DSP-6745, a novel 5-hydroxytryptamine modulator with rapid antidepressant, anxiolytic, antipsychotic and procognitive effects.

BACKGROUND: Current treatment of major depressive disorder is facing challenges, including a low remission rate, late onset of efficacy, and worsening severity due to comorbid symptoms such as psychosis and cognitive dysfunction. Serotonin (5-HT) neurotransmission is involved in a wide variety of psychiatric diseases and its potential as a drug target continues to attract attention. OBJECTIVES: The present study elucidates the effects of a novel 5-HT modulator, DSP-6745, on depression and its comorbid symptoms. RESULTS: In vitro radioligand binding and functional assays showed that DSP-6745 is a potent inhibitor of 5-HT transporter and 5-HT2A, 5-HT2C, and 5-HT7 receptors. In vivo, DSP-6745 (6.4 and 19.1 mg/kg as free base, p.o.) increased the release of not only 5-HT, norepinephrine, and dopamine, but also glutamate in the medial prefrontal cortex. The results of in vivo mouse phenotypic screening by SmartCube® suggested that DSP-6745 has a behavioral signature combined with antidepressant-, anxiolytic-, and antipsychotic-like signals. A single oral dose of DSP-6745 (6.4 and 19.1 mg/kg) showed rapid antidepressant-like efficacy in the rat forced swim test, even at 24 h post-dosing, and anxiolytic activity in the rat social interaction test. Moreover, DSP-6745 (12.7 mg/kg, p.o.) led to an improvement in the apomorphine-induced prepulse inhibition deficit in rats. In the marmoset object retrieval with detour task, which is used to assess cognitive functions such as attention and behavioral inhibition, DSP-6745 (7.8 mg/kg, p.o.) enhanced cognition. CONCLUSIONS: These data suggest that DSP-6745 is a multimodal 5-HT receptor antagonist and a 5-HT transporter inhibitor and has the potential to be a rapid acting antidepressant with efficacies in mitigating the comorbid symptoms of depression.

A Case of Odontoid Osteomyelitis.

Odontoid osteomyelitis is a rare infectious disease that manifests as fever and posterior neck pain, while crowned dens syndrome is a relatively common inflammatory disorder with similar signs and symptoms. We describe the case of a 90-year-old woman presenting with fever, posterior neck pain, throat pain, and headache. Crowned dens syndrome was initially diagnosed based on the clinical picture and calcification around the odontoid process on cervical spine CT. However, the diagnosis was revised to odontoid osteomyelitis following the detection of Staphylococcus aureus in blood cultures that were performed due to the presence of headache. Infectious complications included spinal epidural abscess extending to the hypoglossal canal and osteomyelitis spreading to the clivus. Nonetheless, the patient achieved complete recovery after 13 weeks of antimicrobial therapy. No reports of odontoid osteomyelitis with calcification around the odontoid process have been reported. This case underscores the importance of avoiding a hasty diagnosis of crowned dens syndrome when calcification around the odontoid process is observed in patients presenting with fever and posterior neck pain. It is crucial to perform a thorough medical history review and physical examination to exclude other conditions. In cases where infection is suspected, blood cultures and cervical spine MRI are essential to investigate odontoid osteomyelitis and other complications.

[A Case of Small Intestinal GIST Associated with Neurofibromatosis Type 1].

A 41-year-old male, with a history of neurofibromatosis type 1(NF1)was referred for further evaluation of positive fecal occult blood test. Abdominal contrast-enhanced CT incidentally showed a mass lesion with early darkening in the small intestine. It was suspected to be a small intestinal arteriovenous malformation, and surgery was performed. The tumor in the small intestine was resected under laparoscopic assistance. The histopathological diagnosis was gastrointestinal stromal tumors associated with NF1, which are usually located in the small intestine and relatively favorable prognosis.

[A Case of Combined Single Incisional Laparoscopic and Endoscopic Approaches to Neoplasia with Non-Exposure Technique(CLEAN-NET)for Gastric GIST].

An 80-year-old female patient visited our hospital with scrutinizing acid reflex after meals. Esophagogastroendoscopy (EGD)and computed tomography(CT)showed a 25 mm submucosal tumor of the stomach in the lesser curvature of the middle stomach. A pathological examination using endoscopic ultrasound-guided fine needle aspiration(EUS-FNA) revealed gastrointestinal stromal tumor(GIST). The combination of laparoscopic and endoscopic approaches to neoplasia with the non-exposure technique(CLEAN-NET)was undertaken via a 2.5 cm single umbilical incision. The tumor was identified by EGD, and the scheduled dissected line was confirmed both from the mucosal and serosa side with an endoscope and laparoscope, respectively. The serosal and muscular layers were incised with an electric knife from outside the gastric wall; hypertonic saline solution was locally injected to swell the submucosal layer; partial gastric resection was performed with an automatic suture device without capsule damage. The operation time was 61 minutes, and there was blood less. The patient was discharged 6 days after the operation without complications. Histopathological findings showed GIST, 16× 14 mm, c-kit(+), CD34(+). The tumor was very low risk, as per the modified-Fletcher classification. Currently, 5 months after the operation, follow-up is underway without recurrence.

Performance Evaluation of a Voice-Based Depression Assessment System Considering the Number and Type of Input Utterances.

It is empirically known that mood changes affect facial expressions and voices. In this study, the authors have focused on the voice to develop a method for estimating depression in individuals from their voices. A short input voice is ideal for applying the proposed method to a wide range of applications. Therefore, we evaluated this method using multiple input utterances while assuming a unit utterance input. The experimental results revealed that depressive states could be estimated with sufficient accuracy using the smallest number of utterances when positive utterances were included in three to four input utterances.

Risk of Seasonal Influenza by Occupation in a Railway Company in a Metropolitan Area of Japan during Three Influenza Seasons.

Station staff may be at high risk for influenza due to high frequency contact with other people. We examined the risk of influenza by occupational group in a railway company. A retrospective observational study was conducted among employees at a branch office of a railway company in eastern Japan, located in a metropolitan area, for 2012/13, 2013/14, and 2014/15 influenza seasons. The study population included employees who had received influenza vaccination for the season in question and the previous season. Outcome was defined as self-reported influenza illness during the respective season, identified through the vaccine screening questionnaire in the following season. Study participants included employees whose outcome information could be obtained. Standardized morbidity ratios (SMRs) by occupational group (station staff, engineers, train crew) for each season were calculated. For 2012/13, 2013/14, and 2014/15 seasons, attack rates were 4.7% (19/403), 5.2% (21/407), and 7.8% (31/397), respectively. Among the participants, SMRs of station staff were lower in the 2012/13 (SMR = 57; 95% confidence intervals [CI] = 18-133) and 2014/15 (SMR = 75; 95%CI = 36-138) seasons and similar to other groups in the 2013/14 season. Enhanced countermeasures, regardless of occupational group, may be effective in preventing the spread of influenza infection.

Inflammation-induced endothelial cell-derived extracellular vesicles modulate the cellular status of pericytes.

Emerging lines of evidence have shown that extracellular vesicles (EVs) mediate cell-to-cell communication by exporting encapsulated materials, such as microRNAs (miRNAs), to target cells. Endothelial cell-derived EVs (E-EVs) are upregulated in circulating blood in different pathological conditions; however, the characteristics and the role of these E-EVs are not yet well understood. In vitro studies were conducted to determine the role of inflammation-induced E-EVs in the cell-to-cell communication between vascular endothelial cells and pericytes/vSMCs. Stimulation with inflammatory cytokines and endotoxin immediately induced release of shedding type E-EVs from the vascular endothelial cells, and flow cytometry showed that the induction was dose dependent. MiRNA array analyses revealed that group of miRNAs were specifically increased in the inflammation-induced E-EVs. E-EVs added to the culture media of cerebrovascular pericytes were incorporated into the cells. The E-EV-supplemented cells showed highly induced mRNA and protein expression of VEGF-B, which was assumed to be a downstream target of the miRNA that was increased within the E-EVs after inflammatory stimulation. The results suggest that E-EVs mediate inflammation-induced endothelial cell-pericyte/vSMC communication, and the miRNAs encapsulated within the E-EVs may play a role in regulating target cell function. E-EVs may be new therapeutic targets for the treatment of inflammatory diseases.

Expression of annexin-A1 and galectin-1 anti-inflammatory proteins and mRNA in chronic gastritis and gastric cancer.

OBJECTIVE: The anti-inflammatory proteins annexin-A1 and galectin-1 have been associated with tumor progression. This scenario prompted us to investigate the relationship between the gene and protein expression of annexin-A1 (ANXA1/AnxA1) and galectin-1 (LGALS1/Gal-1) in an inflammatory gastric lesion as chronic gastritis (CG) and gastric adenocarcinoma (GA) and its association with H. pylori infection. METHODS: We analyzed 40 samples of CG, 20 of GA, and 10 of normal mucosa (C) by the quantitative real-time PCR (qPCR) technique and the immunohistochemistry assay. RESULTS: High ANXA1 mRNA expression levels were observed in 90% (36/40) of CG cases (mean relative quantification RQ = 4.26 ± 2.03) and in 80% (16/20) of GA cases (mean RQ = 4.38 ± 4.77). However, LGALS1 mRNA levels were high (mean RQ = 2.44 ± 3.26) in 60% (12/20) of the GA cases, while low expression was found in CG (mean RQ = 0.43 ± 3.13; P < 0.01). Normal mucosa showed modest immunoreactivity in stroma but not in epithelium, while stroma and epithelium displayed an intense immunostaining in CG and GA for both proteins. CONCLUSION: These results have provided evidence that galectin-1 and mainly annexin-A1 are overexpressed in both gastritis and gastric cancer, suggesting a strong association of these proteins with chronic gastric inflammation and carcinogenesis.

[A case of lung cancer with endometrial metastasis].

A 50-year-old woman was admitted because of chest pain and lumbago. A chest X-ray film showed a 4-cm mass in the left lower lung field. Computed tomography scans revealed a nodule with spicule formation in the left lower lobe, and therefore we strongly suspected lung cancer. Fiberoptic bronchoscopy yielded a diagnosis of adenocarcinoma. However, since she had metrorrhagia we performed an endometrial biopsy. Histologically, the endometrium was similar to the lung lesion, with positive staining for thyroid transcription factor-1 (TTF-1), and lung cancer with endometrial metastasis was therefore diagnosed. Although lung cancer with endometrial metastasis is rare, it should be included in the differential diagnosis in patients with gynecological symptoms such as metrorrhagia.

[A case of Wegener granulomatosis after treatment for non-tuberculous mycobacteriosis].

A 67-year-old woman with a history of non-tuberculous mycobacteriosis due to Mycobacterium avium in 2002 and 2006 was admitted because of low-grade fever and cavitary lesions in both lungs on chest computed tomography (CT). Bronchial brushing cytology by bronchoscopy did not yield mycobacterium. Her serum antineutrophil cytoplasmic antibodies (MPO-ANCA) level was elevated and PR3 ANCA was negative. A surgical lung biopsy revealed basophilic necrosis with cavitary lesions and necrotizing vasculitis compatible with Wegener granulomatosis (WG). In the resected specimen, several hyalinizing nodules with caseous necrosis, probably associated with previous mycobacterium infection, were also seen. Because her disease was limited to the lungs, we successfully treated her with corticosteroids and methotrexate. WG is considered to be a type of ANCA-associated vasculitis, and microbial infections have been associated with initiation and relapse of WG. This case clearly demonstrated that lesions of WG occurred after mycobacterial infection in the lung. We believe that this case was ANCA production in association with bacterial infection.

[Sputum smear-positive tuberculosis diagnosed after hospitalization].

A tuberculosis infection-control program is based on a 3-level hierarchy of measures, including administrative, environmental, and respiratory protection. The most important level of tuberculosis controls is the use of administrative measures to reduce the risk of exposure to patients who might have infectious tuberculosis. In 126 patients tuberculosis was given diagnosed in our hospital over 10 years, and 51 patients among them had bacilli-positive sputum smears, of whom 26 were given diagnoses after admission, and 15 patients of these, in whom tuberculosis was not strongly suspected on admission, were considered to be at risk of nosocomial infection. The mean age of these 15 patients was 66.3 years, and 9 of them were given diagnoses of pneumonia. Duration until tuberculosis diagnosis was within 3 days in 11 patients, 4 to 7 days in 2, and more than 8 days in 2 patients. Twelve patients were admitted from the emergency room or belonged to other departments than respiratory medicine. We believe that it is important to consider tuberculosis when deciding about admission, in order not to delay diagnosis. Furthermore, ventilation of general rooms must be sufficient when dealing with the unexpected admission of patients with tuberculosis, and ability of health-care workers to use N95 respirators should be confirmed.

[Patient with MPO-ANCA-associated disease with interstitial pneumonia and lower cranial nerves palsy who was previously exposed to asbestos].

A 65-year-old man who was occupationally exposed to asbestos for 40 years was admitted to our hospital with fever and cough. Chest CT revealed paraseptal emphysema, subpleural fibrosis in both lungs, and pleural plaques. On bronchoalveolar lavage fluid contained elevated levels of lymphocytes and neutrophils (15% and 17%, respectively), and asbestos bodies were also found. Because serum MPO-ANCA titer was elevated to 188 EU, we suspected ANCA-associated disease with interstitial pneumonia. Prednisolone was begun at 30 mg/day and his lung opacities partially disappeared. Six weeks later, he complained of headache, dysphagia and hoarseness, and was admitted to the neurology department of the hospital. Under a diagnosis of either hypertrophic pachymeningitis or neuritis due to angiitis of the lower cranial nerves, steroid pulse therapy was performed. Asbestos exposure may have been a contributing factor for ANCA generation in this case. Furthermore, the fact that cranial nerves palsy occurred in spite of steroid therapy may also be important.

kDNA gene signatures of Trypanosoma cruzi in blood and oesophageal mucosa from chronic chagasic patients.

Trypanosoma cruzi presents a high degree of intraspecific variability, with possible implications for the pathogenesis of Chagas disease. The aim of this study was to evaluate T. cruzi kDNA minicircle gene signatures using the low-stringency single-specific-primer PCR technique in both peripheral blood and oesophageal mucosa from chronic chagasic patients, with or without megaesophagus, alone or in combination with cardiopathy and megacolon. It was not possible to identify a uniform pattern of shared bands between blood and oesophageal mucosa samples from individuals with the same clinical form or mixed forms, suggesting multiple T. cruzi infections with differential tissue tropism. Thus, the results indicate that there is an intense intraspecific variability in the hypervariable regions of T. cruzi kDNA, which has so far made it impossible to correlate the genetic profile of this structure with the clinical manifestations of Chagas disease.

Protein expression profile characteristic to hepatocellular carcinoma revealed by 2D-DIGE with supervised learning.

Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies. Although several major risks related to HCC, e.g., hepatitis B and/or hepatitis C virus infection, aflatoxin B1 exposure, alcohol drinking and genetic defects have been revealed, the molecular mechanisms leading to the initiation and progression of HCC have not been clarified. To reduce the mortality and improve the effectiveness of therapy, it is important to detect the proteins which are associated with tumor progression and may be useful as potential therapeutic or diagnosis targets. However, previous studies have not yet revealed the associations among HCC cells, histological grade and AFP. Here, we performed two-dimensional difference gel electrophoresis (2D-DIGE) combined with MS for 18 HCC patients. To focus not on individual proteins but on multiple proteins associated with pathogenesis, we introduce the supervised feature selection based on stochastic gradient boosting (SGB) for identifying protein spots that discriminate HCC/non HCC, histological grade of moderate/well and high alpha-fetoprotein (AFP)/low AFP level without arbitrariness. We detected 18, 25 and 27 protein spots associated with HCC, histological grade and AFP level, respectively. We confirmed that SGB is able to identify the known HCC-related proteins, e.g., heat shock proteins, carbonic anhydrase 2. Moreover, we identified the differentially expressed proteins associated with histological grade of HCC and AFP level and found that aldo-keto reductase 1B10 (AKR1B10) is related to well differentiated HCC, keratin 8 (KRT8) is related to both histological grade and AFP level and protein disulfide isomerase-associated 3 (PDIA3) is associated with both HCC and AFP level. Our pilot study provides new insights on understanding the pathogenesis of HCC, histological grade and AFP level.

Comparative proteomic and transcriptomic profiling of the human hepatocellular carcinoma.

Proteome analysis of human hepatocellular carcinoma (HCC) was done using two-dimensional difference gel electrophoresis. To gain an understanding of the molecular events accompanying HCC development, we compared the protein expression profiles of HCC and non-HCC tissue from 14 patients to the mRNA expression profiles of the same samples made from a cDNA microarray. A total of 125 proteins were identified, and the expression profiles of 93 proteins (149 spots) were compared to the mRNA expression profiles. The overall protein expression ratios correlated well with the mRNA ratios between HCC and non-HCC (Pearson's correlation coefficient: r=0.73). Particularly, the HCC/non-HCC expression ratios of proteins involved in metabolic processes showed significant correlation to those of mRNA (r=0.9). A considerable number of proteins were expressed as multiple spots. Among them, several proteins showed spot-to-spot differences in expression level and their expression ratios between HCC and non-HCC poorly correlated to mRNA ratios. Such multi-spotted proteins might arise as a consequence of post-translational modifications.

Analysis of acidic peptides with a matrix-assisted laser desorption/ionization mass spectrometry using positive and negative ion modes with additive monoammonium phosphate.

Acidic PTMs such as phosphorylation and sulfonation of proteins are known to play important roles in many cellular processes including signal transductions and protein-protein interactions. In MS, the acidic modified peptides, that have negative charge, are observable in negative ion mode rather than in positive ion mode. Moreover, addition of ammonium salt into MALDI matrix solution improves the relative intensity of ionization of the phosphorylated peptide to unmodified one. We demonstrate that a combination of the negative ion mode and addition of ammonium salt is more effective in the ionization of the acidic modified peptides. We applied this method to 2-DE separated proteins of Caenorhabditis elegans. As a result, 42 spots were identified as modified proteins, of which 34 proteins were nonoverlapping unique proteins. Furthermore, our study revealed that pI shifts of the DIM-1 and MLC-1 proteins in the 2-DE gel were attributed to the presence of the acidic modifications. The negative ion mode together with the addition of ammonium salt provides us a useful method to detect the phosphorylation and/or sulfonation of protein in a simple manner.

C-terminal sequencing method for proteins in polyacrylamide gel by the reaction of acetic anhydride.

A successive C-terminal amino acid truncation reaction with acetic anhydride was applied on proteins in polyacrylamide gel. Protein bands separated by conventional SDS-PAGE were excised, partially fixed in the gel with glutaraldehyde ethanol solution, dehydrated with ACN and subjected to the truncation reaction with acetic anhydride formamide solution. Pre-treatment of the gel with pyridine aqueous solution was found to enhance the truncation reaction yields. After the truncation reaction, the products were treated with an aqueous solution of dimethylaminoethanol to hydrolyze oxazolone rings at the C termini of the truncated products and O-acetylated products of serine, threonine and/or tyrosine. Several commercially available proteins of 10-40 kDa, as determined by SDS-PAGE, such as myoglobin, trypsin inhibitor, alpha-hemolysin, cytochrome c, chymotrypsin C chain, elastase, acylase and histone H4, were subjected to the C-terminal analysis. The truncated proteins were in-gel digested with trypsin and the extracted peptides were analyzed by MALDI-TOF MS, giving rise to a series of molecular mass ions of the C-terminal truncated fragments corresponding to the C-terminal amino acid sequence of the relevant protein.

High-throughput and high-resolution two dimensional mapping of pI and m/z using a microchip in a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer.

We have developed a high-throughput, two-dimensional-mapping (isoelectric point [pI], mass-to-charge ratio [m/z]) method by combining a capillary isoelectric focusing chip sealed with removable resin tape and a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. Sample proteins are separated in a meandering channel on the chip and immediately frozen. The tape is then removed and the proteins are freeze-dried. The freeze-drying maintains the separation state of the proteins and prevents movement of the sample solution, which can reduce pI resolution. A matrix solution is then applied and mass spectrometry is carried out by laser irradiation. The whole process takes less than 70 min, more than 10 times faster than with two-dimensional, polyacrylamide gel electrophoresis.

Two-dimensional electrophoretic profiling of normal human kidney glomerulus proteome and construction of an extensible markup language (XML)-based database.

To contribute to physiology and pathophysiology of the glomerulus of human kidney, we have launched a proteomic study of human glomerulus, and compiled a profile of proteins expressed in the glomerulus of normal human kidney by two-dimensional gel electrophoresis (2-DE) and identification with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and/or liquid chromatography-tandem mass spectrometry (LC-MS/MS). Kidney cortices with normal appearance were obtained from patients under surgical nephrectomy due to renal tumor, and glomeruli were highly purified by a standard sieving method followed by picking-up under a phase-contrast microscope. The glomerular proteins were separated by 2-DE with 24 cm immobilized pH gradient strips in the 3-10 range in the first dimension and 26 x 20 cm sodium dodecyl sulfate polyacrylamide electrophoresis gels of 12.5% in the second dimension. Gels were silver-stained, and valid spots were processed for identification through an integrated robotic system that consisted of a spot picker, an in-gel digester, and a MALDI-TOF MS and / or a LC-MS/MS. From 2-DE gel images of glomeruli of four subjects with no apparent pathologic manifestations, a synthetic gel image of normal glomerular proteins was created. The synthetic gel image contained 1713 valid spots, of which 1559 spots were commonly observed in the respective 2-DE gels. Among the 1559 spots, 347 protein spots, representing 212 proteins, have so far been identified, and used for the construction of an extensible markup language (XML)-based database. The database is deposited on a web site (http://www.sw.nec.co.jp/bio/rd/hgldb/index.html) in a form accessible to researchers to contribute to proteomic studies of human glomerulus in health and disease.

C-terminal sequencing method for peptides and proteins by the reaction with a vapor of perfluoric acid in acetic anhydride.

A successive C-terminal amino acid truncation reaction of peptides and proteins with a vapor generated from a low-concentrated perfluoric acid in acetic anhydride is presented. The reaction products were analyzed with matrix-assisted laser desorption/ionization-time of flight mass-spectrometry giving molecular mass ions of the C-terminal truncated peptides or proteins from which the C-terminal sequence information can be deduced. Acetylation reaction preceded the truncation reaction in order to protect the amino groups and other reactive groups in peptides and proteins, and after the truncation reaction, hydration reaction was carried out to afford cleaner mass spectra.