A Communication-Efficient Secure Routing Protocol for IoT Networks.

This paper proposes a secure routing protocol based on an ad hoc on-demand distance vector to simultaneously achieve communication efficiency and security. Many studies have discussed secure protocols. However, conventional protocols tend to exhibit low communication efficiencies owing to the long packets required by digital signatures, specifically in large-scale networks. Hence, our proposed method aims to allow the intermediate node to initiate a route reply (RREP), which is prohibited in conventional protocols because of digital signature restrictions. Based on an ID-based signature, the proposed protocol allows each intermediate node to hold a packet received from a specific node in the past. Each node then appends it to the route request of another node and generates its own signed RREP. This procedure guarantees that a third party holds the route to the destination. Theoretical evaluations demonstrate that the proposed method outperforms the communication efficiency of conventional secure protocols. We measured the time required for routing (i.e., the sum of communication and cryptographic calculation times) using a Raspberry Pi with C language. We show that the proposed protocol can improve the average routing time by more than 3× compared with conventional methods when 30 relay nodes are randomly distributed in a 300-square meter area.

Assessment of metabolic activation of felbamate in chimeric mice with humanized liver in combination with in vitro metabolic assays.

Felbamate (FBM) is an antiepileptic drug that has minimal toxicity in preclinical toxicological species but has a serious idiosyncratic drug toxicity (IDT) in humans. The formation of reactive metabolites is common among most drugs associated with IDT, and 2-phenylpropenal (2-PP) is believed to be the cause of IDT by FBM. It is important to consider the species difference in susceptibility to IDT between experimental animals and humans. In the present study, we used an in vitro and in vivo model system to reveal species difference in IDT of FBM. Human cytochrome P450 (CYP) and carboxylesterase (CES) expressing microsomes were used to clarify the isozymes involved in the metabolism of FBM. The remaining amount of FBM was significantly reduced in incubation with microsomes expressing human CYP2C8, 2C9, 2E1, and CES1c isozymes. Chimeric mice with humanized liver are expected to predict IDT in humans. Therefore, metabolite profiles in chimeric mice with humanized liver were investigated after administration of FBM. Metabolites after glutathione (GSH) conjugation of 2-phenylpropenal (2-PP), which is the reactive metabolite responsible for FBM-induced IDT, were detected in chimeric mice plasma and liver homogenate. Mass spectrometry imaging (MSI) visualizes distribution of FBM and endogenous GSH, and GSH levels in human hepatocyte were decreased after administration of FBM. In this study, we identified CYP and CES isozymes involved in the metabolism of FBM and confirmed reactive metabolite formation and subsequent decrease in GSH using humanized animal model. These results would provide useful information for the susceptibility to IDT between experimental animals and humans.

Crowdsourcing-Assisted Radio Environment Database for V2V Communication.

In order to realize reliable Vehicle-to-Vehicle (V2V) communication systems for autonomous driving, the recognition of radio propagation becomes an important technology. However, in the current wireless distributed network systems, it is difficult to accurately estimate the radio propagation characteristics because of the locality of the radio propagation caused by surrounding buildings and geographical features. In this paper, we propose a measurement-based radio environment database for improving the accuracy of the radio environment estimation in the V2V communication systems. The database first gathers measurement datasets of the received signal strength indicator (RSSI) related to the transmission/reception locations from V2V systems. By using the datasets, the average received power maps linked with transmitter and receiver locations are generated. We have performed measurement campaigns of V2V communications in the real environment to observe RSSI for the database construction. Our results show that the proposed method has higher accuracy of the radio propagation estimation than the conventional path loss model-based estimation.

Murine Cyp3a knockout chimeric mice with humanized liver: prediction of the metabolic profile of nefazodone in humans.

Chimeric mice with humanized livers (PXB mice) are used to investigate the metabolism and pharmacokinetics of drugs in humans. However, residual murine enzymatic activities derived from the liver and the presence of mouse small intestinal metabolism can hamper the prediction of human drug metabolism. Recently murine Cytochrome P450 3a gene knockout chimeric mice with humanized livers (Cyp3a KO CM) were developed. To evaluate the prediction of drug metabolism, nefazodone (NEF) was administered orally at 10 mg/kg to the following mouse strains: Cyp3a KO CM, murine Cyp3a gene knockout (Cyp3a KO), PXB and severe combined immunodeficiency (SCID) mice. Liquid chromatography-mass spectrometry was used for metabolic profiling of plasma, urine and bile. The prediction of human metabolite levels such as hydroxy nefazodone (OH-NEF), triazoledione form (TD), m-chlorophenylpiperazine and dealkyl metabolites in Cyp3a KO CM was superior to that in Cyp3a KO, PXB or SCID mice. Further, clinical exposure levels of NEF, OH-NEF and TD were reproduced in Cyp3a KO CM. In contrast, NEF was rapidly metabolized to TD in both PXB and SCID mice but not in Cyp3a KO mice, suggesting that murine CYP3A is involved in the elimination of NEF in these mice. These findings demonstrate that the metabolic profile of NEF in Cyp3a KO CM differs qualitatively and quantitatively from that in PXB mice due to the higher metabolic rate of NEF and its metabolites via murine CYP3A. Therefore Cyp3a KO CM might be useful in predicting the metabolic profiles of drug candidates in humans.

Development of Murine Cyp3a Knockout Chimeric Mice with Humanized Liver.

We developed murine CYP3A knockout ko chimeric mice with humanized liver expressing human P450S similar to those in humans and whose livers and small intestines do not express murine CYP3A this: approach may overcome effects of residual mouse metabolic enzymes like Cyp3a in conventional chimeric mice with humanized liver, such as PXB-mice [urokinase plasminogen activator/severe combined immunodeficiency (uPA/SCID) mice repopulated with over 70% human hepatocytes] to improve the prediction of drug metabolism and pharmacokinetics in humans. After human hepatocytes were transplanted into Cyp3a KO/uPA/SCID host mice, human albumin levels logarithmically increased until approximately 60 days after transplantation, findings similar to those in PXB-mice. Quantitative real-time-polymerase chain reaction analyses showed that hepatic human P450s, UGTs, SULTs, and transporters mRNA expression levels in Cyp3a KO chimeric mice were also similar to those in PXB-mice and confirmed the absence of Cyp3a11 mRNA expression in mouse liver and intestine. Findings for midazolam and triazolam metabolic activities in liver microsomes were comparable between Cyp3a KO chimeric mice and PXB-mice. In contrast, these activities in the intestine of Cyp3a KO chimeric mice were attenuated compared with PXB-mice. Owing to the knockout of murine Cyp3a, hepatic Cyp2b10 and 2c55 mRNA levels in Cyp3a KO/uPA/SCID mice (without hepatocyte transplants) were 8.4- and 61-fold upregulated compared with PXB-mice, respectively. However, human hepatocyte transplantation successfully restored Cyp2b10 level nearly fully and Cyp2c55 level partly (still 13-fold upregulated) compared with those in PXB-mice. Intestinal Cyp2b10 and 2c55 were also repressed by human hepatocyte transplantation in Cyp3a KO chimeric mice.

Predictability of plasma concentration-time curves in humans using single-species allometric scaling of chimeric mice with humanized liver.

1. We used chimeric mice (PXB mice®), which were repopulated with human hepatocytes, to evaluate their predictabilities of human pharmacokinetics. 2. The relationships of total clearance (CLt) and the volume of distribution at steady state (Vdss) between that predicted from single-species allometric scaling (SSS) of PXB mice and the observed human values indicated good correlations for various drugs metabolized by cytochrome P450s (CYPs) and non-CYPs. 3. We examined the Dedrick plot with which the plasma concentration-time curves can exhibit superimposability using SSS of PXB mice for CLt and Vdss. The predicted plasma concentration-time curves using the complex Dedrick plot from PXB mice were generally superimposed with the observed human data. 4. However, the predicted curve of diazepam was not superimposable with the observed profile. Residual mouse hepatocytes in the livers of PXB mice may affect predictability of CLt of diazepam because significant discrepancy of in vitro intrinsic clearance in PXB mouse liver microsomes consisted of low and high replacement of human hepatocytes were observed. 5. The complex Dedrick plot with SSS from PXB mice is useful for predicting the plasma concentration-time curve in drug discovery, although there are some limitations.

Megalin/cubilin-mediated uptake of FITC-labeled IgG by OK kidney epithelial cells.

In this paper, we characterize the uptake mechanism of fluorescein isothiocyanate-labeled human immunoglobulin G (FITC-hIgG) in opossum kidney (OK) epithelial cells, which have been shown to express megalin and cubilin. Confocal immunofluorescence microscopy showed the punctate expression of the neonatal Fc receptor FcRn in the cytoplasm, but not on the cell surface membrane. Temperature- and energy-dependent uptake of FITC-hIgG was observed at pH 7.4 but not at pH 6.0, indicating that the internalization of FITC-hIgG might not be due to FcRn, which has a binding affinity for IgG under acidic conditions. Under physiological pH conditions, human and bovine serum γ-globulin decreased FITC-hIgG uptake in a concentration-dependent manner. In addition, FITC-hIgG uptake was inhibited by various megalin and/or cubilin ligands including albumin, cytochrome c, transferrin and gentamicin. Endosomal acidification inhibitors (bafilomycin A(1) and chloroquine) significantly decreased the uptake of FITC-hIgG. Clathrin-dependent endocytosis inhibitors (phenylarsine oxide and chlorpromazine) decreased FITC-hIgG uptake. Potassium depletion and hypertonicity, conditions known to inhibit clathrin-dependent endocytosis, also decreased FITC-hIgG uptake. In contrast, caveolin-dependent endocytosis inhibitors (nystatin and methyl-ß-cyclodextrin) did not decrease, but rather increased the uptake of FITC-hIgG. These observations suggest that the internalization of FITC-hIgG in OK cells might be, at least in part, due to megalin/cubilin-mediated, clathrin-dependent endocytosis.

Function and expression of ATP-binding cassette transporters in cultured human Y79 retinoblastoma cells.

The aim of this study was to reveal the expression and function of P-glycoprotein and multidrug resistance-associated proteins (MRP), members of the ATP-binding cassette (ABC) superfamily of drug transporters, in cultured human Y79 retinoblastoma cells. ABC transporter mRNA expression was evaluated by conventional reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR analyses. Cellular accumulation of rhodamine 123 (P-glycoprotein substrate), calcein (MRP substrate), and doxorubicin (P-glycoprotein/MRP substrate) was analyzed by fluorometry. Conventional RT-PCR analysis showed the expression of multidrug resistance 1 (MDR1), MRP1, MRP2 and lung resistance-related protein (LRP) mRNAs. Real-time RT-PCR analysis revealed that the expression levels of the MDR1 and MRP2 genes in Y79 cells were much lower than those in human intestinal cell line Caco-2, while the expression level of MRP1 was higher than that in Caco-2 cells. The accumulation of rhodamine 123 was not enhanced by verapamil or reversin 205, inhibitors of P-glycoprotein, indicating no function of P-glycoprotein in Y79 cells. The accumulation of calcein was significantly increased by various MRP inhibitors including probenecid, indicating that MRP functions in Y79 cells. The accumulation of doxorubicin was increased in the presence of metabolic inhibitors (10 mM 2-deoxyglucose and 5 mM sodium azide). However, most MRP inhibitors such as probenecid and indomethacin did not affect doxorubicin accumulation, while cyclosporin A and taclorimus significantly increased doxorubicin accumulation. These results suggest that MRP, but not P-glycoprotein, functions in Y79 cells, and that the efflux of doxorubicin from Y79 cells may be due to an ATP-dependent transporter, which has not been identified yet.

Effects of endocytosis inhibitors on internalization of human IgG by Caco-2 human intestinal epithelial cells.

AIMS: The purpose of this study was to characterize the internalization mechanism of human IgG into the epithelial cells of human small intestine, employing human intestinal epithelial cell line Caco-2 as an in vitro model system. MAIN METHODS: Real-time PCR analysis and uptake studies of fluorescein isothiocyanate-labeled IgG (FITC-IgG) from human serum were performed using Caco-2 cells. KEY FINDINGS: Real-time PCR analysis showed that mRNA level of the neonatal Fc receptor (FcRn) was increased during the differentiation process in Caco-2 cells. The binding of FITC-labeled human IgG to the membrane surface of Caco-2 cells increased with a decrease in pH of incubation buffer. The uptake of FITC-IgG was also stimulated at acidic pH and was time-dependent. The binding and uptake of FITC-IgG at pH 6.0 was partially, but significantly, decreased by human gamma-globulin in a concentration-dependent manner. A mixture of metabolic inhibitors (sodium azide and 2-deoxyglucose) significantly inhibited the uptake, but not the binding, of FITC-IgG. In addition, endosomal acidification inhibitors such as bafilomycin A(1) and chloroquine significantly increased the accumulation of FITC-IgG. Clathrin-dependent endocytosis inhibitors (phenylarsine oxide and chlorpromazine) and caveolin-dependent endocytosis inhibitors (nystatin and indomethacin) did not decrease the uptake of FITC-IgG at pH 6.0. In contrast, macropinocytosis inhibitors such as cytochalasin B and 5-(N-ethyl-N-isopropyl) amiloride significantly decreased the uptake of FITC-IgG at pH 6.0. SIGNIFICANCE: The internalization of human IgG in human intestine might be, at least in part, due to FcRn-mediated endocytosis, which could occur by a process other than clathrin- and caveolin-dependent mechanisms.

Effects of exercise training on physical activity in older people: a randomized controlled trial.

BACKGROUND: Recent randomized controlled trials indicated that exercise training for elderly significantly increased their physical fitness. However, very few studies have examined changes in physical activity after exercise training. The purpose of this study was to investigate whether six-month exercise training for older adults can increase and maintain their physical activity in daily life. METHODS: Sixty-two men and women aged 60 to 81 years (mean age 67.1 years), living in communities, were randomly allocated into an exercise group (n = 32) or a control group (n = 33). The intervention started in April 1998 and lasted for 25 weeks. The exercise regimen consisted of endurance training and resistance exercises in a two-hour class conducted at least twice a week. The subjects completed a physical activity diary at each pre-intervention (March 1998), post-intervention (September 1998) and follow-up (April 1999) measurement of physical activity. Physical activity, expressed as total daily energy expenditure, was calculated by multiplying the amount of time spent in each activity and the corresponding METs. RESULTS: Total daily energy expenditure significantly increased from 40.8 kcal/kg/day to 43.5 kcal/kg/day in the exercise group (p = 0.03), but did not change in the control group. At the follow-up measurement, the mean total daily energy expenditure in the exercise group remained significantly higher, by 1.7 kcal/kg/day, than that at the pre-intervention (p = 0.05). CONCLUSIONS: This randomized controlled trial indicated that exercise training for elderly was effective in increasing physical activity in daily life.