Core clock gene, Bmal1, is required for optimal second-level interval production.

Perception and production of second-level temporal intervals are critical in several behavioral and cognitive processes, including adaptive anticipation, motor control, and social communication. These processes are impaired in several neurological and psychological disorders, such as Parkinson's disease and attention-deficit hyperactivity disorder. Although evidence indicates that second-level interval timing exhibit circadian patterns, it remains unclear whether the core clock machinery controls the circadian pattern of interval timing. To investigate the role of core clock molecules in interval timing capacity, we devised a behavioral assay called the interval timing task to examine prospective motor interval timing ability. In this task, the mouse produces two separate nose pokes in a pretrained second-level interval to obtain a sucrose solution as a reward. We discovered that interval perception in wild-type mice displayed a circadian pattern, with the best performance observed during the late active phase. To investigate whether the core molecular clock is involved in the circadian control of interval timing, we employed Bmal1 knockout mice (BKO) in the interval timing task. The interval production of BKO did not display any difference between early and late active phase, without reaching the optimal interval production level observed in wild-type. In summary, we report that the core clock gene Bmal1 is required for the optimal performance of prospective motor timing typically observed during the late part of the active period.

Advantage of extracellular vesicles in hindering the CD47 signal for cancer immunotherapy.

The cluster of differentiation 47 (CD47) protein is abundantly expressed on various malignant cells and suppresses the phagocytic function of macrophages and dendritic cells. High CD47 expression levels are correlated with poor cancer survival. Antagonizing CD47 antibodies with potent antitumor effects have been developed in clinical trials, but have critical side effects, inducing anemia and thrombocytopenia. To develop a safe and potent CD47 blockade, we designed extracellular vesicles (EVs) harboring signal regulatory protein alpha (SIPRα)-EV-SIRPα (EVs that express SIPRα). EV-SIRPα showed minimal toxic effects on hematologic parameters and utilized RBCs as delivery vehicles to tumors rather than inducing anemia. EV-SIRPα inhibited ligation of residual CD47 molecules, which attribute to the EV-endocytosis-mediated CD47 depletion and steric hindrance of EV. In an immunologically cold tumor model, EV-SIRPα induced tumor-specific T-cell-mediated antitumor effects. When directly administered to the accessible lesions, EV-SIRPα monotherapy elicited an abscopal effect in the B16F10 tumor model by increasing immune cell infiltration and CD8+-mediated immunity against non-treated tumors. The combinational approach by loading doxorubicin into the EV-SIRPα dramatically reduced the tumor burden and led to 80% complete remission rate. Thus, a potent EV-based CD47 blockade that is hematologically safe, has efficient signaling blocking efficacy, and has systemic antitumor immunity against cancer is recommended.

Nanocages displaying SIRP gamma clusters combined with prophagocytic stimulus of phagocytes potentiate anti-tumor immunity.

Antigen-presenting cells (APCs), including macrophages and dendritic cells (DCs), play a crucial role in bridging innate and adaptive immunity; thereby, innate immune checkpoint blockade-based therapy is an attractive approach for the induction of sustainable tumor-specific immunity. The interaction between the cluster of differentiation 47 (CD47) on tumor and signal-regulatory protein alpha (SIRPα) on phagocytic cells inhibits the phagocytic function of APCs, acting as a "don't eat me" signal. Accordingly, CD47 blockade is known to increase tumor cell phagocytosis, eliciting tumor-specific CD8+ T-cell immunity. Here, we introduced a nature-derived nanocage to deliver SIRPγ for blocking of antiphagocytic signaling through binding to CD47 and combined it with prophagocytic stimuli using a metabolic reprogramming reagent for APCs (CpG-oligodeoxynucleotides). Upon delivering the clustered SIRPγ variant, the nanocage showed enhanced CD47 binding profiles on tumor cells, thereby promoting active engulfment by phagocytes. Moreover, combination with CpG potentiated the prophagocytic ability, leading to the establishment of antitumorigenic surroundings. This combination treatment could competently inhibit tumor growth by invigorating APCs and CD8+ T-cells in TMEs in B16F10 orthotopic tumor models, known to be resistant to CD47-targeting therapeutics. Collectively, enhanced delivery of an innate immune checkpoint antagonist with metabolic modulation stimuli of immune cells could be a promising strategy for arousing immune responses against cancer.

Functionalized exosome harboring bioactive molecules for cancer therapy.

Exosomes are nanosized vesicles with a lipid membrane that are secreted by most cells and play a crucial role as intermediates of intercellular communication because they carry bioactive molecules. Exosomes are promising for drug delivery of chemicals, proteins, and nucleic acids owing to their inherent properties such as excellent biocompatibility, high tumor targetability, and prolonged circulation in vivo. In this review, we cover recent approaches and advances made in the field of exosome-mediated delivery of bioactive molecules for cancer therapy and factors that affect the clinical use of exosomes. This review can be used as a guideline for further study in expanding the utility of therapeutic exosomes.

Degradation of tumour stromal hyaluronan by small extracellular vesicle-PH20 stimulates CD103+ dendritic cells and in combination with PD-L1 blockade boosts anti-tumour immunity.

Highly accumulated hyaluronan (HA) not only provides a physiological barrier but also supports an immune-suppressive tumour microenvironment. High-molecular-weight (HMW)-HA inhibits the activation of immune cells and their access into tumour tissues, whereas, low-molecular-weight oligo-HA is known to potentially activate dendritic cells (DCs). In this paper, we investigated whether small extracellular vesicle (EVs)-PH20 hyaluronidase induces tumour HA degradation, which, in turn, activates DCs to promote anti-cancer immune responses. Informed by our previous work, we used a small EV carrying GPI-anchored PH20 hyaluronidase (Exo-PH20) that could deeply penetrate into tumour foci via HA degradation. We found that Exo-PH20-treatment successfully activates the maturation and migration of DCs in vivo, particularly CD103+ DCs leading to the activation of tumour-specific CD8+ T cells, which work together to inhibit tumour growth. Moreover, combination with anti-PD-L1 antibody provided potent tumour-specific CD8+ T cell immune responses as well as elicited prominent tumour growth inhibition both in syngenic and spontaneous breast cancer models, and this anti-tumour immunity was durable. Together, these results present new insights for HA degradation by Exo-PH20, providing a better understanding of oligo HA-triggered immune responses to cancer.

Intrinsic cancer vaccination.

Immunotherapy is revolutionizing the treatment of cancer, and the current immunotherapeutics have remarkably improved the outcomes for some cancer patients. However, we still need answers for patients with immunologically cold tumors that do not benefit from the current immunotherapy treatments. Here, we suggest a novel strategy that is based on using a very old and sophisticated system for cancer immunotherapy, namely "intrinsic cancer vaccination", which seeks to awaken our own immune system to activate tumor-specific T cells. To do this, we must take advantage of the genetic instability of cancer cells and the expression of cancer cell neoantigens to trigger immunity against cancer cells. It will be necessary to not only enhance the phagocytosis of cancer cells by antigen presenting cells but also induce immunogenic cancer cell death and the subsequent immunogenic clearance, cross-priming and generation of tumor-specific T cells. This strategy will allow us to avoid using known tumor-specific antigens, ex vivo manipulation or adoptive cell therapy; rather, we will efficiently present cancer cell neoantigens to our immune system and propagate the cancer-immunity cycle. This strategy simply follows the natural cycle of cancer-immunity from its very first step, and therefore could be combined with any other treatment modality to yield enhanced efficacy.

An optimized protocol to determine the engulfment of cancer cells by phagocytes using flow cytometry and fluorescence microscopy.

The engulfment of cancer cells by macrophages is an important cellular process in innate cancer immunity. Antitumor immunotherapy that utilizes the enhanced engulfment of cancer cells by phagocytic cells has attracted much attention. Therefore, there is a growing demand for methods of measuring cancer cell phagocytosis. Quantifying the various stages of phagocytosis is invaluable for elucidating cancer-immune responses during this process. Here, we describe two phagocytosis assays, a flow cytometric assay and a fluorescent microscopic assay; the flow cytometric method utilizing CellTracker dye provides a simple, measurable, and highly reproducible functional assay to measure the phagocytosis efficiency of cancer cells by bone marrow-derived macrophages. As an alternative method of evaluating various states of cancer cell phagocytosis, a fluorescent microscopic method that employs a pH-sensitive dye (pHrodo-SE dye) is also described in this paper. Image-based analysis using this labeling approach enables researchers to measure phagocytic indices that indicate the number of cancer cells engulfed by each macrophage. We have highlighted that these assays can be applied to multiple tumor types and used as selection tools for a variety of phagocytosis agonist types. The results of this study may facilitate a better understanding of the interactions between tumor cells and phagocytes, which could lead to the identification of new therapeutic targets against cancer.

Designed trimer-mimetic TNF superfamily ligands on self-assembling nanocages.

Presentation of an endogenous bioactive ligand in its native form is a key factor in controlling and determining its bioactivity, stability, and therapeutic efficacy. In this study, we developed a novel strategy for presenting trimeric ligands on nanocages by designing, optimizing and testing based on the rational design, high-resolution structural analysis and agonistic activity assays in vitro and in vivo. We successfully designed a nanocage that presents the TNF superfamily member, TRAIL (TNF-related apoptosis-inducing ligand) in its native-like trimeric structure. The native structure of TRAIL complexes was mimicked on the resulting trimeric TRAIL-presenting nanocages (TTPNs) by inserting sufficient spacing, determined from three-dimensional structural models, to provide optimal access to the corresponding receptors. The efficacy of TTPNs as an anti-tumor agent was confirmed in preclinical studies, which revealed up to 330-fold increased affinity, 62.5-fold enhanced apoptotic activity, and improved pharmacokinetic characteristics and stability compared with the monomeric form of TRAIL (mTRAIL). In this latter context, TTPNs exhibited greater than 90% stability over 1 mo, whereas ∼50% of mTRAIL aggregated within 2 d. Consistent with their enhanced stability and ultra-high affinity for the TRAIL receptor, TTPNs effectively induced apoptosis of tumor cells in vivo, leading to effective inhibition of tumor growth. Although TRAIL was used here as a proof-of-concept, all members of the TNF superfamily share the TNF homology domain (THD) and have similar distances between ecto-domain C-termini. Thus, other TNF superfamily ligands could be genetically substituted for the TRAIL ligand on the surface of this biomimetic delivery platform.

Combined Rho-kinase inhibition and immunogenic cell death triggers and propagates immunity against cancer.

Activation of T cell immune response is critical for the therapeutic efficacy of cancer immunotherapy. Current immunotherapies have shown remarkable clinical success against several cancers; however, significant responses remain restricted to a minority of patients. Here, we show a therapeutic strategy that combines enhancing the phagocytic activity of antigen-presenting cells with immunogenic cell death to trigger efficient antitumour immunity. Rho-kinase (ROCK) blockade increases cancer cell phagocytosis and induces antitumour immunity through enhancement of T cell priming by dendritic cells (DCs), leading to suppression of tumour growth in syngeneic tumour models. Combining ROCK blockade with immunogenic chemotherapy leads to increased DC maturation and synergistic CD8+ cytotoxic T cell priming and infiltration into tumours. This therapeutic strategy effectively suppresses tumour growth and improves overall survival in a genetic mouse mammary tumour virus/Neu tumour model. Collectively, these results suggest that boosting intrinsic cancer immunity using immunogenic killing and enhanced phagocytosis is a promising therapeutic strategy for cancer immunotherapy.

Comparison of exosomes and ferritin protein nanocages for the delivery of membrane protein therapeutics.

Exosomes are small membrane vesicles secreted by most cell types that play an important role in intercellular communication. Due to the characteristic of transferring their biomacromolecules, exosomes have potential as a new alternative for delivering protein therapeutics. Here, we investigate whether exosomes provide crucial advantages over other nanoparticles, in particular protein nanocage formulations, as a delivery system for membrane protein therapeutics. We characterized membrane-scaffold-based exosomes and protein-scaffold-based ferritin nanocages, both harboring SIRPα (signal regulatory protein α), an antagonist of CD47 on tumor cells. The efficacy of these two systems in delivering protein therapeutics was compared by testing their ability to enhance phagocytosis of tumor cells by bone-marrow-derived macrophages and subsequent inhibition of in vivo tumor growth. These analyses allowed us to comprehensively conclude that the therapeutic index of exosome-mediated CD47 blockade against tumor growth inhibition was higher than that of the same dose of ferritin-SIRPα. The results of this analysis reveal the importance of the unique characteristics of exosomes, in particular their membrane scaffold, in improving therapeutic protein delivery compared with protein-scaffold-based nanocages.

Nanocage-Therapeutics Prevailing Phagocytosis and Immunogenic Cell Death Awakens Immunity against Cancer.

A growing appreciation of the relationship between the immune system and the tumorigenesis has led to the development of strategies aimed at "re-editing" the immune system to kill tumors. Here, a novel tactic is reported for overcoming the activation-energy threshold of the immunosuppressive tumor microenvironment and mediating the delivery and presentation of tumor neoantigens to the host's immune system. This nature-derived nanocage not only efficiently presents ligands that enhance cancer cell phagocytosis, but also delivers drugs that induce immunogenic cancer cell death. The designed nanocage-therapeutics induce the release of neoantigens and danger signals in dying tumor cells, and leads to enhancement of tumor cell phagocytosis and cross-priming of tumor specific T cells by neoantigen peptide-loaded antigen-presenting cells. Potent inhibition of tumor growth and complete eradication of tumors is observed through systemic tumor-specific T cell responses in tumor draining lymph nodes and the spleen and further, infiltration of CD8+ T cells into the tumor site. Remarkably, after removal of the primary tumor, all mice treated with this nanocage-therapeutics are protected against subsequent challenge with the same tumor cells, suggesting development of lasting, tumor-specific responses. This designed nanocage-therapeutics "awakens" the host's immune system and provokes a durable systemic immune response against cancer.

The effect of cilostazol on electrophysiologic changes in non-proliferative diabetic retinopathy patients.

PURPOSE: To evaluate the effects of cilostazol, an antiplatelet and vasodilation agent, on the retinal function of patients with non-proliferative diabetic retinopathy (NPDR) using a full-field electroretinogram (ffERG). METHODS: A total of 20 eyes from 20 patients were enrolled as the cilostazol-treated group, and 16 eyes from 16 patients were enrolled as the control group to assess the functional effects of cilostazol. Ophthalmologic examinations including fundus fluorescein angiography (FFA), fundus color photography, optical coherence tomography (OCT), and ffERG responses were recorded at baseline and after 1 year of cilostazol treatment. The number of microaneurysms on FFA, the number of exudates on fundus photographs, and central macular thickness (CMT) on OCT were compared between the two groups. Recording of ffERG was also performed at baseline and repeated after 1 year of treatment. The mean implicit times and amplitudes of a- and b-waves in each ffERG response were analyzed to evaluate the retinal function. RESULTS: CMT and the numbers of microaneurysms and exudates showed no significant change in the cilostazol-treated group. There was no significant change in ffERG parameters between baseline and 1 year after the treatment in each group. The mean changes in implicit times from the cilostazol-treated group were significantly less than in the control group in b-waves from dark-adapted 3 ERG (p = 0.017) and 10 ERG responses (p = 0.047). On the other hand, the mean changes in amplitudes were not significant after 1 year of cilostazol treatment, but there were slight increases in amplitudes of dark-adapted 0.01 ERG and 10 ERG in the cilostazol-treated group. CONCLUSIONS: These results suggest that cilostazol administration could reduce the implicit times of ffERG in patients with NPDR. It may be beneficial to preserve the retinal function in the diabetic retina, and additional research with larger populations and extended duration are needed to clarify the efficacy and safety of cilostazol for these patients.

The ketogenic diet suppresses the cathepsin E expression induced by kainic acid in the rat brain.

PURPOSE: The ketogenic diet has long been used to treat epilepsy, but its mechanism is not yet clearly understood. To explore the potential mechanism, we analyzed the changes in gene expression induced by the ketogenic diet in the rat kainic acid (KA) epilepsy model. MATERIALS AND METHODS: KA-administered rats were fed the ketogenic diet or a normal diet for 4 weeks, and microarray analysis was performed with their brain tissues. The effects of the ketogenic diet on cathepsin E messenger ribonucleic acid (mRNA) expression were analyzed in KA-administered and normal saline-administered groups with semi-quantitative and real-time reverse transcription polymerase chain reaction (RT-PCR). Brain tissues were dissected into 8 regions to compare differential effects of the ketogenic diet on cathepsin E mRNA expression. Immunohistochemistry with an anti-cathepsin E antibody was performed on slides of hippocampus obtained from whole brain paraffin blocks. RESULTS: The microarray data and subsequent RT-PCR experiments showed that KA increased the mRNA expression of cathepsin E, known to be related to neuronal cell death, in most brain areas except the brain stem, and these increases of cathepsin E mRNA expression were suppressed by the ketogenic diet. The expression of cathepsin E mRNA in the control group, however, was not significantly affected by the ketogenic diet. The change in cathepsin E mRNA expression was greatest in the hippocampus. The protein level of cathepsin E in the hippocampus of KA-administered rat was elevated in immunohistochemistry and the ketogenic diet suppressed this increase. CONCLUSION: Our results showed that KA administration increased cathepsin E expression in the rat brain and its increase was suppressed by the ketogenic diet.

A novel c.-22T>C mutation in GALK1 promoter is associated with elevated galactokinase phenotype.

BACKGROUND: Many genetic variations of GALK1 have been identified in the patients with galactokinase (GALK1) deficiency. However, the molecular characteristics of GALK1 in individuals with elevated GALK1 activity are relatively unknown. METHODS: We investigated the relationship between elevated GALK1 activity and the molecular GALK1 gene variations, and the molecular mechanism underlying elevated GALK1 activity. PCR products from 63 subjects, without any attenuation of galactose degradation enzymes, were sequenced to screen for nucleotide alterations in the GALK1 promoter. RESULTS: Three nucleotide substitutions were identified: c.-179A>G, c.-27A>C, and c.-22T>C. With respect to the c.-22T>C mutation, GALK1 activity in 13 subjects with the T/C or C/C genotype was significantly higher than those in 50 subjects with the T/T genotype (p < 0.001). The dual luciferase reporter assay in Hep3B cells showed that the luciferase activity with the GALK1 promoter with the c.-22C mutant allele increased approximately 2.5-fold, compared to that with the c.-22T. A specific DNA-protein complex was observed in an electrophoretic mobility shift assay, with slightly higher affinity to c.-22C than to c.-22T. CONCLUSION: The c.-22T>C mutation, which was observed frequently in individuals with elevated GALK1 activity, increased the expression of a reporter gene through enhanced binding of a currently unidentified nuclear protein. These results suggest that the elevated GALK1 activity resulted from enhanced gene expression, due to nucleotide variation within GALK1 promoter.

Sterol-dependent regulation of proprotein convertase subtilisin/kexin type 9 expression by sterol-regulatory element binding protein-2.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the subtilases that promotes the internalization and degradation of LDL receptor in liver and thereby controls the level of LDL cholesterol in plasma. Here, we show that the expression of PCSK9 in HepG2 cells is completely dependent on the absence or presence of sterols. The minimal promoter region of the PCSK9 gene contains a sterol-regulatory element (SRE), which makes the transcription of PCSK9 dependent on sterols. Expression of nuclear forms of sterol-regulatory element binding protein-1 (SREBP-1) and SREBP-2 dramatically increased the promoter activity of PCSK9. In vitro-translated nuclear forms of SREBPs showed interactions with SRE, whereas mutations in SRE abolished their binding. In vivo studies in mice showed that Pcsk9 protein and mRNA were decreased significantly by fasting and increased by refeeding. However, supplementation with 2% cholesterol in the diet prevented the increase in Pcsk9. The amounts of Pcsk9 mRNA in livers of refed mice showed correlated regulation by the changes in the nuclear form of Srebp-2. In summary, it is suggested that the expression of PCSK9 is regulated by sterol at the transcriptional level in HepG2 cells and that both SREBP-1 and SREBP-2 can transcriptionally activate PCSK9 via SRE in its proximal promoter region in vitro. However, in vivo, it is suggested that the sterol-dependent regulation of PCSK9 is mediated predominantly by SREBP-2.

Electrolyzed-reduced water protects against oxidative damage to DNA, RNA, and protein.

The generation of reactive oxygen species is thought to cause extensive oxidative damage to various biomolecules such as DNA, RNA, and protein. In this study, the preventive, suppressive, and protective effects of in vitro supplementation with electrolyzed-reduced water on H2O2-induced DNA damage in human lymphocytes were examined using a comet assay. Pretreatment, cotreatment, and posttreatment with electrolyzed-reduced water enhanced human lymphocyte resistance to the DNA strand breaks induced by H2O2 in vitro. Moreover, electrolyzed-reduced water was much more effective than diethylpyrocarbonate-treated water in preventing total RNA degradation at 4 and 25 degrees C. In addition, electrolyzed-reduced water completely prevented the oxidative cleavage of horseradish peroxidase, as determined using sodium dodecyl sulfate-polyacrylamide gels. Enhancement of the antioxidant activity of ascorbic acid dissolved in electrolyzed-reduced water was about threefold that of ascorbic acid dissolved in nonelectrolyzed deionized water, as measured by a xanthine-xanthine oxidase superoxide scavenging assay system, suggesting an inhibitory effect of electrolyzedreduced water on the oxidation of ascorbic acid.

Purification and characterization of an anionic isoperoxidase from scented-geranium callus.

Secretory anionic isoperoxidase (EC 1.11.1.7), named PA1, was 68-fold purified from scented-geranium (Pelargonium graveolense) callus by using ion exchange chromatography and gel filtration. Isoperoxidase PA1 was a glycoprotein with an isoelectric point (pI) of 4.0. The molecular weight of PA1 was approximately 42.5 and 44 kDa, estimated by SDS-PAGE and Sephadex G-150 gel filtration, respectively. The optimum pH of the enzyme was 5.0 for guaiacol and H2O2, and the Km values for guaiacol and H2O2 were 1.96 and 8.5mM, respectively. Substrate studies in terms of optimum pHs and Km values with various synthetic and naturally occurring phenolic compounds were performed. In comparison with cationic isoperoxidase, PC3, which has been already characterized, anionic isoperoxidase PA1 had much lower Km values for synthetic phenolic compounds and much higher Km values for naturally occurring phenolic compounds than PC3. Moreover, anionic isoperoxidase PA1 could utilize ferulic acid as a substrate very well, while cationic isoperoxidase PC3 could not utilize ferulic acid as a substrate.