Self-assembled adhesive biomaterials formed by a genetically designed fusion protein.

Here we report a recombinant protein (MS) obtained by genetic fusion of a mussel foot protein (Mfp3) motif into a silk spidroin (MaSp1). The MS not only self-assembled into a supramolecular fibre, as does the parent MaSp1, but also showed enhanced adhesiveness resulting from the DOPA-containing Mfp3 portion. The successful incorporation of the wet adhesiveness of Mfp3 into the well-structured assembly of MaSp1 may provide a new insight for the genetic design of underwater adhesive recombinant proteins by utilizing the structural features of a spidroin protein.

Effects of Preferential Counterion Interactions on the Specificity of RNA Folding.

The real-time search for native RNA structure is essential for the operation of regulatory RNAs. We previously reported that a fraction of the Azoarcus ribozyme achieves a compact structure in less than a millisecond. To scrutinize the forces that drive initial folding steps, we used time-resolved SAXS to compare the folding dynamics of this ribozyme in thermodynamically isostable concentrations of different counterions. The results show that the size of the fast-folding population increases with the number of available counterions and correlates with the flexibility of initial RNA structures. Within 1 ms of folding, Mg2+ exhibits a smaller preferential interaction coefficient per charge, ΔΓ+/ Z, than Na+ or [Co(NH3)6]3+. The lower ΔΓ+/ Z corresponds to a smaller yield of folded RNA, although Mg2+ stabilizes native RNA more efficiently than other ions at equilibrium. These results suggest that strong Mg2+-RNA interactions impede the search for globally native structure during early folding stages.

Iodide-Selective Synthetic Ion Channels Based on Shape-Persistent Organic Cages.

We report here a synthetic ion channel developed from a shape-persistent porphyrin-based covalent organic cage. The cage was synthesized by employing a synthetically economical dynamic covalent chemistry (DCC) approach. The organic cage selectively transports biologically relevant iodide ions over other inorganic anions by a dehydration-driven, channel mechanism as evidenced by vesicle-based fluorescence assays and planar lipid bilayer-based single channel recordings. Furthermore, the organic cage appears to facilitate iodide transport across the membrane of a living cell, suggesting that the cage could be useful as a biological tool that may replace defective iodide channels in living systems.

Self-Healable Supramolecular Hydrogel Formed by Nor-Seco-Cucurbit[10]uril as a Supramolecular Crosslinker.

A supramolecular hydrogel was formed by a simple mixing of solutions of nor-seco-cucurbit[10]uril (NS-CB[10]) and adamantylamine-terminated 4-armed polyethylene glycol (AdA-4-arm-PEG). In the formation of the hydrogel, NS-CB[10] acted as a noncovalent crosslinker to form a ternary complex with two AdA moieties. The dynamic and selective nature of the host-guest interaction between NS-CB[10] and AdA enabled the supramolecular hydrogel to rapidly recover its physical properties after it was damaged. In addition, the recovered hydrogel retained its physical properties with negligible differences from those of the pristine material, even after multiple self-healing cycles. The NS-CB[10]-based hydrogel with the self-healing property may be useful for various biological applications such as drug delivery, cell therapy and tissue engineering.

Charge screening in RNA: an integral route for dynamical enhancements.

Electrostatic interactions of RNA are at the center of determining the dynamical flexibility and structural stability. By analysing neutron scattering spectroscopy, we show that fast dynamics of hydrated tRNA on ps to ns timescales increases with stronger charge screening, while its structural stability either increases or remains largely unchanged. An unprecedented electrostatic threshold for the onset of additional flexibility is induced from the correlation between the charge-screening density of counterions and the promoted dynamical properties. The results demonstrate that the enhanced dynamical flexibility of tRNA originates from local conformational relaxation coupled with stabilized charge screening rather than governed by fluctuation of hydrated counterions. The present study casts light on the specificity of electrostatic interactions in the thermodynamic balance between the dynamical flexibility and structural stability of RNA.

A clinical analysis of bilateral orbital fracture.

Although bilateral orbital fracture can cause serious eyeball and facial skeletal problems, few reports have been issued on the topic. We analyzed the clinical features of bilateral orbital fracture by reviewing the medical records of 147 patients and compared bilateral and unilateral fractures by reviewing the literature.Bilateral orbital fracture was most common in men aged between 50 and 59 years. A traffic accident was the leading cause of trauma, and average time between trauma and surgery was 12.2 days. Bilateral medial fracture accompanied by nasal fracture accounted for the overwhelming majority, and impure blowout fracture in at least 1 eye occurred in 69.4% of the 147 patients. Associated ocular injuries seemed to be similar for bilateral and unilateral fracture. Thirty-five patients (23.8%) had other multiple traumas affecting other than the eyes, and this significantly increased the need for surgery (P < 0.05). Of the 48 patients who underwent surgery, including 4 cases of bilateral surgery, 21 patients who had ocular motility restriction with central diplopia within 30 degrees almost completely recovered. No significant relation between the timing of surgery and improvement was found. Although unilateral surgery was performed in most cases, facial asymmetry related to enophthalmos was unclear at 6 months postoperatively.In summary, bilateral orbital fracture was found to be clinically distinguishable from unilateral fracture in several aspects. We hope these findings provide a reference guide to the approach and management of bilateral orbital fracture.

Crowders perturb the entropy of RNA energy landscapes to favor folding.

Biological macromolecules have evolved to fold and operate in the crowded environment of the cell. We have shown previously that molecular crowding stabilizes folded RNA structures. Here we report SAXS measurements on a 64 kDa bacterial group I ribozyme in the presence of mono- and divalent ions and PEG crowders of different molecular weight. These experiments show that crowders always stabilize the folded RNA, but this stabilization is weaker in NaCl solutions than MgCl2 solutions. Additionally, we find that RNAs with the same global structure, parametrized by Rg, have different scattering functions depending upon the ratio of electrostatic and entropic stabilization by ions and crowders, respectively. We quantify this difference using the scattering length per scattering volume and find that this ratio is larger for RNAs that fold in lower ionic strength solutions due to the higher crowder content. We conclude that lower RNA flexibility, or reduced configurational entropy, widens the free energy gap between the unfolded and folded RNA in crowded MgCl2 solutions.

Cooperative tertiary interaction network guides RNA folding.

Noncoding RNAs form unique 3D structures, which perform many regulatory functions. To understand how RNAs fold uniquely despite a small number of tertiary interaction motifs, we mutated the major tertiary interactions in a group I ribozyme by single-base substitutions. The resulting perturbations to the folding energy landscape were measured using SAXS, ribozyme activity, hydroxyl radical footprinting, and native PAGE. Double- and triple-mutant cycles show that most tertiary interactions have a small effect on the stability of the native state. Instead, the formation of core and peripheral structural motifs is cooperatively linked in near-native folding intermediates, and this cooperativity depends on the native helix orientation. The emergence of a cooperative interaction network at an early stage of folding suppresses nonnative structures and guides the search for the native state. We suggest that cooperativity in noncoding RNAs arose from natural selection of architectures conducive to forming a unique, stable fold.

The dynamics of unfolded versus folded tRNA: the role of electrostatic interactions.

The dynamics of RNA contributes to its biological functions such as ligand recognition and catalysis. Using quasielastic neutron scattering spectroscopy, we show that Mg(2+) greatly increases the picosecond to nanosecond dynamics of hydrated tRNA while stabilizing its folded structure. Analyses of the atomic mean-squared displacement, relaxation time, persistence length, and fraction of mobile atoms showed that unfolded tRNA is more rigid than folded tRNA. This same result was found for a sulfonated polystyrene, indicating that the increased dynamics in Mg(2+) arises from improved charge screening of the polyelectrolyte rather than specific interactions with the folded tRNA. These results are opposite to the relationship between structural compactness and internal dynamics for proteins in which the folded state is more rigid than the denatured state. We conclude that RNA dynamics are strongly influenced by the electrostatic environment, in addition to the motions of local waters.

Multistage collapse of a bacterial ribozyme observed by time-resolved small-angle X-ray scattering.

Ribozymes must fold into compact, native structures to function properly in the cell. The first step in forming the RNA tertiary structure is the neutralization of the phosphate charge by cations, followed by collapse of the unfolded molecules into more compact structures. The specificity of the collapse transition determines the structures of the folding intermediates and the folding time to the native state. However, the forces that enable specific collapse in RNA are not understood. Using time-resolved SAXS, we report that upon addition of 5 mM Mg(2+) to the Azoarcus group I ribozyme up to 80% of chains form compact structures in less than 1 ms. In 1 mM Mg(2+), the collapse transition produces extended structures that slowly approach the folded state, while > or = 1.5 mM Mg(2+) leads to an ensemble of random coils that fold with multistage kinetics. Increased flexibility of molecules in the intermediate ensemble correlates with a Mg(2+)-dependent increase in the fast folding population and a previously unobserved crossover in the collapse kinetics. Partial denaturation of the unfolded RNA with urea also increases the fraction of chains following the fast-folding pathway. These results demonstrate that the preferred collapse mechanism depends on the extent of Mg(2+)-dependent charge neutralization and that non-native interactions within the unfolded ensemble contribute to the heterogeneity of the ribozyme folding pathways at the very earliest stages of tertiary structure formation.

Molecular crowding stabilizes folded RNA structure by the excluded volume effect.

Crowder molecules in solution alter the equilibrium between folded and unfolded states of biological macromolecules. It is therefore critical to account for the influence of these other molecules when describing the folding of RNA inside the cell. Small angle X-ray scattering experiments are reported on a 64 kDa bacterial group I ribozyme in the presence of polyethylene-glycol 1000 (PEG-1000), a molecular crowder with an average molecular weight of 1000 Da. In agreement with expected excluded volume effects, PEG favors more compact RNA structures. First, the transition from the unfolded to the folded (more compact) state occurs at lower MgCl(2) concentrations in PEG. Second, the radius of gyration of the unfolded RNA decreases from 76 to 64 A as the PEG concentration increases from 0 to 20% wt/vol. Changes to water and ion activities were measured experimentally, and theoretical models were used to evaluate the excluded volume. We conclude that the dominant influence of the PEG crowder on the folding process is the excluded volume effect.

Efficacy of dye disappearance test and tear meniscus height in diagnosis and postoperative assessment of nasolacrimal duct obstruction.

OBJECTIVE: To evaluate the efficacy of the fluorescein dye disappearance test (FDDT) and measurement of tear meniscus height (TMH) in the diagnosis and postoperative assessment of nasolacrimal duct obstruction (NLDO). METHODS: The study group included 42 eyes of 42 patients who had a diagnosis of primary acquired nasolacrimal duct obstruction (PANDO) or functional nasolacrimal duct obstruction (FNDO) and underwent endoscopic transnasal dacryocystorhinostomy. The control group included 38 eyes of 38 people without tearing. The values of the FDDT, TMH and tearing symptom score (TSS), which was evaluated in five levels based on patients' history taking, were measured before surgery, and 1 day, 2 weeks, 1, 3 and 6 months after surgery. The values of the FDDT, TMH and TSS in the study group were compared with those of the control group. RESULTS: The preoperative values of the FDDT, TMH and TSS were 0.7 +/- 0.4, 0.22 +/- 0.08 mm and 1.6, respectively, in the control group and 3.0 +/- 0.8, 0.53 +/- 0.15 mm and 4.4, respectively, in the study group. These differences were statistically significant. Also, at each follow-up, the postoperative values of the FDDT and TMH decreased significantly compared to their preoperative values in the study group. The postoperative TSS decreased significantly compared to the preoperative values at each follow-up except at postoperative 1-day follow-up. There were no significant differences in the preoperative values of the FDDT, TMH and TSS between the types of NLDO. The TSS had positive correlations with the FDDT and TMH before and after surgery. CONCLUSION: These results suggest that the FDDT and TMH measurement might be effective in the diagnosis and postoperative assessment of NLDO.

Comparison of posterior capsular opacification between a combined procedure and a sequential procedure of pars plana vitrectomy and cataract surgery.

PURPOSE: To compare posterior capsular opacification (PCO) between a combined procedure and a sequential procedure of pars plana vitrectomy (PPV) and cataract surgery (CS). METHODS: The medical records of 89 eyes of 85 patients who underwent PPV and CS were retrospectively reviewed. There were 56 eyes of 52 patients with a combined PPV and CS (the combined surgery group), and 33 eyes of 33 patients with CS in a previously vitrectomized eye (the sequential surgery group). The control group was comprised of 130 eyes of 102 patients who underwent CS alone. All patients were followed up for at least 1 year after CS. The major outcome measures were the PCO rate and the interval between CS and PCO formation. There were no significant differences in age and the incidence of diabetes among the 3 groups. RESULTS: The PCO rate checked at 1 year after CS was 12.5% (7/56) in the combined surgery group, 24.2% (8/33) in the sequential surgery group, and 4.6% (6/130) in the control group. The differences in the PCO rate between the subgroups as well as among the 3 groups were statistically significant (p < 0.05). There were no significant differences in the interval between CS and PCO formation among the 3 groups. CONCLUSION: This study demonstrates that the PCO rate may be lower in patients who have a combined procedure of PPV and CS than in those who have a sequential procedure.

Different expression of vascular endothelial growth factor and pigment epithelium-derived factor between diabetic and non-diabetic epiretinal membranes.

PURPOSE: To determine whether there is a difference in protein levels of expression of vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), platelet-derived growth factor (PDGF) and transforming growth factor beta(1) (TGF-beta(1)) between diabetic and non-diabetic epiretinal membranes (ERMs). METHODS: ERMs and vitreous were surgically removed from the eyes of 8 patients with proliferative diabetic retinopathy and from 6 patients with proliferative vitreoretinopathy. Concentrations of VEGF, PEDF, PDGF and TGF-beta(1) were investigated by an enzyme-linked immunosorbent assay. RESULTS: The concentrations of VEGF were higher in ERMs from patients with diabetes (9.2 +/- 8.0 pg/microg protein) compared to those without diabetes (1.2 +/- 3.0 pg/microg protein; p = 0.026), while the concentrations of PEDF were higher in ERMs from patients without diabetes (7.5 +/- 1.5 ng/microg protein) compared to those with diabetes (3.5 +/- 1.5 ng/microg protein; p = 0.002). In addition, the VEGF-to-PEDF ratio was higher in diabetic ERMs (2.80 +/- 1.83) than in non-diabetic ones (0.17 +/- 0.09; p = 0.019). However, the concentrations of PDGF and TGF-beta(1) in membranes were not different between patients with diabetes and those without diabetes. CONCLUSION: This study found the difference in protein levels of expression of VEGF and PEDF between diabetic and non-diabetic ERMs. These results suggest that a disturbance in the balance between angiogenic and anti-angiogenic factors may play an important role in angiogenesis in diabetic ERMs.