General Design Strategy to Precisely Control the Emission of Fluorophores via a Twisted Intramolecular Charge Transfer (TICT) Process.

Fluorogenic probes for bioimaging have become essential tools for life science and medicine, and the key to their development is a precise understanding of the mechanisms available for fluorescence off/on control, such as photoinduced electron transfer (PeT) and Förster resonance energy transfer (FRET). Here we establish a new molecular design strategy to rationally develop activatable fluorescent probes, which exhibit a fluorescence off/on change in response to target biomolecules, by controlling the twisted intramolecular charge transfer (TICT) process. This approach was developed on the basis of a thorough investigation of the fluorescence quenching mechanism of N-phenyl rhodamine dyes (commercially available as the QSY series) by means of time-dependent density functional theory (TD-DFT) calculations and photophysical evaluation of their derivatives. To illustrate and validate this TICT-based design strategy, we employed it to develop practical fluorogenic probes for HaloTag and SNAP-tag. We further show that the TICT-controlled fluorescence off/on mechanism is generalizable by synthesizing a Si-rhodamine-based fluorogenic probe for HaloTag, thus providing a palette of chemical dyes that spans the visible and near-infrared range.

Mode-Specific Vibrational Analysis of Exciton Delocalization and Structural Dynamics in Conjugated Oligomers.

Exciton delocalization in organic semiconducting polymers, affected by structures at a molecular level, plays a crucial role in modulating relaxation pathways, such as charge generation and singlet fission, which can boost device efficiency. However, the structural diversity of polymers and broad signals from typical electronic spectroscopy have their limits when it comes to revealing the interplay between local structures and exciton delocalization. To tackle these problems, we apply femtosecond stimulated Raman spectroscopy in archetypical conjugated oligothiophenes with different chain lengths. We observed Raman frequency dispersions of symmetric bond stretching modes and mode-specific kinetics in the S1 Raman spectra, which underpins the subtle and complex interplay between exciton delocalization and bond length alternation along the conjugation coordinate. Our results provide a more general picture of exciton delocalization in the context of molecular structures for conjugated materials.

Oligoarginine-Bearing Tandem Repeat Penetration-Accelerating Sequence Delivers Protein to Cytosol via Caveolae-Mediated Endocytosis.

To facilitate the cytosolic delivery of larger molecules such as proteins, we developed a new cell-penetrating peptide sequence, named Pas2r12, consisting of a repeated Pas sequence (FFLIG-FFLIG) and d-dodeca-arginine (r12). This peptide significantly enhanced the cellular uptake and cytosolic release of enhanced green fluorescent protein and immunoglobulin G as cargos. We found that simply mixing Pas2r12 with cargos could generate cytosolic introducible forms. The cytosolic delivery of cargos by Pas2r12 was found to be an energy-requiring process, to rely on actin polymerization, and to be suppressed by caveolae-mediated endocytosis inhibitors (genistein and methyl-ß-cyclodextrin) and small interfering RNA against caveolin-1. These results suggest that Pas2r12 enhances membrane penetration of cargos without the need for cross-linking and that caveolae-mediated endocytosis may be the route by which cytosolic delivery is enhanced.

Demonstration of a Light-Driven SO42- Transporter and Its Spectroscopic Characteristics.

In organisms, ion transporters play essential roles in the generation and dissipation of ion gradients across cell membranes. Microbial rhodopsins selectively transport cognate ions using solar energy, in which the substrate ions identified to date have been confined to monovalent ions such as H+, Na+, and Cl-. Here we report a novel rhodopsin from the cyanobacterium Synechocystis sp. PCC 7509, which inwardly transports a polyatomic divalent sulfate ion, SO42-, with changes of its spectroscopic properties in both unphotolyzed and photolyzed states. Upon illumination, cells expressing the novel rhodopsin, named Synechocystis halorhodopsin (SyHR), showed alkalization of the medium only in the presence of Cl- or SO42-. That alkalization signal was enhanced by addition of a protonophore, indicating an inward transport of Cl- and SO42- with a subsequent secondary inward H+ movement across the membrane. The anion binding to SyHR was suggested by absorption spectral shifts from 542 to 536 nm for Cl- and from 542 to 556 nm for SO42-, and the affinities of Cl- and SO42- were estimated as 0.112 and 5.81 mM, respectively. We then performed time-resolved spectroscopic measurements ranging from femtosecond to millisecond time domains to elucidate the structure and structural changes of SyHR during the photoreaction. Based on the results, we propose a photocycle model for SyHR in the absence or presence of substrate ions with the timing of their uptake and release. Thus, we demonstrate SyHR as the first light-driven polyatomic divalent anion (SO42-) transporter and report its spectroscopic characteristics.

Synthesis of perfluoroalkylated pentacenes and evaluation of their fundamental physical properties.

Symmetrical and unsymmetrical pentacenes carrying two perfluoroalkyl (Rf) chains, at the 6 and 13 positions, were synthesized from easily available pentacene-6,13-quinone via facile three or four step reactions. After extensive evaluation, it was clearly found that the control of both the electron density of the aromatic rings on the pentacene core and molecular alignment in the crystalline state nicely affected their physical properties. Thus, we successfully prove in this article that (1) their anti-oxidation ability was significantly enhanced due to a decrease in the HOMO and LUMO energy and (2) a distinct difference in charge-transporting properties was observed between the symmetrical and unsymmetrical pentacenes.

One-stage umbilicus reconstruction after resection of urachal cyst.

Umbilical reconstruction after total excision of the umbilicus represents a challenging problem for reconstructive surgeons. We describe herein a new method for one-stage umbilical reconstruction after resection of a urachal cyst. This case series included 6 patients, with laparoscopic urachal cyst removal in 5 and conventional transcutaneous surgery in 1. One-stage umbilical reconstruction was performed in all cases. When a conventional transcutaneous approach is indicated, umbilical reconstruction can be undertaken through the same skin incision used for total resection of the urachal cyst. Two triangular flaps were designed just below the umbilical defect. Flaps were rotated 180 degrees and sutured together to form one big triangular flap. This flap was then folded to create the new umbilicus. A deep umbilicus with good shape was constructed in all cases, and all patients were satisfied with the outcome. This method is simple, easy, and produces a natural-looking umbilicus.

Label-free autofluorescence lifetime reveals the structural dynamics of ataxin-3 inside droplets formed via liquid-liquid phase separation.

Liquid-liquid phase separation is a phenomenon that features the formation of liquid droplets containing concentrated solutes. The droplets of neurodegeneration-associated proteins are prone to generate aggregates and cause diseases. To uncover the aggregation process from the droplets, it is necessary to analyze the protein structure with keeping the droplet state in a label-free manner, but there was no suitable method. In this study, we observed the structural changes of ataxin-3, a protein associated with Machado-Joseph disease, inside the droplets, using autofluorescence lifetime microscopy. Each droplet showed autofluorescence due to tryptophan (Trp) residues, and its lifetime increased with time, reflecting structural changes toward aggregation. We used Trp mutants to reveal the structural changes around each Trp and showed that the structural change consists of several steps on different timescales. We demonstrated that the present method visualizes the protein dynamics inside a droplet in a label-free manner. Further investigations revealed that the aggregate structure formed in the droplets differs from that formed in dispersed solutions and that a polyglutamine repeat extension in ataxin-3 hardly modulates the aggregation dynamics in the droplets. These findings highlight that the droplet environment facilitates unique protein dynamics different from those in solutions.

Postoperative Showering for Patients With Closed Suction Drainage: A Retrospective Cohort Study of Deep Inferior Epigastric Perforator Flap Breast Reconstructions.

Background The deep inferior epigastric perforator (DIEP) flap has been widely used in breast reconstruction. During surgery, many surgeons use closed suction drainage for both the donor site and the reconstructed breast. However, the criteria for drainage removal depend on the surgeon's preference and remain controversial. Moreover, it is well known that early postoperative showering is harmless to the surgical site and is recommended in many reports. However, it has not been discussed whether it is acceptable for patients with closed suction drainage to take a shower. Methodology We conducted a retrospective study of postoperative showering in 30 patients who underwent breast reconstruction with a DIEP flap. During the surgery, a total of three closed suction drains were connected to the patient's body (one was connected to the reconstructed breast, and the other two were connected to the abdominal donor site). After the surgery, patients were allowed to shower when the number of connected drainage tubes was ≤2. Results The patients were divided into three groups according to the number of remaining drainage tubes connected to their bodies when they started postoperative showering. Group A included patients with no drainage tubes. Group B included patients with one remaining drainage tube. Group C included patients with two drainage tubes. No significant differences in the incidence of postoperative individual complications were observed among the three groups. Conclusions Postoperative showering for patients with closed suction drainage is safe and does not increase the incidence of postoperative complications, including surgical site infection.

SOD1 gains pro-oxidant activity upon aberrant oligomerization: change in enzymatic activity by intramolecular disulfide bond cleavage.

Copper-zinc superoxide dismutase (SOD1) has been proposed as one of the causative proteins of amyotrophic lateral sclerosis (ALS). The accumulation of non-native conformers, oligomers, and aggregates of SOD1 in motor neurons is considered responsible for this disease. However, it remains unclear which specific feature of these species induces the onset of ALS. In this study, we showed that disulfide-linked oligomers of denatured SOD1 exhibit pro-oxidant activity. Substituting all the cysteine residues in the free thiol state with serine resulted in the loss of both the propensity to oligomerize and the increase in pro-oxidant activity after denaturation. In contrast, these cysteine mutants oligomerized and acquired the pro-oxidant activity after denaturation in the presence of a reductant that cleaves the intramolecular disulfide bond. These results indicate that one of the toxicities of SOD1 oligomers is the pro-oxidant activity induced by scrambling of the disulfide bonds. Small oligomers such as dimers and trimers exhibit stronger pro-oxidant activity than large oligomers and aggregates, consistent with the trend of the cytotoxicity of oligomers and aggregates reported in previous studies. We propose that the cleavage of the intramolecular disulfide bond accompanied by the oligomerization reduces the substrate specificity of SOD1, leading to the non-native enzymatic activity.

A modified transmetatarsal amputation.

The incidence of the diabetic foot is increasing worldwide. Because evidence has shown that transmetatarsal amputation is associated with fewer failures in amputations of the diabetic foot with or without peripheral arterial disease, improving its management and surgical technique is a mission for the surgeon. Conventional transmetatarsal amputation has held firm, however, for more than 150 years. With a new concept for the transmetatarsal amputation method aimed at a better outcome, we propose a modified procedure for preserving the soft tissue between the metatarsal bones (the vasculature complex with the muscles, periostea, and vessels) and applying it to the distal bone stumps. The purpose of this method is to secure a functional foot by preserving the longitudinal arch. The new method was applied to 11 patients with diabetes mellitus or peripheral arterial disease, or both. All wounds closed successfully. Of the 11 patients, 8 were still alive with no complications. Of these 8 patients, 6 were able to ambulate with a custom-made shoe and 2 used a wheelchair, just as preoperatively. Of the 3 patients who died, 1 died a natural death, 1 died of sepsis, and 1 of cerebral infarction. We believe that the modified transmetatarsal amputation that we have described in this report is a potential breakthrough in the care of patients with forefoot gangrene and may gain acceptance over time.

Congenital blepharoptosis co-occurring with VATER association.

VATER association is the tendency for 5 specific anomalies (vertebral and vascular anomalies [V], anal atresia [A], esophageal atresia and/or tracheoesophageal fistula [TE], and radial and renal anomalies [R]) to occur together in one individual. Recently, malformations and abnormalities, other than those of diagnostic criteria of VATER association, have been considered significant for clarifying the nature of VATER association and for establishing guidelines for the treatment of infants with VATER association because they are supposed to be determinants of prognosis. Malformations associated with the eye have, however, scarcely been highlighted in VATER association, although several occurrences have been reported. The authors describe the first case of congenital blepharoptosis co-occurring in a patient with VATER association. The co-occurrence of several malformations in the ipsilateral face and hand of the patient were indicative of their arising from the congeneric pathogenesis. Surgical repair of the blepharoptosis was carried out by eyebrow suspension with fascia lata when the patient was 5 years old; a favorable outcome was achieved in both function and aesthetics.

Nonbonded Atomic Contacts Drive Ultrafast Helix Motions in Myoglobin.

The association and dissociation of small ligands regulate the functions of proteins through structural changes in the protein. Such structural changes propagate long distances, and this allostery plays a key role in molecular functions. However, the mechanism by which structural changes are transmitted is poorly understood. Here we show that nonbonded atomic contacts play an essential role in driving the displacement of a helix in picosecond time scale primary structural changes following the dissociation of carbon monoxide from the heme group in myoglobin. The present time-resolved ultraviolet resonance Raman study revealed that the amplitude of this helix displacement was reduced upon substitution of Val68, which contacts the heme in wild-type myoglobin, with a less bulky side chain (Ala). Our findings provided the first direct evidence that structural changes are transmitted not only by covalent bonds, salt bridges and hydrogen bonds but also by nonbonded atomic contacts in the primary protein response upon ligand dissociation. Furthermore, the present results indicate the importance of dense atomic packing in a protein structure for responding to the association and dissociation of small molecules. The high compactness of protein structures makes possible the propagation of structural changes, providing useful clues to the design of molecular machines.

Reconstruction of the orbital floor with sheets of autogenous iliac cancellous bone.

PURPOSE: Various autografts and synthetic materials such as titanium plates have been used for orbital floor reconstruction; however, no consensus on reconstruction methods has been reached. We have used iliac cancellous bone for this reconstruction with good results. PATIENTS AND METHODS: Patients (n = 101) enrolled in this study underwent orbital floor reconstruction of pure blowout fracture with an approximate 1 mm thinned and trimmed iliac cancellous bone without fixation of the graft. The results were assessed by CT; diplopia exceeding an angle of 45 degrees was evaluated as a poor outcome. RESULTS: Postoperatively, diplopia appeared in 15 patients and disappeared in 86 patients. At 6-month follow-up, CT showed that the morphology of the orbit was well maintained and no shedding or improper positioning of the grafted bone was observed in any of the patients. Moreover, CT images demonstrated ossification of the transplanted bone graft. CONCLUSIONS: The method encompassing the use of the medulla of the iliac bone has several merits: 1) the graft bone is sufficiently pliable and flexible to fit the gently curving orbital floor; 2) being soft, it is easy to cut. Furthermore, no complication attributable to its fragility is observed at short- and long-term follow-up. The 1 mm thin medullar bone graft derives its robustness from its beamed structure. We consider the medulla of the iliac bone fitting autosomal material for the reconstruction of the orbital floor.

Protein Dynamics Preceding Photoisomerization of the Retinal Chromophore in Bacteriorhodopsin Revealed by Deep-UV Femtosecond Stimulated Raman Spectroscopy.

Bacteriorhodopsin is a prototypical photoreceptor protein that functions as a light-driven proton pump. The retinal chromophore of bacteriorhodopsin undergoes C13═C14 trans-to-cis isomerization upon photoexcitation, and it has been believed to be the first event that triggers the cascaded structural changes in bacteriorhodopsin. We investigated the protein dynamics of bacteriorhodopsin using deep-ultraviolet resonance femtosecond stimulated Raman spectroscopy. It was found that the stimulated Raman signals of tryptophan and tyrosine residues exhibit significant changes within 0.2 ps after photoexcitation while they do not noticeably change during the isomerization process. This result implies that the protein environment changes first, and its change is small during isomerization. The obtained femtosecond stimulated Raman data indicate that ultrafast change is induced in the protein part by the sudden creation of the large dipole of the excited-state chromophore, providing an environment that realizes efficient and selective isomerization.

Ultrafast Dynamics of Heliorhodopsins.

Heliorhodopsins (HeR) constitute a new rhodopsin family and show only <15% sequence identities with type-1 and type-2 rhodopsins. The large difference in amino acid sequence between HeRs and other rhodopsins raises a question whether their biological function is triggered by efficient and rapid photoisomerization of the retinal chromophore as in the case of other rhodopsins. We performed femtosecond time-resolved absorption measurements of two HeRs, HeR 48C12 and HeR from Thermoplasmatales archaeon SG8-52-1. Both HeRs exhibit excited-state absorption around 480 nm and stimulated emission in the >650 nm region, and these transient signals decay concomitantly with appearance of photoproduct absorption on a subpicosecond time scale. The observed spectral change indicates that ultrafast retinal photoisomerization proceeds in the femtosecond time region. The transient spectra and dynamics of HeRs are surprisingly similar to those of type-1 rhodopsins, despite remarkable differences in amino acid arrangement in the hydrophobic region of the retinal binding site.

Importance of skin perfusion pressure in treatment of critical limb ischemia.

UNLABELLED:  The authors studied whether skin perfusion pressure (SPP) measurements can be used to accurately predict wound healing in critical limb ischemia (CLI) and to select peripheral arterial reconstructive procedures. METHODS: Forty-seven patients (33 men and 14 women, age 36-83 years) with 69 ischemic limbs with foot ulcers or gangrene were studied retrospectively. Skin perfusion pressure was compared to the treatment outcomes (ulcers healed and ulcers that failed to heal). As a diagnostic test for CLI, the sensitivity, specificity, and the positive and the negative predictive values (PPV, NPV) of SPP measurement were calculated; the data was then analyzed by the receiver operation characteristic (ROC) curve. RESULTS: According to the ROC curve, the best SPP measurement performance was at 35 mmHg. CONCLUSION: Wound healing at the appropriate amputation level must be predicted to minimize invasive debridement. Skin perfusion pressure measurement is useful for predicting wound healing in the presence of CLI. Skin perfusion pressure ≥ 35 mmHg is requisite for wound healing; at SPP <35 mmHg, peripheral arterial reconstruction is necessary before debridement.

Spectroscopic Study of Proton-Transfer Mechanism of Inward Proton-Pump Rhodopsin, Parvularcula oceani Xenorhodopsin.

Parvularcula oceani xenorhodopsin is the first light-driven inward proton pump. To understand the mechanism of inward proton transport, comprehensive transient absorption spectroscopy was conducted. Ultrafast pump-probe spectroscopy revealed that the isomerization time of retinal is 1.2 ps, which is considerably slower than those of other microbial rhodopsins (180-770 fs). Following the production of J, the K intermediate was formed at 4 ps. Proton transfer occurred on a slower timescale. Proton release and uptake were observed on the L/M-to-M and M decay, respectively, by monitoring transient absorption changes of pH-indicating dye, pyranine. Although a proton was released from Asp216 into the cytoplasmic medium, no proton-donating residue was identified on the extracellular side in mutation experiments. We revealed that a branched retinal isomerization (from 13-cis-15-anti to 13-cis-15-syn and all-trans-15-anti) occurred simultaneously with proton uptake. Furthermore, although the proton release showed a large kinetic isotope effect (KIE), the KIE of proton uptake was negligible. These results suggest that retinal isomerization is the rate-limiting process in proton uptake and that the regulation of p Ka of the retinal Schiff base by thermal isomerization enables the uptake from extracellular medium. This proton uptake mechanism differs from that of the outward proton pump with an internal proton donor and is important for understanding how the direction of ion transport by membrane proteins is determined.

Origin of the Reactive and Nonreactive Excited States in the Primary Reaction of Rhodopsins: pH Dependence of Femtosecond Absorption of Light-Driven Sodium Ion Pump Rhodopsin KR2.

KR2 is the first light-driven Na+-pumping rhodopsin discovered. It was reported that the photoexcitation of KR2 generates multiple S1 states, i.e., "reactive" and "nonreactive" S1 states at physiological pH, but their origin remained unclear. In this study, we examined the S1 state dynamics of KR2 using femtosecond time-resolved absorption spectroscopy at different pH's in the range from 4 to 11. It was found that the reactive S1 state is predominantly formed at pH >9, but its population drastically decreases with decreasing pH while the population of the nonreactive S1 state(s) increases. The pH dependence of the relative population of the reactive S1 state correlates very well with the pH titration curve of Asp116, which is the counterion of the protonated retinal Schiff base (PRSB) in KR2. This strongly indicates that the deprotonation/protonation of Asp116 is directly related to the generation of the multiple S1 states in KR2. The quantitative analysis of the time-resolved absorption data led us to conclude that the reactive and nonreactive S1 states of KR2 originate from KR2 proteins having a hydrogen bond between Asp116 and PRSB or not, respectively. In other words, it is the ground-state inhomogeneity that is the origin of the coexistence of the reactive and nonreactive S1 states in KR2. So far, the generation of multiple S1 states having a different photoreactivity of rhodopsins has been mainly explained with the branching of the relaxation pathway in the Franck-Condon region in the S1 state. The present study shows that the structural inhomogeneity in the ground state, in particular that of the hydrogen-bond network, is the more plausible origin of the reactive and nonreactive S1 states which have been widely observed for various rhodopsins.

Congenital melanocytic nevi and nevus spilus have a tendency to follow the lines of Blaschko: an examination of 200 cases.

There have been few reports about the characteristics of the pigmentary arrangement of congenital melanocytic nevi (CMN) and nevus spilus (NS). Though the lines of Blaschko, which don't correspond to other lines such as Langer's lines and skin dermatomes, are well known to be followed by some rare pigmentary disorders, the relationship between these two disorders and the lines of Blaschko has not been discussed. Therefore, through an examination of 200 cases, we investigated whether or not the arrangement of CMN and NS follow the lines of Blaschko. Thirty-nine out of 92 cases (42.4%) of CMN and 67 out of 108 cases (62.0%) of NS were considered to follow the lines of Blaschko. Today, the idea is widely accepted that two functionally different cells proliferate during early embryogenesis of the skin which is caused from mosaicism and, as a result, the lines of Blaschko appear visually. In our results, we found that there were some cases of CMN and NS which showed a relationship with the lines of Blaschko, which is meaningful considering that the lesions and the lines are both related to pigmentary gene anomalies. The result was compared with CMN and NS by the chi(2) test. NS showed a predominant tendency to follow the lines with a significant difference (P = 0.0055). This may indicate that the lines of Blaschko reflect more clearly the proliferation of the epidermal cells than other cells in growth of the skin of the embryo. Further studies are required to elucidate the reason why some cases follow these lines and others do not.

Orthognathic surgery for occlusal reconstruction of old malunited jaw fracture.

Old malunited jaw fractures of nine patients who underwent orthognathic surgery for occlusal reconstruction were clinically evaluated. Early surgery on fractures of the jaw is the optimal treatment when due attention must be paid to occlusion. Since occlusal revision surgery subsequent to inaccurate diagnosis and inappropriate surgery is certainly very difficult and often unsuccessful, surgeons need to pay special attention to this situation.