Functional quantification of oral motor cortex at rest and during tasks using activity phase ratio: A zero-setting vector functional near-infrared spectroscopy study.

Oral frailty associated with oral hypokinesia may cause dementia. Functional near-infrared spectroscopy (fNIRS) can be used while the participants are in seating position with few restrictions. Thus, it is useful for assessing brain function, particularly oral motor activity. However, methods for identifying oral motor cortex (OMC) activation via the scalp have not been established. The current study aimed to detect OMC activation, an indicator of activity phase ratio (APR), which reflects increased oxygen consumption (0 < [deoxyhemoglobin (ΔDeoxyHb) or 0 < {[ΔDeoxyHb- oxyhemoglobin (ΔOxyHb)/√2]}, via fNIRS to accurately identify local brain activity. The APR, calculated via zero-set vector analysis, is a novel index for quantifying brain function both temporally and spatially at rest and during tasks. In total, 14 healthy participants performed bite tasks for 3 s per side for 10 times while in the sitting position. Then, time-series data on concentration changes in ΔOxyHb and ΔDeoxyHb were obtained via fNIRS. The anatomical location of the OMC was determined using a pooled data set of three-dimensional magnetic resonance images collected in advance from 40 healthy adults. In the zero-set vector analysis, the average change in ΔOxyHb and ΔDeoxyHb concentrations was utilized to calculate the APR percentage in 140 trials. The significant regions (z-score of ≥2.0) of the APR and ΔOxyHb in the task were compared. During the bite task, the APR significantly increased within the estimated OMC region (56-84 mm lateral to Cz and 4-20 mm anterior to Cz) in both the right and left hemispheres. By contrast, the ΔOxyHb concentrations increased on the bite side alone beyond the OMC region. The mean APR at rest for 2 s before the task showed 59.5%-62.2% in the left and right OMCs. The average APR for 3 s during the task showed 75.3% for the left OMC and 75.7% for the right OMC during the left bite task, and 65.9% for the left OMC and 80.9% for the right OMC during the right bite task. Interestingly, the average increase in APR for the left and right OMCs for the left bite task and the right bite task was 13.9% and 13.7%, respectively, showing almost a close match. The time course of the APR was more limited to the bite task segment than that of ΔOxyHb or ΔDexyHb concentration, and it increased in the OMC. Hence, the APR can quantitatively monitor both the resting and active states of the OMC in the left and right hemispheres. Using the zero-set vector-based fNIRS, the APR can be a valid indicator of oral motor function and bite force.

Development and characterization of a plant-derived rotavirus-like particle vaccine.

BACKGROUND: Virus-like particles (VLPs) are unable to replicate in the recipient but stimulate the immune system through recognition of repetitive subunits. Parenterally delivered rotavirus-VLP (Ro-VLP) vaccine could have the potential to overcome the weaknesses of licensed oral live-attenuated rotavirus vaccines, namely, low efficacy in low-income and high mortality settings and a potential risk of intussusception. METHODS: A monovalent Ro-VLP composed of viral protein (VP) 7, VP6 and VP2 of G1 genotype specificity was produced in Nicotiana benthamiana using Agrobacterium tumefaciens infiltration-based transient recombinant expression system. Plants expressing recombinant G1 Ro-VLP were harvested, then the resultant biomass was processed through a series of clarification and purification steps including standard extraction, filtration, ultrafiltration and chromatography. The purified G1 Ro-VLP was subsequently examined for its immunogenicity and toxicological profile using animal models. RESULTS: G1 Ro-VLP had a purity of ≥90% and was structurally similar to triple-layered rotavirus particles as determined by cryogenic transmission electron microscopy. Two doses of aluminum hydroxide-adjuvanted G1 Ro-VLP (1 µg, 5 µg or 30 µg), administered intramuscularly, elicited a robust homotypic neutralizing antibody response in rats. Also, rabbits administered G1 Ro-VLP (10 µg or 30 µg) four times intramuscularly with aluminum hydroxide adjuvant did not show any significant toxicity. CONCLUSIONS: Plant-derived Ro-VLP composed of VP7, VP6 and VP2 structural proteins would be a plausible alternative to live-attenuated oral rotavirus vaccines currently distributed worldwide.

A novel strategy of selective gene delivery by using a uniform magnetic field.

The application of a magnetic field to enhance the transfection efficiency has been reported to be mainly dependent on the magnetic force generated by a magnetic field gradient to attract paramagnetic bead-conjugated carrier and polynucleotide complexes. This strategy has the advantage of targeting a point or an area on the culture vessel. However, it is difficult to target deeply placed tissues in vivo. Uniform magnetic field-correlated effect is applicable to such a purpose. Here, we attempted to establish a novel procedure for uniform magnetic field-dependent enhancement of transfection efficiency. We examined the effect of a 1.5 mT uniform magnetic field on cellular reactive oxygen species (ROS) level and transfection efficiency mediated by a ROS-sensitive transfection carrier. Our experimental results revealed that a 1.5 mT uniform magnetic field transiently decreased cellular ROS levels and strongly enhanced transfection efficiency mediated by polyethylenimine (PEI). The uniform magnetic field-dependent enhancement of PEI-mediated in vivo transfection was confirmed in the livers of mice. Local intensification of a uniform magnetic field in a culture dish resulted in selective gene delivery into cells on the target area. Although further examination and improvement are necessary for this procedure, our findings provide a novel option for spatial control of gene delivery.

PEPITEM/Cadherin 15 Axis Inhibits T Lymphocyte Infiltration and Glomerulonephritis in a Mouse Model of Systemic Lupus Erythematosus.

Control of lymphocyte infiltration in kidney is a potential therapeutic strategy for lupus nephritis, considering that control of lymphocyte migration by sphingosine 1 phosphate has been implicated in inflammation-related pathology. The peptide inhibitor of the transendothelial migration (PEPITEM)/cadherin (CDH) 15 axis was recently reported to promote sphingosine 1 phosphate secretion. In this study, we investigated whether CDH15 is expressed in the kidney of MRL/lpr mice and whether lymphocyte infiltration is suppressed by exogenously administered PEPITEM. Mice (18 wk old) were randomized into 4-wk treatment groups that received PEPITEM or PBS encapsulated in dipalmitoylphosphatidylcholine liposomes. Enlargement of the kidney, spleen, and axillary lymph nodes was suppressed by PEPITEM treatment, which also blocked infiltration of double-negative T lymphocytes into the kidney and glomerular IgG/C3 deposition, reduced proteinuria, and increased podocyte density. Immunohistochemical analysis revealed that the PEPITEM receptor CDH15 was expressed on vascular endothelial cells of glomeruli and kidney arterioles, skin, and peritoneum in lupus mice at 22 wk of age but not in 4-wk-old mice. These results suggest that PEPITEM inhibits lymphocyte migration and infiltration into the kidney, thereby preserving the kidney structure and reducing proteinuria. Thus, PEPITEM administration may be considered as a potential therapeutic tool for systemic lupus erythematosus.

Donepezil-induced oligodendrocyte differentiation is mediated through estrogen receptors.

Loss of oligodendrocytes, the myelin-forming cells of the central nervous system, and subsequent failure of myelin development result in serious neurological disorders such as multiple sclerosis. Using primary mouse embryonic neural stem cells (NSCs), we previously demonstrated that donepezil, an acetylcholinesterase inhibitor developed for the treatment of Alzheimer's disease, stimulates the differentiation of NSCs into oligodendrocytes and neurons, albeit at the expense of astrogenesis. However, the precise mechanisms underlying donepezil-induced differentiation remain unclear. In this study, we aimed at elucidating the molecular pathways contributing to donepezil-induced differentiation of mouse-induced pluripotent stem cell-derived neural stem cells (miPSC-NSCs). We used cell-based reporter gene arrays to investigate effects of donepezil on differentiation of miPSC-NSCs. Subsequently, we assessed the molecular pathway underlying donepezil action on differentiation of miPSC-NSCs into mature oligodendrocytes. Donepezil increased the transcriptional activity of estrogen response element under differentiating conditions. Moreover, estrogen receptors α (ERα) and ß (ERß) were highly expressed in MBP-positive mature oligodendrocytes. The ER antagonist ICI 182,780 abrogated the number of MBP-positive oligodendrocytes induced by donepezil, but showed no effect on the differentiation of miPSC-NSCs into Tuj1-positive neurons and GFAP-positive astrocytes. Furthermore, the donepezil-induced generation of mature oligodendrocytes from miPSC-NSC was significantly attenuated by antagonists and siRNA targeting ERα and ERß. In conclusion, we demonstrated, for the first time, that donepezil-induced oligodendrogenesis is mediated through both ER subtypes, ERα and ERß. Cover Image for this issue: https://doi.org/10.1111/jnc.14771.

Neuronal Ca2+ -dependent activator protein 1 (NCDAP1) induces neuronal cell death by activating p53 pathway following traumatic brain injury.

Overactivation of N-methyl-d-aspartate glutamate receptors (NMDARs) after traumatic brain injury (TBI) contributes to excitotoxic cell death. The hyperactivation of NMDARs results in toxic levels of intracellular Ca2+ and in the activation of p53-mediated apoptosis pathway. Neuronal Ca2+ -dependent activator protein 1 (NCDAP1) was identified as an epileptogenic gene of unknown function in our laboratory. In this study, we investigated the expression and cellular localization of NCDAP1 in rat models of fluid percussion-induced TBI. NCDAP1 expression increased in the ipsilateral cortex and hippocampus adjacent to the lesion of the TBI rats compared with that in the sham-operated controls. In addition, NCDAP1 was co-expressed with neuronal marker (NeuN), and the results of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining suggest that NCDAP1 is involved in neuronal apoptosis that occurs after brain injury. In addition, the expression levels of p53, Bax, and active caspase-3 correlated with those of NCDAP1. To further investigate the function of NCDAP1, primary cultured neurons were employed to establish an apoptosis model. The expression of NCDAP1 was induced by NMDA-induced Ca2+ influx, and the knockdown of NCDAP1 by siRNA decreased apoptosis caused by treatment with NMDA. Silencing of NCDAP1 also reduced p53 expression, whereas the over-expression of NCDAP1 induced cell death and up-regulated the expression of p53. The inhibition of p53 with pifithrin alpha or siRNA counteracted the effects of NCDAP1. Based on our data, we suggest that NCDAP1 plays an important role in p53-mediated neuronal apoptosis following TBI.

Proposal for a screening questionnaire for detecting habitual mouth breathing, based on a mouth-breathing habit score.

BACKGROUND: When mouth breathing becomes habitual, it can cause sleep disorders and abnormal maxillofacial growth, thus early detection of habitual mouth breathing is important. We created a questionnaire for early detection of habitual mouth breathing using a score based on a spectrum of factors found to be characteristic of mouth breathers. METHODS: First, a draft 50-question questionnaire was given to 101 random dental clinic patients, classified by dental professionals into habitual mouth breathers (n = 28) and nose breathers (n = 73). The 10 questions that significantly differentiated mouth and nose breathers (p < 0.05) were identified from this questionnaire. These questions, regarding nasal obstruction, open mouth at rest, awareness of mouth breathing, gum swelling and dental staining of the front teeth, bad breath, maxillary protrusion, nasal obstruction in childhood, bottle-feeding, and history of asthma, formed the basis for a second questionnaire. This second survey was completed by another 242 participants, separately classified into mouth breathing (n = 26), suspected mouth breathing (n = 40), and nose breathing groups (n = 176). RESULTS: Receiver operating characteristic curve analysis of the resulting mouth breathing habit scores, representing the responses to the 10-question survey, showed moderate checklist diagnosability. Sensitivity of cut-off values was 61.5% (specificity 92.0%) for the mouth-breathing group, and 77.5% (specificity 56.3%) for the suspected mouth-breathing group. Information was also obtained from visual assessment of maxillofacial characteristics. We found that the mouth-breathing and suspected mouth-breathing groups showed significantly high odds ratios for 7 items: discomfort while breathing and increased chin muscle tonus with lip closure, maxillary protrusion, tongue thrust, open mouth at rest, open bite, and childhood asthma. For 94.6% of the nose breathing group, ≥1 of these items applied. CONCLUSIONS: These findings were then used together to create a sample screening form. We believe that screening of this kind can facilitate more accurate diagnosis of habitual mouth breathing and contribute to its early detection.

Donepezil promotes differentiation of neural stem cells into mature oligodendrocytes at the expense of astrogenesis.

Oligodendrocytes are the myelin-forming cells of the central nervous system. Oligodendrocyte loss and failure of myelin development result in serious human disorders, including multiple sclerosis. Previously, using oligodendrocyte progenitor cells, we have shown that donepezil, which is an acetylcholinesterase inhibitor developed for the treatment of Alzheimer's disease, stimulates myelin gene expression and oligodendrocyte differentiation. Here, we aimed to analyze the effects of donepezil on primary mouse embryonic neural stem cells (NSCs). Donepezil treatment led to impaired self-renewal ability and increased apoptosis. These effects appeared to be mediated through the Akt/Bad signaling pathway. Using neurosphere differentiation analysis, we observed that donepezil leads to reduced numbers of astrocytes and increased numbers of oligodendrocytes and neurons. Consistent with this finding, mRNA and protein levels for the oligodendrocyte markers myelin-associated glycoprotein, 2', 3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase), and myelin basic protein, as well as the neuronal marker ß-tubulin type III (Tuj1) were up-regulated. In contrast, the expression of the astrocyte marker glial fibrillary acidic protein (GFAP) was down-regulated by donepezil in a dose- and time-dependent manner. Moreover, donepezil increased oligodendrocyte differentiation, resulting in a reduction in the differentiation of NSCs into astrocytes, by suppressing the activation of signal transducer and activator of transcription 3 (STAT3), SMAD1/5/9, and the downstream target gene GFAP, even under astrocyte-inducing conditions. These results suggest that efficient differentiation of NSCs into oligodendrocytes by donepezil may indicate a novel therapeutic role for this drug in promoting repair in demyelinated lesions in addition to its role in preventing astrogenesis.

A novel strategy for selective gene delivery by using the inhibitory effect of blue light on jetPRIME-mediated transfection.

Photodynamic control of gene delivery is a new technology with growing applications in gene therapy and basic cell research. Main approaches of light-selective gene delivery rely on the light-dependent enhancement of transfection efficiency. Studies focused on light-stimulated inhibitory regulation of transfection have rarely been reported. Here, we tried to establish a novel procedure of light-dependent inhibition of transfection. Our experiments, conducted with several types of commercial transfection reagents, revealed that jetPRIME-mediated transfection was strongly inhibited by blue light. Although the uptake of reagent-DNA complex was drastically reduced, preliminary exposure of cells or reagent-DNA complex to blue light had no inhibitory effect on the transfection efficiency. The inhibitory effect was wavelength-dependent and mediated by reactive oxygen species. Partial exposure of a culture vessel to blue light resulted in selective gene delivery into cells grown on the unexposed area of the vessel. By using this approach, different types of plasmid DNA were delivered into different areas in the culture vessel. This novel approach to the inhibitory control of transfection provides practical options for research and therapeutics. Biotechnol. Bioeng. 2016;113: 1560-1567. © 2015 Wiley Periodicals, Inc.

Nicotinic acetylcholine receptors mediate donepezil-induced oligodendrocyte differentiation.

Oligodendrocytes are the myelin-forming cells of the central nervous system (CNS). Failure of myelin development and oligodendrocyte loss results in serious human disorders, including multiple sclerosis. Here, we show that donepezil, an acetlycholinesterase inhibitor developed for the treatment of Alzheimer's disease, can stimulate oligodendrocyte differentiation and maturation of neural stem cell-derived oligodendrocyte progenitor cells without affecting proliferation or cell viability. Transcripts for essential myelin-associated genes, such as PLP, MAG, MBP, CNPase, and MOG, in addition to transcription factors that regulate oligodendrocyte differentiation and myelination, were rapidly increased after treatment with donepezil. Furthermore, luciferase assays confirmed that both MAG and MBP promoters display increased activity upon donepezil-induced oligodendrocytes differentiation, suggesting that donepezil increases myelin gene expression mainly through enhanced transcription. We also found that the increase in the number of oligodendrocytes observed following donepezil treatment was significantly inhibited by the nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine, but not by the muscarinic acetylcholine receptor antagonist scopolamine. Moreover, donepezil-induced myelin-related gene expression was suppressed by mecamylamine at both the mRNA and protein level. These results suggest that donepezil stimulates oligodendrocyte differentiation and myelin-related gene expression via nAChRs in neural stem cell-derived oligodendrocyte progenitor cells. We show that donepezil, a drug for the treatment of Alzheimer disease, can stimulate oligodendrocyte differentiation and maturation of oligodendrocyte progenitor cells. Transcripts for essential myelin-associated genes, such as PLP, MAG, MBP, CNPase and MOG in addition to transcripton factors that regulate oligodendrocyte differentiation and myelination were rapidly increased after treatment with donepezil. These effects were partly dependent on nicotinic acetylcholine receptor (nAChR).

In vivo therapeutic potential of Dicer-hunting siRNAs targeting infectious hepatitis C virus.

The development of RNA interference (RNAi)-based therapy faces two major obstacles: selecting small interfering RNA (siRNA) sequences with strong activity, and identifying a carrier that allows efficient delivery to target organs. Additionally, conservative region at nucleotide level must be targeted for RNAi in applying to virus because hepatitis C virus (HCV) could escape from therapeutic pressure with genome mutations. In vitro preparation of Dicer-generated siRNAs targeting a conserved, highly ordered HCV 5' untranslated region are capable of inducing strong RNAi activity. By dissecting the 5'-end of an RNAi-mediated cleavage site in the HCV genome, we identified potent siRNA sequences, which we designate as Dicer-hunting siRNAs (dh-siRNAs). Furthermore, formulation of the dh-siRNAs in an optimized multifunctional envelope-type nano device inhibited ongoing infectious HCV replication in human hepatocytes in vivo. Our efforts using both identification of optimal siRNA sequences and delivery to human hepatocytes suggest therapeutic potential of siRNA for a virus.

Resistance to cyclosporin A derives from mutations in hepatitis C virus nonstructural proteins.

Cyclosporine A (CsA) is an immunosuppressive drug that targets cyclophilins, cellular cofactors that regulate the immune system. Replication of hepatitis C virus (HCV) is suppressed by CsA, but the molecular basis of this suppression is still not fully understood. To investigate this suppression, we cultured HCV replicon cells (Con1, HCV genotype 1b, FLR-N cell) in the presence of CsA and obtained nine CsA-resistant FLR-N cell lines. We determined full-length HCV sequences for all nine clones, and chose two (clones #6 and #7) of the nine clones that have high replication activity in the presence of CsA for further analysis. Both clones showed two consensus mutations, one in NS3 (T1280V) and the other in NS5A (D2292E). Characterization of various mutants indicated that the D2292E mutation conferred resistance to high concentrations of CsA (up to 2 µM). In addition, the missense mutation T1280V contributed to the recovery of colony formation activity. The effects of these mutations are also evident in two established HCV replicon cell lines-HCV-RMT ([1], genotype 1a) and JFH1 (genotype 2a). Moreover, three other missense mutations in NS5A-D2303H, S2362G, and E2414K-enhanced the resistance to CsA conferred by D2292E; these double or all quadruple mutants could resist approximately 8- to 25-fold higher concentrations of CsA than could wild-type Con1. These four mutations, either as single or combinations, also made Con1 strain resistant to two other cyclophilin inhibitors, N-methyl-4-isoleucine-cyclosporin (NIM811) or Debio-025. Interestingly, the changes in IC50 values that resulted from each of these mutations were the lowest in the Debio-025-treated cells, indicating its highest resistant activity against the adaptive mutation.

Isolation and characterization of highly replicable hepatitis C virus genotype 1a strain HCV-RMT.

Multiple genotype 1a clones have been reported, including the very first hepatitis C virus (HCV) clone called H77. The replication ability of some of these clones has been confirmed in vitro and in vivo, although this ability is somehow compromised. We now report a newly isolated genotype 1a clone, designated HCV-RMT, which has the ability to replicate efficiently in patients, chimeric mice with humanized liver, and cultured cells. An authentic subgenomic replicon cell line was established from the HCV-RMT sequence with spontaneous introduction of three adaptive mutations, which were later confirmed to be responsible for efficient replication in HuH-7 cells as both subgenomic replicon RNA and viral genome RNA. Following transfection, the HCV-RMT RNA genome with three adaptive mutations was maintained for more than 2 months in HuH-7 cells. One clone selected from the transfected cells had a high copy number, and its supernatant could infect naïve HuH-7 cells. Direct injection of wild-type HCV-RMT RNA into the liver of chimeric mice with humanized liver resulted in vigorous replication, similar to inoculation with the parental patient's serum. A study of virus replication using HCV-RMT derivatives with various combinations of adaptive mutations revealed a clear inversely proportional relationship between in vitro and in vivo replication abilities. Thus, we suggest that HCV-RMT and its derivatives are important tools for HCV genotype 1a research and for determining the mechanism of HCV replication in vitro and in vivo.

Synthetic lipophilic antioxidant BO-653 suppresses HCV replication.

The influence of the intracellular redox state on the hepatitis C virus (HCV) life cycle is poorly understood. This study demonstrated the anti-HCV activity of 2,3-dihydro-5-hydroxy-2,2-dipentyl-4,6-di-tert-butylbenzofuran (BO-653), a synthetic lipophilic antioxidant, and examined whether BO-653's antioxidant activity is integral to its anti-HCV activity. The anti-HCV activity of BO-653 was investigated in HuH-7 cells bearing an HCV subgenomic replicon (FLR3-1 cells) and in HuH-7 cells infected persistently with HCV (RMT-tri cells). BO-653 inhibition of HCV replication was also compared with that of several hydrophilic and lipophilic antioxidants. BO-653 suppressed HCV replication in FLR3-1 and RMT-tri cells in a concentration-dependent manner. The lipophilic antioxidants had stronger anti-HCV activities than the hydrophilic antioxidants, and BO-653 displayed the strongest anti-HCV activity of all the antioxidants examined. Therefore, the anti-HCV activity of BO-653 was examined in chimeric mice harboring human hepatocytes infected with HCV. The combination treatment of BO-653 and polyethylene glycol-conjugated interferon-α (PEG-IFN) decreased serum HCV RNA titer more than that seen with PEG-IFN alone. These findings suggest that both the lipophilic property and the antioxidant activity of BO-653 play an important role in the inhibition of HCV replication.

Monoclonal antibody 2-152a suppresses hepatitis C virus infection through betaine/GABA transporter-1.

BACKGROUND: We recently established a monoclonal antibody (2-152a MAb) that binds to 3ß-hydroxysterol-Δ24-reductase (DHCR24) by immunizing mice with cells (RzM6-LC) persistently expressing hepatitis C virus (HCV). Here, we aimed to analyze the activity of 2-152a MAb against HCV replication and explore the molecular mechanism underlying the antiviral activity. METHODS: We characterized the effects of 2-152a MAb on HCV replication and performed a microarray analysis of antibody-treated HCV replicon cells. The molecules showing a significant change after the antibody treatment were screened to examine their relationship with HCV replication. RESULTS: The antibody had antiviral activity both in vitro and in vivo (chimeric mice). In the microarray analysis, 2-152a MAb significantly suppressed the expression of betaine/GABA transporter-1 (BGT-1) in 2 HCV replicon cell lines but not in HCV-cured cells. Silencing of BGT-1 expression by small interfering RNA (siRNA) revealed significant suppression of HCV replication and infection without cytotoxicity. Further, BGT-1 expression was significantly increased in the presence of HCV (P < .05). CONCLUSIONS: Our results suggest that 2-152a MAb suppresses HCV replication and infection through BGT-1. These findings highlight important roles of BGT-1 in HCV replication and reveal a possible target for anti-HCV therapy.

Establishment of infectious HCV virion-producing cells with newly designed full-genome replicon RNA.

Hepatitis C virus (HCV) replicon systems enable in-depth analysis of the life cycle of HCV. However, the previously reported full-genome replicon system is unable to produce authentic virions. On the basis of these results, we constructed newly designed full-genomic replicon RNA, which is composed of the intact 5'-terminal-half RNA extending to the NS2 region flanked by an extra selection marker gene. Huh-7 cells harboring this full-genomic RNA proliferated well under G418 selection and secreted virion-like particles into the supernatant. These particles, which were round and 50 nm in diameter when analyzed by electron microscopy, had a buoyant density of 1.08 g/mL that shifted to 1.19 g/mL after NP-40 treatment; these figures match the putative densities of intact virions and nucleocapsids without envelope. The particles also showed infectivity in a colony-forming assay. This system may offer another option for investigating the life cycle of HCV.

Sphingomyelin activates hepatitis C virus RNA polymerase in a genotype-specific manner.

Hepatitis C virus (HCV) replication and infection depend on the lipid components of the cell, and replication is inhibited by inhibitors of sphingomyelin biosynthesis. We found that sphingomyelin bound to and activated genotype 1b RNA-dependent RNA polymerase (RdRp) by enhancing its template binding activity. Sphingomyelin also bound to 1a and JFH1 (genotype 2a) RdRps but did not activate them. Sphingomyelin did not bind to or activate J6CF (2a) RdRp. The sphingomyelin binding domain (SBD) of HCV RdRp was mapped to the helix-turn-helix structure (residues 231 to 260), which was essential for sphingomyelin binding and activation. Helix structures (residues 231 to 241 and 247 to 260) are important for RdRp activation, and 238S and 248E are important for maintaining the helix structures for template binding and RdRp activation by sphingomyelin. 241Q in helix 1 and the negatively charged 244D at the apex of the turn are important for sphingomyelin binding. Both amino acids are on the surface of the RdRp molecule. The polarity of the phosphocholine of sphingomyelin is important for HCV RdRp activation. However, phosphocholine did not activate RdRp. Twenty sphingomyelin molecules activated one RdRp molecule. The biochemical effect of sphingomyelin on HCV RdRp activity was virologically confirmed by the HCV replicon system. We also found that the SBD was the lipid raft membrane localization domain of HCV NS5B because JFH1 (2a) replicon cells harboring NS5B with the mutation A242C/S244D moved to the lipid raft while the wild type did not localize there. This agreed with the myriocin sensitivity of the mutant replicon. This sphingomyelin interaction is a target for HCV infection because most HCV RdRps have 241Q.

Traumatic dissection of extracranial vertebral artery with massive subtentorial cerebral infarction: report of an autopsy case.

We present an extremely rare autopsy case with traumatic dissection of the extracranial vertebral artery due to blunt injury caused by a traffic accident. The patient complained of nausea and numbness of the hands at the scene of the accident. His consciousness deteriorated and he fell into a coma within 12h, then died 4 days after the collision. Brain CT/MRI disclosed massive infratentorial cerebral infarction while MRA imaged neither of the vertebral arteries. Autopsy revealed a seatbelt mark on the right side of the lower neck, with fracture of the right transverse process of the sixth cervical vertebra. The right extracranial vertebral artery (V2) showed massive medial dissection from the portion adjacent to the fracture throughout the upper end of the extracranial part of the artery and was occluded by a thrombus. An intimal tear was confirmed near the starting point of the dissection. The brain disclosed massive infarction of posterior circulation territories with changes to the so-called respirator brain. The victim's left vertebral artery was considerably hypoplastic. We concluded that a massive infratentorial infarction was caused by dissection of the right extracranial vertebral artery and consecutive thrombus formation brought about by impact with the seatbelt at the time of the collision.

The knockdown of endogenous replication factor C4 decreases the growth and enhances the chemosensitivity of hepatocellular carcinoma cells.

AIMS: To identify differentially expressed genes and thereby detect potential molecular targets for future therapies directed against hepatocellular carcinoma (HCC). METHODS: To isolate differentially expressed genes between HCC and adjacent non-cancerous liver tissues, cDNA microarray and quantitative reverse transcriptase polymerase chain reaction analyses were performed. Gene knockdown experiments in HepG2 cells were also performed using small interfering RNAs (siRNAs). Proteins were detected by immunostaining, and cell proliferation was analysed using the MTT/WST-8 assay. Apoptosis and cell cycle analyses were performed using flow cytometry. RESULTS: After an intensive screening for differentially expressed genes in HCC tissues, we isolated 23 upregulated genes in these lesions. Among these, we focused on the replication factor C4 (RFC4) gene. The expression of endogenous RFC4 proteins in HepG2 cells was found to be significantly reduced by RFC4-specific siRNA. This inhibition of RFC4 expression correlated with a decrease in cellular proliferation, increased levels of apoptosis and a sensitizing of the cells to the DNA-damaging chemotherapeutic agents, doxorubicin and camptothecin. CONCLUSION: The replication factor C4 gene may be a novel target for developing cancer therapeutics, which can enhance the antitumour activity of chemotherapeutic agents that induce DNA damage.

Carbonyl Reductase 3 (CBR3) Mediates 9-cis-Retinoic Acid-Induced Cytostatis and is a Potential Prognostic Marker for Oral Malignancy.

The molecular mechanisms of growth suppression by retinoic acid (RA) were examined. Our results suggest that the cytostatic effects of RA could be mediated by the activation of endogenous CBR3 gene in oral squamous cell carcinomas (OSCCs), and the expression is a potential marker for oral malignancy.