in vitro, thereby facilitating research in this field.

Enhanced differentiation of mesenchymal stem cells into NP-like cells via 3D co-culturing with mechanical stimulation.

This study proposes a three-dimensional co-culturing system of mesenchymal stem cells (MSCs) and nucleus pulposus (NP) cells from New Zealand white male rabbits to differentiate MSCs into NP-like cells. The preferable ratio of MSCs to NP cells and the effects of mechanical stimulation were investigated without biochemical reagents. The preferable ratio was investigated without mechanical stimulation using five groups: Group I (MSC control); Group II (NP cell control); and Groups III, IV, and V, for which the ratios of NP cells to MSCs were 1:1, 1:2, and 2:1, respectively. During culture for 10 days without stimulation, the proliferation of MSCs did not increase after day 4. NP cells proliferated more when co-cultured as in Group V. However, the degree of differentiation of MSCs increased significantly in Group V. The differentiation of NP cells decreased gradually over time. When mechanical stimulation was applied to Groups I, II, and V, it contributed to the differentiation of MSCs into NP-like cells, as well as to that of NP cells, but did not contribute to the proliferation of either cell type. The contribution of mechanical stimulation to differentiation was also confirmed by RT-PCR.

Manufacturing of multi-layered nanofibrous structures composed of polyurethane and poly(ethylene oxide) as potential blood vessel scaffolds.

One of the current limitations in using electrospun nanofibrous materials for tissue engineering is that cells have difficulty penetrating into the materials. For this, multi-layered electrospun structures composed of polyurethane (PU) and poly(ethylene oxide) (PEO) were fabricated and tested in vitro. A 20% (w/v) PU solution was electrospun for 30 min, while a 20% (w/v) PEO solution was electrospun for 5, 15 or 30 min, alternatively. Then, the PEO was extracted by immersing the structure in distilled water to make multi-layered structure. The characteristics of fabricated structures were examined by SEM, FT-IR spectroscopy, mechanical tests and cell penetration test. The bioactivities of smooth muscle cells (SMCs) on these scaffolds were assessed by quantifying DNA, collagen and glycosaminoglycan (GAG) levels. Although hybrid PEO-extracted scaffolds had a little of residual PEO, they were more penetrable than PU alone scaffolds. Also, they showed higher bioactivity than PU-alone scaffolds. The results of this study provided potential of this structure in the application not only to the development of artificial blood vessels but also to other types for tissue engineering.

In vitro and animal study of novel nano-hydroxyapatite/poly(epsilon-caprolactone) composite scaffolds fabricated by layer manufacturing process.

The purpose of this study was to propose a computer-controllable scaffold structure made by a layer manufacturing process (LMP) with addition of nano- or micro-sized particles and to investigate the effects of particle size in vitro. In addition, the superiority of this LMP method over the conventional scaffolds made by salt leaching and gas forming process was investigated through animal study. Using the LMP, we have created a new nano-sized hydroxyapatite/poly(epsilon-caprolactone) composite (n-HPC) scaffold and a micro-sized hydroxyapatite/poly(epsilon-caprolactone) composite (m-HPC) scaffold for bone tissue engineering applications. The scaffold macropores were well interconnected, with a porosity of 73% and a pore size of 500 microm. The compressive modulus of the n-HPC and m-HPC scaffolds was 6.76 and 3.18 MPa, respectively. We compared the cellular responses to the two kinds of scaffolds. Both n-HPC and m-HPC exhibited good in vitro biocompatibility. Attachment and proliferation of mesenchymal stem cells were better on the n-HPC than on the m-HPC scaffold. Moreover, significantly higher alkaline phosphatase activity and calcium content were observed on the n-HPC than on the m-HPC scaffold. In an animal study, the LMP scaffolds enhanced bone formation, owing to their well-interconnected pores. Radiological and histological examinations confirmed that the new bony tissue had grown easily into the entire n-HPC scaffold fabricated by LMP. We suggest that the well-interconnected pores in the LMP scaffolds might encourage cell attachment, proliferation, and migration to stimulate cell functions, thus enhancing bone formation in the LMP scaffolds. This study shows that bioactive and biocompatible n-HPC composite scaffolds prepared using an LMP have potential applications in bone tissue engineering.

Enhancement of adipose tissue formation by implantation of adipogenic-differentiated preadipocytes.

Engineered adipose tissue could be used for the reconstruction or augmentation of soft tissues lost due to mastectomy or lumpectomy in plastic and reconstructive surgery. Preadipocytes are a feasible cell source for adipose tissue regeneration. However, the enhancement of the in vivo adipogenic conversion of preadipocytes remains a major task. In vitro, the adipogenic differentiation of preadipocytes prior to implantation might enhance the adipose tissue regeneration. In the present study, we investigated whether implantation of adipogenic-differentiated preadipocytes enhances the adipose tissue formation compared with implantation of undifferentiated preadipocytes. We also investigated whether basic fibroblast growth factor (bFGF) further enhances the adipose tissue formation mediated by the implantation of adipogenic-differentiated preadipocytes. A fibrin matrix containing human preadipocytes cultured in adipogenic differentiation-inducing conditions with (group 1) or without (group 2) bFGF was injected into the subcutaneous spaces of athymic mice. Fibrin matrices containing undifferentiated human preadipocytes with (group 3) or without (group 4) bFGF were also implanted. Six weeks after implantation, the implanted cells formed new tissues in all groups. Importantly, the implantation of adipogenic-differentiated preadipocytes resulted in more extensive adipogenesis than the implantation of undifferentiated preadipocytes, as evaluated by adipose tissue area and human adipocyte-specific gene expression in the newly formed tissues. In addition, bFGF enhanced neovascularization in the newly formed tissues and further enhanced the adipogenesis mediated by the adipogenic-differentiated preadipocytes. The present study demonstrates that the implantation of adipogenic-differentiated preadipocytes enhances adipose tissue regeneration, as compared with the implantation of undifferentiated preadipocytes, and that cell transplantation-mediated adipogenesis can be further enhanced by the delivery of bFGF.