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1.  Bidirectional role of IL-6 signal in pathogenesis of lung fibrosis 
Respiratory Research  2015;16(1):99.
Various signals are known to participate in the pathogenesis of lung fibrosis. Our aim was to determine which signal is predominantly mobilized in the early inflammatory phase and thereafter modulates the development of lung fibrosis.
Mice received a single dose of 3 mg/kg body weight of bleomycin (BLM) and were sacrificed at designated days post-instillation (dpi). Lung homogenates and sections from mice in the early inflammatory phase were subjected to phospho-protein array analysis and immunofluorescence studies, respectively. Bronchoalveolar lavage fluid (BALF) from mice was subjected to an enzyme-linked immunosorbent assay (EIA) for interleukin (IL)-6 and evaluation of infiltrated cell populations. The effects of endogenous and exogenous IL-6 on the BLM-induced apoptotic signal in A549 cells and type 2 pneumocytes were elucidated. In addition, the effect of IL-6-neutralizing antibody on BLM-induced lung injury was evaluated.
Phospho-protein array revealed that BLM induced phosphorylation of molecules downstream of the IL-6 receptor such as Stat3 and Akt in the lung at 3 dpi. At 3 dpi, immunofluorescence studies showed that signals of phospho-Stat3 and -Akt were localized in type 2 pneumocytes, and that BLM-induced IL-6-like immunoreactivity was predominantly observed in type 2 pneumocytes. Activation of caspases in BLM-treated A549 cells and type 2 pneumocytes was augmented by application of IL-6-neutralizing antibody, a PI3K inhibitor or a Stat3 inhibitor. EIA revealed that BLM-induced IL-6 in BALF was biphasic, with the first increase from 0.5 to 3 dpi followed by the second increase from 8 to 10 dpi. Blockade of the first increase of IL-6 by IL-6-neutralizing antibody enhanced apoptosis of type 2 pneumocytes and neutrophilic infiltration and markedly accelerated fibrosis in the lung. In contrast, blockade of the second increase of IL-6 by IL-6-neutralizing antibody ameliorated lung fibrosis.
The present study demonstrated that IL-6 could play a bidirectional role in the pathogenesis of lung fibrosis. In particular, upregulation of IL-6 at the early inflammatory stage of BLM-injured lung has antifibrotic activity through regulating the cell fate of type 2 pneumocytes in an autocrine/paracrine manner.
Electronic supplementary material
The online version of this article (doi:10.1186/s12931-015-0261-z) contains supplementary material, which is available to authorized users.
PMCID: PMC4546032  PMID: 26289430
2.  Long-term complete remission in a patient with intravascular large B-cell lymphoma with central nervous system involvement 
OncoTargets and therapy  2014;7:2133-2136.
This report describes a patient with intravascular large B-cell lymphoma (IVLBCL) with central nervous system involvement at the time of diagnosis who achieved complete remission for over 5 years in response to therapy. The patient, a 71 year-old woman, was previously healthy with the exception of taking verapamil for paroxysmal supraventricular tachycardia. She had presented with pyrexia and gradually progressive anemia. Brain magnetic resonance imaging revealed an infarct-like lesion in the pons, although no paralysis was observed. She was diagnosed with IVLBCL on the basis of random skin biopsy. After eight cycles of rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone therapy, abnormal laboratory data had normalized, and no pontine lesion was evident on magnetic resonance imaging without receiving any intrathecal chemotherapy. IVLBCL is associated with poor prognosis, particularly in patients with central nervous system involvement. Early initiation of rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone therapy and drug interactions between anticancer agents and verapamil as a p-glycoprotein inhibitor were considered the possible reasons for favorable outcome in the present case.
PMCID: PMC4242899  PMID: 25429230
intravascular large B-cell lymphoma; random skin biopsy; CNS involvement; rituximab; verapamil; blood–brain barrier
3.  Necrotizing Keratitis Caused by Acyclovir-Resistant Herpes Simplex Virus 
Case Reports in Ophthalmology  2014;5(3):325-328.
We report a case of necrotizing keratitis caused by acyclovir (ACV)-resistant herpes simplex virus (HSV) with a clinical appearance similar to a previous fungal keratitis infection.
Observational case report.
Penetrating keratoplasty was performed in the left eye with a history of herpetic keratitis that resolved with periodic treatment with ACV ointment and a topical steroid. The left eye was painful and red with an abscess and corneal erosion in the peripheral donor cornea. Examination of the scraped corneal epithelium by light microscopy and culturing identified Candida albicans; polymerase chain reaction (PCR) was negative for human herpes viruses. After antifungal treatment, the ocular pain gradually decreased and the lesions slowly improved but recurred with a similar clinical appearance. A second light microscopy examination and cultures were negative for pathogens including C. albicans. PCR was positive for HSV-1 DNA; treatment with 3% topical ACV ointment was unsuccessful. A third examination showed only HSV-1 DNA. Despite antiviral ACV ointment, no clinical improvement occurred based on the HSV DNA copy numbers, which were the same before and after treatment, indicating a possible ACV-resistant strain. When topical trifluorothymidine was substituted for ACV, clinical improvement occurred and the HSV DNA copy numbers decreased.
Necrotizing keratitis induced by ACV-resistant HSV occurred independently after fungal keratitis, with a similar clinical appearance in this case, making diagnosis and treatment difficult. Monitoring the HSV DNA load by real-time PCR could be useful for refractory cases even with atypical clinical appearances.
PMCID: PMC4241636  PMID: 25473399
Herpes simplex virus; Acyclovir-resistant herpes simplex virus; Necrotizing keratitis; Fungal infection; Real-time polymerase chain reaction
4.  Transient receptor potential ankyrin 1 in spinal cord dorsal horn is involved in neuropathic pain in nerve root constriction rats 
Molecular Pain  2014;10:58.
Lumbar radicular pain is categorized as a type of neuropathic pain, but its pathophysiological mechanisms are not fully understood. The substantia gelatinosa (SG) in the spinal cord dorsal horn receives primary afferent inputs and is considered to be a therapeutic target for treating neuropathic pain. In vivo patch-clamp recording is a useful procedure for analyzing the functional properties of synaptic transmission in SG neurons. Transient receptor potential ankyrin 1 (TRPA1) has been widely identified in the central and peripheral nervous systems, such as in the peripheral nociceptor, dorsal root ganglion, and spinal cord dorsal horn and is involved in synaptic transmission of pain. However, its functional role and mechanism of pain transmission in the spinal cord dorsal horn are not well understood. The purpose of this study was to use in vivo patch-clamp analysis to examine changes in the excitatory synaptic transmission of SG neurons treated with TRPA1 antagonist and to clarify the potential role of TRPA1 in the rat spinal cord dorsal horn.
The rats with root constriction (RC) showed mechanical hypersensitivity, hyperalgesia, and thermal hyperalgesia. In addition, pin pricks elicited pain-related behavior even in the sham and naïve rats. These pain-related behaviors were significantly attenuated by intrathecal injection of a TRPA1 antagonist. The degrees of intrathecal injection efficacy were equivalent among the 3 groups (RC, sham, and naïve groups). In an electrophysiological study, the frequencies and amplitudes of excitatory postsynaptic currents (EPSCs) were significantly increased in the RC rats compared with those in the sham and naïve rats. Spontaneous EPSCs and evoked-EPSCs by non-noxious and noxious stimuli were significantly decreased by TRPA1 antagonist. As in the behavioral study, there were no statistically significant differences among the 3 groups.
These data showed that the TRPA1 antagonist had an inhibitory effect on mechanical hypersensitivity and hyperalgesia as well as on physiological pain transmission in the spinal cord dorsal horn. This suggests that TRPA1 is consistently involved in excitatory synaptic transmission even in the physiological state and has a role in coordinating pain transmission.
PMCID: PMC4163170  PMID: 25192906
Transient receptor potential ankyrin 1 (TRPA1); Neuropathic pain; Root constriction; Spinal cord; Substantia gelatinosa neuron; In vivo patch-clamp
5.  Evaluation of side effects of radiofrequency capacitive hyperthermia with magnetite on the blood vessel walls of tumor metastatic lesion surrounding the abdominal large vessels: an agar phantom study 
Vascular Cell  2014;6:15.
Magnetite used in an 8-MHz radiofrequency (RF) capacitive heating device can increase the temperature of a specific site up to 45°C. When treating a metastatic lesion around large abdominal vessels via hyperthermia with magnetite, heating-induced adverse effects on these vessels need to be considered. Therefore, this study examined hyperthermia-induced damage to blood vessel walls in vitro.
A large agar phantom with a circulatory system consisting of a swine artery and vein connected to a peristaltic pump was prepared. The blood vessels were placed on the magnetite-containing agar piece. Heating was continued for 30 min at 45°C. After heating, a histological study for injury to the blood vessels was performed.
The inner membrane temperature did not reach 45°C due to the cooling effect of the blood flow. In the heated vessels, vascular wall collagen degenerated and smooth muscle cells were narrowed; however, no serious changes were noted in the vascular endothelial cells or vascular wall elastic fibers. The heated vessel wall was not severely damaged; this was attributed to cooling by the blood flow.
Our findings indicate that RF capacitive heating therapy with magnetite may be used for metastatic lesions without injuring the surrounding large abdominal vessels.
PMCID: PMC4128615  PMID: 25114787
Magnetic cationic liposome; Interstitial hyperthermia injury; Large vessels; Vascular cell damage; Agar phantom study
6.  Nonstructural Protein σ1s Mediates Reovirus-Induced Cell Cycle Arrest and Apoptosis 
Journal of Virology  2013;87(23):12967-12979.
Reovirus nonstructural protein σ1s is implicated in cell cycle arrest at the G2/M boundary and induction of apoptosis. However, the contribution of σ1s to these effects in an otherwise isogenic viral background has not been defined. To evaluate the role of σ1s in cell cycle arrest and apoptosis, we used reverse genetics to generate a σ1s-null reovirus. Following infection with wild-type virus, we observed an increase in the percentage of cells in G2/M, whereas the proportion of cells in G2/M following infection with the σ1s-null mutant was unaffected. Similarly, we found that the wild-type virus induced substantially greater levels of apoptosis than the σ1s-null mutant. These data indicate that σ1s is required for both reovirus-induced cell cycle arrest and apoptosis. To define sequences in σ1s that mediate these effects, we engineered viruses encoding C-terminal σ1s truncations by introducing stop codons in the σ1s open reading frame. We also generated viruses in which charged residues near the σ1s amino terminus were replaced individually or as a cluster with nonpolar residues. Analysis of these mutants revealed that amino acids 1 to 59 and the amino-terminal basic cluster are required for induction of both cell cycle arrest and apoptosis. Remarkably, viruses that fail to induce cell cycle arrest and apoptosis also are attenuated in vivo. Thus, identical sequences in σ1s are required for reovirus-induced cell cycle arrest, apoptosis, and pathogenesis. Collectively, these findings provide evidence that the σ1s-mediated properties are genetically linked and suggest that these effects are mechanistically related.
PMCID: PMC3838159  PMID: 24067959
7.  Various pAQU plasmids possibly contribute to disseminate tetracycline resistance gene tet(M) among marine bacterial community 
Emergence of antibiotic-resistant bacteria in the aquaculture environment is a significant problem for disease control of cultured fish as well as in human public health. Conjugative mobile genetic elements (MGEs) are involved in dissemination of antibiotic resistance genes (ARGs) among marine bacteria. In the present study, we first designed a PCR targeting traI gene encoding essential relaxase for conjugation. By this new PCR, we demonstrated that five of 83 strains isolated from a coastal aquaculture site had traI-positive MGEs. While one of the five strains that belonged to Shewanella sp. was shown to have an integrative conjugative element of the SXT/R391 family (ICEVchMex-like), the MGEs of the other four strains of Vibrio spp. were shown to have the backbone structure similar to that of previously described in pAQU1. The backbone structure shared by the pAQU1-like plasmids in the four strains corresponded to a ~100-kbp highly conserved region required for replication, partition and conjugative transfer, suggesting that these plasmids constituted “pAQU group.” The pAQU group plasmids were shown to be capable of conjugative transfer of tet(M) and other ARGs from the Vibrio strains to E. coli. The pAQU group plasmid in one of the examined strains was designated as pAQU2, and its complete nucleotide sequence was determined and compared with that of pAQU1. The results revealed that pAQU2 contained fewer ARGs than pAQU1 did, and most of the ARGs in both of these plasmids were located in the similar region where multiple transposases were found, suggesting that the ARGs were introduced by several events of DNA transposition into an ancestral plasmid followed by drug selection in the aquaculture site. The results of the present study indicate that the “pAQU group” plasmids may play an important role in dissemination of ARGs in the marine environment.
PMCID: PMC4026752  PMID: 24860553
pAQU group; pAQU2; transferable plasmid; tet(M); antimicrobial resistance genes; SXT/R391 ICEs; aquaculture; traI
8.  Imported Case of Acute Respiratory Tract Infection Associated with a Member of Species Nelson Bay Orthoreovirus 
PLoS ONE  2014;9(3):e92777.
A Japanese man suffered from acute respiratory tract infection after returning to Japan from Bali, Indonesia in 2007. Miyazaki-Bali/2007, a strain of the species of Nelson Bay orthoreovirus, was isolated from the patient's throat swab using Vero cells, in which syncytium formation was observed. This is the sixth report describing a patient with respiratory tract infection caused by an orthoreovirus classified to the species of Nelson Bay orthoreovirus. Given the possibility that all of the patients were infected in Malaysia and Indonesia, prospective surveillance on orthoreovirus infections should be carried out in Southeast Asia. Furthermore, contact surveillance study suggests that the risk of human-to-human infection of the species of Nelson Bay orthoreovirus would seem to be low.
PMCID: PMC3965453  PMID: 24667794
9.  Hyaluromycin, a New Hyaluronidase Inhibitor of Polyketide Origin from Marine Streptomyces sp. 
Marine Drugs  2014;12(1):491-507.
Hyaluromycin (1), a new member of the rubromycin family of antibiotics, was isolated from the culture extract of a marine-derived Streptomyces sp. as a HAase inhibitor on the basis of HAase activity screening. The structure of 1 was elucidated through the interpretation of NMR data for the compound and its 3″-O-methyl derivative in combination with an incorporation experiment with [1,2-13C2]acetate. The compound’s absolute configuration was determined by the comparison of its circular dichroism (CD) spectrum with those of other rubromycins. Hyaluromycin (1) consists of a γ-rubromycin core structure possessing a 2-amino-3-hydroxycyclopent-2-enone (C5N) unit as an amide substituent of the carboxyl function; both structural units have been reported only from actinomycetes. Hyaluromycin (1) displayed approximately 25-fold more potent hyaluronidase inhibitory activity against hyaluronidase than did glycyrrhizin, a known inhibitor of plant origin.
PMCID: PMC3917283  PMID: 24451191
rubromycin; hyaluronidase inhibitor; marine actinomycetes; Streptomyces; 2-amino-3-hydroxycyclopent-2-enone
10.  Construction and Characterization of an Infectious Molecular Clone of Koala Retrovirus 
Journal of Virology  2013;87(9):5081-5088.
Koala retrovirus (KoRV) is a gammaretrovirus that is currently endogenizing into koalas. Studies on KoRV infection have been hampered by the lack of a replication-competent molecular clone. In this study, we constructed an infectious molecular clone, termed plasmid pKoRV522, of a KoRV isolate (strain Aki) from a koala reared in a Japanese zoo. The virus KoRV522, derived from pKoRV522, grew efficiently in human embryonic kidney (HEK293T) cells, attaining 106 focus-forming units/ml. Several mutations in the Gag (L domain) and Env regions reported to be involved in reduction in viral infection/production in vitro are found in pKoRV522, yet KoRV522 replicated well, suggesting that any effects of these mutations are limited. Indeed, a reporter virus pseudotyped with pKoRV522 Env was found to infect human, feline, and mink cell lines efficiently. Analyses of KoRV L-domain mutants showed that an additional PPXY sequence, PPPY, in Gag plays a critical role in KoRV budding. Altogether, our results demonstrate the construction and characterization of the first infectious molecular clone of KoRV. The infectious clone reported here will be useful for elucidating the mechanism of endogenization of the virus in koalas and screening for antiretroviral drugs for KoRV-infected koalas.
PMCID: PMC3624308  PMID: 23427161
11.  Macrophage Migration Inhibitory Factor and Stearoyl-CoA Desaturase 1: Potential Prognostic Markers for Soft Tissue Sarcomas Based on Bioinformatics Analyses 
PLoS ONE  2013;8(10):e78250.
The diagnosis and treatment of soft tissue sarcomas (STSs) has been particularly difficult, because STSs are a group of highly heterogeneous tumors in terms of histopathology, histological grade, and primary site. Recent advances in genome technologies have provided an excellent opportunity to determine the complete biological characteristics of neoplastic tissues, resulting in improved diagnosis, treatment selection, and investigation of therapeutic targets. We had previously developed a novel bioinformatics method for marker gene selection and applied this method to gene expression data from STS patients. This previous analysis revealed that the extracted gene combination of macrophage migration inhibitory factor (MIF) and stearoyl-CoA desaturase 1 (SCD1) is an effective diagnostic marker to discriminate between subtypes of STSs with highly different outcomes. In the present study, we hypothesize that the combination of MIF and SCD1 is also a prognostic marker for the overall outcome of STSs. To prove this hypothesis, we first analyzed microarray data from 88 STS patients and their outcomes. Our results show that the survival rates for MIF- and SCD1-positive groups were lower than those for negative groups, and the p values of the log-rank test are 0.0146 and 0.00606, respectively. In addition, survival rates are more significantly different (p = 0.000116) between groups that are double-positive and double-negative for MIF and SCD1. Furthermore, in vitro cell growth inhibition experiments by MIF and SCD1 inhibitors support the hypothesis. These results suggest that the gene set is useful as a prognostic marker associated with tumor progression.
PMCID: PMC3805525  PMID: 24167613
12.  Transileocolic Vein Obliteration for Bleeding Rectal Varices with Portal Thrombus 
We report a case of rectal varices treated successfully with transileocolic vein obliteration (TIO). A 70-year-old man was admitted to our hospital for evaluation of fresh bloody stools in January 2011. Emergent colonoscopy revealed fresh blood in the rectum and tortuous rectal varices. Three-dimensional computed tomography was used as a non-invasive method for the identification of rectal varices and thrombus in the extrahepatic portal vein. Angiography demonstrated that rectal varices were supplied with backward blood flow by the inferior mesenteric vein. Transileocolic variceal obliteration was performed using coils and 5% ethanolamine oleate with iopamidol. Complete hemostasis was achieved without complications. We conclude that TIO is a safe and effective hemostatic measure for ruptured rectal varices with portal thrombus.
PMCID: PMC3617969  PMID: 23626507
Transileocolic vein obliteration; Rectal varices; Portal thrombus
13.  Melanoma-Targeted Chemothermotherapy and In Situ Peptide Immunotherapy through HSP Production by Using Melanogenesis Substrate, NPrCAP, and Magnetite Nanoparticles 
Journal of Skin Cancer  2013;2013:742925.
Exploitation of biological properties unique to cancer cells may provide a novel approach to overcome difficult challenges to the treatment of advanced melanoma. In order to develop melanoma-targeted chemothermoimmunotherapy, a melanogenesis substrate, N-propionyl-4-S-cysteaminylphenol (NPrCAP), sulfur-amine analogue of tyrosine, was conjugated with magnetite nanoparticles. NPrCAP was exploited from melanogenesis substrates, which are expected to be selectively incorporated into melanoma cells and produce highly reactive free radicals through reacting with tyrosinase, resulting in chemotherapeutic and immunotherapeutic effects by oxidative stress and apoptotic cell death. Magnetite nanoparticles were conjugated with NPrCAP to introduce thermotherapeutic and immunotherapeutic effects through nonapoptotic cell death and generation of heat shock protein (HSP) upon exposure to alternating magnetic field (AMF). During these therapeutic processes, NPrCAP was also expected to provide melanoma-targeted drug delivery system.
PMCID: PMC3595688  PMID: 23533767
14.  Meta-Analyses of Microarrays of Arabidopsis asymmetric leaves1 (as1), as2 and Their Modifying Mutants Reveal a Critical Role for the ETT Pathway in Stabilization of Adaxial–Abaxial Patterning and Cell Division During Leaf Development 
Plant and Cell Physiology  2013;54(3):418-431.
It is necessary to use algorithms to analyze gene expression data from DNA microarrays, such as in clustering and machine learning. Previously, we developed the knowledge-based fuzzy adaptive resonance theory (KB-FuzzyART), a clustering algorithm suitable for analyzing gene expression data, to find clues for identifying gene networks. Leaf primordia form around the shoot apical meristem (SAM), which consists of indeterminate stem cells. Upon initiation of leaf development, adaxial–abaxial patterning is crucial for lateral expansion, via cellular proliferation, and the formation of flat symmetric leaves. Many regulatory genes that specify such patterning have been identified. Analysis by the KB-FuzzyART and subsequent molecular and genetic analyses previously showed that ASYMMETRIC LEAVES1 (AS1) and AS2 repress the expression of some abaxial-determinant genes, such as AUXIN RESPONSE FACTOR3 (ARF3)/ETTIN (ETT) and ARF4, which are responsible for defects in leaf adaxial–abaxial polarity in as1 and as2. In the present study, genetic analysis revealed that ARF3/ETT and ARF4 were regulated by modifier genes, BOBBER1 (BOB1) and ELONGATA3 (ELO3), together with AS1–AS2. We analyzed expression arrays with as2 elo3 and as2 bob1, and extracted genes downstream of ARF3/ETT by using KB-FuzzyART and molecular analyses. The results showed that expression of Kip-related protein (KRP) (for inhibitors of cyclin-dependent protein kinases) and Isopentenyltransferase (IPT) (for biosynthesis of cytokinin) genes were controlled by AS1–AS2 through ARF3/ETT and ARF4 functions, which suggests that the AS1–AS2–ETT pathway plays a critical role in controlling the cell division cycle and the biosynthesis of cytokinin around SAM to stabilize leaf development in Arabidopsis thaliana.
PMCID: PMC3589830  PMID: 23396601
ASYMMETRIC LEAVES2 (AS2); AUXIN RESPONSE FACTOR3/ETTIN; CDK inhibitors; Cytokinin; Shoot apical meristem
15.  Use of 5-Cyano-2,3-Ditolyl-Tetrazolium Chloride Staining as an Indicator of Biocidal Activity in a Rapid Assay for Anti-Acanthamoeba Agents 
Journal of Clinical Microbiology  2012;50(5):1606-1612.
The usefulness of 5-cyano-2,3-ditolyl-tetrazolium chloride (CTC) staining to determine the respiratory activity of Acanthamoeba was evaluated in this study. Acanthamoeba trophozoites and cysts have a red fluorescence after staining with CTC. To determine the effectiveness of CTC staining as a CTC biocidal assay for Acanthamoeba, the trophozoites and cysts of Acanthamoeba castellanii (ATCC 5037) were treated with serial concentrations of disinfectant solutions, namely, polyhexamethylene biguanide (PHMB) and commercial soft contact lens (SCL) disinfectant solutions. The treated Acanthamoeba organisms were stained with CTC, and their respiratory activity was determined by the intensity of fluorescence in a fluorescence microplate reader. The survival rates of the same samples were determined by a culture-dependent biocidal assay using the Spearman-Karber method. Our results showed that the respiratory activities determined by the CTC biocidal assay and the survival rates determined by the culture-dependent biocidal assay for Acanthamoeba trophozoites and cysts decreased in a dose-dependent way after PHMB treatments, and the results were significantly correlated (r = 0.83 and P < 0.01 for trophozoites; r = 0.60 and P < 0.01 for cysts; Spearman rank correlation test). The respiratory activities in the trophozoites and cysts treated with SCL disinfectant solutions were significantly correlated with the survival rate (r = 0.70 and P < 0.01 for trophozoites; r = 0.64 and P < 0.01 for cysts; Spearman rank correlation test). The significant correlation of the results indicated that the CTC biocidal assay can be used as an alternative method to a culture-dependent biocidal assay. The CTC biocidal assay is a rapid and simple method to test the effectiveness of disinfectant solutions against Acanthamoeba trophozoites and cysts.
PMCID: PMC3347101  PMID: 22337974
16.  Reverse Genetics for Mammalian Reovirus 
Methods (San Diego, Calif.)  2011;55(2):109-113.
Mammalian orthoreoviruses (reoviruses) are highly tractable models for studies of viral replication and pathogenesis. The versatility of reovirus as an experimental model has been enhanced by development of a plasmid-based reverse genetics system. Infectious reovirus can be recovered from cells transfected with plasmids encoding cDNAs of each reovirus gene segment using a strategy that does not require helper virus and is independent of selection. In this system, transcription of each gene segment is driven by bacteriophage T7 RNA polymerase, which can be supplied transiently by recombinant vaccinia virus (rDIs-T7pol) or by cells that constitutively express the enzyme. Reverse genetics systems have been developed for two prototype reovirus strains, type 1 Lang (T1L) and type 3 Dearing (T3D). Each reovirus cDNA was encoded on an independent plasmid for the first-generation rescue system. The efficiency of virus recovery was enhanced in a second-generation system by combining the cDNAs for multiple reovirus gene segments onto single plasmids to reduce the number of plasmids from 10 to 4. The reduction in plasmid number and the use of baby hamster kidney cells that express T7 RNA polymerase increased the efficiency of viral rescue, reduced the incubation time required to recover infectious virus, and eliminated potential biosafety concerns associated with the use of recombinant vaccinia virus. Reovirus reverse genetics has been used to introduce mutations into viral capsid and nonstructural components to study viral protein-structure activity relationships and can be exploited to engineer recombinant reoviruses for vaccine and oncolytic applications.
PMCID: PMC3208765  PMID: 21798351
reovirus; reverse genetics; dsRNA; T7 RNA polymerase; reassortment
17.  Does Norepinephrine Influence Pain Behavior Mediated by Dorsal Root Ganglia?: A Pilot Study 
Postganglionic neurons in the sympathetic nervous system reportedly are involved in lumbar radicular pain and release norepinephrine (NE), a neurotransmitter. Increased numbers of sympathetic nerve fibers have been found in dorsal root ganglion (DRG) neurons in a root constriction model. Whether this is a reasonable model for pain, however, is unclear
We asked whether: (1) painful behaviors occurred in the root constriction model; (2) NE enhanced the excitability of DRG neurons in the root constriction model; and (3) which adrenoceptors were related to the mediation of the NE effects.
The L5 root was sutured proximal to the DRG as the root constriction model. Behavioral tests were performed until 28 days after surgery. At 10 to 14 days after the root constriction, DRG neurons were quickly excised and digested with collagenase for electrophysiologic studies. Action potentials were recorded from single DRG neurons using a whole-cell patch clamp technique. NE (10 μmol/L) was directly applied to the DRG neurons. The adrenergic sensitivity was examined in combination with antagonists.
The rats with root constriction exhibited painful behavior. NE increased the excitability of DRG neurons in the root constriction model. The effects of NE were inhibited by pretreatment with an α-antagonist and α2-antagonist but not an α1-antagonist.
Our observations suggest NE plays an important role in generating lumbar radicular pain mainly via α2-adrenoceptors.
Clinical Relevance
An α2-antagonist may be an appropriate agent for trials to treat lumbar radicular pain.
PMCID: PMC3148377  PMID: 21312078
18.  The Reovirus σ1s Protein Is a Determinant of Hematogenous but Not Neural Virus Dissemination in Mice ▿ 
Journal of Virology  2011;85(22):11781-11790.
Nonstructural protein σ1s is a critical determinant of hematogenous dissemination by type 1 reoviruses, which reach the central nervous system (CNS) by a strictly blood-borne route. However, it is not known whether σ1s contributes to neuropathogenesis of type 3 reoviruses, which disseminate by both vascular and neural pathways. Using isogenic type 3 viruses that vary only in σ1s expression, we observed that mice survived at a higher frequency following hind-limb inoculation with σ1s-null virus than when inoculated with wild-type virus. This finding suggests that σ1s is essential for reovirus virulence when inoculated at a site that requires systemic spread to cause disease. Wild-type and σ1s-null viruses produced comparable titers in the spinal cord, suggesting that σ1s is dispensable for invasion of the CNS. Although the two viruses ultimately achieved similar peak titers in the brain, loads of wild-type virus were substantially greater than those of the σ1s-null mutant at early times after inoculation. In contrast, wild-type virus produced substantially higher titers than the σ1s-null virus in peripheral organs to which reovirus spreads via the blood, including the heart, intestine, liver, and spleen. Concordantly, viral titers in the blood were higher following infection with wild-type virus than following infection with the σ1s-null mutant. These results suggest that differences in viral brain titers at early time points postinfection are due to limited virus delivery to the brain by hematogenous pathways. Transection of the sciatic nerve prior to hind-limb inoculation diminished viral spread to the spinal cord. However, wild-type virus retained the capacity to disseminate to the brain following sciatic nerve transection, indicating that wild-type reovirus can spread to the brain by the blood. Together, these results indicate that σ1s is not required for reovirus spread by neural mechanisms. Instead, σ1s mediates hematogenous dissemination within the infected host, which is required for full reovirus neurovirulence.
PMCID: PMC3209282  PMID: 21917967
19.  Involvement of stem cell factor and c-kit in corneal wound healing in mice 
Molecular Vision  2012;18:1505-1515.
To study the roles played by stem cell factor (SCF) and SCF receptor c-kit in wound healing of corneal epithelial cells.
A 2 mm corneal epithelial wound was made in control (WBB6F1+/+), SCF (Sl/Sld)-, and c-kit (W/Wv) mutant mice, and the speed of wound healing, 5-bromo-2’-deoxyuridine (BrdU) incorporation, and scanning electron microscopic (SEM) morphology of the corneas were examined. The incorporation of BrdU and the degree of cell attachment in cultured mouse corneal epithelial cells (MCECs) isolated from WBB6F1+/+, Sl/Sld, and W/Wv mice were examined. Cultured immortalized human corneal epithelial cells (HCECs) were examined by a cell attachment assay after their exposure to anti-SCF antibodies, tyrosine kinase inhibitor (genistein), and competitive Arg-Gly-Asp (RGD) peptide, as well as on cultures treated with extracellular matrix.
The speed of corneal wound healing was slower in Sl/Sld and W/Wv mice than in controls (p<0.01) and the speed of healing in Sl/Sld mice recovered after topical application of SCF (8 ng/ml). No significant difference was found in the BrdU incorporation assay either in vivo or in vitro. Loosened epithelial cells were detected at wound margins in W/Wv mice by SEM. The cell attachment rate was increased by 157% in cells from WBB6F1+/+ and 252% in Sl/Sld MCECs by recombinant mouse SCF; however, no significant difference was found in W/Wv MCECs. Anti-SCF antibodies (Ab), genistein, and RGD peptide reduced the percentage of attached HCECs. Anti-SCF Ab inhibited the attachment of HCECs on fibronectin, laminin, or type IV collagen coated dishes.
These findings indicate that the SCF/c-kit system may play a role in corneal wound healing through epithelial cell attachment.
PMCID: PMC3381705  PMID: 22736941
20.  Crystal Structure of Reovirus Attachment Protein σ1 in Complex with Sialylated Oligosaccharides 
PLoS Pathogens  2011;7(8):e1002166.
Many viruses attach to target cells by binding to cell-surface glycans. To gain a better understanding of strategies used by viruses to engage carbohydrate receptors, we determined the crystal structures of reovirus attachment protein σ1 in complex with α-2,3-sialyllactose, α-2,6-sialyllactose, and α-2,8-di-siallylactose. All three oligosaccharides terminate in sialic acid, which serves as a receptor for the reovirus serotype studied here. The overall structure of σ1 resembles an elongated, filamentous trimer. It contains a globular head featuring a compact β-barrel, and a fibrous extension formed by seven repeating units of a triple β-spiral that is interrupted near its midpoint by a short α -helical coiled coil. The carbohydrate-binding site is located between β-spiral repeats two and three, distal from the head. In all three complexes, the terminal sialic acid forms almost all of the contacts with σ1 in an identical manner, while the remaining components of the oligosaccharides make little or no contacts. We used this structural information to guide mutagenesis studies to identify residues in σ1 that functionally engage sialic acid by assessing hemagglutination capacity and growth in murine erythroleukemia cells, which require sialic acid binding for productive infection. Our studies using σ1 mutant viruses reveal that residues 198, 202, 203, 204, and 205 are required for functional binding to sialic acid by reovirus. These findings provide insight into mechanisms of reovirus attachment to cell-surface glycans and contribute to an understanding of carbohydrate binding by viruses. They also establish a filamentous, trimeric carbohydrate-binding module that could potentially be used to endow other trimeric proteins with carbohydrate-binding properties.
Author Summary
Human reoviruses bind first with low affinity to a carbohydrate receptor that brings the virus in close proximity to the host cell. This interaction then facilitates high-affinity binding to a second receptor, the tight junction component junctional adhesion molecule-A (JAM-A). While all human reoviruses bind JAM-A, they differ in carbohydrate receptor specificity, and this difference may influence the distinct disease patterns of reovirus serotypes. We present here the structure of the attachment protein of type 3 reovirus in complex with carbohydrates that naturally occur on human cells. Our results show that the protein forms an elongated trimer, with the carbohydrate binding site being located close to the midpoint of the molecule in a fiber-like region. Our findings provide insights into mechanisms of reovirus attachment to cell-surface glycans and contribute to an understanding of carbohydrate binding by viruses. They also establish a filamentous, trimeric carbohydrate-binding module that could potentially be used to introduce carbohydrate-binding properties into other trimeric proteins.
PMCID: PMC3150272  PMID: 21829363
21.  An Improved Reverse Genetics System for Mammalian Orthoreoviruses 
Virology  2009;398(2):194-200.
Mammalian orthoreoviruses (reoviruses) are highly useful models for studies of double-stranded RNA virus replication and pathogenesis. We previously developed a strategy to recover prototype reovirus strain T3D from cloned cDNAs transfected into murine L929 fibroblast cells. Here, we report the development of a second-generation reovirus reverse genetics system featuring several major improvements: (1) the capacity to rescue prototype reovirus strain T1L, (2) reduction of required plasmids from ten to four, and (3) isolation of recombinant viruses following transfection of baby hamster kidney cells engineered to express bacteriophage T7 RNA polymerase. The efficiency of virus rescue using the 4-plasmid strategy was substantially increased in comparison to the original 10-plasmid system. We observed full compatibility of T1L and T3D rescue vectors when intermixed to produce a panel of T1L × T3D monoreassortant viruses. Improvements to the reovirus reverse genetics system enhance its applicability for studies of reovirus biology and clinical use.
PMCID: PMC2823833  PMID: 20042210
reverse genetics; reovirus; dsRNA; T7 RNA polymerase; reassortment
22.  Observation of the esophagus, pharynx and lingual root by gastrointestinal endoscopy with a percutaneous retrograde approach 
AIM: To evaluate the efficacy of retrograde observation of the esophagus, pharynx, larynx and lingual root.
METHODS: With the beagle dog under anesthesia, the anterior wall of the stomach was fixed on the abdominal wall in a similar way to percutaneous endoscopic gastrostomy. The gastrointestinal scope was inserted via a 12 mm laparoscopic port for subsequent retrograde observation from stomach to the oral cavity.
RESULTS: With this technique, direct observation of gastric cardia was possible without restriction. The cervical esophagus was dilated well, also allowing clear observation of the hypopharyngo-esophageal junction. If the tongue was manually pulled out forward, observation of the lingual root was possible.
CONCLUSION: This procedure is easy and effective for pre-treatment evaluation of the feasibility of endoscopic resection in cases of superficial carcinoma of head and neck.
PMCID: PMC2999146  PMID: 21160628
Mesopharynx; Lingual root; Percutaneous endoscopic gastrostomy; Gastrointestinal endoscopy; Retrograde observation
23.  Identification of Functional Domains in Reovirus Replication Proteins μNS and μ2▿  
Journal of Virology  2009;83(7):2892-2906.
Mammalian reoviruses are nonenveloped particles containing a genome of 10 double-stranded RNA (dsRNA) gene segments. Reovirus replication occurs within viral inclusions, which are specialized nonmembranous cytoplasmic organelles formed by viral nonstructural and structural proteins. Although these structures serve as sites for several major events in the reovirus life cycle, including dsRNA synthesis, gene segment assortment, and genome encapsidation, biochemical mechanisms of virion morphogenesis within inclusions have not been elucidated because much remains unknown about inclusion anatomy and functional organization. To better understand how inclusions support viral replication, we have used RNA interference (RNAi) and reverse genetics to define functional domains in two inclusion-associated proteins, μNS and μ2, which are interacting partners essential for inclusion development and viral replication. Removal of μNS N-terminal sequences required for association with μ2 or another μNS-binding protein, σNS, prevented the capacity of μNS to support viral replication without affecting inclusion formation, indicating that μNS-μ2 and μNS-σNS interactions are necessary for inclusion function but not establishment. In contrast, introduction of changes into the μNS C-terminal region, including sequences that form a putative oligomerization domain, precluded inclusion formation as well as viral replication. Mutational analysis of μ2 revealed a critical dependence of viral replication on an intact nucleotide/RNA triphosphatase domain and an N-terminal cluster of basic amino acid residues conforming to a nuclear localization motif. Another domain in μ2 governs the capacity of viral inclusions to affiliate with microtubules and thereby modulates inclusion morphology, either globular or filamentous. However, viral variants altered in inclusion morphology displayed equivalent replication efficiency. These studies reveal a modular functional organization of inclusion proteins μNS and μ2, define the importance of specific amino acid sequences and motifs in these proteins for viral replication, and demonstrate the utility of complementary RNAi-based and reverse genetic approaches for studies of reovirus replication proteins.
PMCID: PMC2655549  PMID: 19176625
24.  Reovirus μ2 Protein Inhibits Interferon Signaling through a Novel Mechanism Involving Nuclear Accumulation of Interferon Regulatory Factor 9▿  
Journal of Virology  2008;83(5):2178-2187.
The secreted cytokine alpha/beta interferon (IFN-α/β) binds its receptor to activate the Jak-STAT signal transduction pathway, leading to formation of the heterotrimeric IFN-stimulated gene factor 3 (ISGF3) transcription complex for induction of IFN-stimulated genes (ISGs) and establishment of an antiviral state. Many viruses have evolved countermeasures to inhibit the IFN pathway, thereby subverting the innate antiviral response. Here, we demonstrate that the mildly myocarditic reovirus type 1 Lang (T1L), but not the nonmyocarditic reovirus type 3 Dearing, represses IFN induction of a subset of ISGs and that this repressor function segregates with the T1L M1 gene. Concordantly, the T1L M1 gene product, μ2, dramatically inhibits IFN-β-induced reporter gene expression. Surprisingly, T1L infection does not degrade components of the ISGF3 complex or interfere with STAT1 or STAT2 nuclear translocation as has been observed for other viruses. Instead, infection with T1L or reassortant or recombinant viruses containing the T1L M1 gene results in accumulation of interferon regulatory factor 9 (IRF9) in the nucleus. This effect has not been previously described for any virus and suggests that μ2 modulates IRF9 interactions with STATs for both ISGF3 function and nuclear export. The M1 gene is a determinant of virus strain-specific differences in the IFN response, which are linked to virus strain-specific differences in induction of murine myocarditis. We find that virus-induced myocarditis is associated with repression of IFN function, providing new insights into the pathophysiology of this disease. Together, these data provide the first report of an increase in IRF9 nuclear accumulation associated with viral subversion of the IFN response and couple virus strain-specific differences in IFN antagonism to the pathogenesis of viral myocarditis.
PMCID: PMC2643726  PMID: 19109390
25.  Detectability of Regional Lung Ventilation with Flat-panel Detector-based Dynamic Radiography 
Journal of Digital Imaging  2007;21(1):109-120.
This study was performed to investigate the ability of breathing chest radiography using flat-panel detector (FPD) to quantify relative local ventilation. Dynamic chest radiographs during respiration were obtained using a modified FPD system. Imaging was performed in three different positions, ie, standing and right and left decubitus positions, to change the distribution of local ventilation. We measured the average pixel value in the local lung area. Subsequently, the interframe differences, as well as difference values between maximum inspiratory and expiratory phases, were calculated. The results were visualized as images in the form of a color display to show more or less x-ray translucency. Temporal changes and spatial distribution of the results were then compared to lung physiology. In the results, the average pixel value in each lung was associated with respiratory phase. In all positions, respiratory changes of pixel value in the lower area were greater than those in the upper area (P < 0.01), which was the same tendency as the regional differences in ventilation determined by respiratory physiology. In addition, in the decubitus position, it was observed that areas with large respiratory changes in pixel value moved up in the vertical direction during expiration, which was considered to be airway closure. In conclusion, breathing chest radiography using FPD was shown to be capable of quantifying relative ventilation in local lung area and detecting regional differences in ventilation and timing of airway closure. This method is expected to be useful as a new diagnostic imaging modality for evaluating relative local ventilation.
PMCID: PMC3043825  PMID: 17356803
Digital imaging; functional imaging; computer analysis; chest radiographs; flat-panel detector; FPD; ventilation

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