Five amino acid mismatches in the zinc-finger domains of Cellulose Synthase 5 and Cellulose Synthase 6 cooperatively modulate their functional properties by controlling homodimerization in Arabidopsis.

Cellulose synthase 5 (CESA5) and CESA6 are known to share substantial functional overlap. In the zinc-finger domain (ZN) of CESA5, there are five amino acid (AA) mismatches when compared to CESA6. These mismatches in CESA5 were replaced with their CESA6 counterparts one by one until all were replaced, generating nine engineered CESA5s. Each N-terminal enhanced yellow fluorescent protein-tagged engineered CESA5 was introduced to prc1-1, a cesa6 null mutant, and resulting mutants were subjected to phenotypic analyses. We found that five single AA-replaced CESA5 proteins partially rescue the prc1-1 mutant phenotypes to different extents. Multi-AA replaced CESA5s further rescued the mutant phenotypes in an additive manner, culminating in full recovery by CESA5G43R + S49T+S54P+S80A+Y88F. Investigations in cellulose content, cellulose synthase complex (CSC) motility, and cellulose microfibril organization in the same mutants support the results of the phenotypic analyses. Bimolecular fluorescence complementation assays demonstrated that the level of homodimerization in every engineered CESA5 is substantially higher than CESA5. The mean fluorescence intensity of CSCs carrying each engineered CESA5 fluctuates with the degree to which the prc1-1 mutant phenotypes are rescued by introducing a corresponding engineered CESA5. Taken together, these five AA mismatches in the ZNs of CESA5 and CESA6 cooperatively modulate the functional properties of these CESAs by controlling their homodimerization capacity, which in turn imposes proportional changes on the incorporation of these CESAs into CSCs.

A finite element analysis of stress distribution with various directions of intermaxillary fixation using orthodontic mini-implants and elastics following mandibular advancement with a bilateral sagittal split ramus osteotomy.

OBJECTIVE: This finite element analysis (FEA) aimed to assess the stress distribution in the mandible and fixation system with various directions of the intermaxillary fixation (IMF) using mini-implants (MIs) and elastics following mandibular advancement with a bilateral sagittal split ramus osteotomy (BSSRO). MATERIALS AND METHODS: A total of nine mandibular advancement models were set according to the position of the MIs (1.6 mm in diameter, 8 mm in length) and direction of the IMF elastics (1/4 inch, 5 oz). Major and minor principal stresses in the cortical and cancellous bones, von Mises stresses in the fixation system (miniplate and monocortical screws), and bending angles of the miniplate were analysed. RESULTS: Compressive and tensile stress distributions in the mandible and von Mises stress distributions in the fixation system were greater in models with a Class III IMF elastic direction and a higher IMF elastic force than in models with a Class II IMF elastic direction and a lower IMF elastic force. The bending angle of the miniplate was negligible. CONCLUSIONS: Stress distributions in the bone and fixation system varied depending on the direction, amount of force, and position of IMF elastics and MIs. Conclusively, IMF elastics in the Class II direction with minimal load in the area close to the osteotomy site should be recommended.

The N-terminal zinc finger of CELLULOSE SYNTHASE6 is critical in defining its functional properties by determining the level of homodimerization in Arabidopsis.

Primary cell wall cellulose is synthesized by the cellulose synthase complex (CSC) containing CELLULOSE SYNTHASE1 (CESA1), CESA3 and one of four CESA6-like proteins in Arabidopsis. It has been proposed that the CESA6-like proteins occupy the same position in the CSC, but their underlying selection mechanism remains unclear. We produced a chimeric CESA5 by replacing its N-terminal zinc finger with its CESA6 counterpart to investigate the consequences for its homodimerization, a crucial step in forming higher-order structures during assembly of the CSC. We found that the mutant phenotypes of prc1-1, a cesa6 null mutant, were rescued by the chimeric CESA5, and became comparable to the wild type (WT) and prc1-1 complemented by WT CESA6 in regard to plant growth, cellulose content, cellulose microfibril organization, CSC dynamics and subcellular localization. Bimolecular fluorescence complementation assays were employed to evaluate pairwise interactions between the N-terminal regions of CESA1, CESA3, CESA5, CESA6 and the chimeric CESA5. We verified that the chimeric CESA5 explicitly interacted with all the other CESA partners, comparable to CESA6, whereas interaction between CESA5 with itself was significantly weaker than that of all other CESA pairs. Our findings suggest that the homodimerization of CESA6 through its N-terminal zinc finger is critical in defining its functional properties, and possibly determines its intrinsic roles in facilitating higher-order structures in CSCs.

A mutation in the catalytic domain of cellulose synthase 6 halts its transport to the Golgi apparatus.

Cellulose microfibrils, which form the mechanical framework of the plant cell wall, are synthesized by the cellulose synthase complex in the plasma membrane. Here, we introduced point mutations into the catalytic domain of cellulose synthase 6 (CESA6) in Arabidopsis to produce enhanced yellow fluorescent protein (EYFP)-tagged CESA6D395N, CESA6Q823E, and CESA6D395N+Q823E, which were exogenously produced in a cesa6 null mutant, prc1-1. Comparison of these mutants in terms of plant phenotype, cellulose content, cellulose synthase complex dynamics, and organization of cellulose microfibrils showed that prc1-1 expressing EYFP:CESA6D395N or CESA6D395N+Q823E was nearly the same as prc1-1, whereas prc1-1 expressing EYFP:CESA6Q823E was almost identical to wild type and prc1-1 expressing EYFP:WT CESA6, indicating that CESA6D395N and CESA6D395N+Q823E do not function in cellulose synthesis, while CESA6Q823E is still functionally active. Total internal reflection fluorescence microscopy and confocal microscopy were used to monitor the subcellular localization of these proteins. We found that EYFP:CESA6D395N and EYFP:CESA6D395N+Q823E were absent from subcellular regions containing the Golgi and the plasma membrane, and they appeared to be retained in the endoplasmic reticulum. By contrast, EYFP:CESA6Q823E had a normal localization pattern, like that of wild-type EYFP:CESA6. Our results demonstrate that the D395N mutation in CESA6 interrupts its normal transport to the Golgi and its eventual participation in cellulose synthase complex assembly.

Thin PEGylated Carbon Nitrides: Water-Dispersible Organic Nanodots as Bioimaging Probes.

Fluorescent materials are being used for the optical/fluorescence imaging of living cells and animal models. As such, the development of heavy-metal-free, water-dispersible, and biocompatible imaging probes is still important. Carbon nitride (C3 N4 ) is used as a bioimaging probe due to its suitable optical properties, thus enhancing its biocompatibility and dispersibility in aqueous media is required. In this study, we incorporated short-chain polyethylene glycol (PEG) groups onto a carbon nitride network by the simple N-alkylation of hexaethylene glycolic mesylate with nucleophilic nitrogen atoms on oxidized carbon nitride (OCN). The PEGylated OCN (PEG-OCN) was well dispersed in water as nanodots with a lateral dimension of approximately 30 nm and a thickness of 0.5-1.2 nm and showed strong photoluminescence in the visible region. Cell-viability testing confirmed that these "heavy-metal-free" organic nanodots were highly biocompatible and noncytotoxic. In particular, the developed nanodots could provide clear confocal images of RAW 264.7 cells without weakening cell activity and displaying any aggregation in a range of concentrations (25-100 µg mL-1 ) with bright-green emission in the cytoplasm.

Generation of ultra-high-molecular-weight polyethylene from metallocenes immobilized onto N-doped graphene nanoplatelets.

Catalytic natures of organometallic catalysts are modulated by coordinating organic ligands with proper steric and electronic properties to metal centers. Carbon-based nanomaterials such as graphene nanoplatelets are used with and without N-doping and multiwalled carbon nanotube as a ligand for ethylene polymerizations. Zirconocenes or titanocenes are immobilized on such nanomaterials. Polyethylenes (PEs) produced by such hybrids show a great increase in molecular weight relative to those produced by free catalysts. Specially, ultra-high-molecular-weight PEs are produced from the polymerizations at low temperature using the hybrid with N-doped graphene nanoplatelets. This result shows that such nanomaterials act a crucial role to tune the catalytic natures of metallocenes.

Investigation of Bone Regeneration Efficacy of New Bovine Bone Minerals in a Canine Mandibular Critical Defect Model.

This study aims to investigate the bone regeneration effect of bovine hydroxyapatite-processed biomaterials Bone-XB and S1-XB in a beagle mandibular defect model. A total of four saddle-type critical sizes (15 mm × 10 mm) bone defects are created in each dog: two defects in the left mandible and two defects in the right mandible. The defect control (DC) group is kept unfilled, and the other three defects are filled with three different biomaterials as follows: positive control Bio-Oss (Bio-Oss group), Bone-XB (XB group), and S1-XB (S1-XB group). Bone regeneration is evaluated by radiography, micro-computed tomography, and histological analysis. It is revealed that Bone-XB and S1-XB significantly increase newly formed bone, defect filling percentage, and bone healing score compared to the DC group, which is confirmed by bone microstructure augmentation (bone volume/total volume, trabecular number, and trabecular thickness). Interestingly, no significant differences are observed between the Bone-XB, S1-XB, and Bio-Oss groups. It is suggested that Bone-XB or S1-XB stimulates bone regeneration demonstrated by the increase in newly formed bone and bone microstructure, thereby improving bone defect filling, which is equivalent to the Bio-Oss. Therefore, bovine hydroxyapatite-processed Bone-XB or S1-XB can be considered effective biomaterials for correcting critical-size bone defects or fractures.

The effect of oxidation on properties of graphene and its polycaprolactone nanocomposites.

A functionalized graphene sheet (FGS), which was prepared by the thermal reduction of graphite oxide, was modified by oxidation with H2O2. Elemental analysis, X-ray photoelectron spectroscopy, and Fourier transform infrared (FTIR) spectroscopy showed that additional oxygen functional groups, either doubly or singly bound to carbon, were created by the oxidation. The size and electrical conductivity of the FGS were reduced by the oxidation. During FGS/Polycaprolactone (PCL) nanocomposite preparation by an in situ polymerization method, PCL was grafted onto a FGS by chain growth from a functional group on FGS such as hydroxyl. Thermogravimetry and FTIR spectra demonstrated that the amount of grafted polycaprolactone (PCL) was decreased by the oxidation of FGS, suggesting that PCL chain growth from the FGS surface was inhibited by the neighboring carboxylic acid group on the FGS. Enhanced compatibility between the oxidized FGS and the PCL matrix was observed by optical microscopy and scanning electron microscopy. The reinforcing effect on tensile properties was also enhanced by the oxidized FGS in the FGS/PCL nanocomposites. This suggests that the surface coverage by the grafted PCL chains on the oxidized FGS is higher than that on a pristine FGS, although the grafted PCL chain length is shorter and the amount grafted is smaller.

Microfluidic permeation printing of self-assembled monolayer gradients on surfaces for chemoselective ligand immobilization applied to cell adhesion and polarization.

To study complex cell behavior on model surfaces requires biospecific interactions between the interfacing cell and material. Developing strategies to pattern well-defined molecular gradients on surfaces is difficult but critical for studying cell adhesion, polarization, and directed cell migration. We introduce a new strategy, microfluidic SPREAD (Solute PeRmeation Enhancement And Diffusion) for inking poly(dimethylsiloxane) (PDMS) microfluidic cassettes with a gradient of alkanethiol. Using SPREAD, an oxyamine-terminated alkanethiol is able to permeate into a PDMS microfluidic cassette, creating a chemical gradient, which can subsequently be transfer printed onto a gold surface to form the corresponding chemoselective gradient of oxyamine-alkanethiol self-assembled monolayer (SAM). By first patterning regions of the gold surface with a protective SAM using microfluidic lithography, directional gradients can be stamped exclusively onto unprotected bare gold regions to form single cell gradient microarrays. The microfluidic SPREAD strategy can also be extended to print micrometer-sized islands of radial SAM gradients with excellent geometric resolution. The immobilization of a cell adhesive Arg-Gly-Asp (RGD)-ketone peptide to the SPREAD stamped oxyamine-alkanethiol SAMs provides a stable interfacial oxime linkage for biospecific studies of cell adhesion, polarity, and migration.

Poly(ester amine)-mediated, aerosol-delivered Akt1 small interfering RNA suppresses lung tumorigenesis.

RATIONALE: The low efficiency of conventional therapies in achieving long-term survival of patients with lung cancer calls for the development of novel therapeutic options. Recent advances in aerosol-mediated gene delivery have provided the possibility of an alternative for the safe and effective treatment of lung cancer. OBJECTIVES: To demonstrate the feasibility and emphasize the importance of noninvasive aerosol delivery of Akt1 small interfering RNA (siRNA) as an effective and selective option for lung cancer treatment. METHODS: Nanosized poly(ester amine) polymer was synthesized and used as a gene carrier. An aerosol of poly(ester amine)/Akt1 siRNA complex was delivered into K-ras(LA1) and urethane-induced lung cancer models through a nose-only inhalation system. The effects of Akt1 siRNA on lung cancer progression and Akt-related signals were evaluated. MEASUREMENTS AND MAIN RESULTS: The aerosol-delivered Akt1 siRNA suppressed lung tumor progression significantly through inhibiting Akt-related signals and cell cycle. CONCLUSIONS: The use of poly(ester amine) serves as an effective carrier, and aerosol delivery of Akt1 siRNA may be a promising approach for lung cancer treatment and prevention.

Serum immunoglobulin fused interferon-alpha inhibited tumor growth in athymic mice bearing colon 26 adenocarcinoma cells.

Interferon (IFN) has therapeutic potential for a wide range of infectious and proliferative disorders. However, the half-life of IFN is too short to have a stable therapeutic effect. To overcome this problem, serum immunoglobulin has been fused to IFN. In this study, the efficacy of serum immunoglobulin fused INFs (si-IFN1 and si-IFN2) was evaluated on athymic mice bearing colon 26 adenocarcinoma cells. Seven days after the implantation of tumor cells, each group of mice was injected once a week with si-IFN1 and si-IFN2 at two different concentrations (10 x : 30 microg/kg and 50 x : 150 microg/kg). A slight anti-tumoral effect was observed in all 10 x groups compared to the control. In the 50 x groups, however, si-IFN1 and si-IFN2 showed significant anti- tumoral effects compared to the control. To gain more information on the mechanisms associated with the decrease of tumor size, a Western blot assay of apoptosis-related molecules was performed. The protein expression of cytochrome c, caspase 9, 6, and 3 were increased by si-IFN1 and si-IFN2. These 2 IFNs also increased the expressions of p53, p21, Bax and Bad. Interestingly, si-IFN1 and si-IFN2 decreased the expression of VEGF-beta. Taken together, serum immunoglobulin fused IFNs increased therapeutic efficacy under current experimental condition.

Photosynthetic artificial organelles sustain and control ATP-dependent reactions in a protocellular system.

Inside cells, complex metabolic reactions are distributed across the modular compartments of organelles. Reactions in organelles have been recapitulated in vitro by reconstituting functional protein machineries into membrane systems. However, maintaining and controlling these reactions is challenging. Here we designed, built, and tested a switchable, light-harvesting organelle that provides both a sustainable energy source and a means of directing intravesicular reactions. An ATP (ATP) synthase and two photoconverters (plant-derived photosystem II and bacteria-derived proteorhodopsin) enable ATP synthesis. Independent optical activation of the two photoconverters allows dynamic control of ATP synthesis: red light facilitates and green light impedes ATP synthesis. We encapsulated the photosynthetic organelles in a giant vesicle to form a protocellular system and demonstrated optical control of two ATP-dependent reactions, carbon fixation and actin polymerization, with the latter altering outer vesicle morphology. Switchable photosynthetic organelles may enable the development of biomimetic vesicle systems with regulatory networks that exhibit homeostasis and complex cellular behaviors.

A tissue-engineered scale model of the heart ventricle.

Laboratory studies of the heart use cell and tissue cultures to dissect heart function yet rely on animal models to measure pressure and volume dynamics. Here, we report tissue-engineered scale models of the human left ventricle, made of nanofibrous scaffolds that promote native-like anisotropic myocardial tissue genesis and chamber-level contractile function. Incorporating neonatal rat ventricular myocytes or cardiomyocytes derived from human induced pluripotent stem cells, the tissue-engineered ventricles have a diastolic chamber volume of ~500 µl (comparable to that of the native rat ventricle and approximately 1/250 the size of the human ventricle), and ejection fractions and contractile work 50-250 times smaller and 104-108 times smaller than the corresponding values for rodent and human ventricles, respectively. We also measured tissue coverage and alignment, calcium-transient propagation and pressure-volume loops in the presence or absence of test compounds. Moreover, we describe an instrumented bioreactor with ventricular-assist capabilities, and provide a proof-of-concept disease model of structural arrhythmia. The model ventricles can be evaluated with the same assays used in animal models and in clinical settings.

Aerosol gene delivery using viral vectors and cationic carriers for in vivo lung cancer therapy.

INTRODUCTION: Lung cancer has the highest mortality rate among all cancers in both men and women. Aerosol delivery is a noninvasive method for gene delivery to the lungs, although efficient and biocompatible vectors need to be developed for lung cancer therapy. AREAS COVERED: This review summarizes recent advances in airway gene delivery for lung cancer treatment in animal models using viral vectors or cationic polymers. Viral vectors including lentiviruses and adenoviruses have been used for airway gene delivery because of their high transfection efficiency. Cationic polymers have also been developed for aerosol gene therapy owing to their biocompatibility and ease of modification. EXPERT OPINION: Efficient delivery and specific promoters are needed for lung cancer therapy. Capsid engineering or PEGylation can lower immunogenicity. Moreover, immunotherapy and oncolytic viruses need to be tested with aerosol delivery for lung cancer therapy. Meanwhile, naturally existing cationic materials may allow the development of novel and biocompatible carriers. In combination with various technologies for aerosol delivery, novel and specific carriers could be developed for lung cancer therapy in the future. Finally, standardized protocols for quantifying and manufacturing viral vectors and cationic polymers need to be developed in order to ensure biosafety.

Polymeric nanoparticles containing taxanes enhance chemoradiotherapeutic efficacy in non-small cell lung cancer.

PURPOSE: To reduce the side effects and improve the efficacy of chemoradiation therapy, taxanes were incorporated into polymeric nanoparticles (PNP), and their synergic effect on radiation therapy in non-small cell lung cancer was evaluated. METHODS AND MATERIALS: The properties of PNP-taxanes were characterized by transmission electron microscopy and dynamic light scattering. The chemoradiotherapeutic efficacy of PNP-taxanes was determined by clonogenic assay, cellular morphology, and flow cytometry in A549 cells. In mice bearing A549-derived tumors, the tumor growth delay was examined after the treatment of PNP-taxanes and/or ionizing radiation (IR). RESULTS: The PNP-taxanes were found to be approximately 45 nm in average diameter and to have high solubility in water. They showed the properties of active internalization into cells and preserved the anticancer effect of free taxanes. The survival fraction of A549 cells by clonogenic assay was significantly reduced in the group receiving combined treatment of PNP-taxanes and IR. In addition, in vivo radiotherapeutic efficacy was markedly enhanced by the intravenous injection of PNP-taxanes into the xenograft mice. CONCLUSIONS: We have demonstrated the feasibility of PNP-taxanes to enhance the efficacy of chemoradiation therapy. These results suggest PNP-taxanes can hold an invaluable and promising position in treating human cancers as a novel and effective chemoradiation therapy agent.

Enhancement of radiotherapeutic effectiveness by temperature-sensitive liposomal 1-methylxanthine.

Most of methylxanthine derivatives including caffeine have been known to radiosensitize cancer cells, but the obstacles such as toxicity, request of high dose and poor solubility hinder their preclinical evaluations and clinical applications. In this study, we evaluated the efficacy of 1-methylxanthine (1-MTX), a caffeine metabolite as a radiosensitizer and the in vivo effectiveness of the temperature-sensitive liposomal 1-methylxanthine (tsl-MTX) in combination with ionizing radiation and regional hyperthermia. In human colorectal and lung cancer cells, treatment of 1-MTX sensitized cells to ionizing radiation. To evaluate the in vivo capability of 1-MTX to radiosensitize tumors, we developed temperature-sensitive liposomal 1-MTX using DPPC:DMPC:DSPC (4:1:1 molar ratio) with intention of overcoming lethal toxicity of 1-MTX and controlling drug-release. The particle size of the liposomes was approximately 200 nm in diameter. The release of 1-MTX from the liposomes was responding to increase of temperature. In xenograft tumor-bearing mice, the tsl-MTX administered using the i.p. route showed delay of tumor growth. Importantly, tsl-MTX in combination with radiation and regional hyperthermia exhibited marked delay of tumor growth, suggesting that 1-MTX effectively enhanced radiation-induced suppression of tumor growth. In conclusion, tsl-MTX has highly efficacious anticancer competence in vivo, enhancing radiotherapeutic effectiveness, and feasibility for further clinical applications.