Fluorophore-Decorated Mie Resonant Silicon Nanosphere for Scattering/Fluorescence Dual-Mode Imaging.

Inorganic nanoparticles with multiple functions have been attracting attention as multimodal nanoprobes in bioimaging, biomolecule detection, and medical diagnosis and treatment. A drawback of conventional metallic nanoparticle-based nanoprobes is the Ohmic losses that lead to fluorescence quenching of attached molecules and local heating under light irradiation. Here, metal-free nanoprobes capable of scattering/fluorescence dual-mode imaging are developed. The nanoprobes are composed of a silicon nanosphere core having efficient Mie scattering in the visible to near infrared range and a fluorophore doped silica shell. The dark-field scattering and photoluminescence images/spectra for nanoprobes made from different size silicon nanospheres and different kinds of fluorophores are studied by single particle spectroscopy. The fluorescence spectra are strongly modified by the Mie modes of a silicon nanosphere core. By comparing scattering and fluorescence spectra and calculated Purcell factors, the fluorescence enhancement factor is quantitatively discussed. In vitro scattering/fluorescence imaging studies on human cancer cells demonstrate that the developed nanoparticles work as scattering/fluorescence dual-mode imaging nanoprobes.

Dual real-time in vivo monitoring system of the brain-gut axis.

The brain-gut axis which is an interaction between recognition and emotion and the gut sensory system for food and microbiota is important for health. However, there is no real-time monitoring system of the brain and the gut simultaneously so far. We attempted to establish a dual real-time monitoring system for the brain-gut axis by a combination of intravital Ca2+ imaging of the gut and electroencephalogram. Using a conditional Yellow Cameleon 3.60 expression mouse line, we performed intravital imaging of the gut, electrophysiological recordings of the vagus nerve, and electroencephalogram recordings of the various cortical regions simultaneously upon capsaicin stimuli as a positive control. Upon capsaicin administration into the small intestinal lumen, a simultaneous response of Ca2+ signal in the enteric nervous system and cortical local field potentials (LFPs) was successfully observed. Both of them responded immediately upon capsaicin stimuli. Capsaicin triggered a significant increase in the frequency of vagus nerve spikes and a significant decrease in the slow-wave power of cortical LFPs. Furthermore, capsaicin induced delayed and sustained Ca2+ signal in intestinal epithelial cells and then suppressed intestinal motility. The dual real-time monitoring system of the brain and the gut enables to dissect the interaction between the brain and the gut over time with precision.

Sniffing behaviour-related changes in cardiac and cortical activity in rats.

KEY POINTS: High-frequency (HF) sniffing represents active odour sampling and an increase in the animal's motivation. We examined how HF sniffing affects the physiological activity of the brain-body system. During HF sniffing, heart rates and the ratio of theta to delta critical local field potential power were comparable to those observed during motion periods. Vagus nerve spike rates did not vary depending on HF sniffing. Our results suggest that physiological factors in the central nervous system and the periphery are not simply determined by locomotion but are crucially associated with HF sniffing. ABSTRACT: Sniffing is a fundamental behaviour for odour sampling, and high-frequency (HF) sniffing, generally at a sniff frequency of more than 6 Hz, is considered to represent an animal's increased motivation to explore external environments. Here, we examined how HF sniffing is associated with changes in physiological signals from the central and peripheral organs in rats. During HF sniffing while the rats were stationary, heart rates, the magnitude of dorsal neck muscle contraction, and the ratio of theta to delta local field potential power in the motor cortex were comparable to those observed during motion periods and were significantly higher than those observed during resting respiration periods. No pronounced changes in vagus nerve spike rates were detected in relation to HF sniffing. These results demonstrate that central and peripheral physiological factors are crucially associated with the emergence of HF sniffing, especially during quiescent periods. Behavioural data might be improved to more accurately evaluate an animal's internal psychological state if they are combined with a sniffing pattern as a physiological marker.

Vagus nerve spiking activity associated with locomotion and cortical arousal states in a freely moving rat.

The vagus nerve serves as a central pathway for communication between the central and peripheral organs. Despite traditional knowledge of vagus nerve functions, detailed neurophysiological dynamics of the vagus nerve in naïve behavior remain to be understood. In this study, we developed a new method to record spiking patterns from the cervical vagus nerve while simultaneously monitoring central and peripheral organ bioelectrical signals in a freely moving rat. When the rats transiently elevated locomotor activity, the frequency of vagus nerve spikes was correspondingly increased, and this activity was retained for several seconds after the increase in running speed terminated. Spike patterns of the vagus nerve were not robustly associated with which arms the animals entered on an elevated plus maze. During sniffing behavior, vagus nerve spikes were nearly absent. During stopping, the vagus nerve spike patterns differed considerably depending on external contexts and peripheral activity states associated with cortical arousal levels. Stimulation of the vagus nerve altered rat's running speed and cortical arousal states depending on running speed at the instant of stimulation. These observations are a new step for uncovering the physiological dynamics of the vagus nerve modulating the visceral organs such as cardiovascular, respiratory, and gastrointestinal systems.

Metabolic engineering of the 2-ketobutyrate biosynthetic pathway for 1-propanol production in Saccharomyces cerevisiae.

BACKGROUND: To produce 1-propanol as a potential biofuel, metabolic engineering of microorganisms, such as E. coli, has been studied. However, 1-propanol production using metabolically engineered Saccharomyces cerevisiae, which has an amazing ability to produce ethanol and is thus alcohol-tolerant, has infrequently been reported. Therefore, in this study, we aimed to engineer S. cerevisiae strains capable of producing 1-propanol at high levels. RESULTS: We found that the activity of endogenous 2-keto acid decarboxylase and alcohol/aldehyde dehydrogenase is sufficient to convert 2-ketobutyrate (2 KB) to 500 mg/L 1-propanol in yeast. Production of 1-propanol could be increased by: (i) the construction of an artificial 2 KB biosynthetic pathway from pyruvate via citramalate (cimA); (ii) overexpression of threonine dehydratase (tdcB); (iii) enhancement of threonine biosynthesis from aspartate (thrA, thrB and thrC); and (iv) deletion of the GLY1 gene that regulates a competing pathway converting threonine to glycine. With high-density anaerobic fermentation of the engineered S. cerevisiae strain YG5C4231, we succeeded in producing 180 mg/L 1-propanol from glucose. CONCLUSION: These results indicate that the engineering of a citramalate-mediated pathway as a production method for 1-propanol in S. cerevisiae is effective. Although optimization of the carbon flux in S. cerevisiae is necessary to harness this pathway, it is a promising candidate for the large-scale production of 1-propanol.

Mib1 modulates dynamin 2 recruitment via Snx18 to promote Dll1 endocytosis for efficient Notch signaling.

Notch signaling regulates normal development and tissue homeostasis. Ligand endocytosis plays critical roles in Notch signaling activation. Endocytic proteins such as epsin and dynamin participate in Notch ligand activity by mediating Notch ligand endocytosis. The ubiquitin ligase Mib1 also plays essential roles in Notch signaling via Notch ligand ubiquitination. However, the molecular links between Mib1 and endocytic proteins have not been fully defined. Here, we show that Mib1 is involved in dynamin 2 recruitment to Dll1 and that Snx18, which interacts with dynamin 2, modestly regulates Dll1 endocytosis. Furthermore, the ubiquitin ligase activity of Mib1 is induced by Notch ligand-receptor interactions. Mib1 promotes the interaction between dynamin 2 and Snx18 in an ubiquitin ligase activity-dependent manner. These results suggest that Mib1 modulates dynamin recruitment by regulating the interaction between Snx18 and dynamin 2, thereby helping to ensure the efficient signaling activity of Notch ligands.

Affibody-displaying bio-nanocapsules effective in EGFR, typical biomarker, expressed in various cancer cells.

The expression of epidermal growth factor receptor (EGFR) across a wide range of tumor cells has attracted attention for use as a tumor marker in drug delivery systems. Therefore, binding molecules with the ability to target EGFR have been developed. Among them, we focused on affibodies that are binding proteins derived from staphylococcal protein A. By displaying affibody (ZEGFR) binding to EGFR on the surface of a bio-nanocapsule (BNC) derived from a hepatitis B virus (HBV), we developed an altered BNC (ZEGFR-BNC) with a high specificity to EGFR-expressing cells. We considered two different types of ZEGFR (Z955 and Z1907), and found that the Z1907 dimer-displaying BNC ([Z1907]2-BNC) could effectively bind to EGFR-expressing cells and deliver drugs to the cytosol. Since this study showed that [Z1907]2-BNC could target EGFR-expressing cells, we would use this particle as a drug delivery carrier for various cancer cells expressing EGFR.

Simultaneous Recordings of Central and Peripheral Bioelectrical Signals in a Freely Moving Rodent.

Understanding physiological interactions between the central and peripheral nervous systems requires an experimental strategy to simultaneously monitor activity patterns of the brain and peripheral organs. In this study, we developed a novel method to record extracellular field potential signals from a wide range of brain regions together with electrocardiograms, electromyograms, and breathing signals from a freely moving rodent. This method collects all recorded signals into a single device mounted on an animal's head, allowing the reduction of experimental costs and the simplification of data processing. The methodological concept is applicable to a number of biological research issues of how the brain-body association is altered in response to various environmental changes, emotional challenges, and acute and chronic dysfunction of internal organs.

Mutation of arginine residues to avoid non-specific cellular uptakes for hepatitis B virus core particles.

BACKGROUND: The hepatitis B virus core (HBc) particle is known as a promising new carrier for the delivery of drugs and nucleic acids. However, since the arginine-rich domain that is located in the C-terminal region of the HBc monomer binds to the heparan sulphate proteoglycan on the cell surface due to its positive charge, HBc particles are introduced non-specifically into a wide range of cells. To avoid non-specific cellular uptake with the intent to control the ability of cell targeting, we individually replaced the respective arginine (R) residues of the arginine-rich domain located in amino acid positions 150-159 in glycine (G) residues. RESULTS: The mutated HBc particles in which R154 was replaced with glycine (G) residue (R154G) showed a drastic decrease in the ability to bind to the heparan sulphate proteoglycan and to avoid non-specific cellular uptake by several types of cancer cells. CONCLUSIONS: Because this mutant particle retains most of its C-terminal arginine-rich residues, it would be useful in the targeting of specificity-altered HBc particles in the delivery of nucleic acids.

A display of pH-sensitive fusogenic GALA peptide facilitates endosomal escape from a Bio-nanocapsule via an endocytic uptake pathway.

BACKGROUND: An affibody-displaying bio-nanocapsule (ZHER2-BNC) with a hepatocyte specificity derived from hepatitis B virus (HBV) was converted into an affibody, ZHER2, that recognizes HER2 receptors. This affibody was previously reported to be the result of the endocytosis-dependent specific uptake of proteins and siRNA into target cancer cells. To assist the endosomal escape of inclusions, a helper lipid with pH-sensitive fusogenic ability (1,2-dioleoyl-sn-glycero-3-phos phoethanolamine; DOPE) was conjugated with a ZHER2-BNC. FINDINGS: In this study, we displayed a pH-sensitive fusogenic GALA peptide on the surface of a particle in order to confer the ability of endosomal escape to a ZHER2-BNC. A GALA-displaying ZHER2-BNC purified from yeast uneventfully formed a particle structure. Furthermore, endosomal escape of the particle was facilitated after endocytic uptake and release of the inclusions to the cytoplasm without the cell toxicity. CONCLUSION: The genetic fusion of a GALA peptide to the virus-like particle confers the ability of endosomal escape.

Non-canonical interplay between glutamatergic NMDA and dopamine receptors shapes synaptogenesis.

Direct interactions between receptors at the neuronal surface have long been proposed to tune signaling cascades and neuronal communication in health and disease. Yet, the lack of direct investigation methods to measure, in live neurons, the interaction between different membrane receptors at the single molecule level has raised unanswered questions on the biophysical properties and biological roles of such receptor interactome. Using a multidimensional spectral single molecule-localization microscopy (MS-SMLM) approach, we monitored the interaction between two membrane receptors, i.e. glutamatergic NMDA (NMDAR) and G protein-coupled dopamine D1 (D1R) receptors. The transient interaction was randomly observed along the dendritic tree of hippocampal neurons. It was higher early in development, promoting the formation of NMDAR-D1R complexes in an mGluR5- and CK1-dependent manner, favoring NMDAR clusters and synaptogenesis in a dopamine receptor signaling-independent manner. Preventing the interaction in the neonate, and not adult, brain alters in vivo spontaneous neuronal network activity pattern in male mice. Thus, a weak and transient interaction between NMDAR and D1R plays a structural and functional role in the developing brain.

Targeting cancer cell-specific RNA interference by siRNA delivery using a complex carrier of affibody-displaying bio-nanocapsules and liposomes.

BACKGROUND: Small interfering RNA (siRNA) has attracted attention in the field of nucleic acid medicine as a RNA interference (RNAi) application that leads to gene silencing due to specific messenger RNA (mRNA) destruction. However, since siRNA is unstable in blood and unable to cross the cell membrane, encapsulation of siRNA into a carrier is required. RESULTS: In this study, we used a carrier that combined ZHER2-displaying bio-nanocapsule (derived from hepatitis B virus surface antigen) and liposomes in a complex in order to investigate the feasibility of effective and target-cell-specific RNAi applications. As a result, by observing RNAi only in HER2-expressing breast cancer cells, using our proposed methodology, we successfully demonstrated target-cell-specific delivery and effective function expression of siRNA. CONCLUSIONS: These findings show that, in the field of nucleic acid medicine, ZHER2-BNC/LP can be a useful carrier for siRNA delivery, and could also become a useful tool for gene silencing and to accomplish protein knock-down.

Skeletal myotube-derived extracellular vesicles enhance itaconate production and attenuate inflammatory responses of macrophages.

Introduction: Macrophages play an important role in the innate immunity. While macrophage inflammation is necessary for biological defense, it must be appropriately controlled. Extracellular vesicles (EVs) are small vesicles released from all types of cells and play a central role in intercellular communication. Skeletal muscle has been suggested to release anti-inflammatory factors, but the effect of myotube-derived EVs on macrophages is unknown. As an anti-inflammatory mechanism of macrophages, the immune responsive gene 1 (IRG1)-itaconate pathway is essential. In this study, we show that skeletal muscle-derived EVs suppress macrophage inflammatory responses, upregulating the IRG1-itaconate pathway. Methods: C2C12 myoblasts were differentiated into myotubes and EVs were extracted by ultracentrifugation. Skeletal myotube-derived EVs were administered to mouse bone marrow-derived macrophages, then lipopolysaccharide (LPS) stimulation was performed and inflammatory cytokine expression was measured by RT-qPCR. Metabolite abundance in macrophages after addition of EVs was measured by CE/MS, and IRG1 expression was measured by RT-PCR. Furthermore, RNA-seq analysis was performed on macrophages after EV treatment. Results: EVs attenuated the expression of LPS-induced pro-inflammatory factors in macrophages. Itaconate abundance and IRG1 expression were significantly increased in the EV-treated group. RNA-seq analysis revealed activation of the PI3K-Akt and JAK-STAT pathways in macrophages after EV treatment. The most abundant miRNA in myotube EVs was miR-206-3p, followed by miR-378a-3p, miR-30d-5p, and miR-21a-5p. Discussion: Skeletal myotube EVs are supposed to increase the production of itaconate via upregulation of IRG1 expression and exhibited an anti-inflammatory effect in macrophages. This anti-inflammatory effect was suggested to involve the PI3K-Akt and JAK-STAT pathways. The miRNA profiles within EVs implied that miR-206-3p, miR-378a-3p, miR-30d-5p, and miR-21a-5p may be responsible for the anti-inflammatory effects of the EVs. In summary, in this study we showed that myotube-derived EVs prevent macrophage inflammatory responses by activating the IRG1-itaconate pathway.

Electrical stimulation facilitates NADPH production in pentose phosphate pathway and exerts an anti-inflammatory effect in macrophages.

Macrophages play an important role as effector cells in innate immune system. Meanwhile, macrophages activated in a pro-inflammatory direction alter intracellular metabolism and damage intact tissues by increasing reactive oxygen species (ROS). Electrical stimulation (ES), a predominant physical agent to control metabolism in cells and tissues, has been reported to exert anti-inflammatory effect on immune cells. However, the mechanism underlying the anti-inflammatory effects by ES is unknown. This study aimed to investigate the effect of ES on metabolism in glycolytic-tricarboxylic acid cycle (TCA) cycle and inflammatory responses in macrophages. ES was performed on bone marrow-derived macrophages and followed by a stimulation with LPS. The inflammatory cytokine expression levels were analyzed by real-time polymerase chain reaction and ELISA. ROS production was analyzed by CellRox Green Reagent and metabolites by capillary electrophoresis-mass spectrometry. As a result, ES significantly reduced proinflammatory cytokine expression levels and ROS generation compared to the LPS group and increased glucose-1-phosphate, a metabolite of glycogen. ES also increased intermediate metabolites of the pentose phosphate pathway (PPP); ribulose-5-phosphate, rebose-5 phosphate, and nicotinamide adenine dinucleotide phosphate, a key factor of cellular antioxidation systems, as well as α-Ketoglutarate, an anti-oxidative metabolite in the TCA cycle. Our findings imply that ES enhanced NADPH production with enhancement of PPP, and also decreased oxidative stress and inflammatory responses in macrophages.

Biocompatible polymer-modified gold nanocomposites of different shapes as radiation sensitizers.

Radiation therapy is a powerful approach for cancer treatment due to its low invasiveness. The development of radiation sensitizers is of great importance as they assist in providing radiation therapy at a low dose. In this study, poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC)-modified gold nanocomposites of different shapes were created using the grafting-to approach to serve as a novel radiation sensitizer with high cellular uptake. The effect of the shape of the nanocomposite on cellular uptake by the breast cancer cell line MCF-7 was also investigated. The PMPC-modified gold nanostars showed the highest cellular uptake compared to the other gold nanocomposites (spheres and rods), whereas cell cytotoxicity was negligible among all candidates. Furthermore, the therapeutic effect of radiation of PMPC-modified nanostars was the highest among all the gold nanocomposites. These results clearly indicate that the shape of the gold nanocomposite is an important parameter for cellular uptake and radiation sensitizing effects in breast cancer cells.

Pulsed-Ultrasound Irradiation Induces the Production of Itaconate and Attenuates Inflammatory Responses in Macrophages.

Background: Itaconate is a key metabolite in the innate immune system and exerts strong anti-inflammatory effects in macrophages. For the production of itaconate in macrophages, immune-responsive gene 1 (IRG1) is an imperative enzyme, and activating the IRG1-itaconate pathway is reported to alleviate inflammatory diseases by upregulating nuclear factor-erythroid 2-related factor 2 (NRF2). However, there are very few reports on strategies to increase itaconate production. Ultrasound therapy is a widely used intervention for anti-inflammatory and soft-tissue regeneration purposes. Here we show the effect of ultrasound irradiation on the production of itaconate in macrophages. Methods: Murine bone marrow-derived macrophages (BMDMs) were exposed to pulsed ultrasound (3.0 W/cm2) for 5 minutes. Three hours after irradiation, the intracellular levels of metabolites and mRNA expression levels of Irg1 and Nrf2 were measured using CE/MS and qPCR, respectively. To evaluate macrophage inflammation status, 3 h after irradiation, the cells were stimulated with 100 ng/mL lipopolysaccharide (LPS) for 1.5 h and the mRNA expression levels of pro-inflammatory factors (Il-1ß, Il-6, and Tnf-α) were measured. Student's t-test, one-way ANOVA and Tukey's multiple comparison test were used for statistical processing, and the significance level was set to less than 5%. Results: Ultrasound irradiation significantly increased the intracellular itaconate level and the expression levels of Irg1 and Nrf2 in BMDMs. Upregulation of Il-1ß, Il-6, and Tnf-α by LPS was significantly suppressed in BMDMs treated with ultrasound. Ultrasound irradiation did not affect cell viability and apoptosis. Conclusion: Ultrasound irradiation induces the production of itaconate by upregulating Irg1 expression and attenuates inflammatory responses in macrophages via Nrf2. These results suggest that ultrasound is a potentially useful method to increase itaconate production in macrophages.

Reactive oxygen species-inducing titanium peroxide nanoparticles as promising radiosensitizers for eliminating pancreatic cancer stem cells.

BACKGROUND: Despite recent advances in radiotherapy, radioresistance in patients with pancreatic cancer remains a crucial dilemma for clinical treatment. Cancer stem cells (CSCs) represent a major factor in radioresistance. Developing a potent radiosensitizer may be a novel candidate for the eradication of pancreatic CSCs. METHODS: CSCs were isolated from MIA PaCa-2 and PANC1 human pancreatic cancer cell lines. Titanium peroxide nanoparticles (TiOxNPs) were synthesized from titanium dioxide nanoparticles (TiO2NPs) and utilized as radiosensitizers when added one hour prior to radiation exposure. The antitumor activity of this novel therapeutic strategy was evaluated against well-established pancreatic CSCs model both in vitro and in vivo. RESULTS: It is shown that TiOxNPs combined with ionizing radiation exhibit anti-cancer effects on radioresistant CSCs both in vitro and in vivo. TiOxNPs exhibited a synergistic effect with radiation on pancreatic CSC-enriched spheres by downregulating self-renewal regulatory factors and CSC surface markers. Moreover, combined treatment suppressed epithelial-mesenchymal transition, migration, and invasion properties in primary and aggressive pancreatic cancer cells by reducing the expression of proteins relevant to these processes. Notably, radiosensitizing TiOxNPs suppressed the growth of pancreatic xenografts following primary or dissociating sphere MIA PaCa-2 cell implantation. It is inferred that synergy is formed by generating intolerable levels of reactive oxygen species (ROS) and inactivating the AKT signaling pathway. CONCLUSIONS: Our data suggested the use of TiOxNPs in combination with radiation may be considered an attractive therapeutic strategy to eliminate pancreatic CSCs.

Synthesis of Chiral α-Substituted ß-Aminophosphine Derivatives through Asymmetric Hydrophosphinylation Utilizing Secondary Phosphine Sulfides.

The organocatalytic enantioselective hydrophosphinylation of various secondary phosphine sulfides with aromatic and aliphatic nitroalkenes is presented in this study. The reaction produced chiral ß-nitrophosphine sulfides with excellent yields and enantioselectivities (up to 99% yield and 99% ee). Furthermore, the chiral ß-nitrophosphine sulfides can be easily converted into α-substituted ß-aminophosphine, which is a family of useful P, N-ligands and phosphine catalysts.

In vivo stealthified molecularly imprinted polymer nanogels incorporated with gold nanoparticles for radiation therapy.

Radiation therapy is a representative therapeutic approach for cancer treatment, wherein the development of efficient radiation sensitizers with low side effects is critical. In this study, a novel stealth radiation sensitizer based on Au-embedded molecularly imprinted polymer nanogels (Au MIP-NGs) was developed for low-dose X-ray radiation therapy. Surface plasmon resonance measurements reveal the good affinity and selectivity of the obtained Au MIP-NGs toward the target dysopsonic protein, human serum albumin. The protein recognition capability of the nanogels led to the formation of the albumin-rich protein corona in the plasma. The Au MIP-NGs acquire stealth capability in vivo through protein corona regulation using the intrinsic dysopsonic proteins. The injection of Au MIP-NGs improved the efficiency of the radiation therapy in mouse models of pancreatic cancer. The growth of the pancreatic tumor was inhibited even at low X-ray doses (2 Gy). The novel strategy reported in this study for the synthesis of stealth nanomaterials based on nanomaterial-protein interaction control shows significant potential for application even in other approaches for cancer treatment, diagnostics, and theranostics. This strategy paves a way for the development of a wide range of effective nanomedicines for cancer therapy.

Concurrent recordings of hippocampal neuronal spikes and prefrontal synaptic inputs from an awake rat.

A major challenge in neuroscience is linking synapses to cognition and behavior. Here, we developed an experimental technique to concurrently conduct a whole-cell recording of a prefrontal neuron and a multiunit recording of hippocampal neurons from an awake rat. This protocol includes surgical steps to establish a cranial window and 3D printer-based devices to hold the rat. The data sets allow us to directly compare how subthreshold synaptic transmission is associated with suprathreshold spike patterns of neuronal ensembles. For complete details on the use and execution of this protocol, please refer to Nishimura et al. (2021).