Hepatitis C Virus Infection of Cultured Human Hepatoma Cells Causes Apoptosis and Pyroptosis in Both Infected and Bystander Cells.

Individuals infected with hepatitis C virus (HCV) are at high risk of developing progressive liver disease, including cirrhosis and hepatocellular carcinoma (HCC). How HCV infection causes liver destruction has been of significant interest for many years, and apoptosis has been proposed as one operative mechanism. In this study, we employed a tissue culture-adapted strain of HCV (JFH1T) to test effects of HCV infection on induction of programmed cell death (PCD) in Huh-7.5 cells. We found that HCV infection reduced the proliferation rate and induced caspase-3-mediated apoptosis in the infected cell population. However, in addition to apoptosis, we also observed infected cells undergoing caspase-1-mediated pyroptosis, which was induced by NLRP3 inflammasome activation. By co-culturing HCV-infected Huh-7.5 cells with an HCV-non-permissive cell line, we also demonstrated induction of both apoptosis and pyroptosis in uninfected cells. Bystander apoptosis, but not bystander pyroptosis, required cell-cell contact between infected and bystander cells. In summary, these findings provide new information on mechanisms of cell death in response to HCV infection. The observation that both apoptosis and pyroptosis can be induced in bystander cells extends our understanding of HCV-induced pathogenesis in the liver.

Global gene analysis identifying genes commonly regulated by the Ras/Raf/MEK and type I IFN pathways.

Oncolytic viruses exploit alterations in cancer cells to specifically infect cancer cells but not normal healthy cells. Previous work has shown that oncogenic Ras interferes with interferon (IFN) signaling to promote viral replication. Furthermore, inhibition of the Ras/Raf/MEK/ERK pathway at the level of Ras, MEK, or ERK was sufficient to restore IFN signaling. In order to identify genes that were commonly regulated by the inhibition of the Ras pathway and the IFN pathway, we treated NIH/3T3 cells that overexpress oncogenic Ras with the MEK inhibitor, U0126, or IFN-α for 6 h, and performed DNA microarray analysis (Gene Expression Omnibus accession number GSE49469). Here, we also provide additional information on the experimental and functional analysis of the genes responsive to U0126 and IFN.

Oncogenic Ras inhibits IRF1 to promote viral oncolysis.

Oncolytic viruses exploit common molecular changes in cancer cells, which are not present in normal cells, to target and kill cancer cells. Ras transformation and defects in type I interferon (IFN)-mediated antiviral responses are known to be the major mechanisms underlying viral oncolysis. Previously, we demonstrated that oncogenic RAS/Mitogen-activated protein kinase kinase (Ras/MEK) activation suppresses the transcription of many IFN-inducible genes in human cancer cells, suggesting that Ras transformation underlies type I IFN defects in cancer cells. Here, we investigated how Ras/MEK downregulates IFN-induced transcription. By conducting promoter deletion analysis of IFN-inducible genes, namely guanylate-binding protein 2 and IFN gamma inducible protein 47 (Ifi47), we identified the IFN regulatory factor 1 (IRF1) binding site as the promoter region responsible for the regulation of transcription by MEK. MEK inhibition promoted transcription of the IFN-inducible genes in wild type mouse embryonic fibroblasts (MEFs), but not in IRF1(-/-) MEFs, showing that IRF1 is involved in MEK-mediated downregulation of IFN-inducible genes. Furthermore, IRF1 protein expression was lower in RasV12 cells compared with vector control NIH3T3 cells, but was restored to equivalent levels by inhibition of MEK. Similarly, the restoration of IRF1 expression by MEK inhibition was observed in human cancer cells. IRF1 re-expression in human cancer cells caused cells to become resistant to infection by the oncolytic vesicular stomatitis virus strain. Together, this work demonstrates that Ras/MEK activation in cancer cells downregulates transcription of IFN-inducible genes by targeting IRF1 expression, resulting in increased susceptibility to viral oncolysis.

In vitro and in vivo comparison of viral and cellular internal ribosome entry sites for bicistronic vector expression.

Bicistronic vectors are essential to achieve efficient expression of multiple genes in gene therapy protocols and biomedical applications. Internal ribosome entry site (IRES) elements have been utilized to initiate expression of an additional protein from a bicistronic vector. The IRES element commonly used in current bicistronic vectors originates from the encephalomyocarditis virus (EMCV). As IRES-mediated translation is dependent on availability of IRES trans-acting factors, which vary between cell types and species, adequate gene expression from the EMCV IRES element is not always achieved. To identify a novel IRES element that mediates gene expression consistently with a higher efficiency than the EMCV IRES, we tested 13 bicistronic reporter constructs containing different viral and cellular IRES elements. The in vitro screening in human and mouse fibroblast and hepatocarcinoma cells revealed that the vascular endothelial growth factor and type 1 collagen-inducible protein (VCIP) IRES was the only IRES element that directed translation more efficiently than the EMCV IRES in all cell lines. Furthermore, the VCIP IRES initiated greater reporter expression levels than the EMCV IRES in transfected mouse livers. These results suggest that VCIP-IRES containing vectors improve gene expression compared with those harboring an EMCV-IRES. This could increase the potential benefits of bicistronic vectors for experimental and therapeutic purposes.

Effect of maternal stress on fetal heart rate assessed by vibroacoustic stimulation.

This study was designed to determine whether maternal stress levels, state and trait anxiety levels, and stress hormones affect fetal heart rate (FHR) patterns after vibroacoustic stimulation (VAS) at 30 weeks of gestation. A total of 24 healthy pregnant women with a single fetus pregnancy were enrolled. Corticotropin releasing hormone (CRH) and adrenocorticotropic hormone in maternal plasma and cortisol, and chromogranin A in saliva were measured. The FHR patterns after VAS were divided into three types: type I, a long period of acceleration or one acceleration lasting > 1 min or at least two accelerations lasting > 15 s; type II, a biphasic response with acceleration followed by deceleration; and type III, no response or prolonged deceleration. In the high trait anxiety group, CRH levels were significantly higher than in the low trait anxiety group, and FHR patterns after VAS showed mostly a type II response pattern. These findings suggest that stress in pregnant women with high trait anxiety may influence FHR patterns after VAS.

Distribution of emm genotypes among group A streptococcus isolates from patients with severe invasive streptococcal infections in Japan, 2001-2005.

We surveyed emm genotypes of group A streptococcus (GAS) isolates from patients with severe invasive streptococcal infections during 2001-2005 and compared their prevalence with that of the preceding 5 years. Genotype emm1 remained dominant throughout 2001 to 2005, but the frequency rate of this type decreased compared with the earlier period. Various other emm types have appeared in recent years indicating alterations in the prevalent strains causing severe invasive streptococcal infections. The cover of the new 26-valent GAS vaccine fell from 93.5% for genotypes of isolates from 1996-2000 to 81.8% in 2001-2005.

Brachial-ankle pulse wave velocity measured automatically by oscillometric method is elevated in diabetic patients with incipient nephropathy.

AIMS: To examine whether brachial-ankle pulse wave velocity (baPWV), a possible early marker of atherosclerotic vascular damage, is associated with albuminuria in patients with Type 2 diabetes. METHODS: BaPWV was measured by automatic oscillometric method in 346 Type 2 diabetic patients with normoalbuminuria (a mean level of three times measurements of albumin-to-creatinine (ACR)<30 microg/mg creatinine; n=200), incipient nephropathy (a mean level of ACR> or =30 and <300 microg/mg creatinine; n=119), and clinical nephropathy (a mean level of ACR> or =300 microg/mg creatinine; n=27), and without peripheral vascular disease. RESULTS: BaPWV (cm/s) was significantly higher in patients with incipient nephropathy (1722 +/- 382) and clinical nephropathy (1763 +/- 322) than in patients with normoalbuminuria (1559 +/- 343, P<0.0001, respectively). By univariate analysis it correlated significantly with age (r=0.44, P<0.0001), systolic blood pressure (r=0.55, P<0.0001), diastolic blood pressure (r=0.42, P<0.0001), albuminuria (r=0.24, P<0.0001) and HbA1C (r=0.11, P<0.05). Albuminuria revealed an independent significant association with baPWV (P<0.01) after adjustment for age, sex, smoking, BMI, HbA1C, hyperlipidemia, and hypertension. Multiple regression analysis showed age, diastolic blood pressure and albuminuria were independently associated with baPWV (adjusted R2=0.42, P<0.0001). CONCLUSIONS: The results might indicate a possible link between the pathogenesis of atherosclerosis and diabetic nephropathy. Future studies are needed to clarify the usefulness and its predictable value.

Musculoskeletal disorders among hospital nurses in rural Japan.

INTRODUCTION: Although musculoskeletal disorders (MSD) represent a significant occupational problem for hospital nurses, few investigations target nurses in rural facilities. This study investigated the prevalence and correlates of MSD among nurses within an affiliated hospital conglomerate of rural Japan. METHODS: A self-reporting survey was used as diagnostic tool; the reliability, validity and cost-effectiveness of this method has been established. RESULTS: An initial group of 329 nurses was recruited from three affiliated, rural locations in Yamanashi prefecture, central Japan; 247 usable replies were obtained (final response rate: 75.1%). The 12-month period-prevalence of self-reported MSD at any body site was 91.9% (95% CI 87.8-94.7). Low back pain (LBP) was the most commonly reported MSD with a prevalence of 82.6% (95% CI 77.4-86.8). Next most reported were shoulder (61.1%, 95% CI 54.9-67.0), neck (36.8%, 95% CI 31.1-43.0), upper back (29.1%, 95% CI 23.8-35.1), knee (23.5%, 95% CI 18.6-29.1) and upper leg MSD (19.4%, 95% CI 15.0-24.8). MSD of the wrist (14.2%, 95% CI 10.4-19.1), upper arm (13.0%, 95% CI 9.3 -17.7) and lower arm (11.7% 95% CI 8.3-16.4) were less common. Logistic regression indicated that nurses who were regularly involved in the manual handling of patients had an increased LBP risk of 16.7 (OR 16.7, 95% CI 1.3-412.7, p = 0.0340) when compared with nurses who were not involved in manual handling of patients. CONCLUSION: Overall, our study indicated that MSD is common among rural Japanese nurses and its prevalence is consistent between related facilities. The excessive amount of LBP, however, suggests that Japanese nurses are at higher risk of this condition compared with their foreign counterparts.

Magnetic resonance imaging and clinical findings examined in adulthood-studies on three adults with Rett syndrome.

PURPOSE: To clarify magnetic resonance imaging (MRI) findings in three adult patients with Rett syndrome who had been diagnosed with mental retardation and autism. METHOD: Clinical and MRI findings in three adult cases with Rett syndrome were studied. Ages (in years) in three adult cases with Rett Syndrome were 46 in Case 1, 35 in Case 2 and 20 in Case 3. They were able to walk and their convulsions were well controlled. RESULTS: MRI findings in all patients showed mild cerebral atrophy, especially in the frontal and temporal lobes and two of the cases also had mild cerebellar atrophy. One case also showed a narrowing of the brainstem and thinning of the corpus callosum. CONCLUSIONS: These results indicate that abnormalities in MRI imaging, in cases where there is narrowing of the brainstem and thinning of the corpus callosum, may be due to congenital hypoplasia. It was also seen that cerebellar atrophy became more distinct in older cases.

Increased synchronization of cortical oscillatory activities between human supplementary motor and primary sensorimotor areas during voluntary movements.

In human, both primary and nonprimary motor areas are involved in the control of voluntary movements. However, the dynamics of functional coupling among different motor areas has not been fully clarified yet. Because it has been proposed that the functional coupling among cortical areas might be achieved by the synchronization of oscillatory activity, we investigated the electrocorticographic coherence between the supplementary motor and primary sensorimotor areas (SMA and S1-M1) by means of event-related partial coherence analysis in 11 intractable epilepsy patients. We found premovement increase of coherence between the SMA proper and S1-M1 at the frequency of 0-33 Hz and between the pre-SMA and S1-M1 at 0-18 Hz. Coherence between the SMA proper and M1 started to increase 0.9 sec before the movement onset and peaked 0.3 sec after the movement. There was no systematic difference within the SMA (SMA proper vs pre-SMA) or within the S1-M1, in terms of the time course as well as the peak value of coherence. The phase spectra revealed near-zero phase difference in 57% (20 of 35) of region pairs analyzed, and the remaining pairs showed inconsistent results. This increase of synchronization between multiple motor areas in the preparation and execution of voluntary movements may reflect the multiregional functional interactions in human motor behavior.

Effects of cryopreservation of pronuclear-stage rabbit zygotes on the morphological survival, blastocyst formation, and full-term development after DNA microinjection.

The objectives of this study were to examine the freezing sensitivity of pronuclear-stage rabbit zygotes and to produce transgenic rabbits using the cryopreserved zygotes. Zygotes were cryopreserved either by one of two vitrification protocols or by one of the two conventional freezing protocols. The morphological survival rates of zygotes subjected to two-step freezing in 1.5 M ethylene glycol and 0.1 M sucrose (74%) or to vitrification in 7.2 M ethylene glycol and 1.0 M sucrose (81%) were higher than those subjected to freezing in 1.5 M DMSO (46%) or to vitrification in a mixture of 2.0 M DMSO, 1.0 M acetamide, and 3.0 M propylene glycol (41%). But the in vitro development into blastocysts of zygotes cryopreserved by vitrification (17%) or to a lesser extent by freezing (52%) was impaired, when compared to that of fresh control zygotes (89%). Next, a fusion gene composed from bovine aS1-casein promoter and a human GH structural gene (2.8 kb) was microinjected into the pronucleus of rabbit zygotes frozen-thawed in ethylene glycol and sucrose. Then, the presence of exogenous DNA in the genome of newborn offspring was determined by PCR. The post-injection survival of frozen zygotes (97%) was the same as that of fresh control zygotes (96%). However, of 18 offspring derived from 414 frozen-thawed and DNA-injected zygotes, no transgenic rabbits were produced. Of 52 offspring derived from 403 DNA-injected fresh zygotes, 3 transgenic rabbits were found. Here we report the first rabbit offspring resulting from zygotes cryopreserved at the pronuclear-stage, although the cryopreservation procedure employed must be improved if zygotes are to be used for systematic production of transgenic rabbits.

Microbial desulfurization of alkylated dibenzothiophene and alkylated benzothiophene by recombinant Rhodococcus sp. strain T09.

The dibenzothiophene (DBT) desulfurizing operon, dsz, was introduced into various benzothiophene (BT)-desulfurizing bacteria using a Rhodococcus-E. coli shuttle vector. Of the tested recombinant bacteria, only those from Rhodococcus sp. strain T09 grew with both DBT and BT as the sole sulfur source. These recombinant cells desulfurized not only alkylated BTs, but also various alkylated DBTs, producing alkylated hydroxybiphenyls as the desulfurized products. Recombinant strain T09 also desulfurized alkylated DBT in an oil-water, two-phase resting-cell reaction. The dsz operon had the same desulfurizing activity when inserted into the vector in either orientation, indicating that the promoter region of the operon was functional in strain T09.

A comparative study on the integration of exogenous DNA into mouse, rat, rabbit, and pig genomes.

Transgenic mammals, from small laboratory rodents to domestic animals, have been successfully produced to date, but their production efficiency within or across species has been variable. This is probably due to the differences in the type of injected DNA and/or technical procedures employed in each laboratory, as well as the reproductive characteristics of the species. Here we report the direct comparison of the efficiencies of producing transgenic mice, rats, rabbits and pigs by one technician using a fusion gene composed of the bovine alpha S1-casein promoter and human growth hormone (hGH) gene. Before the fusion gene was injected into the zygotes, high magnitude centrifugation to visualize the pronuclei was necessary for all of the pig zygotes and one-third of the rabbit zygotes, but not for mouse and rat zygotes. Post-injection survival of the mouse zygotes (67.1%) was lower than those of the rat, rabbit and pig zygotes (89.6 to 100%). The volume change of the pronucleus following DNA injection was the lowest in mice (50% increase), moderate in rabbits (148% increase), and the most prominent in rats (238% increase). The data from only 1 pig zygote indicated a 22% increase in the pronucleus volume by DNA injection. The PCR analyses of the tail DNA of new born offspring indicated that 0.8% (4/493), 4.8% (22/463), 0.8% (3/367) and 0.9% (2/221) of the injected eggs in mice, rats, rabbits and pigs, respectively, developed into transgenic offspring. Some of the founder animals in all four species expressed the transgene in the mammary gland which was confirmed in hGH mRNA by RT-PCR and/or hGH peptide in Witch's milk with ELISA. These results suggest that the maximum volume of DNA solution injectable into the pronucleus is a possible factor explaining the species differences in the production of transgenic animals.

Comparison of source localization of interictal epileptic spike potentials in patients estimated by the dipole tracing method with the focus directly recorded by the depth electrodes.

The purpose of the study was to investigate the accuracy of location of equivalent current dipoles estimated by the dipole tracing method (DT) utilizing a realistic 3-shell (scalp-skull-brain) head model (SSB-DT). Three patients with intractable complex partial seizures, diagnosed as having typical temporal seizures were investigated. We recorded the interictal spike potentials with surface electrodes (International 10/20 system) and with intracerebral depth electrodes simultaneously. We compared the location of dipoles of the spikes estimated by the SSB-DT with the focus of the spikes determined by the recording from the depth electrodes. We found that the location of the dipoles estimated by SSB-DT corresponded to the location of the depth electrodes, which could record the epileptic spikes. This finding proved that SSB-DT is reliable and valid for estimating neural activity in deep locations such as the limbic system.

Improvement of desulfurization activity in Rhodococcus erythropolis KA2-5-1 by genetic engineering.

Rhodococcus erythropolis KA2-5-1 can desulfurize dibenzothiophene (DBT) into 2-hydroxybiphenyl. A cryptic plasmid, pRC4, which was derived from R. rhodochrous IFO3338, was combined with an Escherichia coli vector to construct an E. coli-Rhodococcus shuttle vector. The complete nucleotide sequence of 2582-bp pRC4 was analyzed. Based on the characteristics of its putative replication genes, pRC4 was assigned to the family of pAL5000-related replicons. The desulfurization gene cluster, dszABC, and the related reductase gene, dszD, cloned from KA2-5-1, were reintroduced into KA2-5-1 and efficiently expressed. The DBT desulfurization ability of the transformant carrying two dszABC clusters and one dszD on the vector was about 4-fold higher than that of the parent strain, and the transformant also showed improved desulfurization activity for light gas oil (LGO). Sulfur components in LGO before and after the reaction were analyzed with gas chromatography-atomic emission detection.

Kinetic analysis of microbial desulfurization of model and light gas oils containing multiple alkyl dibenzothiophenes.

The reaction mechanism of biodesulfurization was investigated using whole cells of Rhodococcus erythropolis KA2-5-1, which have the ability to convert dibenzothiophene (DBT) into 2-hydroxybiphenyl. The desulfurization patterns of alkyl DBTs were represented by the Michaeis-Menten equation. The values of rate constants, the limiting maximal velocity (Vmax) and Michaelis constant (Km), for desulfurization of alkyl DBTs were calculated. The relative desulfurization activities of various alkyl DBTs were reduced in proportion to the total carbon numbers of alkyl substituent groups. Alkyl DBTs that had a total of six carbons of alkyl substituent groups were not desulfurized. The type or position of alkyl substituent groups had little effect on desulfurization activity. The desulfurization activity of each alkyl DBT, when mixed together, was reduced. This phenomenon was caused by apparent competitive inhibition of substrates. Using the apparent competitive inhibition model, the desulfurization pattern of a multiple components system containing alkyl DBTs was elucidated. This model was also applicable for biodesulfurization of light gas oil.

A new control method of nonlinear systems based on impulse responses of universal learning networks.

A new control method of nonlinear dynamic systems is proposed based on the impulse responses of universal learning networks (ULNs), ULNs form a superset of neural networks. They consist of a number of interconnected nodes where the nodes may have any continuously differentiable nonlinear functions in them and each pair of nodes can be connected by multiple branches with arbitrary time delays. A generalized learning algorithm is derived for the ULNs, in which both the first order derivatives (gradients) and the higher order derivatives are incorporated. One of the distinguished features of the proposed control method is that the impulse response of the systems is considered as an extended part of the criterion function and it can be calculated by using the higher order derivatives of ULNs. By using the impulse response as the criterion function, nonlinear dynamics with not only quick response but also quick damping and small steady state error can be more easily obtained than the conventional nonlinear control systems with quadratic form criterion functions of state and control variables.

Photo-responsive retention behavior of azobenzene-modified cyclodextrin stationary phase in micro-HPLC.

An azobenzene-modified gamma-cyclodextrin stationary phase (Az gamma-CDSP) was prepared and its photo- and temperature-responses for the retention of perylene and pentacene were investigated using a mixture of methanol and water as the mobile phase in micro-HPLC. The retention of perylene slightly increased, whereas that of pentacene significantly decreased by UV light irradiation to Az gamma-CDSP. These retentions recovered upon irradiation with visible light. Both retentions decreased upon an increase in the column temperature. It was presumed that the trans-azobenzene moiety acts as a preventive cap for perylene and a spacing for pentacene in filling the CD cavity. An azobenzene-modified stationary phase changed its retention behavior with the column temperature and the light irradiation. An improvement in the micro-HPLC system and the optimization of the molecular structure of the photo-responsive stationary phase would provide selective retention control by the irradiation of light in micro-separation systems.

Improvement of generalization ability for identifying dynamical systems by using universal learning networks.

This paper studies how the generalization ability of models of dynamical systems can be improved by taking advantage of the second order derivatives of the outputs with respect to the external inputs. The proposed method can be regarded as a direct implementation of the well-known regularization technique using the higher order derivatives of the Universal Learning Networks (ULNs). ULNs consist of a number of interconnected nodes where the nodes may have any continuously differentiable nonlinear functions in them and each pair of nodes can be connected by multiple branches with arbitrary time delays. A generalized learning algorithm has been derived for the ULNs, in which both the first order derivatives (gradients) and the higher order derivatives are incorporated. First, the method for computing the second order derivatives of ULNs is discussed. Then, a new method for implementing the regularization term is presented. Finally, simulation studies on identification of a nonlinear dynamical system with noises are carried out to demonstrate the effectiveness of the proposed method. Simulation results show that the proposed method can improve the generalization ability of neural networks significantly, especially in terms that (1) the robust network can be obtained even when the branches of trained ULNs are destructed, and (2) the obtained performance does not depend on the initial parameter values.