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1.  Cost-effectiveness of adding rituximab to splenectomy and romiplostim for treating steroid-resistant idiopathic thrombocytopenic purpura in adults 
Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disease in which the platelet count falls to <100 × 109/L. Corticosteroids are recommended as the first-line treatment, splenectomy is recommended as the second-line treatment, and thrombopoietin receptor agonists (TPO-RAs) and rituximab are recommended as the third-line treatments for ITP in Japanese ITP treatment guidelines. However, in Japan, rituximab is not eligible for reimbursement for the treatment of ITP. The cost-effectiveness of ITP treatment has not been investigated in Japan. Therefore, in this study, the cost-effectiveness of adding rituximab treatment to the existing treatments indicated for ITP in Japan, namely splenectomy and the TPO-RA romiplostim, was investigated based on the scenario that rituximab is eligible for reimbursement in Japan as a treatment for ITP.
The efficacy endpoint was set as the number of years with a platelet count ≥30 × 109/L. The analysis was conducted from the healthcare payer’s perspective. If the first treatment is ineffective or relapse occurs, then the patient is given the following treatment. The analyzed treatment order consisted of three patterns: splenectomy-romiplostim (sequence 1), splenectomy-romiplostim-rituximab (sequence 2), and splenectomy-rituximab-romiplostim (sequence 3). A Markov model was built for ITP, and the analysis period was set as 2 years. The discount rate was an annual rate of 2%.
Sensitivity analyses of the efficacy of splenectomy, romiplostim, and rituximab; treatment cost; and romiplostim dose were performed.
The expected costs per patient over a 2-year period for sequences 1, 2, and 3 were USD 40,980, USD 39,822, and USD 33,551, respectively. The expected years with a platelet count ≥30 × 109/L for the three sequences were 1.75, 1.79, and 1.78 years, respectively. The sensitivity analyses illustrated that the results of the base case analysis were robust.
Adding rituximab to standard treatment for ITP (sequences 2–3) is less costly and marginally more effective than standard therapy in adults. According to the study results, if rituximab is reimbursed for the treatment of ITP in Japan, medical expenses are expected to decline.
PMCID: PMC4307915  PMID: 25609557
ITP; Splenectomy; Romiplostim; Rituximab; Cost-effectiveness
2.  Molecular Mechanisms of Hepatocellular Apoptosis Induced by Trovafloxacin-Tumor Necrosis Factor-alpha Interaction 
Toxicological Sciences  2013;137(1):91-101.
Idiosyncratic drug-induced liver injury (IDILI) continues to be a significant human health problem. IDILI is characterized as occurring in a minority of individuals exposed to a drug, yet it accounts for as much as 17% of all cases of acute liver failure. Despite these concerns, the mechanisms underlying IDILI remain unknown. Trovafloxacin (TVX), which causes IDILI in humans, also causes hepatocellular death in vitro when combined with tumor necrosis factor-alpha (TNF) treatment. However, the molecular mechanisms involved in this toxicity are not fully characterized. The purpose of this study was to identify mechanisms by which TVX and TNF interact to cause hepatocellular death, with a focus on a human hepatocyte cell line. TVX and TNF interacted to cause cytotoxicity in HepG2 cells at drug concentrations similar to those in people undergoing TVX therapy. TVX/TNF treatment caused apoptosis and DNA damage in HepG2 cells that depended on caspase activation. Prolonged activation of JNK occurred in TVX/TNF-induced cytotoxicity, and treatment with the JNK selective inhibitor SP600125 attenuated cytotoxicity. TVX/TNF cotreatment also caused cytotoxicity in isolated primary murine hepatocytes that was dependent on caspase activation. These results increase understanding of molecular signaling pathways involved in hepatocellular death caused by a drug with idiosyncratic liability in the presence of TNF.
PMCID: PMC3871929  PMID: 24097668
idiosyncratic drug-induced liver injury; hepatotoxicity; caspase; JNK; trovafloxacin.
3.  IL1RAPL1 knockout mice show spine density decrease, learning deficiency, hyperactivity and reduced anxiety-like behaviours 
Scientific Reports  2014;4:6613.
IL-1 receptor accessory protein-like 1 (IL1RAPL1) is responsible for nonsyndromic intellectual disability and is associated with autism. IL1RAPL1 mediates excitatory synapse formation through trans-synaptic interaction with PTPδ. Here, we showed that the spine density of cortical neurons was significantly reduced in IL1RAPL1 knockout mice. The spatial reference and working memories and remote fear memory were mildly impaired in IL1RAPL1 knockout mice. Furthermore, the behavioural flexibility was slightly reduced in the T-maze test. Interestingly, the performance of IL1RAPL1 knockout mice in the rotarod test was significantly better than that of wild-type mice. Moreover, IL1RAPL1 knockout mice consistently exhibited high locomotor activity in all the tasks examined. In addition, open-space and height anxiety-like behaviours were decreased in IL1RAPL1 knockout mice. These results suggest that IL1RAPL1 ablation resulted in spine density decrease and affected not only learning but also behavioural flexibility, locomotor activity and anxiety.
PMCID: PMC4196104  PMID: 25312502
4.  Expression, purification, crystallization and X-ray analysis of 3-quinuclidinone reductase from Agrobacterium tumefaciens  
The purification and crystallization of 3-quinuclidinone reductase from A. tumefaciens allowed the collection of a diffraction data set to 1.72 Å resolution.
(R)-3-Quinuclidinol is a useful chiral building block for the synthesis of various pharmaceuticals and can be produced from 3-quinuclidinone by asymmetric reduction. A novel 3-quinuclidinone reductase from Agrobacterium tumefaciens (AtQR) catalyzes the stereospecific reduction of 3-quinuclidinone to (R)-3-quinuclidinol with NADH as a cofactor. Recombinant AtQR was overexpressed in Escherichia coli, purified and crystallized with NADH using the sitting-drop vapour-diffusion method at 293 K. Crystals were obtained using a reservoir solution containing PEG 3350 as a precipitant. X-ray diffraction data were collected to 1.72 Å resolution on beamline BL-5A at the Photon Factory. The crystal belonged to space group P21, with unit-cell parameters a = 62.0, b = 126.4, c = 62.0 Å, β = 110.5°, and was suggested to contain four molecules in the asymmetric unit (V M = 2.08 Å3 Da−1).
PMCID: PMC3497986  PMID: 23027756
3-quinuclidinone reductase; Agrobacterium tumefaciens
5.  Transient receptor potential ankyrin 1 in spinal cord dorsal horn is involved in neuropathic pain in nerve root constriction rats 
Molecular Pain  2014;10(1):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
6.  Expression, purification, crystallization and preliminary X-ray analysis of a novel N-substituted branched-chain l-amino-acid dioxygenase from Burkholderia ambifaria AMMD 
Diffraction data were collected to a limiting resolution of 2.4 Å from a crystal of selenomethionyl-labelled SadA, an l-amino-acid dioxygenase.
Ferrous ion- and α-ketoglutarate-dependent dioxygenase from Burkholderia ambifaria AMMD (SadA) catalyzes the C3-hydroxylation of N-substituted branched-chain l-amino acids, especially N-succinyl-l-leucine, coupled to the conversion of α-ketoglutarate to succinate and CO2. SadA was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method at 293 K. Crystals of selenomethionine-substituted SadA were obtained using a reservoir solution containing PEG 3000 as the precipitant at pH 9.5 and diffracted X-rays to 2.4 Å resolution. The crystal belonged to space group P212121, with unit-cell parameters a = 49.3, b = 70.9, c = 148.2 Å. The calculated Matthews coefficient (V M = 2.1 Å3 Da−1, 41% solvent content) suggested that the crystal contains two molecules per asymmetric unit.
PMCID: PMC3433199  PMID: 22949196
C3-hydroxylation; dioxygenases; N-succinyl-l-leucine
7.  SIRT1 overexpression ameliorates a mouse model of SOD1-linked amyotrophic lateral sclerosis via HSF1/HSP70i chaperone system 
Molecular Brain  2014;7:62.
Dominant mutations in superoxide dismutase 1 (SOD1) cause degeneration of motor neurons in a subset of inherited amyotrophic lateral sclerosis (ALS). The pathogenetic process mediated by misfolded and/or aggregated mutant SOD1 polypeptides is hypothesized to be suppressed by protein refolding. This genetic study is aimed to test whether mutant SOD1-mediated ALS pathology recapitulated in mice could be alleviated by overexpressing a longevity-related deacetylase SIRT1 whose substrates include a transcription factor heat shock factor 1 (HSF1), the master regulator of the chaperone system.
We established a line of transgenic mice that chronically overexpress SIRT1 in the brain and spinal cord. While inducible HSP70 (HSP70i) was upregulated in the spinal cord of SIRT1 transgenic mice (PrP-Sirt1), no neurological and behavioral alterations were detected. To test hypothetical benefits of SIRT1 overexpression, we crossbred PrP-Sirt1 mice with two lines of ALS model mice: A high expression line that exhibits a severe phenotype (SOD1G93A-H) or a low expression line with a milder phenotype (SOD1G93A-L). The Sirt1 transgene conferred longer lifespan without altering the time of symptomatic onset in SOD1G93A-L. Biochemical analysis of the spinal cord revealed that SIRT1 induced HSP70i expression through deacetylation of HSF1 and that SOD1G93A-L/PrP-Sirt1 double transgenic mice contained less insoluble SOD1 than SOD1G93A-L mice. Parallel experiments showed that Sirt1 transgene could not rescue a more severe phenotype of SOD1G93A-H transgenic mice partly because their HSP70i level had peaked out.
The genetic supplementation of SIRT1 can ameliorate a mutant SOD1-linked ALS mouse model partly through the activation of the HSF1/HSP70i chaperone system. Future studies shall include testing potential benefits of pharmacological enhancement of the deacetylation activity of SIRT1 after the onset of the symptom.
PMCID: PMC4237944  PMID: 25167838
Sirtuin 1 (SIRT1); Cu/Zn-superoxide dismutase (SOD1); Heat shock factor 1 (HSF1); Heat shock protein (HSP); Amyotrophic lateral sclerosis (ALS); Systematic behavioral screening
8.  Expression, purification, crystallization and preliminary X-ray analysis of 4-hydroxy-3-methyl-2-­keto-pentanoate aldolase (asHPAL) from Arthrobacter simplex strain AKU 626 
asHPAL, a member of the HpaI/HpcH subfamily of class II aldolases, was expressed, purified and crystallized in the absence and presence of 2-ketobutyrate as one of its substrates using the sitting-drop vapour-diffusion method. asHPAL crystals grown without and with 2-ketobutyrate diffracted to 1.60 and 1.55 Å resolution, respectively.
4-Hydroxy-3-methyl-2-keto-pentanoate aldolase (asHPAL), an enzyme used in the synthesis of (2S,3R,4S)-4-hydroxyisoleucine, was crystallized in the absence and the presence of 2-ketobutyrate as one of its substrates by the sitting-drop vapour-diffusion method using PEG 400 as a precipitant. Crystals of asHPAL grown without and with 2-ketobutyrate diffracted to 1.60 and 1.55 Å resolution and belonged to space group C2, with unit-cell parameters a = 116.8, b = 88.2, c = 85.3 Å, β = 122.3° and a = 116.2, b = 88.1, c = 85.0 Å, β = 122.3°, respectively.
PMCID: PMC3412783  PMID: 22869132
4-hydroxy-3-methyl-2-keto-pentanoate aldolase; Arthrobacter simplex strain AKU 626; aldolases
9.  Dermatoscopy of folliculosebaceous cystic hamartoma 
A 29-year-old Japanese man presented to our institution with a nodule on his nose that had increased in size since childhood. Physical examination indicated the presence of an elastic, firm, pedunculated red nodule measuring 15 mm in size. Dermatoscopic examination of the nodule indicated a yellowish-white network, yellowish-orange dots/globules at its center, and a pinkish-white structureless peripheral area. Histopathological examination of an excisional biopsy specimen showed a dilated infundibulocystic structure with sebaceous lobules proliferating radially, surrounded by fibrous stroma. Moreover, mature adipocytes and small vessels were noted in the stroma. Based on these histopatho-logic findings, the patient was diagnosed with folliculosebaceous cystic hamartoma.
PMCID: PMC4131998  PMID: 25126458
folliculosebaceous cystic hamartoma; sebaceous trichofolliculoma; yellowish-white network; yellowish-orange dots/globules; dermatoscopy
10.  Phosphodiesterase III inhibitor promotes drainage of cerebrovascular β-amyloid 
Brain amyloidosis is a key feature of Alzheimer's disease (AD). It also incorporates cerebrovascular amyloid β (Aβ) in the form of cerebral amyloid angiopathy (CAA) involving neurovascular dysfunction. We have recently shown by retrospective analysis that patients with mild cognitive impairment receiving a vasoactive drug cilostazol, a selective inhibitor of phosphodiesterase (PDE) III, exhibit significantly reduced cognitive decline. Here, we tested whether cilostazol protects against the disruption of the neurovascular unit and facilitates the arterial pulsation-driven perivascular drainage of Aβ in AD/CAA.
We explored the expression of PDE III in postmortem human brain tissue followed by a series of experiments examining the effects of cilostazol on Aβ metabolism in transgenic mice (Tg-SwDI mice) as a model of cerebrovascular β-amyloidosis, as well as cultured neurons.
We established that PDE III is abnormally upregulated in cerebral blood vessels of AD and CAA subjects and closely correlates with vascular amyloid burden. Furthermore, we demonstrated that cilostazol treatment maintained cerebral hyperemic and vasodilative responses to hypercapnia and acetylcholine, suppressed degeneration of pericytes and vascular smooth muscle cells, promoted perivascular drainage of soluble fluorescent Aβ1-40, and rescued cognitive deficits in Tg-SwDI mice. Although cilostazol decreased endogenous Aβ production in cultured neurons, C-terminal fragment of amyloid precursor protein expression was not altered in cilostazol-treated Tg-SwDI mice.
The predominant action of cilostazol on Aβ metabolism is likely to facilitate Aβ clearance due to the sustained cerebrovascular function in vivo. Our findings mechanistically demonstrate that cilostazol is a promising therapeutic approach for AD and CAA.
PMCID: PMC4184555  PMID: 25356424
11.  Radiation Exposure and the Risk of Pediatric Thyroid Cancer 
It has been more than three years since the unprecedentedly massive earthquake and tsunami struck eastern Japan on March 11, 2011, and the large accident occurred at the Fukushima Daiichi Nuclear Power Plant. To investigate the influence of radiation exposure, thyroid ultrasonography has been provided preliminarily for 360,000 children who lived in Fukushima Prefecture at the time of the accident. As of September 2013, 59 children had been diagnosed with thyroid cancer by fine-needle aspiration cytology, and 34 children had been treated surgically and ultimately diagnosed with papillary thyroid cancer. Here, I would like to describe the characteristics of pediatric thyroid cancer and typical thyroid images obtained by ultrasonography.
PMCID: PMC4125599  PMID: 25110391
radiation exposure; pediatric thyroid cancer; ultrasonography; the accident at the Fukushima Daiichi Nuclear Power Plant
12.  Mechanisms for Interferon-α-Induced Depression and Neural Stem Cell Dysfunction 
Stem Cell Reports  2014;3(1):73-84.
New neurons generated by the neural stem cells (NSCs) in the adult hippocampus play an important role in emotional regulation and respond to the action of antidepressants. Depression is a common and serious side effect of interferon-α (IFN-α), which limits its use as an antiviral and antitumor drug. However, the mechanism(s) underlying IFN-induced depression are largely unknown. Using a comprehensive battery of behavioral tests, we found that mice subjected to IFN-α treatment exhibited a depression-like phenotype. IFN-α directly suppressed NSC proliferation, resulting in the reduced generation of new neurons. Brain-specific mouse knockout of the IFN-α receptor prevented IFN-α-induced depressive behavioral phenotypes and the inhibition of neurogenesis, suggesting that IFN-α suppresses hippocampal neurogenesis and induces depression via its receptor in the brain. These findings provide insight for understanding the neuropathology underlying IFN-α-induced depression and for developing new strategies for the prevention and treatment of IFN-α-induced depressive effects.
•IFN-α-treated mice show a depression-like phenotype in a behavioral test battery•IFN-α directly suppresses NSC proliferation in adult hippocampus•IFN-α suppresses neurogenesis and induced depression via its receptor in the brain
Depression is a common and serious side effect of interferon-α (IFN-α). However, the mechanisms underlying IFN-induced depression are largely unknown. Here, Sawamoto, Kaneko, and colleagues demonstrate that chronic IFN-α treatment caused a depression-like phenotype and directly suppressed neural stem cell proliferation in adult mice via brain IFN receptor. These findings provide new insight for understanding the neuropathology underlying IFN-induced depression.
PMCID: PMC4110771  PMID: 25068123
13.  Definite Differences between In Vitro Actin-Myosin Sliding and Muscle Contraction as Revealed Using Antibodies to Myosin Head 
PLoS ONE  2014;9(6):e93272.
Muscle contraction results from attachment-detachment cycles between myosin heads extending from myosin filaments and actin filaments. It is generally believed that a myosin head first attaches to actin, undergoes conformational changes to produce force and motion in muscle, and then detaches from actin. Despite extensive studies, the molecular mechanism of myosin head conformational changes still remains to be a matter for debate and speculation. The myosin head consists of catalytic (CAD), converter (CVD) and lever arm (LD) domains. To give information about the role of these domains in the myosin head performance, we have examined the effect of three site-directed antibodies to the myosin head on in vitro ATP-dependent actin-myosin sliding and Ca2+-activated contraction of muscle fibers. Antibody 1, attaching to junctional peptide between 50K and 20K heavy chain segments in the CAD, exhibited appreciable effects neither on in vitro actin-myosin sliding nor muscle fiber contraction. Since antibody 1 covers actin-binding sites of the CAD, one interpretation of this result is that rigor actin-myosin linkage is absent or at most a transient intermediate in physiological actin-myosin cycling. Antibody 2, attaching to reactive lysine residue in the CVD, showed a marked inhibitory effect on in vitro actin-myosin sliding without changing actin-activated myosin head (S1) ATPase activity, while it showed no appreciable effect on muscle contraction. Antibody 3, attaching to two peptides of regulatory light chains in the LD, had no significant effect on in vitro actin-myosin sliding, while it reduced force development in muscle fibers without changing MgATPase activity. The above definite differences in the effect of antibodies 2 and 3 between in vitro actin-myosin sliding and muscle contraction can be explained by difference in experimental conditions; in the former, myosin heads are randomly oriented on a glass surface, while in the latter myosin heads are regularly arranged within filament-lattice structures.
PMCID: PMC4053314  PMID: 24918754
14.  Enhanced stability of hippocampal place representation caused by reduced magnesium block of NMDA receptors in the dentate gyrus 
Molecular Brain  2014;7:44.
Voltage-dependent block of the NMDA receptor by Mg2+ is thought to be central to the unique involvement of this receptor in higher brain functions. However, the in vivo role of the Mg2+ block in the mammalian brain has not yet been investigated, because brain-wide loss of the Mg2+ block causes perinatal lethality. In this study, we used a brain-region specific knock-in mouse expressing an NMDA receptor that is defective for the Mg2+ block in order to test its role in neural information processing.
We devised a method to induce a single amino acid substitution (N595Q) in the GluN2A subunit of the NMDA receptor, specifically in the hippocampal dentate gyrus in mice. This mutation reduced the Mg2+ block at the medial perforant path–granule cell synapse and facilitated synaptic potentiation induced by high-frequency stimulation. The mutants had more stable hippocampal place fields in the CA1 than the controls did, and place representation showed lower sensitivity to visual differences. In addition, behavioral tests revealed that the mutants had a spatial working memory deficit.
These results suggest that the Mg2+ block in the dentate gyrus regulates hippocampal spatial information processing by attenuating activity-dependent synaptic potentiation in the dentate gyrus.
PMCID: PMC4073519  PMID: 24893573
Mg2+ block; NMDA receptor; Dentate gyrus; Place cell
15.  Population Based Cohort Study for Pediatric Infectious Diseases Research in Vietnam 
Tropical Medicine and Health  2014;42(2 Suppl):47-58.
A population-based cohort study on pediatric infectious diseases was established at Khanh Hoa Province, central Vietnam in 2006, to determine the etiology and risk factors for severe pediatric infectious diseases (SPID) such as acute respiratory infection (ARI), diarrhea and dengue which are the major causes of under 5 mortality. A population census survey was conducted in Nha-Trang and Ninh-Hoa to collect demographic, social-behavioral data and disease burden on SPID. The study site covered a population of 353,525 residing in 75,826 households with 24,781 children less than 5 years. Hospital databases from two hospitals covering the region were obtained. Linking the census and hospital databases, we were able to investigate on a variety of SPID such as environmental tobacco smoking exposure and increased risked of pediatric pneumonia hospitalization, population density, water supply and risk of dengue fever and animal livestock and risk of hospitalized diarrhea. To determine incidence, viral etiology and risk factors for pediatric ARI/pneumonia, we setup a population based prospective hospitalized Pediatric ARI surveillance at Khanh Hoa General Hospital, Nha-Trang in February 2007. The study has revealed RSV, rhinovirus and influenza A as major viral pathogens, role of multiple viral infection and its interaction with bacteria in the development of pneumonia. In addition, we are also conducting a birth cohort study to investigate the incidence of congenital infection and its impact on physical-neurological development, and role of host genetic polymorphism on SPID hospitalization in Vietnam. Population mobility, high cost of regular census update and low mortality are the challenges.
PMCID: PMC4204059  PMID: 25425951
Population based cohort; pediatric; infectious diseases; Vietnam
16.  Transcriptomic evidence for immaturity of the prefrontal cortex in patients with schizophrenia 
Molecular Brain  2014;7:41.
Schizophrenia, a severe psychiatric disorder, has a lifetime prevalence of 1%. The exact mechanisms underlying this disorder remain unknown, though theories abound. Recent studies suggest that particular cell types and biological processes in the schizophrenic cortex have a pseudo-immature status in which the molecular properties partially resemble those in the normal immature brain. However, genome-wide gene expression patterns in the brains of patients with schizophrenia and those of normal infants have not been directly compared. Here, we show that the gene expression patterns in the schizophrenic prefrontal cortex (PFC) resemble those in the juvenile PFC.
We conducted a gene expression meta-analysis in which, using microarray data derived from different studies, altered expression patterns in the dorsolateral PFC (DLFC) of patients with schizophrenia were compared with those in the DLFC of developing normal human brains, revealing a striking similarity. The results were replicated in a second DLFC data set and a medial PFC (MFC) data set. We also found that about half of the genes representing the transcriptomic immaturity of the schizophrenic PFC were developmentally regulated in fast-spiking interneurons, astrocytes, and oligodendrocytes. Furthermore, to test whether medications, which often confound the results of postmortem analyses, affect on the juvenile-like gene expressions in the schizophrenic PFC, we compared the gene expression patterns showing transcriptomic immaturity in the schizophrenic PFC with those in the PFC of rodents treated with antipsychotic drugs. The results showed no apparent similarities between the two conditions, suggesting that the juvenile-like gene expression patterns observed in the schizophrenic PFC could not be accounted for by medication effects. Moreover, the developing human PFC showed a gene expression pattern similar to that of the PFC of naive Schnurri-2 knockout mice, an animal model of schizophrenia with good face and construct validity. This result also supports the idea that the transcriptomic immaturity of the schizophrenic PFC is not due to medication effects.
Collectively, our results provide evidence that pseudo-immaturity of the PFC resembling juvenile PFC may be an endophenotype of schizophrenia.
PMCID: PMC4066280  PMID: 24886351
Schizophrenia; Transcriptome; Prefrontal cortex; Immaturity; Parvalbumin; Endophenotype
17.  Generation of Rhesus Macaque-Tropic HIV-1 Clones That Are Resistant to Major Anti-HIV-1 Restriction Factors 
Journal of Virology  2013;87(21):11447-11461.
Human immunodeficiency virus type 1 (HIV-1) replication in macaque cells is restricted mainly by antiviral cellular APOBEC3, TRIM5α/TRIM5CypA, and tetherin proteins. For basic and clinical HIV-1/AIDS studies, efforts to construct macaque-tropic HIV-1 (HIV-1mt) have been made by us and others. Although rhesus macaques are commonly and successfully used as infection models, no HIV-1 derivatives suitable for in vivo rhesus research are available to date. In this study, to obtain novel HIV-1mt clones that are resistant to major restriction factors, we altered Gag and Vpu of our best HIV-1mt clone described previously. First, by sequence- and structure-guided mutagenesis, three amino acid residues in Gag-capsid (CA) (M94L/R98S/G114Q) were found to be responsible for viral growth enhancement in a macaque cell line. Results of in vitro TRIM5α susceptibility testing of HIV-1mt carrying these substitutions correlated well with the increased viral replication potential in macaque peripheral blood mononuclear cells (PBMCs) with different TRIM5 alleles, suggesting that the three amino acids in HIV-1mt CA are involved in the interaction with TRIM5α. Second, we replaced the transmembrane domain of Vpu of this clone with the corresponding region of simian immunodeficiency virus SIVgsn166 Vpu. The resultant clone, MN4/LSDQgtu, was able to antagonize macaque but not human tetherin, and its Vpu effectively functioned during viral replication in a macaque cell line. Notably, MN4/LSDQgtu grew comparably to SIVmac239 and much better than any of our other HIV-1mt clones in rhesus macaque PBMCs. In sum, MN4/LSDQgtu is the first HIV-1 derivative that exhibits resistance to the major restriction factors in rhesus macaque cells.
PMCID: PMC3807366  PMID: 23966385
18.  Expression, purification, crystallization and preliminary X-ray analysis of carbonyl reductase S1 from Candida magnoliae  
Carbonyl reductase S1 from C. magnoliae was expressed, purified and crystallized. The crystals obtained diffracted X-rays to 1.90 Å resolution and belonged to space group P6122 or P6522.
The NADPH-dependent carbonyl reductase S1 from Candida magnoliae stereoselectively catalyzes the reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate (CHBE), which is a chiral compound valuable as a building block for pharmaceuticals. Carbonyl reductase S1 was expressed in Escherichia coli and purified by Ni-affinity, ion-exchange and size-exclusion chromatography. Crystals of carbonyl reductase S1 were obtained by the sitting-drop vapour-diffusion method using PEG 400 as a precipitant. X-ray diffraction data were collected to 1.90 Å resolution using a synchrotron-radiation source. The crystals belonged to space group P6122 or P6522, with unit-cell parameters a = b = 77.7, c = 307.5 Å. The asymmetric unit contained two molecules of the protein, with a solvent content of 44.2%.
PMCID: PMC3374508  PMID: 22691783
stereoselectivity; short-chain dehydrogenase/reductases; carbonyl reductase S1; Candida magnoliae
19.  Short-term mechanical stretch fails to differentiate human adipose-derived stem cells into cardiovascular cell phenotypes 
We and others have previously demonstrated that adipose-derived stem cells (ASCs) transplantation improve cardiac dysfunction post-myocardium infarction (MI) under hemodynamic stress in rats. The beneficial effects appear to be associated with pleiotropic factors due to a complex interplay between the transplanted ASCs and the microenvironment in the absence of cell transdifferentiation. In the present work, we tested the hypothesis that mechanical stretch per se could change human ASCs (hASCs) into cardiovascular cell phenotypes that might influence post-MI outcomes.
Human ASCs were obtained from patients undergoing liposuction procedures. These cells were stretched 12%, 1Hz up to 96 hours by using Flexercell 4000 system. Protein and gene expression were evaluated to identify cardiovascular cell markers. Culture medium was analyzed to determine cell releasing factors, and contraction potential was also evaluated.
Mechanical stretch, which is associated with extracellular signal-regulated kinase (ERK) phosphorylation, failed to induce the expression of cardiovascular cell markers in human ASCs, and mesenchymal cell surface markers (CD29; CD90) remained unchanged. hASCs and smooth muscle cells (SMCs) displayed comparable contraction ability. In addition, these cells demonstrated a profound ability to secrete an array of cytokines. These two properties of human ASCs were not influenced by mechanical stretch.
Altogether, our findings demonstrate that hASCs secrete an array of cytokines and display contraction ability even in the absence of induction of cardiovascular cell markers or the loss of mesenchymal surface markers when exposed to mechanical stretch. These properties may contribute to beneficial post-MI cardiovascular outcomes and deserve to be further explored under the controlled influence of other microenvironment components associated with myocardial infarction, such as tissue hypoxia.
PMCID: PMC4012171  PMID: 24885410
Adipose-derived stem cell; Cell therapy; Stretch; Cell differentiation
20.  Aldehyde dehydrogenase, Ald4p, is a major component of mitochondrial fluorescent inclusion bodies in the yeast Saccharomyces cerevisiae 
Biology Open  2014;3(5):387-396.
When Saccharomyces cerevisiae strain 3626 was cultured to the stationary phase in a medium that contained glucose, needle-like structures that emitted autofluorescence were observed in almost all cells by fluorescence microscopy under UV excitation. The needle-like structures completely overlapped with the profile of straight elongated mitochondria. Therefore, these structures were designated as mitochondrial fluorescent inclusion bodies (MFIBs). The MFIB-enriched mitochondrial fractions were successfully isolated and 2D-gel electrophoresis revealed that a protein of 54 kDa was only highly concentrated in the fractions. Determination of the N-terminal amino acid sequence of the 54-kDa protein identified it as a mitochondrial aldehyde dehydrogenase, Ald4p. Immunofluorescence microscopy showed that anti-Ald4p antibody specifically stained MFIBs. Freeze-substitution electron microscopy demonstrated that cells that retained MFIBs had electron-dense filamentous structures with a diameter of 10 nm in straight elongated mitochondria. Immunoelectron microscopy showed that Ald4p was localized to the electron-dense filamentous structures in mitochondria. These results together showed that a major component of MFIBs is Ald4p. In addition, we demonstrate that MFIBs are common features that appear in mitochondria of many species of yeast.
PMCID: PMC4021361  PMID: 24771619
Yeast; Mitochondria; Inclusion body; Aldehyde dehydrogenase; Ald4p
21.  Comprehensive behavioral study of mGluR3 knockout mice: implication in schizophrenia related endophenotypes 
Molecular Brain  2014;7:31.
We previously performed systematic association studies of glutamate receptor gene family members with schizophrenia, and found positive associations of polymorphisms in the GRM3 (a gene of metabotropic glutamate receptor 3: mGluR3) with the disorder. Physiological roles of GRM3 in brain functions and its functional roles in the pathogenesis of schizophrenia remain to be resolved.
We generated mGluR3 knockout (KO) mice and conducted comprehensive behavioral analyses. KO mice showed hyperactivity in the open field, light/dark transition, and 24-hour home cage monitoring tests, impaired reference memory for stressful events in the Porsolt forced swim test, impaired contextual memory in cued and contextual fear conditioning test, and impaired working memory in the T-Maze forced alternation task test. Hyperactivity and impaired working memory are known as endophenotypes of schizophrenia. We examined long-term synaptic plasticity by assessing long-term potentiation (LTP) in the CA1 region in the hippocampi of KO and wild-type (WT) mice. We observed no differences in the amplitude of LTP between the two genotypes, suggesting that mGluR3 is not essential for LTP in the CA1 region of the mouse hippocampus. As hyperactivity is typically associated with increased dopaminergic transmission, we performed in vivo microdialysis measurements of extracellular dopamine in the nucleus accumbens of KO and WT mice. We observed enhancements in the methamphetamine (MAP)-induced release of dopamine in KO mice.
These results demonstrate that a disturbance in the glutamate-dopamine interaction may be involved in the pathophysiology of schizophrenia-like behavior, such as hyperactivity in mGluR3 KO mice.
PMCID: PMC4021612  PMID: 24758191
Metabotropic glutamate receptors; Grm3; Knockout mice; Working memory; Reference memory; Contextual memory; Hyperactivity; LTP; Microdialysis; Schizophrenia
22.  Targeted deletion of the C-terminus of the mouse adenomatous polyposis coli tumor suppressor results in neurologic phenotypes related to schizophrenia 
Molecular Brain  2014;7:21.
Loss of adenomatous polyposis coli (APC) gene function results in constitutive activation of the canonical Wnt pathway and represents the main initiating and rate-limiting event in colorectal tumorigenesis. APC is likely to participate in a wide spectrum of biological functions via its different functional domains and is abundantly expressed in the brain as well as in peripheral tissues. However, the neuronal function of APC is poorly understood. To investigate the functional role of Apc in the central nervous system, we analyzed the neurological phenotypes of Apc1638T/1638T mice, which carry a targeted deletion of the 3′ terminal third of Apc that does not affect Wnt signaling.
A series of behavioral tests revealed a working memory deficit, increased locomotor activity, reduced anxiety-related behavior, and mildly decreased social interaction in Apc1638T/1638T mice. Apc1638T/1638T mice showed abnormal morphology of the dendritic spines and impaired long-term potentiation of synaptic transmission in the hippocampal CA1 region. Moreover, Apc1638T/1638T mice showed abnormal dopamine and serotonin distribution in the brain. Some of these behavioral and neuronal phenotypes are related to symptoms and endophenotypes of schizophrenia.
Our results demonstrate that the C-terminus of the Apc tumor suppressor plays a critical role in cognitive and neuropsychiatric functioning. This finding suggests a potential functional link between the C-terminus of APC and pathologies of the central nervous system.
PMCID: PMC3986642  PMID: 24678719
Adenomatous polyposis coli (Apc); Apc1638T/1638T mice; Hippocampus; Working memory; Locomotor activity; Schizophrenia
23.  Perspectives in the use of tannins as alternative to antimicrobial growth promoter factors in poultry 
Antibiotics have been included in the formulation of feed for livestock production for more than 40 years as a strategy to improve feed conversion rates and to reduce costs. The use of antimicrobials as growth-promoting factors (AGP) in sub-therapeutic doses for long periods is particularly favorable for the selection of antimicrobial resistant microorganisms. In the last years, global concern about development of antimicrobial resistance and transference of resistance genes from animal to human strains has been rising. Removal of AGP from animal diets involves tremendous pressure on the livestock and poultry farmers, one of the main consequences being a substantial increase in the incidence of infectious diseases with the associated increase in the use of antibiotics for therapy, and concomitantly, economic cost. Therefore, alternatives to AGP are urgently needed. The challenge is to implement new alternatives without affecting the production performances of livestock and avoiding the increase of antimicrobial resistant microorganisms. Plant extracts and purified derived substances are showing promising results for animal nutrition, either from their efficacy as well as from an economical point of view. Tannins are plant derived compounds that are being successfully used as additives in poultry feed to control diseases and to improve animal performance. Successful use of any of these extracts as feed additives must ensure a product of consistent quality in enough quantity to fulfill the actual requirements of the poultry industry. Chestnut (hydrolysable) and Quebracho (condensed) tannins are probably the most readily available commercial products that are covering those needs. The present report intends to analyze the available data supporting their use.
PMCID: PMC3973907  PMID: 24723916
tannins; antibiotics; poultry; growth promoting factors; necrotic enteritis; plant extracts; animal health
24.  Sexually Dimorphic Body Color Is Regulated by Sex-Specific Expression of Yellow Gene in Ponerine Ant, Diacamma Sp 
PLoS ONE  2014;9(3):e92875.
Most hymenopteran species exhibit conspicuous sexual dimorphism due to ecological differences between the sexes. As hymenopteran genomes, under the haplodiploid genetic system, exhibit quantitative differences between sexes while remaining qualitatively identical, sexual phenotypes are assumed to be expressed through sex-specific gene usage. In the present study, the molecular basis for expression of sexual dimorphism in a queenless ant, Diacamma sp., which exhibits a distinct color dimorphism, was examined. Worker females of the species appear bluish-black, while winged males exhibit a yellowish-brown body color. Initially, observations of the pigmentation processes during pupal development revealed that black pigmentation was present in female pupae but not in males, suggesting that sex-specific melanin synthesis was responsible for the observed color dimorphism. Therefore, five orthologs of the genes involved in the insect melanin synthesis (yellow, ebony, tan, pale and dopa decarboxylase) were subcloned and their spatiotemporal expression patterns were examined using real-time quantitative RT-PCR. Of the genes examined, yellow, which plays a role in black melanin synthesis in insects, was expressed at higher levels in females than in males throughout the entire body during the pupal stage. RNA interference of yellow was then carried out in order to determine the gene function, and produced females with a more yellowish, brighter body color similar to that of males. It was concluded that transcriptional regulation of yellow was responsible for the sexual color dimorphism observed in this species.
PMCID: PMC3965500  PMID: 24667821
25.  Splenic Stromal Cells from Aged Mice Produce Higher Levels of IL-6 Compared to Young Mice 
Mediators of Inflammation  2014;2014:826987.
Inflamm-aging indicates the chronic inflammatory state resulting from increased secretion of proinflammatory cytokines and mediators such as IL-6 in the elderly. Our principle objective was to identify cell types that were affected with aging concerning IL-6 secretion in the murine model. We compared IL-6 production in spleen cells from both young and aged mice and isolated several types of cells from spleen and investigated IL-6 mRNA expression and protein production. IL-6 protein productions in cultured stromal cells from aged mice spleen were significantly high compared to young mice upon LPS stimulation. IL-6 mRNA expression level of freshly isolated stromal cells from aged mice was high compared to young mice. Furthermore, stromal cells of aged mice highly expressed IL-6 mRNA after LPS injection in vivo. These results suggest that stromal cells play a role in producing IL-6 in aged mice and imply that they contribute to the chronic inflammatory condition in the elderly.
PMCID: PMC3960767  PMID: 24729663

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