[Femtosecond lenticule extraction for correction of myopia: clinical results and recovery of subbasal nerves].

OBJECTIVE: To investigate the clinical efficacy, safety, predictability, corneal sensitivity, tear function and recovery of subbasal nerves after femtosecond lenticule extraction (FLEx) and laser in situ keratomileusis (LASIK). METHODS: In this prospective, nonrandomized, comparative clinical study, 49 patients (98 eyes) were divided into two groups. FLEx was performed to treat myopia by Visumax femtosecond laser system, and LASIK was performed by Allegretto Wave laser system. The patients were followed up for 6 months. Visual acuity, manifest refraction, intraocular pressure, slit-lamp examination, corneal topography by Pentacam, tear break-up time, Schirmer test, corneal sensitivity and confocal microscopy were assessed. RESULTS: Forty-four patients (88 eyes) completed the 6-month follow-up. Best corrected visual acuity (BCVA) was 0.89±0.14 in eyes with FLEx and 0.98±0.08 in eyes with LASIK at 1 day after surgery. After 10 days, BCVA was 0.98±0.09 and 1.02±0.09, respectively. At the final follow-up visit, the efficacy index was 1.09 in the FLEx group and 1.07 in the LASIK group, and the safety index was 1.12 and 1.07, respectively, in the two groups. Mean Schirmer score was (16.92±7.58) mm and (15.03±5.89) mm (t=1.316, P=0.192), mean tear break-up time was (8.94±2.57) s and (8.00±2.39) s (t=1.759, P=0.082), and corneal sensitivity was (56.46±4.49) mm and (51.38±8.16) mm (t=1.316, P=0.001) in the groups of FLEx and LASIK, respectively. At 10 days after surgery, the number of subbasal nerves was significantly decreased in the FLEx group, and in the LASIK group the subbasal nerve fibers were hardly observed. At 6 months, regenerated nerve fibers were evident in the subbasal area, which recovered faster in eyes with FLEx than in those with LASIK. CONCLUSIONS: Femtosecond lenticule extraction appears to be efficient, safe and predictable for myopia. FLEx surgery is superior over LASIK in less reduction of corneal sensation and lower risk of harm to the subbasal nerve fibers.

A Smart Nanopore for Bio-detection.

The molecular scale pore structure, called nanopore, can be formed from protein ion channels by genetic engineering or fabricated on solid substrates using fashion nanotechnology. Target molecules in interaction with the functionalized lumen of nanopore, can produce characteristic changes in the pore conductance, which act as fingerprints, allowing us to identify single molecules and simultaneously quantify each target species in the mixture. Nanopore sensors have been created for tremendous biomedical detections, with targets ranging from metal ions, drug compounds and cellular second messengers, to proteins and DNAs. Recently, we have used the nanopore technique to dissect folding and unfolding mechanism of a single G-quadruplex DNA aptamer regulated by a variety of ions; we also created a portable and durable molecular device that integrated a protein pore sensor with a solidified lipid membrane for real-time detection.

Pharmacokinetic characterization of hydroxylpropyl-beta-cyclodextrin-included complex of cryptotanshinone, an investigational cardiovascular drug purified from Danshen (Salvia miltiorrhiza).

1. The study aimed to investigate the pharmacokinetics of cryptotanshinone in a hydroxylpropyl-beta-cyclodextrin-included complex in dogs and rats. 2. Animals were administrated the inclusion complex of cryptotanshinone and the concentrations of cryptotanshinone and its major metabolite tanshinone IIA were determined by a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. 3. Cryptotanshinone in inclusion complex was absorbed slowly after an oral dose, and the C(max) and AUC(0-)(t) were dose-proportional. The bioavailability of cryptotanshinone in rats was (6.9% +/- 1.9%) at 60 mg kg(-1) and (11.1% +/- 1.8%) in dogs at 53.4 mg kg(-1). The t(1/2) of the compound in rats and dogs was 5.3-7.4 and 6.0-10.0 h, respectively. Cryptotanshinone showed a high accumulation in the intestine, lung and liver after oral administration, while the lung, liver and heart had the highest level following intravenous dose. Excretion data in rats showed that cryptotanshinone and its metabolites were mainly eliminated from faeces and bile, and the dose recovery rate was 0.02, 2.2, and 14.9% in urine, bile, and faeces, respectively. 4. The disposition of cryptotanshinone in an inclusion complex was dose-independent and the bioavailability was increased compared with that without cyclodextrin used to formulate the drug. Cryptotanshinone was distributed extensively into different organs. Excretion of cryptotanshinone and its metabolites into urine was extremely low, and they were mainly excreted into faeces and bile.

Preclinical factors affecting the pharmacokinetic behaviour of tanshinone IIA, an investigational new drug isolated from Salvia miltiorrhiza for the treatment of ischaemic heart diseases.

Tanshinone IIA (TSIIA) is a major active triterpenoid isolated from Salvia miltiorrhiza. The purposes of this study were to investigate various preclinical factors that determined the pharmacokinetics of TSIIA. After oral dosing at 6.7, 20, and 60 mg kg(-1), TSIIA was detected mainly as glucuronidated conjugate (TSIIAG) with only small amounts of the unchanged in the plasma. TSIIA was predominantly excreted into the bile and faeces as TSIIAG, and urine to a minor extent. The C(max) and AUC(0-)(t) of TSIIAG after i.p. administration were significantly lower than those after intragastric administration. The plasma concentration-time profiles of TSIIA following oral dosing of TSIIA showed multiple peaks. The C(max) and AUC(0-)(t) of TSIIA and its glucuronides in rats with intact bile duct were significantly lower than those of rats with bile duct cannulation. Studies from the linked-rat model and intraduodenal injection of bile containing TSIIA and its metabolites indicate that TSIIA glucuronides underwent hydrolysis and the aglycone was reabsorbed from the gut and excreted into the bile as conjugates. TSIIA had a wide tissue distribution, with a very high accumulation in the lung, but very limited penetration into the brain and testes. TSIIA was metabolized by rat CYP2C, 3A and 2D, as ticlopidine, ketoconazole and quinidine all inhibited TSIIA metabolism in rat liver microsomes. Taken collectively, these findings indicate that multiple factors play important roles in determining the pharmacokinetics of TSIIA.

Synthesis, DNA binding and cytotoxicity of new pyrazole emodin derivatives.

A series of new anthrapyrazoles were derived from emodin by attaching various cationic alkyl amino side chains onto a pyrazole ring which had been incorporated into the anthraquinone chromophore. Compared with emodin, the derivatives had significantly higher DNA binding affinity based on interaction with calf thymus DNA, and much more potent cytotoxicity against different tumor cells. The derivatives with a mono-cationic alkyl side chain exhibited the highest DNA binding affinity and cytotoxicity.

Senescence and telomere shortening induced by novel potent G-quadruplex interactive agents, quindoline derivatives, in human cancer cell lines.

Agents stabilizing G-quadruplexes have the potential to interfere with telomere replication by blocking the elongation step catalysed by telomerase or telomerase-independent mechanism and could therefore act as antitumor agents. In this study, we found that quindoline derivatives interacted preferentially with intramolecular G-quadruplex structures and were novel potent telomerase inhibitors. Treatment with quindoline derivatives reproducibly inhibited telomerase activity in human leukemia K562 cells and colon cancer SW620 cells. N'-(10H-Indolo [3,2-b] quinolin-11-yl)-N, N-dimethyl-propane-1,3-diamine (SYUIQ-5), (one of quindoline derivatives), when added to K562 and SW620 cell culture at nonacute cytotoxic concentrations, increased time of population doublings of K562 and SW620 cells, induced a marked cessation in cell growth and cellular senescence phenotype after 35 and 18 days, respectively. Growth cessation was accompanied by a shortening of telomere length, and induction of p16, p21 and p27 protein expression. However, another compound SYUIQ-7 with greater IC(50) for telomerase had no obvious cellular effect in nonacute cytotoxic concentrations. These results indicate that quindoline derivatives as novel potent G-quadruplex interactive agents induce senescence and telomere shortening in cancer cells and therefore are promising agents for cancer treatment.

Prolonged residence time of a noncovalent molecular adapter, beta-cyclodextrin, within the lumen of mutant alpha-hemolysin pores.

Noncovalent molecular adapters, such as cyclodextrins, act as binding sites for channel blockers when lodged in the lumen of the alpha-hemolysin (alphaHL) pore, thereby offering a basis for the detection of a variety of organic molecules with alphaHL as a sensor element. beta-Cyclodextrin (betaCD) resides in the wild-type alphaHL pore for several hundred microseconds. The residence time can be extended to several milliseconds by the manipulation of pH and transmembrane potential. Here, we describe mutant homoheptameric alphaHL pores that are capable of accommodating betaCD for tens of seconds. The mutants were obtained by site-directed mutagenesis at position 113, which is a residue that lies near a constriction in the lumen of the transmembrane beta barrel, and fall into two classes. Members of the tight-binding class, M113D, M113N, M113V, M113H, M113F and M113Y, bind betaCD approximately 10(4)-fold more avidly than the remaining alphaHL pores, including WT-alphaHL. The lower K(d) values of these mutants are dominated by reduced values of k(off). The major effect of the mutations is most likely a remodeling of the binding site for betaCD in the vicinity of position 113. In addition, there is a smaller voltage-sensitive component of the binding, which is also affected by the residue at 113 and may result from transport of the neutral betaCD molecule by electroosmotic flow. The mutant pores for which the dwell time of betaCD is prolonged can serve as improved components for stochastic sensors.

Capture of a single molecule in a nanocavity.

We describe a heptameric protein pore that has been engineered to accommodate two different cyclodextrin adapters simultaneously within the lumen of a transmembrane beta barrel. The volume between the adapters is a cavity of approximately 4400 cubic angstroms. Analysis of single-channel recordings reveals that individual charged organic molecules can be pulled into the cavity by an electrical potential. Once trapped, an organic molecule shuttles back and forth between the adapters for hundreds of milliseconds. Such self-assembling nanostructures are of interest for the fabrication of multianalyte sensors and could provide a means to control chemical reactions.

[Psychology during the process of human hand allograft].

OBJECTIVE: To study the psychology and its management during the process of hand allograft. METHODS: One psychologist participated through the whole process of the present hand allograft. 12 potential candidates of hand transplant were interviewed during the selection of patients to evaluate the state of psychiatry and their abilities to manage stressors like cooperation with medical workers and medical interventions, waiting for donors, adaptation to a new hand and post operation depression. The psychological state of 11 patients were believed to be able to receive hand transplant, and they are further prepared psychologically by the psychologist while waiting for a donor. Two lucky candidates were decided by tissue typing and received hand allograft simultaneously. After the operation, the two patients psychotherapy assisted with effective analgesia, supporting from family and environmental improvement. RESULTS: One out of 12 patients was found not suitable for the transplantation because of psychiatric problem. One week postoperation, the 2 patients were anxious, lack of patience, and horrified at seeing the long-expected grafted hand. After 1 week of treatments and adapation the patients managed to settle with the new hand, and accepted the hand as a whole 1 month postoperation. With the recovery of the hand sensation and motion 4 to 5 months postoperation, the patients held the hand as his own. CONCLUSION: Psychologists are required in the hand transplantation team during the pre-transplant selection of patients and post-transplant rehabilitation.

Interaction of the noncovalent molecular adapter, beta-cyclodextrin, with the staphylococcal alpha-hemolysin pore.

Cyclodextrins act as noncovalent molecular adapters when lodged in the lumen of the alpha-hemolysin (alphaHL) pore. The adapters act as binding sites for channel blockers, thereby offering a basis for the detection of a variety of organic molecules with alphaHL as a biosensor element. To further such studies, it is important to find conditions under which the dwell time of cyclodextrins in the lumen of the pore is extended. Here, we use single-channel recording to explore the pH- and voltage-dependence of the interaction of beta-cyclodextrin (betaCD) with alphaHL. betaCD can access its binding site only from the trans entrance of pores inserted from the cis side of a bilayer. Analysis of the binding kinetics shows that there is a single binding site for betaCD, with an apparent equilibrium dissociation constant that varies by >100-fold under the conditions explored. The dissociation rate constant for the neutral betaCD molecule varies with pH and voltage, a result that is incompatible with two states of the alphaHL pore, one of high and the other of low affinity. Rather, the data suggest that the actual equilibrium dissociation constant for the alphaHL. betaCD complex varies continuously with the transmembrane potential.

Simultaneous stochastic sensing of divalent metal ions.

Stochastic sensing is an emerging analytical technique that relies upon single-molecule detection. Transmembrane pores, into which binding sites for analytes have been placed by genetic engineering, have been developed as stochastic sensing elements. Reversible occupation of an engineered binding site modulates the ionic current passing through a pore in a transmembrane potential and thereby provides both the concentration of an analyte and, through a characteristic signature, its identity. Here, we show that the concentrations of two or more divalent metal ions in solution can be determined simultaneously with a single sensor element. Further, the sensor element can be permanently calibrated without a detailed understanding of the kinetics of interaction of the metal ions with the engineered pore.

Reversal of charge selectivity in transmembrane protein pores by using noncovalent molecular adapters.

In this study, the charge selectivity of staphylococcal alpha-hemolysin (alphaHL), a bacterial pore-forming toxin, is manipulated by using cyclodextrins as noncovalent molecular adapters. Anion-selective versions of alphaHL, including the wild-type pore and various mutants, become more anion selective when beta-cyclodextrin (betaCD) is lodged within the channel lumen. By contrast, the negatively charged adapter, hepta-6-sulfato-beta-cyclodextrin (s(7)betaCD), produces cation selectivity. The cyclodextrin adapters have similar effects when placed in cation-selective mutant alphaHL pores. Most probably, hydrated Cl(-) ions partition into the central cavity of betaCD more readily than K(+) ions, whereas s(7)betaCD introduces a charged ring near the midpoint of the channel lumen and confers cation selectivity through electrostatic interactions. The molecular adapters generate permeability ratios (P(K+)/P(Cl-)) over a 200-fold range and should be useful in the de novo design of membrane channels both for basic studies of ion permeation and for applications in biotechnology.

Stochastic sensing of organic analytes by a pore-forming protein containing a molecular adapter.

The detection of organic molecules is important in many areas, including medicine, environmental monitoring and defence. Stochastic sensing is an approach that relies on the observation of individual binding events between analyte molecules and a single receptor. Engineered transmembrane protein pores are promising sensor elements for stochastic detection, and in their simplest manifestation they produce a fluctuating binary ('on/off') response in the transmembrane electrical current. The frequency of occurrence of the fluctuations reveals the concentration of the analyte, and its identity can be deduced from the characteristic magnitude and/or duration of the fluctuations. Genetically engineered versions of the bacterial pore-forming protein alpha-haemolysin have been used to identify and quantify divalent metal ions in solution. But it is not immediately obvious how versatile binding sites for organic ligands might be obtained by engineering of the pore structure. Here we show that stochastic sensing of organic molecules can be procured from alpha-haemolysin by equipping the channel with an internal, non-covalently bound molecular 'adapter' which mediates channel blocking by the analyte. We use cyclodextrins as the adapters because these fit comfortably inside the pore and present a hydrophobic cavity suitable for binding a variety of organic analytes. Moreover, a single sensing element of this sort can be used to analyse a mixture of organic molecules with different binding characteristics. We envisage the use of other adapters, so that the pore could be 'programmed' for a range of sensing functions.

Detoxification of aflatoxin B1 by enzymes isolated from Armillariella tabescens.

Detoxification of aflatoxin B1 (AFB1) by Armillariella tabescens multienzyme, which was isolated from mycelium pellets of A. tabescens, was confirmed by thin-layer chromatography (TLC) and rat assay. The results of toxicology and pathology studies showed that toxicity of AFB1 was minimized after treatment with A. tabescens multienzyme. The result of the Ames test indicated that the mutagenic activity of multienzyme-treated AFB1 was greatly reduced (or inactivated) compared with that of untreated controls. TLC determinations showed that AFB1 at an initial concentration of 16 microM was completely detoxified (100%) by the fungal multienzyme. The infrared spectrum suggests that the multienzyme is responsible for opening the difuran ring of AFB1.

A hexameric transmembrane pore revealed by two-dimensional crystallization of the large mechanosensitive ion channel (MscL) of Escherichia coli.

We have established a reconstitution method of the detergent-solubilized recombinant large mechanosensitive ion channel of Escherichia coli (MscL) that yielded two-dimensional crystals. For that purpose, we have developed a new protocol using Triton X-100 to solubilize and purify the MscL protein. This protocol not only allowed an increase in the protein yield but also made it possible to obtain a homogeneous delipidated and reproducible preparation of the purified protein. When examined by the patch-clamp method MscL channels were found to be fully functional, exhibiting characteristic conductance and activation by pressure. For electron crystallography the homogeneous Triton X-100-purified recombinant MscL was further reconstituted at low lipid-to-protein ratios using Bio-Beads SM2 to remove the detergent. Two-dimensional crystals, exhibiting a p6 plane group symmetry, have been produced and examined by negative stain electron microscopy. Image processing of selected micrographs yielded a projection map at 15-A resolution that provided the first explicit structural information about the molecular boundary and homohexameric organization of the MscL channels in the membrane bilayer.