Search

The development of effective anticancer drugs is essential for chemotherapy that specifically targets cancer tissues. We recently synthesized a multifunctional water-soluble anticancer polymer drug consisting of styrene-maleic acid copolymer (SMA) conjugated with glucosamine and boric acid (BA) (SGB complex). It demonstrated about 10 times higher tumor-selective accumulation compared with accumulation in normal tissues because of the enhanced permeability and retention effect, and it inhibited tumor growth via glycolysis inhibition, mitochondrial damage, and thermal neutron irradiation. Gaining insight into the anticancer effects of this SGB complex requires a determination of its structure. We therefore investigated the chemical structure of the SGB complex by means of nuclear magnetic resonance, infrared (IR) spectroscopy, and liquid chromatography-mass spectrometry. To establish the chemical structure of the SGB complex, we synthesized a simple model compound─maleic acid-glucosamine (MAG) conjugate─by using a maleic anhydride (MA) monomer unit instead of the SMA polymer. We obtained two MAG-BA complexes (MAGB) with molecular weights of 325 and 343 after the MAG reaction with BA. We confirmed, by using IR spectroscopy, that MAGB formed a stable complex via an amide bond between MA and glucosamine and that BA bound to glucosamine via a diol bond. As a result of this chemical design, identified via analysis of MAGB, the SGB complex can release BA and demonstrate toxicity to cancer cells through inhibition of lactate secretion in mild hypoxia that mimics the tumor microenvironment. For clinical application of the SGB complex, we confirmed that this complex is stable in the presence of serum. These findings confirm that our design of the SGB complex has various advantages in targeting solid cancers and exerting therapeutic effects when combined with neutron irradiation.

Photochemical switching of cytotoxicity by using spiropyran compounds with pyridinium and alkyl groups was investigated. The spiropyran compound, SP6, with a hexyl group as the alkyl group displayed negative photochromism, in which the hydrophilic open merocyanine form (MC form) was stable and isomerized to the hydrophobic closed spiro form (SP form) by visible light irradiation. Both MC and SP forms exhibited amphiphilicity because of the hydrophobic hexyl and hydrophilic pyridinium groups introduced. Cytotoxicity toward HeLa cells was observed for both MC and SP forms of SP6 at concentrations higher than the critical aggregation concentration of the isomers CACMC and CACSP (CACMC > CACSP), respectively. In contrast, cytotoxicity by SP6 was activated by visible light irradiation at concentrations between CACMC and CACSP; thus, photochemical switching of cytotoxicity from the OFF to ON state was achieved. Cytotoxicity was revealed to be caused by disruption of the cell membrane. The results provide an important step in developing novel next-generation photochemotherapy drugs.

Octanethiol (C8-SH) was shaken with an aqueous solution containing Au(III) and/or Ag(I) ions. The C8-SH molecules reacted with the Au(III) ions to form red-luminescent Au thiolates. The addition of...

Non-small cell lung cancer (NSCLC) is the leading cause of lung cancer-related deaths worldwide. Tumor-associated macrophages (TAMs), which can be polarized into tumor-promoting M2 phenotype, overexpress folate receptor beta (FRβ) and are associated with poor prognosis in NSCLC. In addition, calpain-2 (CAPN2) is overexpressed in NSCLC and is involved in tumor growth. To improve the anticancer efficacy of drugs and reduce their side effects in the treatment of NSCLC, it is important to develop smart drug delivery systems with specific targeting ability and controlled release mechanisms. In this study, FRβ-targeted pH-sensitive liposomes were designed as carriers to ensure efficient drug delivery and acid-responsive release in NSCLC cells. Folate-mediated targeting of FRβ in M2 TAMs and NSCLC cells effectively inhibited tumor growth and the stimulus-responsive drug release reduced the toxic side effects of the drug. The combination of doxycycline (anti-CAPN2) and docetaxel (anticancer drug) showed a synergistic inhibitory effect on tumor growth by suppressing CAPN2 expression.

Endocytosis is a cellular process in which substances are engulfed by the cellular membrane and budded off inside the cells to form vesicles. It plays key roles in controlling nutritional component uptake, immune responses, and other biological functions. A comprehensive understanding of endocytosis gives insights into such physiological functions and informs the design of medical nanodevices that need to enter cells. So far, endocytosis has been studied mostly using established cell lines. However, the established cell lines generally originate from cancer cells or are transformed from normal cells into immortalized cells. Therefore, primary cells may give us more reliable information about the endocytosis process of nanoparticles into cells. In this research, we studied the uptake of gold nanorods (AuNRs) with four different surface modifications (anionic/cationic polymers and anionic/cationic silica) by two kinds of primary cells (human monocyte-derived macrophages and human umbilical vein endothelial cells) and two kinds of established cell lines (HeLa cells and RAW 264.7 cells). We found that the surface properties of AuNRs affected their cellular uptake, and the cationic surface tended was advantageous for uptake, but it depended on the cell types. Control experiments using inhibitors of representative endocytosis pathways (macropinocytosis, clathrin-mediated endocytosis, and caveolae-mediated endocytosis) indicated that primary cells had a dominant uptake pathway for internalization of the AuNRs, whereas the established cell lines had multiple pathways. Our results provide us with novel insights into cellular uptake of AuNRs in that they depend not only on surface characters of the nanoparticles but also cell types, such as primary cells and established cell lines.

Magnesium (Mg) alloy has been actively investigated as a bioresorbable scaffold (BRS) for use as a next-generation stent because of its appropriate mechanical properties and biocompatibility. However, Mg alloy quickly degrades in the physiological environment. Hydrofluoric acid (HF) treatment and surface coating with biodegradable polymer are approaches for enhancing the corrosion resistance of Mg alloy. However, there are no studies that focus on the corrosion behavior of the Mg alloy stent after balloon catheter expansion, which results in mechanical stress and is required for stent placement. In this study, the corrosion behavior of a Mg alloy stent after expansion by a balloon catheter was investigated. Compared with the bare Mg alloy stent, the HF-treated Mg alloy stent showed excellent corrosion resistance without expansion. However, balloon catheter expansion caused small fragments and cracks to appear on the surface of the HF-treated Mg alloy stent and accelerated its corrosion rate. The HF-treated Mg alloy stent was therefore further coated with poly(d,l-lactic acid) (PDLLA). As a result, the high corrosion resistance of the coated stent was maintained after its expansion along with higher biocompatibility for endothelial cell adhesion than the stent without the polymer coating. The HF-treated and PDLLA-coated platform is expected to be a BRS candidate for clinical applications.

A preassembled Cas9/single-guide RNA complex (Cas9 ribonucleoprotein; Cas9 RNP) induces genome editing efficiently, with small off-target effects compared with the conventional techniques, such as plasmid DNA and mRNA systems. However, penetration of Cas9 RNP through the cell membrane is low. In particular, the incorporation of Cas9 RNP into neurons and the brain is challenging. In the present study, we have reported the use of a dendrimer (generation 3; G3)/glucuronylglucosyl-β-cyclodextrin conjugate (GUG-β-CDE (G3)) as a carrier of Cas9 RNP and evaluated genome editing activity in the neuron and the brain. A Cas9 RNP ternary complex with GUG-β-CDE (G3) was prepared by only mixing the components. The resulting complex exhibited higher genome editing activity than the complex with the dendrimer (G3), Lipofectamine 3000 or Lipofectamine CRISPRMAX in SH-SY5Y cells, a human neuroblastoma cell line. In addition, GUG-β-CDE (G3) enhanced the genome editing activity of Cas9 RNP in the whole mouse brain after a single intraventricular administration. Thus, GUG-β-CDE (G3) is a useful Cas9 RNP carrier that can induce genome editing in the neuron and brain.

Poly(D,L-lactide-co-glycolic) acid (PLGA) is a synthetic copolymer that has been used to design micro/nanoparticles as a carrier for macromolecules, such as protein and nucleic acids, that can be internalized by the endocytosis pathway. However, it is difficult to control the intracellular delivery to target organelles. Here we report an intracellular delivery system of nanoparticles modified with bacterial cytotoxins to the endoplasmic reticulum (ER) and anti-inflammatory activity of the nanoparticles. Subtilase cytotoxin (SubAB) is a bacterial toxin in certain enterohemorrhagic Escherichia coli (EHEC) strains that cleaves the host ER chaperone BiP and suppresses nuclear factor-kappaB (NF-κB) activation and nitric oxide (NO) generation in macrophages at sub-lethal concentration. PLGA-nanoparticles were modified with oligo histidine-tagged (6 × His-tagged) recombinant SubAB (SubAB-PLGA) through a pH-sensitive linkage, and their translocation to the ER in macrophage cell line J774.1 cells, effects on inducible NO synthase (iNOS), and levels of tumor necrosis factor (TNF)-α cytokine induced by lipopolysaccharide (LPS) were examined. Compared with free SubAB, SubAB-PLGA was significantly effective in BiP cleavage and the induction of the ER stress marker C/EBP homologous protein (CHOP) in J774.1 cells. Furthermore, SubAB-PLGA attenuated LPS-stimulated induction of iNOS and TNF-α. Our findings provide useful information for protein delivery to macrophages and may encourage therapeutic applications of nanoparticles to the treatment of inflammatory diseases.

Magnesium (Mg) alloy-based, bioresorbable scaffolding is a promising candidate for next-generation stents. Rapid corrosion of Mg alloy in the physiological environment, however, hinders its clinical application. Hydrofluoric acid (HF) treatment and biodegradable polymer coating have been widely reported to enhance corrosion resistance of the Mg alloy. Poor biocompatibility of biodegradable polymers, however, is known to promote adverse events such as intimal hyperplasia and thrombosis. We selected silk fibroin (SF) as the polymer for stent coating and evaluated drug release from the SF layer, corrosion resistance of the Mg alloy, and biocompatibility. After the stent was coated with SF, ethanol treatment of the SF layer enriched the β-sheet content. Release of sirolimus (SRL), a drug that prevents intimal hyperplasia, from the SF layer was slower than that with a poly(ε-caprolactone), the conventional biodegradable polymer used on medical devices. Ethanol treatment of the SF-coated stent further slowed SRL release from the SF layer. Crystalline domains in SF formed by the β-sheet structure could contribute to the slow release of SRL. The SF coating suppressed local and deep corrosion of the Mg alloy stent, although total corrosion remained unaffected. Uniform corrosion without local or deep corrosion prolongs the stent's radial strength. The SF coating showed excellent biocompatibility with human umbilical vein endothelial cells and minimal platelet adhesion. SF is expected to replace traditional biodegradable polymers for use on bioresorbable stents.

We coated triangular-shaped silver nanoparticles, a type of anisotropic nanoplate (NPL), with silica (i.e., prepared Ag@SiO

In 1979, development of the first polymer drug SMANCS [styrene-co-maleic acid (SMA) copolymer conjugated to neocarzinostatin (NCS)] by Maeda and colleagues was a breakthrough in the cancer field. When SMANCS was administered to mice, drug accumulation in tumors was markedly increased compared with accumulation of the parental drug NCS. This momentous result led to discovery of the enhanced permeability and retention effect (EPR effect) in 1986. Later, the EPR effect became known worldwide, especially in nanomedicine, and is still believed to be a universal mechanism for tumor-selective accumulation of nanomedicines. Some research groups recently characterized the EPR effect as a controversial concept and stated that it has not been fully demonstrated in clinical settings, but this erroneous belief is due to non-standard drug design and use of inappropriate tumor models in investigations. Many research groups recently provided solid evidence of the EPR effect in human cancers (e.g., renal and breast), with significant diversity and heterogeneity in various patients. In this review, we focus on the dynamics of the EPR effect and restoring tumor blood flow by using EPR effect enhancers. We also discuss new applications of EPR-based nanomedicine in boron neutron capture therapy and photodynamic therapy for solid tumors.

Silver nanoparticles are known to have strong antibacterial activity and constitute a novel material to treat infectious diseases caused by pathogenic bacteria. The antimicrobial activity of silver...

For more than three decades, enhanced permeability and retention (EPR)-effect-based nanomedicines have received considerable attention for tumor-selective treatment of solid tumors. However, treatment of advanced cancers remains a huge challenge in clinical situations because of occluded or embolized tumor blood vessels, which lead to so-called heterogeneity of the EPR effect. We previously developed a method to restore impaired blood flow in blood vessels by using nitric oxide donors and other agents called EPR-effect enhancers. Here, we show that two novel EPR-effect enhancers—isosorbide dinitrate (ISDN, Nitrol®) and sildenafil citrate—strongly potentiated delivery of three macromolecular drugs to tumors: a complex of poly(styrene-co-maleic acid) (SMA) and cisplatin, named Smaplatin® (chemotherapy), poly(N-(2-hydroxypropyl)methacrylamide) polymer-conjugated zinc protoporphyrin (photodynamic therapy and imaging), and SMA glucosamine-conjugated boric acid complex (boron neutron capture therapy). We tested these nanodrugs in mice with advanced C26 tumors. When these nanomedicines were administered together with ISDN or sildenafil, tumor delivery and thus positive therapeutic results increased two- to four-fold in tumors with diameters of 15 mm or more. These results confirmed the rationale for using EPR-effect enhancers to restore tumor blood flow. In conclusion, all EPR-effect enhancers tested showed great potential for application in cancer therapy.

Controlled drug release in response to light irradiation is an important technique for focusing drug elution to specific sites and reducing the side effects of drugs in normal tissue. In one example, we used double-stranded DNA to modify gold nanorods. When the gold nanorods were heated by irradiation with near-infrared light, single-stranded DNA was released. Thus, we successfully prepared a controlled release system that responds to near-infrared irradiation by combining heat-labile linkers such as double-stranded DNA. However, the drug-loading capacity on the surface of the nanoparticles was limited. To improve the loading efficiency, we encapsulated gold nanorods in poly(lactic-co-glycolic acid) (PLGA) nanoparticles, where PLGA acted as a drug payload. When the gold nanorod-containing PLGA nanoparticles were irradiated with a near-infrared laser, the PLGA nanoparticles were destroyed and significant drug release was observed. In another example, silver nanoplates were used as a near-infrared responsive photothermal nanodevice. Silver nanoparticles show antimicrobial activity that we expected could be controlled by light irradiation. First, we coated the silver nanoplates with gold atoms to mask the antimicrobial activity. When the gold-coated silver nanoplates were irradiated with a near-infrared pulsed laser, the shape of the silver nanoplates changed from plate-like to spherical, and silver ions were released. As a result, the antibacterial activity of the silver nanoplates was recovered. In this review, we outline examples of controlled release systems that respond to light irradiation. We believe that this review will contribute to improving the efficiency and safety of chemotherapy.

We synthesized unique water-soluble synthetic-polymer, styrene-maleic acid copolymer (SMA) conjugated glucosamine (SG); which formed a stable complex with boric acid (BA). This complex had a mean particle size of 15 nm by light scattering, and single peak in gel permeation chromatography. The particles were taken up by tumor cells five times faster than free BA in vitro and liberated BA at acidic tumor pH (5–7). Liberated BA inhibited glycolysis and resulted in tumor suppression in vivo. Intravenously injected SGB-complex did bind with albumin, and plasma half-life was about 8 h in mice, and accumulated to tumor tissues about 10 times more than in normal organs. IC50 of SGB-complex for HeLa cells under pO2 of 6–9% was about 20 μg/ml (free BA equivalent), 150 times more potent than free BA. Neutron irradiation of human oral cancer cells with SGB-complex resulted in 16 times greater cell-killing than that without SGB-complex. In vivo antitumor effect was evaluated after neutron irradiation only once in SCC VII tumor bearing mice and significant tumor suppression was confirmed. These results indicate that SGB-complex is a unique multifunctional anticancer agent with much more potent activity under low pO2 conditions as in large advanced cancers.

ErbB2 is a type of receptor tyrosine kinase, which is known to be involved in tumorigenesis, tumor aggressiveness, and clinical outcome. ErbB2-targeting therapy using therapeutic antibodies has been successful in breast cancer treatment. However, the need for repeated treatments and the high cost are major disadvantages with monoclonal antibody therapies. Compared with antibodies, peptides are cheap, relatively stable, and have low immunogenicity. We have developed a highly specific cancer-targeting drug delivery system using a targeting peptide to maximize the therapeutic efficiency of rapamycin and to help prevent drug resistance in ErbB2-positive breast cancer. Physicochemical characterization confirmed the successful construction of ErbB2-targeting liposomes (ErbB2Lipo). A comparison of a scrambled peptide (ScrErbB2) with the ErbB2-targeting peptide confirmed that these peptides had similar properties except for the targeting ability. The ErbB2Lipo exhibited higher delivery efficiency in ErbB2 positive BT-474 cells than non-targeting liposomes conjugated with ScrErbB2 (ScrErbB2Lipo). This peptide-targeting strategy has the potential to improve the efficacy of chemotherapy in ErbB2-positive cancers.

The cell membrane-coating strategy has opened new opportunities for the development of biomimetic and multifunctional drug delivery platforms. Recently, a variety of gold nanoparticles, which can combine with blood cell membranes, have been shown to provide an effective approach for cancer therapy. Meanwhile, this class of hybrid nanostructures can deceive the immunological system to exhibit synergistic therapeutic effects. Here, we synthesized red blood cell (RBC) and platelet membrane-coated gold nanostars containing curcumin (R/P-cGNS) and evaluated whether R/P-cGNS had improved anticancer efficacy. We also validated a controlled release profile under near-infrared irradiation for the ability to target melanoma cells and to have an immunomodulatory effect on macrophages. RBC membrane coating provided self-antigens; therefore, it could evade clearance by macrophages, while platelet membrane coating provided targetability to cancer cells. Additionally, the nutraceutical curcumin provided anticancer and anti-inflammatory effects. In conclusion, the results presented in this study demonstrated that R/P-cGNS can deliver drugs to the target region and enhance anticancer effects while avoiding macrophage phagocytosis. We believe that R/P-cGNS can be a new design of the cell-based hybrid system for effective cancer therapy.

Subtilase cytotoxin (SubAB) is a member of bacterial AB5 toxin produced by certain enterohemorrhagic E. coli strains which cleaves host chaperone BiP in endoplasmic reticulum (ER), leading to ER stress-mediated cytotoxicity. Previous study suggested that protein disulfide isomerase (PDI), an enzyme which catalyzes the formation and breakage of disulfide bonds in proteins, regulates AB5 toxin such as cholera toxin by unfolding of A subunit, leading to its translocation into cytosol to induce disease. Although SubAB targets ER and has similar A subunit to that of other AB5 toxins, it is unclear whether PDI can modulate the SubAB function. Here we determined the role of PDI on SubAB-induced BiP cleavage, ER stress response and cytotoxicity in HeLa cells. We found that PDI knockdown significantly suppressed SubAB-induced BiP cleavage and eIF2α phosphorylation. The accumulation of SubAB in ER was perturbed upon PDI knockdown. Finally, cell viability assay showed that PDI knockdown and PDI inhibitor canceled the SubAB-induced cytotoxicity. Present results suggested that SubAB, after cellular uptake, translocates into ER and interacts with BiP that might be modulated by PDI. Identification of pivotal role of host proteins on bacterial toxin to elicit its pathogenesis is necessary basis for development of potential chemotherapy and new diagnostic strategy for control of toxin-producing bacterial infections.

Gold nano-rods, rod-shaped gold nanoparticles, act as contrast agents for in vivo bioimaging, drug delivery vehicles and thermal converters for photothermal therapy. Pro-inflammatory cytokines play critical roles in the development of heart failure. We examined the delivery of GNRs into the failing heart of a transgenic (TG) mouse model of inflammatory cardiomyopathy with the cardiac-specific overexpression of TNF-α. We modified GNRs with polyethylene glycol (PEG) to avoid cytotoxicity and reduce the rapid clearance of nanoparticles from blood. PEG-modified GNRs (4.5 mM as gold atoms, 200 μL) were administered intravenously to TG (n = 7) and wild-type (WT) mice (n = 5). These were killed 24 h later, and the heart, lung, liver, kidney and spleen were excised. A quantitative analysis of gold was performed using inductively coupled plasma mass or optical emission spectrometry. The amount of gold (ng) in the TG heart (3.24 ± 1.56 ng/mg heart weight) was significantly greater than that in the WT heart (1.01 ± 0.19; p

Transdermal protein delivery is a powerful and attractive method for protein therapy and dermal vaccination compared with other administrations. However, this delivery method is restricted by the low permeability of the stratum corneum (SC), a hydrophobic barrier that restricts the entry of hydrophilic molecules such as proteins. In this study, we developed an improved gel patch system carrying ovalbumin and ovalbumin epitope peptide, and then compared their permeability into the skin. First, the gel patch was placed on mouse skin to allow contact with the polymer coated gold nanorods and then irradiated by a continuous-wave laser. Thermal ablation of the SC improved the permeability and translocation of ovalbumin and the peptide. Fluorescence images showed the translocation was enhanced when the skin was treated with the FITC-modified ovalbumin epitope peptide. However, induction of anti-OVA IgG production after treatment with the FITC-modified ovalbumin epitope peptide was lower than that with FITC-OVA.

Previous studies have indicated pro-tumor functions of macrophages in tumor progression in different types of malignant tumors. The detailed mechanisms of cell-cell interaction between macrophages and tumor cells have been investigated by means of in vitro co-culture experiments. The present study developed magnetite nanoparticles modified with gelatin that are specifically engulfed by macrophages and investigated methods to deplete these macrophages in co-culture experiments using a magnet. T98G glioma cell line and human monocyte-derived macrophages were mixed and co-cultured for 2 days. The T98G cells were isolated by depletion of the macrophages using the magnetite nanoparticles. mRNA expression of a number of pro-tumor molecules in the isolated T98G cells, with or without co-culture with macrophages, was then evaluated. The mRNA expression levels of chemokine (CC motif) ligand 2, interleukin-6 and macrophage-colony stimulating factor receptor (M-CSFR) were significantly upregulated in T98G cells by co-culture with macrophages (P

Gold nanorods are promising metals in several biomedical applications such as bioimaging, thermal therapy, and drug delivery. Gold nanorods have strong absorption bands in near-infrared (NIR) light region and show photothermal effects. Since NIR light can penetrate deeply into tissues, their unique optical, chemical, and biological properties have attracted considerable clinical interest. Gold nanorods are expected to act not only as on-demand thermal converters for photothermal therapy but also as mediators of a controlled drug-release system responding to light irradiation. In this review, we discuss current progress using gold nanorods as bioimaging platform, phototherapeutic agents, and drug delivery vehicles.

To provide a general overview of the microbial communities in environmentally conserved estuaries, the top 5 cm of sediment was sampled from the sandy estuary of the Shirakawa River and from the muddy estuary of the Midorikawa River. Higher amounts of organic matter were detected in the Midorikawa estuary sample than in the Shirakawa estuary sample. Measurement of redox potential revealed that the Shirakawa estuary was aerobic and the Midorikawa estuary was much less aerobic. Clone analysis was performed by targeting partial 16S rRNA gene sequences and using extracted DNA from the samples as a template. Various bacteria were detected, among which Gammaproteobacteria was dominant at both estuaries. Unclassified clones were detected in the Gammaproteobacteria group, mainly among samples from the Midorikawa estuary. Other detected bacterial groups were Alphaproteobacteria , Deltaproteobacteria , Chloroflexi , Actinobacteria , and Bacteroidetes . All the Deltaproteobacteria clones were anaerobic sulfate-reducing bacteria. Those aerobic and anaerobic bacteria coexisted in the top 5 cm of the estuary sediments indicating the surface layer have active sulfur and carbon cycle. Abundance of aerobic Gammaproteobacteria may be an indicator for conserved estuaries. Keywords: conserved environment, clone analysis, estuary, microbial community, 16S rRNA gene.

A PLGA-AgNPL film was fabricated to demonstrate antibiofilm formation activity against Staphylococcus aureus. The film with a PLGA concentration of 25 mg mL−1 with 0.1% silver nanoplates showed promising (94%) reduction of biofilm formation. The PLGA-AgNPL film did not produce many silver ions. The mode of action of the film was hypothesized to be contact killing when bacteria were attached to the surface to commence biofilm formation.

Soft materials, including elastic polymers and hydrogels, are promising materials for soft robots, implantable devices, and wearable devices owing to their flexibility and biocompatibility. In recent years, the scope of use of such soft materials is expanding further because of their integration with metal micro- and nanostructures, which exhibit attractive optical and electrical properties. In this study, we demonstrate the formation of metal microstructures inside a hydrogel by photoreduction of metal ion by using femtosecond laser. Because the hydrogels have permeability to fluids, metal ions are able to be added by simply immersing the hydrogel into the metal ion solution. Femtosecond laser pulses were focused and scanned in the hydrogel to fabricate metal microstructures [1, 2]. The optical diffraction pattern obtained with the silver grating inside the hydrogel showed spaced diffraction spots, which indicated that a regular, periodic silver grating was formed. Notably, the distance between the diffraction spots changed when the water content in the hydrogel was reduced. The grating period decreased when the hydrogel shrank owing to the loss of water (Fig. 1(a)), but the straight shapes of the line structures were preserved, which demonstrated the optical tunability of the fabricated structure.

The spatially-targeted fabrication of bimetallic microstructures coexisting in the supporting hydrogel is demonstrated by multi-photon photoreduction. Microstructures composed of gold and silver were fabricated along a predefined trajectory by taking advantages of the hydrogel's ionic permeability. Different resonant wavelengths of optical absorption were obtained for gold, silver, and their bimetallic structures. Transmission electron microscopy and energy dispersive X-ray analysis revealed that the optical properties are attributable to the formation of bimetallic structure consisted of core-shell nanoparticles. The fabrication of dissimilar metal structures within hydrogel is a promising technique for optically driven actuators in soft robotics and sensing applications by allowing for site-selective optical properties. © 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

A denitrification hotspot was detected in Kumamoto groundwater in a previous study, little information is concerned with the microbial community profile including denitrifier. Seven samples from inside and outside of the hotspot were collected to analyze the bacterial, archaeal, and denitrifier community using a cloning library approach. The results showed that the microbial diversity and distribution were distinct among the zones. Major operational taxonomic units closely related to Proteobacteria, Firmicutes, and Actinobacteria were found in the denitrification hotspot. Additionally, type I methylotrophic bacteria, such as Methylobacter and Methylomonas, only inhabited the hotspot zone. A positive association between methanogenic archaea and methylotrophic bacteria was observed. Interestingly, acidic ammonia-oxidizing archaea were detected in the neutral groundwater environment. In addition, gene-specific analysis targeting nirS indicated that the majority of denitrifiers belonged to the Proteobacteria including Sulfuritalea hydrogenivorans, Pseudomonas balearica and Sulfuricella denitrificans. Based on the comparative analysis of microbial communities and physiochemical parameters inside and outside of the denitrification hotspot, the anaerobic environment with organic compounds and nitrate could support the biotic reduction of nitrate in confined groundwater.