Polymicrobial periprosthetic joint infection and osteomyelitis of the tibia with circumferential abscess and skin ulcer 11 years after total knee arthroplasty.

We report the case of a 71-year-old woman with a skin ulcer derived from an abscess around the tibia. The abscess resulted in periprosthetic joint infection and osteomyelitis 11 years after total knee arthroplasty. The first symptom was a skin ulcer of the lower leg. Magnetic resonance imaging revealed a circumferential mass around the proximal tibia. A skin biopsy taken around the ulcer showed thrombosis and degenerated collagen. Contrast-enhanced computed tomography showed a circumferential mass around the proximal tibia with ring enhancement. Biopsies of the skin ulcer and circumferential mass showed an abscess caused by Enterococcus faecium and methicillin-resistant Staphylococcus epidermis. We conducted debridement of the abscess, a gastrocnemius flap and split-thickness skin grafting and a 2-stage revision of the total knee component with a hinged prosthesis. Two years later, the infection did not reoccur and the patient can walk without a cane. This case is unique as abscess around proximal tibia caused necrotic skin ulcer and appearance of abscess was fibrous and different from typical bacterial abscesses containing pus or fluid. Contrast-enhanced computed tomography was effective for differentiation of the pathological condition.

Water-Soluble Tris(cyclometalated) Iridium(III) Complexes for Aqueous Electron and Energy Transfer Photochemistry.

Cyclometalated iridium(III) complexes are frequently employed in organic light emitting diodes, and they are popular photocatalysts for solar energy conversion and synthetic organic chemistry. They luminesce from redox-active excited states that can have high triplet energies and long lifetimes, making them well suited for energy transfer and photoredox catalysis. Homoleptic tris(cyclometalated) iridium(III) complexes are typically very hydrophobic and do not dissolve well in polar solvents, somewhat limiting their application scope. We developed a family of water-soluble sulfonate-decorated variants with tailored redox potentials and excited-state energies to address several key challenges in aqueous photochemistry.First, we aimed at combining enzyme with photoredox catalysis to synthesize enantioenriched products in a cyclic reaction network. Since the employed biocatalyst operates best in aqueous solution, a water-soluble photocatalyst was needed. A new tris(cyclometalated) iridium(III) complex provided enough reducing power for the photochemical reduction of imines to racemic mixtures of amines and furthermore was compatible with monoamine oxidase (MAO-N-9), which deracemized this mixture through a kinetic resolution of the racemic amine via oxidation to the corresponding imine. This process led to the accumulation of the unreactive amine enantiomer over time. In subsequent studies, we discovered that the same iridium(III) complex photoionizes under intense irradiation to give hydrated electrons as a result of consecutive two-photon excitation. With visible light as energy input, hydrated electrons become available in a catalytic fashion, thereby allowing the comparatively mild reduction of substrates that would typically only be reactive under harsher conditions. Finally, we became interested in photochemical upconversion in aqueous solution, for which it was desirable to obtain water-soluble iridium(III) compounds with very high triplet excited-state energies. This goal was achieved through improved ligand design and ultimately enabled sensitized triplet-triplet annihilation upconversion unusually far into the ultraviolet spectral range.Studies of photoredox catalysis, energy transfer catalysis, and photochemical upconversion typically rely on the use of organic solvents. Water could potentially be an attractive alternative in many cases, but photocatalyst development lags somewhat behind for aqueous solution compared to organic solvent. The purpose of this Account is to provide an overview of the breadth of new research perspectives that emerged from the development of water-soluble fac-[Ir(ppy)]3 complexes (ppy = 2-phenylpyridine) with sulfonated ligands. We hope to inspire the use of some of these or related coordination compounds in aqueous photochemistry and to stimulate further conceptual developments at the interfaces of coordination chemistry, photophysics, biocatalysis, and sustainable chemistry.

Intracellular Unnatural Catalysis Enabled by an Artificial Metalloenzyme.

Artificial metalloenzymes, constructed by incorporating a synthetic catalyst into the internal spaces of a protein scaffold, can perform noncanonical chemical transformations that are not possible using natural enzymes. The addition of cell-permeable modules to artificial metalloenzymes allows for noncanonical catalysis to be implemented as a function of mammalian cells. In this chapter, we describe a protocol for controlling cellular function through a cascade consisting of an artificial metalloenzyme and a gene-circuit engineered via synthetic biology.

Artificial Metalloenzymes: From Selective Chemical Transformations to Biochemical Applications.

Artificial metalloenzymes (ArMs) comprise a synthetic metal complex in a protein scaffold. ArMs display performances combining those of both homogeneous catalysts and biocatalysts. Specifically, ArMs selectively catalyze non-natural reactions and reactions inspired by nature in water under mild conditions. In the past few years, the construction of ArMs that possess a genetically incorporated unnatural amino acid and the directed evolution of ArMs have become of great interest in the field. Additionally, biochemical applications of ArMs have steadily increased, owing to the fact that compartmentalization within a protein scaffold allows the synthetic metal complex to remain functional in a sea of inactivating biomolecules. In this review, we present updates on: 1) the newly reported ArMs, according to their type of reaction, and 2) the unique biochemical applications of ArMs, including chemoenzymatic cascades and intracellular/in vivo catalysis. We believe that ArMs have great potential as catalysts for organic synthesis and as chemical biology tools for pharmaceutical applications.

Meta-analysis for the efficacy of bisphosphonates on hip fracture prevention.

The aim of this study was to investigate the effect of bisphosphonates on preventing osteoporotic hip fracture in patients with or without prior major osteoporotic fracture. Randomized controlled trials (RCTs) and observational studies (OSs) based on electronic health records were used to assess bisphosphonate efficacy and were searched using PubMed, Scopus, and the Cochrane Library databases. Eight RCT studies and 14 OSs were extracted from the studies and quantitatively combined by random-effects meta-analysis. The odds ratio (OR) for all hip fractures in RCTs of 0.66, with a 95% confidence interval (CI) of 0.55-0.79, was lower than that in the OSs (OR 0.83; CI 0.74-0.94). The OR in patients with prior fracture was significantly reduced by bisphosphonates in both RCTs and OSs. Conversely, significant fracture reduction was not apparent in patients without prior fracture. A moderate relationship between prior major fracture rates and OR in hip fractures was defined. In patients with an average age of over 80 years, similar results were confirmed. In this meta-analysis, the efficacy of bisphosphonates was significant in patients with prior major fracture, recommending to prescribe for such patients. Their effect in patients without prior fracture, in contrast, remains unclear.

A Visible-Light Promoted Amine Oxidation Catalyzed by a Cp*Ir Complex.

Through a rapid screening of Cp*Ir complexes based on a turn-on type fluorescence readout, a [Cp*Ir(dipyrido[3,2-a : 2',3'-c]phenazine)Cl]+ complex was found to catalyze the blue-light promoted dehydrogenation of N-heterocycles under physiological conditions. In the dehydrogenation of tetrahydroisoquinolines, the catalyst preferentially yielded the monodehydrogenated product, accompanying H2O2 generation. We surmise that this mechanism may be reminiscent of flavin-dependent oxidases.

Structural basis for the heme transfer reaction in heme uptake machinery from Corynebacteria.

The crystal structures of the conserved region domains of HtaA and HtaB, which act as heme binding/transport proteins in the heme uptake machinery in Corynebacterium glutamicum, are determined for the first time. The molecular mechanism of heme transfer among these proteins is proposed based on the spectroscopic and structural analyses.

Cell-Penetrating Dynamic-Covalent Benzopolysulfane Networks.

Cyclic oligochalcogenides (COCs) are emerging as promising systems to penetrate cells. Clearly better than and different to the reported diselenolanes and epidithiodiketopiperazines, we introduce the benzopolysulfanes (BPS), which show efficient delivery, insensitivity to inhibitors of endocytosis, and compatibility with substrates as large as proteins. This high activity coincides with high reactivity, selectively toward thiols, exceeding exchange rates of disulfides under tension. The result is a dynamic-covalent network of extreme sulfur species, including cyclic oligomers, from dimers to heptamers, with up to nineteen sulfurs in the ring. Selection from this unfolding adaptive network then yields the reactivities and selectivities needed to access new uptake pathways. Contrary to other COCs, BPS show high retention on thiol affinity columns. The identification of new modes of cell penetration is important because they promise new solutions to challenges in delivery and beyond.

Diselenolane-Mediated Cellular Uptake: Efficient Cytosolic Delivery of Probes, Peptides, Proteins, Artificial Metalloenzymes and Protein-Coated Quantum Dots.

Cyclic oligochalcogenides are emerging as powerful tools to penetrate cells. With disulfide ring tension maximized, selenium chemistry had to be explored next to enhance speed and selectivity of dynamic covalent exchange on the way into the cytosol. We show that diseleno lipoic acid (DiSeL) delivers a variety of relevant substrates. DiSeL-driven uptake of artificial metalloenzymes enables bioorthogonal fluorophore uncaging within cells. Binding of a bicyclic peptide, phalloidin, to actin fibers evinces targeted delivery to the cytosol. Automated tracking of diffusive compared to directed motility and immobility localizes 79 % of protein-coated quantum dots (QDs) in the cytosol, with little endosomal capture (0.06 %). These results suggest that diselenolanes might act as molecular walkers along disulfide tracks in locally denatured membrane proteins, surrounded by adaptive micellar membrane defects. Miniscule and versatile, DiSeL tags are also readily available, stable, soluble, and non-toxic.

Enantioselective synthesis of amines by combining photoredox and enzymatic catalysis in a cyclic reaction network.

Visible light-driven reduction of imines to enantioenriched amines in aqueous solution is demonstrated for the first time. Excitation of a new water-soluble variant of the widely used [Ir(ppy)3] (ppy = 2-phenylpyridine) photosensitizer in the presence of a cyclic imine affords a highly reactive α-amino alkyl radical that is intercepted by hydrogen atom transfer (HAT) from ascorbate or thiol donors to afford the corresponding amine. The enzyme monoamine oxidase (MAO-N-9) selectively catalyzes the oxidation of one of the enantiomers to the corresponding imine. Upon combining the photoredox and biocatalytic processes under continuous photo-irradiation, enantioenriched amines are obtained in excellent yields. To the best of our knowledge, this is the first demonstration of a concurrent photoredox- and enzymatic catalysis leading to a light-driven asymmetric synthesis of amines.

A cell-penetrating artificial metalloenzyme regulates a gene switch in a designer mammalian cell.

Complementing enzymes in their native environment with either homogeneous or heterogeneous catalysts is challenging due to the sea of functionalities present within a cell. To supplement these efforts, artificial metalloenzymes are drawing attention as they combine attractive features of both homogeneous catalysts and enzymes. Herein we show that such hybrid catalysts consisting of a metal cofactor, a cell-penetrating module, and a protein scaffold are taken up into HEK-293T cells where they catalyze the uncaging of a hormone. This bioorthogonal reaction causes the upregulation of a gene circuit, which in turn leads to the expression of a nanoluc-luciferase. Relying on the biotin-streptavidin technology, variation of the biotinylated ruthenium complex: the biotinylated cell-penetrating poly(disulfide) ratio can be combined with point mutations on streptavidin to optimize the catalytic uncaging of an allyl-carbamate-protected thyroid hormone triiodothyronine. These results demonstrate that artificial metalloenzymes offer highly modular tools to perform bioorthogonal catalysis in live HEK cells.

Artificial Metalloenzymes: Reaction Scope and Optimization Strategies.

The incorporation of a synthetic, catalytically competent metallocofactor into a protein scaffold to generate an artificial metalloenzyme (ArM) has been explored since the late 1970's. Progress in the ensuing years was limited by the tools available for both organometallic synthesis and protein engineering. Advances in both of these areas, combined with increased appreciation of the potential benefits of combining attractive features of both homogeneous catalysis and enzymatic catalysis, led to a resurgence of interest in ArMs starting in the early 2000's. Perhaps the most intriguing of potential ArM properties is their ability to endow homogeneous catalysts with a genetic memory. Indeed, incorporating a homogeneous catalyst into a genetically encoded scaffold offers the opportunity to improve ArM performance by directed evolution. This capability could, in turn, lead to improvements in ArM efficiency similar to those obtained for natural enzymes, providing systems suitable for practical applications and greater insight into the role of second coordination sphere interactions in organometallic catalysis. Since its renaissance in the early 2000's, different aspects of artificial metalloenzymes have been extensively reviewed and highlighted. Our intent is to provide a comprehensive overview of all work in the field up to December 2016, organized according to reaction class. Because of the wide range of non-natural reactions catalyzed by ArMs, this was done using a functional-group transformation classification. The review begins with a summary of the proteins and the anchoring strategies used to date for the creation of ArMs, followed by a historical perspective. Then follows a summary of the reactions catalyzed by ArMs and a concluding critical outlook. This analysis allows for comparison of similar reactions catalyzed by ArMs constructed using different metallocofactor anchoring strategies, cofactors, protein scaffolds, and mutagenesis strategies. These data will be used to construct a searchable Web site on ArMs that will be updated regularly by the authors.

Cross-Regulation of an Artificial Metalloenzyme.

Cross-regulation of complex biochemical reaction networks is an essential feature of living systems. In a biomimetic spirit, we report on our efforts to program the temporal activation of an artificial metalloenzyme via cross-regulation by a natural enzyme. In the presence of urea, urease slowly releases ammonia that reversibly inhibits an artificial transfer hydrogenase. Addition of an acid, which acts as fuel, allows to maintain the system out of equilibrium.

From Photodriven Charge Accumulation to Fueling Enzyme Cascades in Molecular Factories.

In a multi-disciplinary team effort we gather experts on light-to-chemical energy conversion, artificial metalloenzymes, and bio-inspired polymer vesicles in order to construct molecular factories which produce added-value chemicals in an overall process fueled by solar energy. We outline our long-term vision and discuss specific challenges associated with this endeavor.

An NAD(P)H-Dependent Artificial Transfer Hydrogenase for Multienzymatic Cascades.

Enzymes typically depend on either NAD(P)H or FADH2 as hydride source for reduction purposes. In contrast, organometallic catalysts most often rely on isopropanol or formate to generate the reactive hydride moiety. Here we show that incorporation of a Cp*Ir cofactor possessing a biotin moiety and 4,7-dihydroxy-1,10-phenanthroline into streptavidin yields an NAD(P)H-dependent artificial transfer hydrogenase (ATHase). This ATHase (0.1 mol%) catalyzes imine reduction with 1 mM NADPH (2 mol%), which can be concurrently regenerated by a glucose dehydrogenase (GDH) using only 1.2 equiv of glucose. A four-enzyme cascade consisting of the ATHase, the GDH, a monoamine oxidase, and a catalase leads to the production of enantiopure amines.

H2O2-dependent substrate oxidation by an engineered diiron site in a bacterial hemerythrin.

The O2-binding carboxylate-bridged diiron site in DcrH-Hr was engineered in an effort to perform the H2O2-dependent oxidation of external substrates. A His residue was introduced near the diiron site in place of a conserved residue, Ile119. The I119H variant promotes the oxidation of guaiacol and 1,4-cyclohexadiene upon addition of H2O2.

Crystal structure, exogenous ligand binding, and redox properties of an engineered diiron active site in a bacterial hemerythrin.

A nonheme diiron active site in a 13 kDa hemerythrin-like domain of the bacterial chemotaxis protein DcrH-Hr contains an oxo bridge, two bridging carboxylate groups from Glu and Asp residues, and five terminally ligated His residues. We created a unique diiron coordination sphere containing five His and three Glu/Asp residues by replacing an Ile residue with Glu in DcrH-Hr. Direct coordination of the carboxylate group of E119 to Fe2 of the diiron site in the I119E variant was confirmed by X-ray crystallography. The substituted Glu is adjacent to an exogenous ligand-accessible tunnel. UV-vis absorption spectra indicate that the additional coordination of E119 inhibits the binding of the exogenous ligands azide and phenol to the diiron site. The extent of azide binding to the diiron site increases at pH ≤ 6, which is ascribed to protonation of the carboxylate ligand of E119. The diferrous state (deoxy form) of the engineered diiron site with the extra Glu residue is found to react more slowly than wild type with O2 to yield the diferric state (met form). The additional coordination of E119 to the diiron site also slows the rate of reduction from the met form. All these processes were found to be pH-dependent, which can be attributed to protonation state and coordination status of the E119 carboxylate. These results demonstrate that modifications of the endogenous coordination sphere can produce significant changes in the ligand binding and redox properties in a prototypical nonheme diiron-carboxylate protein active site.

Crystal structure and spectroscopic studies of a stable mixed-valent state of the hemerythrin-like domain of a bacterial chemotaxis protein.

The bacterial chemotaxis protein of Desulfovibrio vulgaris DcrH (DcrH-Hr) functions as an O(2)-sensing protein. This protein has a hemerythrin-like domain that includes a nonheme diiron center analogous to the diiron center of the hemerythrin (Hr) family. Interestingly, the O(2) affinity of DcrH-Hr is 3.3 × 10(6) M(-1), a value 25-fold higher than that of the Pectinaria gouldii Hr. This high affinity arises from the fast association of the O(2) ligand with DcrH-Hr (k(on) = 5.3 × 10(8) M(-1) s(-1)), which is made possible by a hydrophobic tunnel that accelerates the passage of the O(2) ligand to the diiron site. Furthermore, the autoxidation kinetics indicate that the rate of autoxidation of DcrH-Hr is 54-fold higher than that of P. gouldii Hr, indicating that the oxy form of DcrH-Hr is not stable toward autoxidation. More importantly, a mixed-valent state, semimet(R), which was spectroscopically observed in previous Hr studies, was found to be stable for over 1 week and isolable in the case of DcrH-Hr. The high-resolution crystal structures of the semimet(R)- (1.8 Å) and met-DcrH-Hr (1.4 Å) indicate that the semimet(R)- and met-DcrH-Hr species have very similar coordination geometry at the diiron site.

[Approach to alternative therapies in community pharmacy].

The growth of the dietary supplement in Japanese market suggests that the patient's need for assistance with self-care will also continue to grow. Patients' burgeoning acceptance and use of alternative therapies is another indication that patients seeking more from the health care system. The questionnaire showed that the most of them are expecting the pharmacists to provide their knowledge of dietary supplements. However, only limited amount of information is available. We founded the group Alterna in 2003, that compose the pharmacists working in community pharmacies accompanied with those in pharmaceutical universities. We have published the journal named "Alterna" that includes the information of dietary supplements, and it attained Vol. 9 in 2008. In the past studies, we evaluated the content and solubility of coenzyme Q10 dietary supplements in Japanese markets, some of which showed poor solubility. In others, we had taken up the information about vitamin and minerals, tea catechins, DHA and EPA, cooking oils to reduce body fat, collagen, etc. Findings in these studies present the opportunities for the pharmacists to provide the significant positive impact on health care outcomes and costs to patients.