Distinct manifold encoding of navigational information in the subiculum and hippocampus.

The subiculum (SUB) plays a crucial role in spatial navigation and encodes navigational information differently from the hippocampal CA1 area. However, the representation of subicular population activity remains unknown. Here, we investigated the neuronal population activity recorded extracellularly from the CA1 and SUB of rats performing T-maze and open-field tasks. The trajectory of population activity in both areas was confined to low-dimensional neural manifolds homoeomorphic to external space. The manifolds conveyed position, speed, and future path information with higher decoding accuracy in the SUB than in the CA1. The manifolds exhibited common geometry across rats and regions for the CA1 and SUB and between tasks in the SUB. During post-task ripples in slow-wave sleep, population activity represented reward locations/events more frequently in the SUB than in CA1. Thus, the CA1 and SUB encode information distinctly into the neural manifolds that underlie navigational information processing during wakefulness and sleep.

Reinstating olfactory bulb-derived limbic gamma oscillations alleviates depression-like behavioral deficits in rodents.

Although the etiology of major depressive disorder remains poorly understood, reduced gamma oscillations is an emerging biomarker. Olfactory bulbectomy, an established model of depression that reduces limbic gamma oscillations, suffers from non-specific effects of structural damage. Here, we show that transient functional suppression of olfactory bulb neurons or their piriform cortex efferents decreased gamma oscillation power in limbic areas and induced depression-like behaviors in rodents. Enhancing transmission of gamma oscillations from olfactory bulb to limbic structures by closed-loop electrical neuromodulation alleviated these behaviors. By contrast, silencing gamma transmission by anti-phase closed-loop stimulation strengthened depression-like behaviors in naive animals. These induced behaviors were neutralized by ketamine treatment that restored limbic gamma power. Taken together, our results reveal a causal link between limbic gamma oscillations and depression-like behaviors in rodents. Interfering with these endogenous rhythms can affect behaviors in rodent models of depression, suggesting that restoring gamma oscillations may alleviate depressive symptoms.

Carboxy-terminal dendrimers with phenylalanine for a pH-sensitive delivery system into immune cells including T cells.

Although T cells play important roles in various immune reactions, there are only a few reports on delivery systems into T cells. Our previous study showed that carboxy-terminal phenylalanine (Phe)-modified polyamidoamine (PAMAM) dendrimers have both temperature- and pH-sensitive properties, which are affected by the chemical structure. The self-assembled structures of Phe, observed in phenylketonuria, enhance the protein aggregation, the association with the cell membrane and the membrane permeability. In this study, we applied the Phe-modified dendrimers to a pH-sensitive drug delivery system into T cells. Dendrimers with different amino acids and acid anhydrides were synthesized, and their pH-responsive association with T cells and their subsets was investigated. The dendrimers modified with Phe and cyclohexanedicarboxylic acid (CHex) showed higher uptake into various cells, including Jurkat cells, CD3+ T cells, CD3 + CD4+ helper T cells and CD3 + CD8+ killer T cells. These dendrimers were internalized into T cells via endocytosis, and their cellular uptake was enhanced under weak acidic conditions (pH 6.5). Our results showed that Phe- and CHex-modified dendrimers have a delivery potential to T cells and their subsets, which may be useful for cancer immunotherapy.

Macropinocytosis-Inducible Extracellular Vesicles Modified with Antimicrobial Protein CAP18-Derived Cell-Penetrating Peptides for Efficient Intracellular Delivery.

The antimicrobial protein CAP18 (approximate molecular weight: 18 000), which was first isolated from rabbit granulocytes, comprises a C-terminal fragment that has negatively charged lipopolysaccharide binding activity. In this study, we found that CAP18 (106-121)-derived (sC18)2 peptides have macropinocytosis-inducible biological functions. In addition, we found that these peptides are highly applicable for use as extracellular vesicle (exosomes, EV)-based intracellular delivery, which is expected to be a next-generation drug delivery carrier. Here, we demonstrate that dimerized (sC18)2 peptides can be easily introduced on EV membranes when modified with a hydrophobic moiety, and that they show high potential for enhanced cellular uptake of EVs. By glycosaminoglycan-dependent induction of macropinocytosis, cellular EV uptake in targeted cells was strongly increased by the peptide modification made to EVs, and intriguingly, our herein presented technique is efficiently applicable for the cytosolic delivery of the biologically cell-killing functional toxin protein, saporin, which was artificially encapsulated in the EVs by electroporation, suggesting a useful technique for EV-based intracellular delivery of biofunctional molecules.

Size and surface modification of silica nanoparticles affect the severity of lung toxicity by modulating endosomal ROS generation in macrophages.

BACKGROUND: As the application of silica nanomaterials continues to expand, increasing chances of its exposure to the human body and potential harm are anticipated. Although the toxicity of silica nanomaterials is assumed to be affected by their physio-chemical properties, including size and surface functionalization, its molecular mechanisms remain unclear. We hypothesized that analysis of intracellular localization of the particles and subsequent intracellular signaling could reveal a novel determinant of inflammatory response against silica particles with different physico-chemical properties. RESULTS: We employed a murine intratracheal instillation model of amorphous silica nanoparticles (NPs) exposure to compare their in vivo toxicities in the respiratory system. Pristine silica-NPs of 50 nm diameters (50 nm-plain) induced airway-centered lung injury with marked neutrophilic infiltration. By contrast, instillation of pristine silica particles of a larger diameter (3 µm; 3 µm-plain) significantly reduced the severity of lung injury and neutrophilic infiltration, possibly through attenuated induction of neutrophil chemotactic chemokines including MIP2. Ex vivo analysis of alveolar macrophages as well as in vitro assessment using RAW264.7 cells revealed a remarkably lower cellular uptake of 3 µm-plain particles compared with 50 nm-plain, which is assumed to be the underlying mechanism of attenuated immune response. The severity of lung injury and neutrophilic infiltration was also significantly reduced after intratracheal instillation of silica NPs with an amine surface modification (50 nm-NH2) when compared with 50 nm-plain. Despite unchanged efficacy in cellular uptake, treatment with 50 nm-NH2 induced a significantly attenuated immune response in RAW264.7 cells. Assessment of intracellular redox signaling revealed increased reactive oxygen species (ROS) in endosomal compartments of RAW264.7 cells treated with 50 nm-plain when compared with vehicle-treated control. In contrast, augmentation of endosomal ROS signals in cells treated with 50 nm-NH2 was significantly lower. Moreover, selective inhibition of NADPH oxidase 2 (NOX2) was sufficient to inhibit endosomal ROS bursts and induction of chemokine expressions in cells treated with silica NPs, suggesting the central role of endosomal ROS generated by NOX2 in the regulation of the inflammatory response in macrophages that endocytosed silica NPs. CONCLUSIONS: Our murine model suggested that the pulmonary toxicity of silica NPs depended on their physico-chemical properties through distinct mechanisms. Cellular uptake of larger particles by macrophages decreased, while surface amine modification modulated endosomal ROS signaling via NOX2, both of which are assumed to be involved in mitigating immune response in macrophages and resulting lung injury.

Association of Hydrophobic Carboxyl-Terminal Dendrimers with Lymph Node-Resident Lymphocytes.

Delivery systems to lymph node-resident T cells around tumor tissues are essential for cancer immunotherapy, in order to boost the immune responses. We previously reported that anionic dendrimers, such as carboxyl-, sulfonyl-, and phosphate-terminal dendrimers, were efficiently accumulated in lymph nodes via the intradermal injection. Depending on the terminal structure, their cell association properties were different, and the carboxyl-terminal dendrimers did not associate with any immune cells majorly. In this study, we investigated the delivery of carboxyl-terminal dendrimers with different hydrophobicity to lymph node-resident lymphocytes. Four types of carboxyl-terminal dendrimers-succinylated (C) and 2-carboxy-cyclohexanoylated (Chex) dendrimers with and without phenylalanine (Phe)-were synthesized and named C-den, C-Phe-den, Chex-den, and Chex-Phe-den, respectively. Chex-Phe-den was well associated with lymphocytes, but others were not. Chex-Phe-den, intradermally injected at the footpads of mice, was accumulated in the lymph node, and was highly associated with the lymphocytes, including T cells. Our results suggest that Chex-Phe-den has the potential for delivery to the lymph node-resident T cells, without any specific T cell-targeted ligands.

Effects of gefitinib treatment on cellular uptake of extracellular vesicles in EGFR-mutant non-small cell lung cancer cells.

Extracellular vesicles (exosomes, EVs) are cell membrane particles (30-200 nm) secreted by virtually all cells. During intercellular communication in the body, secreted EVs play crucial roles by carrying functional biomolecules (e.g., microRNAs and enzymes) into other cells to affect cellular function, including disease progression. We previously reported that the macropinocytosis pathway contributes greatly to the efficient cellular uptake of EVs. The activation of growth factor receptors, such as epidermal growth factor receptor (EGFR), induces macropinocytosis. In this study, we demonstrated the effects of gefitinib, a tyrosine kinase inhibitor of EGFR, on the cellular uptake of EVs. In EGFR-mutant HCC827 non-small cell lung cancer (NSCLC) cells, which are sensitive to gefitinib, macropinocytosis was suppressed by gefitinib treatment. However, the cellular uptake of EVs was increased by gefitinib treatment, whereas that of liposomes was reduced. In accordance with the results of the cellular uptake studies, the anti-cancer activity of doxorubicin (DOX)-loaded EVs in HCC827 cells was significantly increased in the presence of gefitinib, whereas the activity of DOX-loaded liposomes was reduced. The digestion of EV proteins by trypsin did not affect uptake, suggesting that the cellular uptake of EVs might not be mediated by EV proteins. These results suggest that gefitinib can enhance cell-to-cell communication via EVs within the tumor microenvironment. In addition, EVs show potential as drug delivery vehicles in combination with gefitinib for the treatment of patients harboring EGFR-mutant NSCLC tumors.

[Factors associated with successful smoking cessation among adolescent smokers undergoing a smoking cessation program involving nicotine replacement therapy].

OBJECTIVE: To identify factors associated with successful smoking cessation among adolescent smokers in a smoking cessation program involving nicotine replacement therapy. METHODS: We recruited adolescent smokers who were prepared to quit smoking and participated in the smoking cessation program in Kanagawa prefecture. All participants fulfilled a questionnaire beforehand, covering gender, age at the beginning of the study, age at onset of smoking, the number of quit attempts, the number of cigarettes per day and the smoking status of their families and friends. Seven nicotine patches (nicotine 52.5 mg/day) were given to them free of charge for daily use. Their smoking status and the use of nicotine patches were confirmed by telephone or postcard at the 1 and 6 month follow-ups. The relationships between successful smoking cessation and different factors among eligible participants were analyzed using the Fisher's exact test and the Mann-Whitney U test. RESULTS: The subjects included 39 adolescent smokers (mean 16.4 years). The mean age at onset of smoking and the mean duration of smoking were 13.3 years and 2.3 years, respectively. The average daily number of cigarettes smoked was 12.8. Of 39 eligible participants at the one month follow up, 14 (35.9%) were found to be abstaining from smoking. The subjects treated with nicotine patches were significantly more likely to be abstinent than those without them (P<0.05). However, no significant associations with other factors were found. Of 39 participants at the six months follow up, 10 (25.6%) were still abstinent but there were no significant associations with any of the factors, including use of nicotine patches. Subjects living with smokers were significantly less likely to be successful in their efforts to quit than those living with non-smokers (P< 0.05). There were no significant associations with other factors, including using nicotine patches. No adverse events relating to the use of the nicotine patches were encountered during the study period. CONCLUSION: The smoking cessation program involving NRT provided for adolescent smokers appeared effective at the one month follow up. Those adolescents living with smokers had more difficulties in quitting smoking than those with non-smokers at the 6 month follow up. In order to increase the number of adolescent smokers in the smoking cessation study, the need to obtain parental consent might be considered as a barrier to be overcome. Additionally, more effective follow-up procedures should be considered for the purpose of avoiding dropouts during the study.

[Predictors of abstinence from smoking among participants in a smoking cessation program in a health center in Japan].

PURPOSE: To identify predictors of successful smoking abstinence. METHOD: A prospective analysis was conducted of a group of 687 individuals (mean age +/- SD 49.5 +/- 14.6 years, range 20 to 81 years) comprising 518 males and 169 females, who attempted to stop smoking by participating in smoking cessation programs provided by Kamakura Health Center in Japan from September 2002 to September 2003. They were provided with free nicotine patches and referred to medical clinics for follow-up. Baseline characteristics such as age, gender, program settings, daily cigarette consumption and time to the first cigarette of the day were recorded at the start of the program. Smoking status was confirmed at the one-year follow up. In addition, the participants were asked whether they had used free nicotine patches or follow-up nicotine patches and gum. The relationships between characteristics used to assess smoking abstinence rates were first analyzed using a chi2 test. Logistic regression analysis was then employed to identify independent predictors of smoking abstinence at the one-year follow up. RESULTS: A total of 687 smokers who participated in the program were registered at the start of this study. Out of these, 528 (76.9%) were eligible because their smoking status could be confirmed at the one-year follow up. The rate of smoking abstinence at the one-year follow up was 39.2%. No significant differences were observed in the rates of smoking abstinence among the participants at the one-year follow up based on gender, daily cigarette consumption or time to the first cigarette of the day. When participants were over fifty years of age (abstinence rate 47.0%), were provided with the program conducted at the Health Center (abstinence rate 50.3%), used free nicotine patches (abstinence rate 50.7%) and received follow-up nicotine replacement therapy (abstinence rate 57.7%). they were significantly more likely to have stopped smoking than the reference groups. With the above-mentioned four predictors, adjusted odds ratios of smoking abstinence observed in the logistic regression analysis were 1.68, 1.80, 2.01 and 1.79, respectively. CONCLUSION: We found a 39.2% smoking abstinence rate at one-year follow up among smokers who were treated with free nicotine patches in the smoking cessation program by the Kamakura Health Center. It was indicated that predictors of smoking abstinence are age (over fifty years), location (at the Health Center), use of free nicotine patches and receipt of follow-up nicotine replacement therapy.

C.EcoO109I, a regulatory protein for production of EcoO109I restriction endonuclease, specifically binds to and bends DNA upstream of its translational start site.

The EcoO109I restriction-modification system, which recognizes 5'-(A/G)GGNCC(C/T)-3', has been cloned, and contains convergently transcribed endonuclease and methylase. The role and action mechanism of the gene product, C.EcoO109I, of a small open reading frame located upstream of ecoO109IR were investigated in vivo and in vitro. The results of deletion analysis suggested that C.EcoO109I acts as a positive regulator of ecoO109IR expression but has little effect on ecoO109IM expression. Assaying of promoter activity showed that the expression of ecoO109IC was regulated by its own gene product, C.EcoO109I. C.EcoO109I was overproduced as a His-tag fusion protein in recombinant Escherichia coli HB101 and purified to homogeneity. C.EcoO109I exists as a homodimer, and recognizes and binds to the DNA sequence 5'-CTAAG(N)(5)CTTAG-3' upstream of the ecoO109IC translational start site. It was also shown that C.EcoO109I bent the target DNA by 54 +/- 4 degrees.