Efficient production of natural sunscreens shinorine, porphyra-334, and mycosporine-2-glycine in Saccharomyces cerevisiae.

Mycosporine-like amino acids (MAAs) are promising natural sunscreens mainly produced in marine organisms. Until now, metabolic engineering efforts to produce MAAs in heterologous hosts have mainly focused on shinorine production, and the low production levels are still not suitable for industrial applications. In this study, we successfully developed Saccharomyces cerevisiae strains that can efficiently produce various disubstituted MAAs, including shinorine, porphyra-334, and mycosporine-2-glycine (M2G), which are formed by conjugating serine, threonine, and glycine to mycosporine-glycine (MG), respectively. We first generated an MG-producing strain by multiple integration of the biosynthetic genes from cyanobacteria and applying metabolic engineering strategies to increase sedoheptulose-7-phosphate pool, a substrate for MG production. Next, five mysD genes from cyanobacteria, which encode D-Ala-D-Ala ligase homologues that conjugate an amino acid to MG, were introduced into the MG-producing strain to determine the substrate preference of each MysD enzyme. MysDs from Lyngbya sp., Nostoclinckia, and Euhalothece sp. showed high specificity toward serine, threonine, and glycine, resulting in efficient production of shinorine, porphyra-334, and M2G, respectively. This is the first report on the production of porphyra-334 and M2G in S. cerevisiae. Furthermore, we identified that the substrate specificity of MysD was determined by the omega loop region of 43-45 amino acids predicted based on its structural homology to a D-Ala-D-Ala ligase from Thermus thermophilus involved in peptidoglycan biosynthesis. The substrate specificities of two MysD enzymes were interchangeable by swapping the omega loop region. Using the engineered strain expressing mysD from Lyngbya sp. or N. linckia, up to 1.53 g/L shinorine or 1.21 g/L porphyra-334 was produced by fed-batch fermentation in a 5-L bioreactor, the highest titer reported so far. These results suggest that S. cerevisiae is a promising host for industrial production of different types of MAAs, providing a sustainable and eco-friendly alternative for the development of natural sunscreens.

Evaluation of conductivity-based osmolality measurement in urine using the Sysmex UF5000.

BACKGROUND: Automated flow cytometry-based urine analyzer is increasingly being used to identify and enumerate cells and particles in urine specimens. It measures electrical conductivity which could be transformed to osmolality. Using this machine, all urine specimens could be screened for osmolality without requiring a separate dedicated device. We evaluated the performance of the new instrument, the UF-5000 (Sysmex Corporation), in the measurement of urine osmolality. METHODS: The precision of urine osmolality measurement by the UF-5000 was evaluated for 20 days and 4 times a day for 2 concentrations. The linearity and detection capability were evaluated according to the Clinical and Laboratory Standards Institute guidelines. For comparison, 270 random urine specimens from patients were tested simultaneously using the UF5000 and the OsmoPro micro-osmometer (Advanced instruments). RESULTS: The laboratory-based coefficient variations were less than 5%. Urine osmolality using the UF-5000 has a verified linear range (y = 1.097x + 16.91, R2  = .997). Within the comparison analysis, the mean difference was not large (-7.72%) but each differences were largely dispersed with 95% limits of agreement (LoA) from -70.5 to 55.06%, and the mean absolute difference -28.3 mOsm/kg with 95% LoA from -295.13 to 238.45 mOsm/kg. Cohen's kappa value was 0.54 (95% CI, 0.45-0.63). CONCLUSIONS: The UF-5000 measured conductivity and generated an acceptable quantitative analysis of urine osmolality. When compared with the results of the freezing point depression method used by the OsmoPro, a percentage of the measured urine osmolality by the UF-5000 was outside the allowable limit.

Distance-dependent magnetic resonance tuning as a versatile MRI sensing platform for biological targets.

Nanoscale distance-dependent phenomena, such as Förster resonance energy transfer, are important interactions for use in sensing and imaging, but their versatility for bioimaging can be limited by undesirable photon interactions with the surrounding biological matrix, especially in in vivo systems. Here, we report a new type of magnetism-based nanoscale distance-dependent phenomenon that can quantitatively and reversibly sense and image intra-/intermolecular interactions of biologically important targets. We introduce distance-dependent magnetic resonance tuning (MRET), which occurs between a paramagnetic 'enhancer' and a superparamagnetic 'quencher', where the T1 magnetic resonance imaging (MRI) signal is tuned ON or OFF depending on the separation distance between the quencher and the enhancer. With MRET, we demonstrate the principle of an MRI-based ruler for nanometre-scale distance measurement and the successful detection of both molecular interactions (for example, cleavage, binding, folding and unfolding) and biological targets in in vitro and in vivo systems. MRET can serve as a novel sensing principle to augment the exploration of a wide range of biological systems.

Magnetic Tandem Apoptosis for Overcoming Multidrug-Resistant Cancer.

Multidrug resistance (MDR) is a leading cause of failure in current chemotherapy treatment and constitutes a formidable challenge in therapeutics. Here, we demonstrate that a nanoscale magnetic tandem apoptosis trigger (m-TAT), which consists of a magnetic nanoparticle and chemodrug (e.g., doxorubicin), can completely remove MDR cancer cells in both in vitro and in vivo systems. m-TAT simultaneously activates extrinsic and intrinsic apoptosis signals in a synergistic fashion and downregulates the drug efflux pump (e.g., P-glycoprotein) which is one of the main causes of MDR. The tandem apoptosis strategy uses low level of chemodrug (in the nanomolar (nM) range) to eliminate MDR cancer cells. We further demonstrate that apoptosis of MDR cancer cells can be achieved in a spatially selective manner with single-cell level precision. Our study indicates that nanoscale tandem activation of convergent signaling pathways is a new platform concept to overcome MDR with high efficacy and specificity.

Colloidal Single-Layer Quantum Dots with Lateral Confinement Effects on 2D Exciton.

Controlled lateral quantum confinement in single-layer transition-metal chalcogenides (TMCs) can potentially combine the unique properties of two-dimensional (2D) exciton with the size-tunability of exciton energy, creating the single-layer quantum dots (SQDs) of 2D TMC materials. However, exploring such opportunities has been challenging due to the limited ability to produce well-defined SQDs with sufficiently high quality and size control, in conjunction with the commonly observed inconsistency in the optical properties. Here, we report an effective method to synthesize high-quality and size-controlled SQDs of WSe2 via multilayer quantum dots (MQDs) precursors, which enables grasping a clear picture of the role of lateral confinement on the optical properties of the 2D exciton. From the single-particle optical spectra and polarization anisotropy of WSe2 SQDs of varying sizes in addition to their ensemble data, we reveal how the properties of 2D exciton in single-layer TMCs evolve with increasing lateral quantum confinement.

Colloidal synthesis of single-layer MSe2 (M = Mo, W) nanosheets via anisotropic solution-phase growth approach.

The generation of single-layer 2-dimensional (2D) nanosheets has been challenging, especially in solution-phase, since it requires highly anisotropic growth processes that exclusively promote planar directionality during nanocrystal formation. In this study, we discovered that such selective growth pathways can be achieved by modulating the binding affinities of coordinating capping ligands to the edge facets of 2D layered transition-metal chalcogenides (TMCs). Upon changing the functional groups of the capping ligands from carboxylic acid to alcohol and amine with accordingly modulated binding affinities to the edges, the number of layers of nanosheets is controlled. Single-layer MSe2 (M = Mo, W) TMC nanosheets are obtained with the use of oleic acid, while multilayer nanosheets are formed with relatively strong binding ligands such as oleyl alcohol and oleylamine. With the choice of appropriate capping ligands in the 2D anisotropic growth regime, our solution-based synthetic method can serve a new guideline for obtaining single-layer TMC nanosheets.

Chemical synthetic strategy for single-layer transition-metal chalcogenides.

A solution-phase synthetic protocol to form two-dimensional (2D) single-layer transition-metal chalcogenides (TMCs) has long been sought; however, such efforts have been plagued with the spontaneous formation of multilayer sheets. In this study, we discovered a solution-phase synthetic protocol, called "diluted chalcogen continuous influx (DCCI)", where controlling the chalcogen source influx (e.g., H2S) during its reaction with the transition-metal halide precursor is the critical parameter for the formation of single-layer sheets as examined for the cases of group IV TMCs. The continuous influx of dilute H2S throughout the entire growth period is necessary for large sheet formation through the exclusive a- and b-axial growth processes. By contrast, the burst influx of highly concentrated H2S in the early stages of the growth process forms multilayer TMC nanodiscs. Our DCCI protocol is a new synthetic concept for single-layer TMCs and, in principle, can be operative for wide range of TMC nanosheets.

Detection of α-Thrombin with Platelet Glycoprotein Ibα (GP1bα) for the Development of a Coagulation Marker.

The detection of prothrombotic markers is crucial for understanding thromboembolism and assessing the effectiveness of anticoagulant drugs. α-Thrombin is a marker that plays a critical role in the coagulation cascade process. However, the detection of this enzymatic molecule was hindered by the absence of an efficient modality in the clinical environment. Previously, we reported that one α-thrombin interacts with two α-chains of glycoprotein Ib (GPIbα), i.e., multivalent protein binding (MPB), using bioresponsive hydrogel nanoparticles (nanogels) and optical microscopy. In this study, we demonstrated that GPIbα-mediated platforms led to the highly sensitive and quantitative detection of α-thrombin in various diagnostic systems. Initially, a bioresponsive nanogel-based surface plasmon resonance (nSPR) assay was developed that responds to the MPB of α-thrombin to GPIbα. The use of GPIbα for the detection of α-thrombin was further validated using the enzyme-linked immunosorbent assay, which is a gold-standard protein detection technique. Additionally, GPIbα-functionalized latex beads were developed to perform latex agglutination (LA) assays, which are widely used with hospital diagnostic instruments. Notably, the nSPR and LA assays exhibited a nearly 1000-fold improvement in sensitivity for α-thrombin detection compared to our previous optical microscopy method. The superiority of our GPIbα-mediated platforms lies in their stability for α-thrombin detection through protein-protein interactions. By contrast, assays relying on α-thrombin enzymatic activity using substrates face the challenge of a rapid decrease in postsample collection. These results suggested that the MPB of α-thrombin to GPIbα is an ideal mode for clinical α-thrombin detection, particularly in outpatient settings.

Unveiling chemical reactivity and structural transformation of two-dimensional layered nanocrystals.

Two-dimensional (2D) layered nanostructures are emerging fast due to their exceptional materials properties. While the importance of physical approaches (e.g., guest intercalation and exfoliation) of 2D layered nanomaterials has been recognized, an understanding of basic chemical reactions of these materials, especially in nanoscale regime, is obscure. Here, we show how chemical stimuli can influence the fate of reaction pathways of 2D layered nanocrystals. Depending on the chemical characteristics (Lewis acid ((1)O2) or base (H2O)) of external stimuli, TiS2 nanocrystal is respectively transformed to either a TiO2 nanodisc through a "compositional metathesis" or a TiO2 toroid through multistage "edge-selective structural transformation" processes. These chemical reactions can serve as the new design concept for functional 2D layered nanostructures. For example, TiS2(disc)-TiO2(shell) nanocrystal constitutes a high performance type II heterojunction which not only a wide range solar energy coverage (~80%) with near-infrared absorption edge, but also possesses enhanced electron transfer property.

A magnetic switch for the control of cell death signalling in in vitro and in vivo systems.

The regulation of cellular activities in a controlled manner is one of the most challenging issues in fields ranging from cell biology to biomedicine. Nanoparticles have the potential of becoming useful tools for controlling cell signalling pathways in a space and time selective fashion. Here, we have developed magnetic nanoparticles that turn on apoptosis cell signalling by using a magnetic field in a remote and non-invasive manner. The magnetic switch consists of zinc-doped iron oxide magnetic nanoparticles (Zn(0.4)Fe(2.6)O(4)), conjugated with a targeting antibody for death receptor 4 (DR4) of DLD-1 colon cancer cells. The magnetic switch, in its On mode when a magnetic field is applied to aggregate magnetic nanoparticle-bound DR4s, promotes apoptosis signalling pathways. We have also demonstrated that the magnetic switch is operable at the micrometre scale and that it can be applied in an in vivo system where apoptotic morphological changes of zebrafish are successfully induced.

Phosphorylation of chloramphenicol by a recombinant protein Yhr2 from Streptomyces avermitilis MA4680.

Although phosphorylation of chloramphenicol has been shown to occur in the chloramphenicol producer, Streptomyces venezuelae, there are no reports on the existence of chloramphenicol phosphorylase in other Streptomyces species. In the present study, we report the modification of chloramphenicol by a recombinant protein, designated as Yhr2 (encoded by SAV_877), from Streptomyces avermitilis MA4680. Recombinant Yhr2 was expressed in Escherichia coli BL21 (DE3) and the cells expressing this recombinant protein were shown to phosphorylate chloramphenicol to a 3'-O-phosphoryl ester derivative, resulting in an inactivated form of the antibiotic. Expression of yhr2 conferred chloramphenicol resistance to E. coli cells up to 25 µg/mL and in an in vitro reaction, adenosine triphosphate (ATP), guanosine triphosphate (GTP), adenosine diphosphate (ADP) and guanosine diphosphate (GDP) were shown to be the phosphate donors for phosphorylation of chloramphenicol. This study highlights that antibiotic resistance conferring genes could be easily expressed and functionalized in other organisms that do not produce the respective antibiotic.

Recent developments in texaphyrin chemistry and drug discovery.

Texaphyrins are pentaaza expanded porphyrins with the ability to form stable complexes with a variety of metal cations, particularly those of the lanthanide series. In biological milieus, texaphyrins act as redox mediators and mediate the production of reactive oxygen species (ROS). In this review, newer studies involving texaphyrin complexes targeting several different applications in anticancer therapy are described. In particular, the preparation of bismuth and lead texaphyrin complexes as potential α-core emitters for radiotherapy is detailed, as are gadolinium texaphyrin functionalized magnetic nanoparticles with features that make them of interest as dual-mode magnetic resonance imaging contrast agents and as constructs with anticancer activity mediated through ROS-induced sensitization and concurrent hyperthermia. Also discussed are gadolinium texaphyrin complexes as possible carrier systems for the targeted delivery of platinum payloads.

Magnetically triggered dual functional nanoparticles for resistance-free apoptotic hyperthermia.

Overcoming resistance: Heat-treated cancer cells possess a protective mechanism for resistance and survival. Resistance-free apoptosis-inducing magnetic nanoparticles (RAINs) successfully promote hyperthermic apoptosis, obstructing cell survival by triggering two functional units of heat generation and the release of geldanamycin (GM) for heat shock protein (Hsp) inhibition under an alternating magnetic field (AMF).

Strategic design of gold nanocatalysts for effective photocatalytic organic transformation.

We demonstrate the design strategy of free-standing Au nanocatalysts by correlating their physicochemical characteristics with photocatalytic performance. By tailoring the particle size and surface characteristics, we found that small Au nanocatalysts called Au nanoclusters with discrete energy levels are more effective than large metallic Au nanoparticles, while the microenvironments (e.g., charge status and hydrophilicity/hydrophobicity) around the surface of Au-nanoclusters are crucial in determining the performance. With the optimized Au nanocatalyst, under visible light, decarboxylative radical addition reactions for C-C bond formation (i.e., Giese reaction) were first achieved with high yields and further utilized for the preparation of one of the bioactive γ-aminobutyric acid derivatives, pregabalin (Lyrica®), demonstrating its potential in pharmaceutical applications.

Orthogonal Dual Photocatalysis of Single Atoms on Carbon Nitrides for One-Pot Relay Organic Transformation.

Single-atom photocatalysis has shown potential in various single-step organic transformations, but its use in multistep organic transformations in one reaction systems has rarely been achieved. Herein, we demonstrate atomic site orthogonality in the M1/C3N4 system (where M = Pd or Ni), enabling a cascade photoredox reaction involving oxidative and reductive reactions in a single system. The system utilizes visible-light-generated holes and electrons from C3N4, driving redox reactions (e.g., oxidation and fluorination) at the surface of C3N4 and facilitating cross-coupling reactions (e.g., C-C and C-O bond formation) at the metal site. The concept is generalized to different systems of Pd and Ni, thus making the catalytic site-orthogonal M1/C3N4 system an ideal photocatalyst for improving the efficiency and selectivity of multistep organic transformations.

Well-defined colloidal 2-D layered transition-metal chalcogenide nanocrystals via generalized synthetic protocols.

While interesting and unprecedented material characteristics of two dimensionality (2-D) layered nanomaterials are emerging, their reliable synthetic methodologies are not well developed. In this study we demonstrate general applicability of synthetic protocols to a wide range of colloidal 2-D layered transition-metal chalcogenide (TMC) nanocrystals. As distinctly different from other nanocrystals, we discovered that 2-D layered TMC nanocrystals are unstable in the presence of reactive radicals from elemental chalcogen during the crystal formation. We first introduce the synthesis of titanium sulfide and selenide where well-defined single crystallinity and lateral size controllability are verified, and then such synthetic protocols are extended to all of group IV and V transition-metal sulfide (TiS(2), ZrS(2), HfS(2), VS(2), NbS(2), and TaS(2)) and selenide (TiSe(2), ZrSe(3), HfSe(3), VSe(2), NbSe(2), and TaSe(2)) nanocrystals. The use of appropriate chalcogen source is found to be critical for the successful synthesis of 2-D layered TMC nanocrystals. CS(2) is an efficient chalcogen precursor for metal sulfide nanocrystals, whereas elemental Se is appropriate for metal selenide nanocrystals. We briefly discuss the effects of reactive radical characteristics of elemental S and Se on the formation of 2-D layered TMC nanocrystals.

Theranostic magnetic nanoparticles.

Early detection and treatment of disease is the most important component of a favorable prognosis. Biomedical researchers have thus invested tremendous effort in improving imaging techniques and treatment methods. Over the past decade, concepts and tools derived from nanotechnology have been applied to overcome the problems of conventional techniques for advanced diagnosis and therapy. In particular, advances in nanoparticle technology have created new paradigms for theranostics, which is defined as the combination of therapeutic and diagnostic agents within a single platform. In this Account, we examine the potential advantages and opportunities afforded by magnetic nanoparticles as platform materials for theranostics. We begin with a brief overview of relevant magnetic parameters, such as saturation magnetization, coercivity, and magnetocrystalline anisotropy. Understanding the interplay of these parameters is critical for optimizing magnetic characteristics needed for effective imaging and therapeutics, which include magnetic resonance imaging (MRI) relaxivity, heat emission, and attractive forces. We then discuss approaches to constructing an MRI nanoparticle contrast agent with high sensitivity. We further introduce a new design concept for a fault-free contrast agent, which is a T1 and T2 dual mode hybrid. Important capabilities of magnetic nanoparticles are the external controllability of magnetic heat generation and magnetic attractive forces for the transportation and movement of biological objects. We show that these functions can be utilized not only for therapeutic hyperthermia of cancer but also for controlled release of cancer drugs through the application of an external magnetic field. Additionally, the use of magnetic nanoparticles to drive mechanical forces is demonstrated to be useful for molecular-level cell signaling and for controlling the ultimate fate of the cell. Finally, we show that targeted imaging and therapy are made possible by attaching a variety of imaging and therapeutic components. These added components include therapeutic genes (small interfering RNA, or siRNA), cancer-specific ligands, and optical reporting dyes. The wide range of accessible features of magnetic nanoparticles underscores their potential as the most promising platform material available for theranostics.

Inactivation of phosphomannose isomerase gene abolishes sporulation and antibiotic production in Streptomyces coelicolor.

Phosphomannose isomerases (PMIs) in bacteria and fungi catalyze the reversible conversion of D-fructose-6-phosphate to D-mannose-6-phosphate during biosynthesis of GDP-mannose, which is the main intermediate in the mannosylation of important cell wall components, glycoproteins, and certain glycolipids. In the present study, the kinetic parameters of PMI from Streptomyces coelicolor were obtained, and its function on antibiotic production and sporulation was studied. manA (SCO3025) encoding PMI in S. coelicolor was deleted by insertional inactivation. Its mutant (S. coelicolor∆manA) was found to exhibit a bld-like phenotype. Additionally, S. coelicolor∆manA failed to produce the antibiotics actinorhodin and red tripyrolle undecylprodigiosin in liquid media. To identify the function of manA, the gene was cloned and expressed in Escherichia coli BL21 (DE3). The purified recombinant ManA exhibited PMI activity (K(cat)/K(m) (mM(-1) s(-1) = 0.41 for D-mannose-6-phosphate), but failed to show GDP-D-mannose pyrophosphorylase [GMP (ManC)] activity. Complementation analysis with manA from S. coelicolor or E. coli resulted in the recovery of bld-like phenotype of S. coelicolor∆manA. SCO3026, another ORF that encodes a protein with sequence similarity towards bifunctional PMI and GMP, was also tested for its ability to function as an alternate ManA. However, the purified protein of SCO3026 failed to exhibit both PMI and GMP activity. The present study shows that enzymes involved in carbohydrate metabolism could control cellular differentiation as well as the production of secondary metabolites.