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The phase properties and structural characteristics of stratum corneum (SC) lipid lamellae have been a subject of considerable interest. To clarify the individual role of the stratum corneum constituent lipids, such as ceramides, free fatty acids, and cholesterol, we investigated the thermotropic properties and aggregation structures of a pseudo-ceramide/stearic acid (1/1 mole ratio)-cholesterol system, which is a simplified model for the natural lipids. Differential scanning calorimetry (DSC) detected decreases of melting entropies (delta Sm) by the incorporation of cholesterol into both anhydrous and hydrated equimolar mixture of pseudo-ceramide (SLE) and stearic acid. Moreover, there was a linear relationship between the cholesterol content and the melting entropies in the region of 0-33 mol% cholesterol for both the anhydrous and hydrate lipids. In addition, as the concentration of cholesterol increased, a liquid lateral packing (4.5 A) appeared in the wide-angle X-ray diffraction and the intensity of a hexagonal packing (4.15 A) decreased. The results from the present study strongly follow the idea that cholesterol can regulate the mobility of hydrocarbon chains of the natural stratum corneum lipid bilayer, which is primarily responsible for the barrier properties.

RA-III acetate was treated with Lawesson's reagent to afford [Tyr-3-Ψ(CS-NH)-Ala-4]RA-III acetate. Treatment of this product with Hg(OAc) 2 /Li 2 CO 3 and then methanol gave an oxazole intermediate. Acid-catalyzed arylation of the methylene carbon atom on the oxazole ring and subsequent partial hydrolysis of the oxazole ring in the resultant compounds afforded RA-VII analogues having an aromatic amino acid at residue 2. [5-Fluoro-d-Trp-2]RA-VII showed IC 50 values of 0.038 and 0.077 μM against the HL-60 and HCT-116 cell lines, respectively.

Background: Long tubular duplication is a rare congenital intestinal disease, that can lead to emergency situations marked by massive hemorrhage. However, preoperative diagnosis and surgical treatment are challenging. This report presents preoperative images and details a surgical procedure for long tubular intestinal duplications with massive hemorrhage., Case Presentation: A 3-year-old boy presented to the emergency department with melena. Despite undergoing a Tc-99m pertechnetate scintigraphy one year prior, which revealed nonspecific findings with enhancement of some parts of the intestine, enhanced abdominal CT revealed an edematous small intestine with luminal extravasation. The patient received a transfusion of red blood cells; however, his hemoglobin level did not improve. Arterial angiography and double-balloon endoscopy revealed no remarkable findings. Exploratory laparotomy revealed a long tubular duplication in half of the small intestine. Utilizing the Wrenn procedure, we successfully removed all duplicate mucosa. Pathological findings showed that almost all duplications contained gastric mucosa and revealed an ulcer with a ruptured arterial vessel. His symptoms were resolved, and the hemoglobin level stabilized. At 2 months postoperatively, no surgical complications were present., Conclusions: Effective management of long tubular duplications with massive hemorrhage involves timely application of the Wrenn procedure. Recognition of specific imaging findings is crucial to prompt exploratory laparotomy, ensuring optimal outcomes and preventing delays in treatment., (© 2024. The Author(s).)

Background: Prolonged grief disorder (PGD) is a new diagnostic entity. However, treatment for PGD is not yet available. Interpersonal psychotherapy (IPT) may be effective for PGD., Case Presentation: A single 27-year-old Japanese woman lost her brother to suicide. However, she did not express her grief to anyone or visit a psychiatric clinic. After experiencing strong depressive symptoms triggered by work stress, she visited a psychiatric clinic, where she was diagnosed with depression as well as PGD. Through pharmacotherapy, her depressive symptoms improved, but no improvement was noted in her PGD symptoms. She therefore began IPT for PGD at our hospital, 5 years after her brother's suicide and after 4 years of PGD symptoms. In the introductory phase of IPT, she was diagnosed with comorbid persistent depressive disorder (PDD). After this diagnosis, through psychoeducation on PDD, she could identify the symptoms that reflected her personality traits as "PDD symptoms." Furthermore, she could affirm her positive and negative feelings and share them with others. Consequently, the grieving process was facilitated, and her interests and relationships were re-established. Her PGD and PDD symptoms improved., Conclusion: IPT may be effective for PGD comorbid with PDD among Japanese., Competing Interests: Tatsuo Akechi is the Vice Editor‐in‐Chief of Psychiatry and Clinical Neurosciences Reports and a co‐author of this article. He was excluded from editorial decision‐making related to the acceptance and publication of this article. The other authors declare no conflict of interest., (© 2023 The Authors. Psychiatry and Clinical Neurosciences Reports published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Psychiatry and Neurology.)

Background: Sessile serrated lesions (SSLs) are precursors of colon cancer, especially in cases of large, right colon. However, they are difficult to not only detect, but only clarify the margin of the lesion, which can lead to the poor endoscopic treatment outcomes., Aims: This study evaluated the usefulness of acetic acid spray with narrow-band imaging (A-NBI) for the better visualization of the margin of SSLs., Methods: From January 2013 to March 2022, patients with superficial elevated polyps suspected of being SSLs ≥ 10 mm with an endoscopic diagnosis that had been endoscopically resected at Hiroshima City Hiroshima Citizens Hospital were enrolled. Endoscopic images with white-light imaging (WLI), narrow-band imaging (NBI), indigo-carmine (IC), and A-NBI were recorded in each lesion and were randomly arranged and assessed by 10 endoscopists. We compared the visibility score (1 to 4) and color differences (ΔE) between inside and outside of the lesions among WLI, NBI, IC, and A-NBI., Results: Forty-one lesions in 33 cases were included, and a total of 164 images were evaluated. As for the visibility score, most of the lesions were scored as 1 or 2 on WLI, whereas most were scored 4 on A-NBI. The median ΔE of A-NBI was also significantly higher than that of WLI, NBI, or IC (20.5 vs. 8.3 vs. 8.2 vs. 12.3, P < 0.01). A significant correlation was observed between the color difference and visibility score (r = 0.53, P < 0.01)., Conclusions: A-NBI may be a useful modality for identifying the margin of SSLs., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Aim: Patients' and spouses' neurodevelopmental traits may influence marital relationships, which are significantly associated with depressive symptoms. However, no studies have examined marital relationships in persistent depressive disorder (PDD) in terms of neurodevelopmental traits. This study aimed to explore the association between the autism spectrum disorder (ASD) or attention-deficit/hyperactivity disorder (ADHD) traits of female PDD patients and both partners' (patient and husband) marital satisfaction., Methods: A cross-sectional online survey was administered during two predetermined consecutive months at seven institutions. Participants were female outpatients who fulfilled the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition criteria for PDD and their husbands. The instruments of the study were the following validated surveys: the Quality Marriage Index (QMI), the Autism-Spectrum Quotient Japanese version-21 (AQ-J-21), and the Adult ADHD Self-Report Scale Part A (ASRS Part A)., Results: The patients' AQ-J-21 showed a positive significant association with their QMI in all patients who responded to this study's questionnaire, whereas among couples wherein both patient and husband responded, the ASRS Part A exhibited a positive significant association with the patients' QMI. Conversely, the husbands' ASRS Part A exhibited a negative significant association with the patients' QMI., Conclusion: The patients' ASD and ADHD traits may play a positive role in the marital satisfaction of female PDD patients, while their husbands' ADHD traits may play a negative role. For female PDD patients with low marital satisfaction, it may be important to consider whether their husbands have ADHD traits; if so, it may be necessary to develop intervention strategies focused on the traits for improving the low marital satisfaction. However, our conclusions are not sufficiently convincing., Competing Interests: The authors declare no conflict of interest., (© 2023 The Authors. Psychiatry and Clinical Neurosciences Reports published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Psychiatry and Neurology.)

Pleural metastasis in rectal cancer is often due to secondary invasion or dissemination from intrapulmonary metastases. To date, there are no reports on solitary pleural metastasis. Here, we report a rare case of lower rectal cancer that recurred as pleural metastasis 4 years after surgical resection of the primary tumor. He was a 65-year-old man who visited our department with an abnormal shadow on his chest X-ray. He had a history of lower rectal cancer and had undergone laparoscopic low anterior resection of the rectum and bilateral lymph node dissection after neoadjuvant chemotherapy. Pathological ypT3N1M0 stage IIIA tumor was diagnosed, and adjuvant chemotherapy was administered. According to the computed tomography scan, a pleural tumor or pulmonary metastasis was suspected. Thoracoscopic partial resection of the lung and a partial pleurectomy were performed for diagnostic and therapeutic purposes. Histopathological examination revealed a highly differentiated tubular adenocarcinoma, consistent with metastatic rectal cancer. The nodule arose from the visceral pleura and invaded the parietal pleura with few malignant cells in the lung parenchyma. The lesion was surgically resected. However, 3 months after the second surgery, tumor recurrence with pleural dissemination was observed, and chemotherapy was initiated., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Norovirus infection is a major cause of acute gastroenteritis, although some infected individuals are asymptomatic. GII.4 is the predominant genotype worldwide and, since 2000, has been the most prevalent in patients in Thailand with acute gastroenteritis. We screened stool samples for norovirus in 786 patients with acute gastroenteritis who were admitted to a hospital in Bangkok from 2017 to early 2019 and detected it in 136 specimens (17.3%). Eight and 124 specimens were positive for the GI and GII genogroups, respectively, and the remaining 4 specimens were double-positive. Nine genotypes (GI.3, GI.5, GII.2, GII.3, GII.4, GII.6, GII.8, GII.13, and GII.17) were identified from 140 strains, and 72 strains (51.4%) were GII.4. We had previously conducted a one-year survey of norovirus infection in residents of a community in Bangkok from May 2018 to April 2019 and found that a substantial portion of the residents were infected asymptomatically. The 9 genotypes identified in the patients were also commonly identified in the community residents. To investigate the relationship between noroviruses identified in the acute gastroenteritis patients and those identified in the community residents, phylogenetic tree analysis was conducted. Of the 9 genotypes, 8 showed similarities in both their genomic sequences and their deduced amino acid sequences. In addition, strain replacement of GI.3 was observed in both the patients and the community residents within the overlapping period. These results suggested that norovirus spreads efficiently to the community by simultaneously causing symptomatic and asymptomatic infections., Competing Interests: The authors declare no conflict of interest., (© 2021 The Author(s).)

Norovirus is a leading cause of acute gastroenteritis worldwide. Norovirus shedding typically lasts one week to one month after the onset of diarrhea in immunocompetent hosts. The occurrence of mutations in the genome during infection has contributed to the evolution of norovirus. It has been suggested that genomic mutations in the P2-domain of capsid protein VP1, the major antigenic site for virus clearance, are involved in the evasion of host immunity and prolonged shedding of norovirus. In our previous study, we found a case of long-term shedding of GII.14 norovirus in a post-symptomatic immunocompetent individual that lasted about three months. In this study, we characterized the genomic sequence of the GII.14 strain to gain insight into the context of long-term shedding. By sequencing a 4.8 kb region of the genome corresponding to half of ORF1 and the entire ORF2 and ORF3, which encode several non-structural proteins and the structural proteins VP1 and VP2, the GII.14 strain was found to be classified as recombinant GII.14[P7]. Six point-mutations occurred during the three-month period of infection in a time-dependent manner in the genomic regions encoding RNA-dependent RNA polymerase, VP1, and VP2. Three of the six mutations were sense mutations, but no amino acid substitution was identified in the P2-domain of VP1. These results suggest that there is a mechanism by which long-term shedding of norovirus occurs in immunocompetent individuals independent of P2-domain mutations., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The toxicity of mercury (Hg), is generally known, and around 90% of Hg exist as methylmercury (CH 3 Hg + ) in marine organism. Mercury concentrates in sharks and whales, which are at the top of the food chain as predators to cephalopods. The concentrations of Hg in liver and muscle of blue shark, caught in Sagami Bay, and in digestive gland and mantles of Todarodes pacificus, Sepia madokai, and Uroteuthis edulis caught in East China Sea were measured and analyzed. The Hg concentrations in the sharks, squids, and cuttlefishes determined in this study were almost same as those in the other sea regions. In addition, the Hg concentration in the blue shark was higher in the muscle than in the liver. In S. madokai and U. edulis, Hg accumulated in the digestive gland but not in the mantle. Although the Hg concentration in the digestive gland of T. pacificus is lower than those of S. madkai and U edulis, Hg concentration in the mantle is critically higher. More than 90% of Hg is present as CH 3 Hg + in muscle of blue shark and mantle of T. pacificus. This feature is explained due to amino acids with the thiol groups and chain genes in the muscle of blue shark as well as in the mantle of T. pacificus. Myosin in the mantle of T. pacificus and blue shark enhances the stability of CH 3 Hg + . The amount of Hg in the digestive gland of T. pacificus could be too large to store; thus, Hg is released to the mantle, whereas the nutrients in the digestive gland of T. pacificus are supplied to other tissues. It is considered that the muscle fiber of T. pacificus is strong; therefore, large amounts of myosin levels may be present in T. pacificus than in S. madokai and U. edulis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Chikungunya fever, a mosquito-borne disease manifested by fever, rash, myalgia, and arthralgia, is caused by chikungunya virus (CHIKV), which belongs to the genus Alphavirus of the family Togaviridae Anti-CHIKV IgG from convalescent patients is known to directly neutralize CHIKV, and the state of immunity lasts throughout life. Here, we examined the epitope of a neutralizing mouse monoclonal antibody against CHIKV, CHE19, which inhibits viral fusion and release. In silico docking analysis showed that the epitope of CHE19 was localized in the viral E2 envelope and consisted of two separate segments, an N-linker and a β-ribbon connector, and that its bound Fab fragment on E2 overlapped the position that the E3 glycoprotein originally occupied. We showed that CHIKV-E2 is lost during the viral internalization and that CHE19 inhibits the elimination of CHIKV-E2. These findings suggested that CHE19 stabilizes the E2-E1 heterodimer instead of E3 and inhibits the protrusion of the E1 fusion loop and subsequent membrane fusion. In addition, the antigen-bound Fab fragment configuration showed that CHE19 connects to the CHIKV spikes existing on the two individual virions, leading us to conclude that the CHE19-CHIKV complex was responsible for the large virus aggregations. In our subsequent filtration experiments, large viral aggregations by CHE19 were trapped by a 0.45-μm filter. This virion-connecting characteristic of CHE19 could explain the inhibition of viral release from infected cells by the tethering effect of the virion itself. These findings provide clues toward the development of effective prophylactic and therapeutic monoclonal antibodies against the Alphavirus infection. IMPORTANCE Recent outbreaks of chikungunya fever have increased its clinical importance. Neither a specific antiviral drug nor a commercial vaccine for CHIKV infection are available. Here, we show a detailed model of the docking between the envelope glycoprotein of CHIKV and our unique anti-CHIKV-neutralizing monoclonal antibody (CHE19), which inhibits CHIKV membrane fusion and virion release from CHIKV-infected cells. Homology modeling of the neutralizing antibody CHE19 and protein-protein docking analysis of the CHIKV envelope glycoprotein and CHE19 suggested that CHE19 inhibits the viral membrane fusion by stabilizing the E2-E1 heterodimer and inhibits virion release by facilitating the formation of virus aggregation due to the connecting virions, and these predictions were confirmed by experiments. Sequence information of CHE19 and the CHIKV envelope glycoprotein and their docking model will contribute to future development of an effective prophylactic and therapeutic agent., (Copyright © 2020 American Society for Microbiology.)

The transmission of human norovirus excreted from infected persons occasionally causes sporadic infections and outbreaks. Both symptomatic patients and asymptomatic carriers have been reported to contribute to norovirus transmission, but little is known about the magnitude of the contribution of asymptomatic carriers. We carried out a 1-year survey of residents of a district of Bangkok, Thailand to determine the percentage of norovirus transmissions originating from asymptomatic individuals. We screened 38 individuals recruited from 16 families from May 2018 to April 2019 for GI and GII genotypes. Norovirus was detected every month, and 101 of 716 stool samples (14.1%) from individuals with no symptoms of acute gastroenteritis were norovirus-positive. The average infection frequency was 2.4 times per person per year. Fourteen genotypes were identified from the positive samples, with GII.4 being detected most frequently. Notably, 89.1% of the norovirus-positive samples were provided by individuals with no diarrhea episode. Similar to cases of symptomatic infections in Thailand, asymptomatic infections were observed most frequently in December. We detected 4 cases of NV infection caused by household transmission, and 3 of the 4 transmissions originated from asymptomatic individuals. We also identified a case in which norovirus derived from an asymptomatic individual caused diarrhea in a family member. These results suggest that asymptomatic individuals play a substantial role in both the maintenance and spreading of norovirus in a community through household transmission., Competing Interests: The authors have declared no competing interest exist.

Japan is undergoing a major population health transition as its society ages, and it continues to experience low birth rates. An aging Japan will bring new challenges to its public health system, highlighted as a model for universal health coverage (UHC) around the world. Specific challenges Japan's health care system will face include an increase in national public health expenditures, higher demand for health care services, acute need for elder and long-term care, shortage of health care workers, and disparities between health care access in rural versus urban areas. Blockchain technology has the potential to address some of these challenges, but only if a health blockchain is conceptualized, designed, localized, and deployed in a way that is compatible with Japan's centralized UHC-centric public health system. Blockchain solutions must also be adaptive to opportunities and barriers unique to Japan's national health and innovation policy, including its regulatory sandbox system, while also seeking to learn from blockchain adoption in the private sector and in other countries. This viewpoint outlines the major opportunities and potential challenges to blockchain adoption for the future of Japan's health care., (©Tim Ken Mackey, Hirofumi Bekki, Tokio Matsuzaki, Hiroshi Mizushima. Originally published in the Journal of Medical Internet Research (http://www.jmir.org), 09.01.2020.)

To visually examine the early phase of chikungunya virus (CHIKV) infection in target cells, we constructed a virus-like particle (VLP) in which the envelope protein E1 is fused with green fluorescent protein (GFP). This chikungunya VLP-GFP (CHIK-VLP-EGFP), purified by density gradient fractionation, was observed as 60-70 nm-dia. particles and was detected as tiny puncta of fluorescence in the cells. CHIK-VLP-EGFP showed binding properties similar to those of the wild-type viruses. Most of the fluorescence signals that had bound on Vero cells disappeared within 30 min at 37 °C, but not in the presence of anti-CHIKV neutralizing serum or an endosomal acidification inhibitor (bafilomycin A1), suggesting that the loss of fluorescence signals is due to the disassembly of the viral envelope following the internalization of CHIK-VLP-EGFP. In addition to these results, the fluorescence signals disappeared in highly susceptible Vero and U251MG cells but not in poorly susceptible A549 cells. Thus, CHIK-VLP-EGFP is a useful tool to examine the effects of the CHIKV neutralizing antibodies and antiviral compounds that are effective in the entry phase of CHIKV., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Changes in the 11th revision of the International Classification of Diseases (ICD-11) from ICD-10 may significantly impact coding quality. We conducted a field trial in 2017 to evaluate the line coding quality of 19 cases coded using the coding method of the World Health Organization. The cases with low agreement between the accuracy rates of ICD-10 and ICD-11 were cases that required the extension code. We should prepare effective educational content about how to use the extension code for proper coding in ICD-11.

Norovirus (NoV) is the leading cause of viral acute gastroenteritis among all age groups in the world. We performed a molecular epidemiological study of the NoVs prevalent in Bangkok between November 2014 and July 2016 to investigate the emergence of new NoV variants in Thailand. A total of 332 stool specimens were collected from hospitalized pediatric patients with acute gastroenteritis in Bangkok, Thailand. NoVs were detected by real-time PCR. The genome of the N-terminal/shell domain was amplified, the nucleotide sequence was determined, and phylogenetic analyses were performed. GII NoV was detected in 58 (17.5%) of the 332 specimens. GII.17, a genotype strain prevalent from 2014 to mid-2015, was hardly detected and replaced by the GII.3 genotype strain. Entire genome sequencing followed by phylogenetic analysis of the GII.3 genotype strains indicated that they are new recombinant viruses, because the genome encoding ORF1 is derived from a GII.12 genotype strain, whereas that encoding ORF2-3 is from a GII.3 genotype strain. The putative recombination breakpoints with the highest statistical significance were located around the border of 3D pol and ORF2. The change in the prevalent strain of NoV seems to be linked to the emergence of new forms of recombinant viruses. These findings suggested that the swapping of the structural and non-structural proteins of NoV is a common mechanism by which new epidemic variants are generated in nature., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Objective: Despite the application of skin care treatments, many infants have skin problems such as dryness and erythema. We proposed a new combination skin care for infants which consisted of a foaming cleanser with lower surfactant activity and moisturizers that contained pseudo-ceramide., Subjects and Methods: A total of 50 infants (age: 3-24 months) with insignificant levels of dry skin were enrolled in this usage trial. The parents washed the infants with the test cleanser while bathing and then applied the moisturizer (lotion or cream) containing pseudo-ceramide. Prior to and following the 4-week usage period, visual evaluation of the skin condition was conducted by a dermatologist, in addition to instrumental analysis., Results: Erythema and papule, accompanied by dryness, were commonly observed at week 0. However, by week 4, these symptoms significantly improved; the condition of none of the subjects deteriorated. The number of infants with lower cutaneous barrier function and higher skin pH decreased. The parents of the infants recognized improvements in the skin symptoms and were appreciative of the test materials., Conclusion: The combined usage of the foaming cleanser with lower surfactant activity and a moisturizer containing pseudo-ceramide may be effective in maintaining healthy infant skin and ameliorating the skin symptoms., Competing Interests: Disclosure Naoko Okamoto, Kaori Umehara, Junko Sonoda, Hiroki Mizushima, and Yutaka Takagi are employees of Skin Care Products Research Laboratories, Kao Corporation, Tokyo, Japan. Mitsuyuki Hotta is an employee of Biological Science Research Laboratories, Kao Corporation, Tochigi, Japan. Keiko Matsuo is an employee of Kao Corporation, Office of the President, Tokyo, Japan. Naoko Baba is an employee of Department of Dermatology, Kanagawa Children’s Medical Center, Kanagawa, Japan. The authors report no other conflicts of interest in this work.

The modification of the Au(111) Shockley surface state (SS) by an n-alkane molecule (n-tetratetracontane) monolayer was observed by angle-resolved ultraviolet photoemission spectroscopy. Although there is little chance of chemical interaction in this ideal physisorption system, the volume of the Fermi surface of the SS was significantly reduced accompanied by the formation of large interface electric dipoles. Moreover, Rashba splitting of the SS by spin-orbit interactions was slightly increased upon n-tetratetracontane adsorption, which arose from the decrease in the symmetry of the wave function around the Au nuclei at the surface. The detailed information about the simple physisorption system presented in this paper provides basic knowledge for understanding the electronic structure at the interface between other organic molecules and metal substrates.

Heparin-binding EGF-like growth factor (HB-EGF) plays an indispensable role in suppression of cell proliferation during mouse valvulogenesis. However, ligands of the EGF receptor (EGFR/ErbB1), including HB-EGF, are generally considered as growth-promoting factors, as shown in cancers. HB-EGF binds to and activates ErbB1 and ErbB4. We investigated the role of ErbB receptors in valvulogenesis in vivo using ErbB1- and ErbB4-deficient mice, and an ex vivo model of endocardial cushion explants. We show that HB-EGF suppresses valve mesenchymal cell proliferation through a heterodimer of ErbB1 and ErbB4, and an ErbB1 ligand (or ligands) promotes cell proliferation through a homodimer of ErbB1. Moreover, a rescue experiment with cleavable or uncleavable isoforms of ErbB4 in ERBB4 -null cells indicates that the cleavable JM-A, but not the uncleavable JM-B, splice variant of ErbB4 rescues the defect of the null cells. These data suggest that the cytoplasmic intracellular domain of ErbB4, rather than the membrane-anchored tyrosine kinase, achieves this suppression. Our study demonstrates that opposing signals generated by different ErbB dimer combinations function in the same cardiac cushion mesenchymal cells for proper cardiac valve formation., (© 2017. Published by The Company of Biologists Ltd.)

Background: BK-UM (CRM197) is a mutant form of diphtheria toxin and a specific inhibitor of heparin-binding epidermal growth factor-like growth factor (HB-EGF). We assessed the safety, pharmacokinetics, recommended dose, and efficacy of BK-UM in patients with recurrent ovarian cancer (OC) or peritoneal cancer (PC), and measured HB-EGF levels in serum and abdominal fluid after BK-UM administration., Methods: Eleven patients with advanced or recurrent OC or PC were enrolled and treated with BK-UM via the intraperitoneal route. The dose was escalated (1.0, 2.0, 3.3, and 5.0 mg/m 2 ) using a 3 + 3 design., Results: Eight of 11 patients completed treatment. No dose-limiting toxicity (DLT) was experienced at dose levels 1 (1.0 mg/m 2 ) and 2 (2.0 mg/m 2 ). Grade 3 transient hypotension as an adverse event (defined as a DLT in the present study) was observed in two of four patients at dose level 3 (3.3 mg/m 2 ). Treatment with BK-UM was associated with decreases in HB-EGF levels in serum and abdominal fluid in seven of 11 patients and five of eight patients, respectively. Clinical outcomes included a partial response in one patient, stable disease in five patients, and progressive disease in five patients., Conclusions: BK-UM was well tolerated at doses of 1.0 and 2.0 mg/m 2 , with evidence for clinical efficacy in patients with recurrent OC or PC. A dose of 2.0 mg/m 2 BK-UM is recommended for subsequent clinical trials., Trial Registration: This trial was prospectively performed as an investigator-initiated clinical trial. The trial numbers are UMIN000001002 and UMIN000001001, with registration dates of 1/30/2008 and 2/4/2008, respectively. UMIN000001001 was registered as a trial for the continuous administration of BK-UM after UMIN000001002 .

Japan promotes research related to intractable diseases and financially supports patients with these diseases. Intractable diseases are designated as those that fulfill the following criteria: (1) rarity (affecting less than 0.1% of the population in Japan), (2) unknown etiology, (3) lack of effective treatment, (4) necessity of long-term treatment, and (5) existence of objective diagnostic criteria and not necessarily equal to rare diseases in other countries. The construction of a national database is required to promote research to clarify the pathogenesis of these diseases and to develop pharmaceutical products and medical devices. The Ministry of Health, Labor, and Welfare launched an online registration system in 2001, but many problems associated with gathering and utilizing information on patients with intractable diseases remain. In this paper, we describe the present status of the national registry of designated intractable diseases in Japan and discuss future prospects., Competing Interests: The authors declare no conflicts of interest.

A New legal structure for rare disease (nambyo) has been established in Japan this year, after 42 years of measures of nambyo. We have been accumulating registry for nambyo from 2003, however, as it was based on paper registration, quality was not enough. Our new registry system will be based under ISO13606 which is a new medical international standard. Authorized doctors can put in data On Line by the new system, which has data cleaning filter for accurate data entry. Patients will be supported their medical expense by authorization by this system, so the registry will be efficient.

Background Schwannomas are the most common primary solitary tumor among peripheral nerve sheath tumors. The occurrence of transient or permanent neurological deficits after schwannoma resection is more common than previously recognized. Here, the neurological deficits before and after surgical resection of schwannomas in the upper extremities were examined. Methods The study included 43 upper-extremity schwannomas that were treated surgically between January 2000 and July 2013. The neurological status of each patient (such as pain, sensory disturbances, and motor disturbances) was evaluated preoperatively, immediately postoperatively, and at the final postoperative follow-up. Results Out of the 43 cases, 34 cases exhibited neurological symptoms before the operation, and in 31 of the 34 cases, neurological symptoms were either reduced or disappeared after the surgery. In 20 of the 43 cases, new neurological deficits that had not been observed preoperatively were noted immediately postoperatively; the newly acquired neurological deficits disappeared over time in 5 of the 20 cases. Significantly, more newly acquired neurological deficits remained in cases where the tumor was located in the upper arm and elbow than in cases where the tumor was located in the distal forearm. Conclusion New neurological deficits occurred after surgery in about half of the cases. This ratio was higher than expected, suggesting that schwannoma resection is not always a complication-free operation. Therefore, patients should be informed preoperatively about the possibility of neurological deficits. Furthermore, extreme care should be taken not to damage the affected and uninvolved nerves during surgery., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

We aimed to identify a novel prognostic biomarker related to recurrence in stage II and III colorectal cancer (CRC) patients. Stage II and III CRC tissue mRNA expression was profiled using an Affymetrix Gene Chip, and copy number profiles of 125 patients were generated using an Affymetrix 250K Sty array. Genes showing both upregulated expression and copy number gains in cases involving recurrence were extracted as candidate biomarkers. The protein expression of the candidate gene was assessed using immunohistochemical staining of tissue from 161 patients. The relationship between protein expression and clinicopathological features was also examined. We identified 9 candidate genes related to recurrence of stage II and III CRC, whose mRNA expression was significantly higher in CRC than in normal tissue. Of these proteins, the S100 calcium-binding protein A2 (S100A2) has been observed in several human cancers. S100A2 protein overexpression in CRC cells was associated with significantly worse overall survival and relapse-free survival, indicating that S100A2 is an independent risk factor for stage II and III CRC recurrence. S100A2 overexpression in cancer cells could be a biomarker of poor prognosis in stage II and III CRC recurrence and a target for treatment of this disease.

N-Glycosylation is an important post-translational modification in most secreted and membrane-bound proteins in eukaryotic cells. However, the insect N-glycosylation pathway and the potentials contributing to the N-glycan synthesis are still unclear because most of the studies on these subjects have focused on mammals and plants. Here, we identified Bombyx mori sialyltransferase (BmST), which is a Golgi-localized glycosyltransferase and which can modify N-glycans. BmST was ubiquitously expressed in different organs and in various stages of development and localized at the Golgi. Biochemical analysis using Sf9-expressed BmST revealed that BmST encoded α2,6-sialyltransferase and transferred N-acetylneuraminic acid (NeuAc) to the nonreducing terminus of Galβ1-R, but exhibited the highest activity toward GalNAcβ1,4-GlcNAc-R. Unlike human α2,6-sialyltransferase, BmST required the post-translational modification, especially N-glycosylation, for its full activity. N-Glycoprotein analysis of B. mori fifth instar larvae revealed that high-mannose-type structure was predominant and GlcNAc-linked and fucosylated structures were observed but endogenous galactosyl-, N-acetylgalactosaminyl- and sialyl-N-glycoproteins were undetectable under the standard analytical approach. These results indicate that B. mori genome encodes an α2,6-sialyltransferase, but further investigations of the sialylation potentials are necessary., (© The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Amyotrophic lateral sclerosis (ALS) is a motor neuron disease characterized by serious muscle atrophy and weakness. The purpose of this study was to find prognostic factors in patients with mild ALS using application forms for the Specified Disease Treatment Research Program in Japan. We classified ALS as mild, moderate and severe. The subjects consisted of 363 patients with mild ALS who underwent needle electromyography at registration and were followed for more than one year. Time to progression to severe ALS and time to deterioration of activities of daily living such as speech dysfunction, upper limb dysfunction, and walking disability were used as outcomes. Cox proportional hazards model analysis was performed to identify prognostic factors. Of the patients with initially mild ALS, 38.3% (139/363) had progressed severe ALS at the last follow-up. In multivariate analysis of time to progression to severe ALS, bulbar onset (hazard ratio [95% confidence interval]: 1.68 [1.13-2.49], p = 0.010), tongue atrophy (1.69 [1.14-2.51], p = 0.009), dyspnea (1.57 [1.02-2.41], p = 0.042) and active denervation findings (ADFs) of the cervical-upper limb area (1.81 [1.25-2.63], p = 0.002) emerged as prognostic factors. Furthermore ADFs in the trunk area were prognostic factors for upper limb dysfunction and walking disability (1.72 [1.05-2.81], p = 0.031, and 1.97 [1.09-3.59], p = 0.026). In conclusion ADFs of the cervical-upper limb area and trunk area were prognostic factors in ALS patients.

Background: The potential of expression profiling using microarray analysis as a tool to predict the prognosis for different types of cancer has been realized. This study aimed to identify a novel biomarker for colorectal cancer (CRC)., Methods: The expression profiles of cancer cells in 152 patients with stage I-III CRC were examined using microarray analysis. High expression in CRC cells, especially in patients with distant recurrences, was a prerequisite to select candidate genes. Thus, we identified seventeen candidate genes, and selected Traf2- and Nck-interacting kinase (TNIK), which was known to be associated with progression in CRC through Wnt signaling pathways. We analyzed the protein expression of TNIK using immunohistochemistry (IHC) and investigated the relationship between protein expression and patient characteristics in 220 stage I-III CRC patients., Results: Relapse-free survival was significantly worse in the TNIK high expression group than in the TNIK low expression group in stage II (p = 0.028) and stage III (p = 0.006) patients. In multivariate analysis, high TNIK expression was identified as a significant independent risk factor of distant recurrence in stage III patients., Conclusion: This study is the first to demonstrate the prognostic significance of intratumoral TNIK protein expression in clinical tissue samples of CRC, in that high expression of TNIK protein in primary tumors was associated with distant recurrence in stage II and III CRC patients. This TNIK IHC study might contribute to practical decision-making in the treatment of these patients.

A cutinase-like enzyme from Saccharomonospora viridis AHK190, Cut190, hydrolyzes the inner block of polyethylene terephthalate (PET); this enzyme is a member of the lipase family, which contains an α/β hydrolase fold and a Ser-His-Asp catalytic triad. The thermostability and activity of Cut190 are enhanced by high concentrations of calcium ions, which is essential for the efficient enzymatic hydrolysis of amorphous PET. Although Ca(2+)-induced thermostabilization and activation of enzymes have been well explored in α-amylases, the mechanism for PET-degrading cutinase-like enzymes remains poorly understood. We focused on the mechanisms by which Ca(2+) enhances these properties, and we determined the crystal structures of a Cut190 S226P mutant (Cut190(S226P)) in the Ca(2+)-bound and free states at 1.75 and 1.45 Å resolution, respectively. Based on the crystallographic data, a Ca(2+) ion was coordinated by four residues within loop regions (the Ca(2+) site) and two water molecules in a tetragonal bipyramidal array. Furthermore, the binding of Ca(2+) to Cut190(S226P) induced large conformational changes in three loops, which were accompanied by the formation of additional interactions. The binding of Ca(2+) not only stabilized a region that is flexible in the Ca(2+)-free state but also modified the substrate-binding groove by stabilizing an open conformation that allows the substrate to bind easily. Thus, our study explains the structural basis of Ca(2+)-enhanced thermostability and activity in PET-degrading cutinase-like enzyme for the first time and found that the inactive state of Cut190(S226P) is activated by a conformational change in the active-site sealing residue, F106.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a ligand of EGF receptor, is involved in the growth and malignant progression of cancers. Cross-reacting material 197, CRM197, a non-toxic mutant of diphtheria toxin (DT), specifically binds to the EGF-like domain of HB-EGF and inhibits its mitogenic activity, thus CRM197 is currently under evaluation in clinical trials for cancer therapy. To develop more potent DT mutants than CRM197, we screened various mutant proteins of R domain of DT, the binding site for HB-EGF. A variety of R-domain mutant proteins fused with maltose-binding protein were produced and their inhibitory activity was evaluated in vitro. We found four R domain mutants that showed much higher inhibitory activity against HB-EGF than wild-type (WT) R domain. These R domain mutants suppressed HB-EGF-dependent cell proliferation more effectively than WT R domain. Surface plasmon resonance revealed their higher affinity to HB-EGF than WT R domain. CRM197(R460H) carrying the newly identified mutation showed increased cell proliferation inhibitory activity and affinity to HB-EGF. These results suggest that CRM197(R460H) or other recombinant proteins carrying newly identified mutation(s) in the R domain are potential therapeutics targeting HB-EGF., (© The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Background: Tongue squamous cell carcinoma (TSCC) is highly diverse, even in its early stages. This cancer is classified into three subtypes (superficial, exophytic, and endophytic) based on macroscopic appearance. Of these subtypes, the endophytic tumours have the worst prognosis because of their invasiveness and higher frequency of metastasis., Methods: To understand the molecular mechanism underlying the endophytic subtype and to identify biomarkers, we performed a comprehensive gene expression microarray analysis of clinical biopsy samples and also confirmed the clinical relevance of differential gene expression., Results: Expression of the parvin-beta (PARVB) gene and its encoded protein was significantly upregulated in endophytic-type TSCC. PARVB is known to play a critical role in actin reorganization and focal adhesions. Knockdown of PARVB expression in vitro caused apparent decreases in cell migration and wound healing, implying that PARVB has a crucial role in cell motility. Moreover, metastasis-free survival was significantly lower in patients with higher tumour expression of PARVB., Conclusions: These findings suggest that PARVB overexpression is a candidate biomarker for endophytic tumours and metastasis. This protein may be a clinically useful target for adjuvant TSCC therapy.

Only two polyethylene glycol terephthalate (PET)-degrading enzymes have been reported, and their mechanism for the biochemical degradation of PET remains unclear. To identify a novel PET-degrading enzyme, a putative cutinase gene (cut190) was cloned from the thermophile Saccharomonospora viridis AHK190 and expressed in Escherichia coli Rosetta-gami B (DE3). Mutational analysis indicated that substitution of Ser226 with Pro and Arg228 with Ser yielded the highest activity and thermostability. The Ca(2+) ion enhanced the enzyme activity and thermostability of the wild-type and mutant Cut190. Circular dichroism suggested that the Ca(2+) changes the tertiary structure of the Cut190 (S226P/R228S), which has optimal activity at 65-75 °C and pH 6.5-8.0 in the presence of 20 % glycerol. The enzyme was stable over a pH range of 5-9 and at temperatures up to 65 °C for 24 h with 40 % activity remaining after incubation for 1 h at 70 °C. The Cut190 (S226P/R228S) efficiently hydrolyzed various aliphatic and aliphatic-co-aromatic polyester films. Furthermore, the enzyme degraded the PET film above 60 °C. Therefore, Cut190 is the novel-reported PET-degrading enzyme with the potential for industrial applications in polyester degradation, monomer recycling, and PET surface modification. Thus, the Cut190 will be a useful tool to elucidate the molecular mechanisms of the PET degradation, Ca(2+) activation, and stabilization.

We identified a novel prognostic biomarker for the distant metastasis of colorectal cancer (CRC) using comprehensive combined copy number and gene expression analyses. Expression of mRNA in CRC tissue was profiled in 115 patients using an Affymetrix Gene Chip, and copy number profiles were generated for 122 patients using an Affymetrix 250K Sty array. Genes showing both upregulated expression and copy number gains in cases involving distant CRC metastasis were extracted as candidate biomarkers. Expression of the candidate gene mRNA was validated in 86 patients using quantitative reverse transcription polymerase chain reaction assays. Expression of the protein encoded by the candidate gene was assessed using immunohistochemical staining of tissue from 269 patients. The relationship between protein expression and clinicopathologic features was also examined. Following combined copy number and gene expression analyses, three genes linked to distant metastasis of CRC were extracted as candidate biomarkers. The expression of NUCKS1, reportedly overexpressed in several cancers other than CRC, was significantly higher in CRC tissue than in normal tissue. Overexpression of the NUCKS1 protein in CRC cells was found to be associated with significantly worse overall survival and relapse-free survival, indicating that NUCKS1 is an independent risk factor for CRC recurrence. The overexpression of NUCKS1 in cancer cells could be used as a CRC prognostic marker and might also be a target for treatment of this disease., (Copyright © 2012 UICC.)

Purpose: Metabolic syndrome (MS) is a group of recognized risk factors for the development of hepatocellular carcinoma (HCC) in patients with chronic liver disease. The aim of this study was to analyze the clinicopathological characteristics of HCC patients with MS and the risk factors for recurrence. Also, the aim was to investigate the cold shock protein: DNA-binding protein A (dbpA) expression in HCC patients with MS., Methods: A total of 243 patients who underwent curative resections for HCC were classified into two groups. dbpA expression was investigated in 66 HCC patients with MS and in 30 patients without MS by using real-time RT-PCR. Promoter methylation status was examined by using MS-PCR., Results: The incidence of metabolic factors affect the HCC significantly higher in non-B non-C patients than in hepatitis B virus (HBV) or hepatitis C virus (HCV) patients (P < 0.001). Univariate analysis of HCC patients with MS recurrence revealed aspartate amino transferase (AST), multiple tumors, liver damage, hepatic vein invasion, advanced cancer stages (P < 0.01), alpha-fetoprotein (AFP) and diabetes mellitus type II (P < 0.05) as risk factors. Multivariate analysis, AST, multiple tumors, and hepatic vein invasion (P < 0.01) were identified as independent factors for the recurrence. dbpA mRNA was higher in patients with MS than in those without MS (P = 0.016), and it was mostly upregulated in non-B non-C HCC patients with MS than in non-B non-C HCC patients without HBV or HCV. Especially, in HCC patients with diabetes mellitus type II, the mRNA and protein levels were highly upregulated. The dbpA expression was regulated by promoter methylation status (P < 0.05)., Conclusions: This study identifies that dbpA may accelerate the hepatocarcinogenesis in HCC patients with MS via inflammation-induced and oxidative stress pathways. The demethylation-related epigenetic activation may be one of the regulating factors for HCC patients with MS.

Background: Protein tyrosine phosphatase type IVA member 3 (PTP4A3/PRL-3), a metastasis-associated phosphatase, plays multiple roles in cancer metastasis. We investigated PTP4A3/PRL-3 expression and its correlation with the clinicopathological features and prognosis in hepatocellular carcinoma (HCC)., Methods: Gene expression profiles of PTP4A3/PRL-3 were obtained in poorly differentiated HCC tissues. The results were validated independently by TaqMan gene expression assays and immunohistochemical analysis., Results: According to the microarray profiles, PTP4A3/PRL-3 was upregulated in patients with poorly differentiated disease compared to patients with well-differentiated disease with hepatic backgrounds associated with hepatitis B or C. Validation analysis showed that the PTP4A3/PRL-3 mRNA and protein levels were significantly associated with poor differentiation (P<0.0001), high serum α-fetoprotein (P<0.01), high serum protein induced by vitamin K absence/antagonist-II (PIVKA-II), and hepatic vascular invasion (P<0.05). The expression of PTP4A3/PRL-3 protein was also correlated with advanced cancer stages (P<0.01); this resulted in a significantly poorer prognosis in both overall (P=0.0024) and recurrence-free survival (P=0.0227). According Cox regression univariate analysis, the positive expression of PTP4A3/PRL-3 was a poor risk prognostic factor (OS, P=0.0031; recurrence-free survival, P=0.0245). Cox regression multivariate analysis indicated that high PTP4A3/PRL-3 expression was an independent, unfavorable prognostic factor for overall survival (hazard ratio 0.542; P=0.048)., Conclusions: PTP4A3/PRL-3 might be closely associated with HCC progression, invasion, and metastasis. Its high expression had a negative impact on the prognosis of HCC patients. This strongly suggests that PTP4A3/PRL-3 should be considered as a prognostic factor. Further analysis should be pursued to evaluate it as a novel prognostic target.

Background/aims: Imidazoline α2-adrenergic agents exert complex effects on mammalian platelet aggregation. Although non-adrenergic, imidazoline (I) receptors have been revealed in human platelets, there is limited information about imidazoline's action on platelet aggregation. This study aimed to investigate aggregatory and anti-aggregatory effects of various imidazoline or non-imidazoline α-adrenergic agents on rabbit platelets., Methods: Aggregatory responses of agents on rabbit platelets were examined by turbidimetric method. Radioligand binding assay to platelet I1 and I2 receptors was performed using [(3)H]-clonidine and [(3)H]-idazoxan, respectively., Results: Aggregation was not induced by α-adrenoceptor agonists alone. Adrenaline and noradrenaline produced dose-dependent potentiation of ADP- or collagen-induced aggregation. Imidazoline adrenoceptor agonists clonidine and p-aminoclonidine also potentiated ADP-induced platelet aggregation. The α2-adrenoceptor antagonists and/or certain imidazoline adrenergic agents inhibited adrenaline-potentiated aggregation in a dose-dependent manner, whereas α1-adrenoceptor antagonists and non-imidazoline α-adrenergic agents were either ineffective or less effective in inhibiting adrenaline-potentiated aggregation. Rabbit platelets did not have I1 receptors, but had I2 receptors, indicating that adrenaline-potentiated platelet aggregation was inhibited by idazoxan, but not by imidazoline compounds clonidine and oxymetazoline., Conclusions/implications: These results demonstrated that α2-adrenoceptor-blocking agents and/or imidazoline α-adrenergic agents effectively inhibit adrenaline-potentiated platelet aggregation. It is proposed that imidazoline structure in part plays a role in the inhibition of adrenaline-potentiated aggregation., (Copyright © 2013 S. Karger AG, Basel.)

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors of the liver. Since postoperative recurrence and intrahepatic metastases occur frequently, the postoperative 5-year survival rate is low. To investigate the molecular mechanisms of HCC progression, mRNA as well as microRNA (miRNA) expression levels have been profiled in various studies. However, no previous study has comprehensively compared the expression of miRNAs in HCC patients with various clinical features using the tumor and surrounding non-tumor tissues and normal liver samples. In this study, we profiled the expression of miRNAs in tumor and non-tumor tissues from 40 HCC patients with heterogeneous pathogenesis and 6 surrounding non-tumor tissues from patients with metastatic liver cancer. To identify miRNAs specific to each disease state, we comprehensively compared the expression of miRNAs in various combinations. The results indicate that the expression of many known as well as novel miRNAs was altered in patients with the hepatitis C virus infection compared with those with the hepatitis B virus and without any virus infection. The following miRNAs were downregulated in the tumor and non-tumor tissues, and thus could serve as novel biomarkers for chronic liver diseases: miR-18b*, miR-296-5p, miR-557, miR-581, miR-625*, miR-1228, miR-1249 and miR-2116*. Similarly, miR-129*, miR-146b-3p and miR-448 are novel candidates for HCC biomarkers regardless of virus infection.

The prognostic assessment of patients with hepatocellular carcinoma (HCC) after resection is an important clinical issue. The present study investigated those genes associated with high serum alpha-fetoprotein (AFP), and their clinical significance, including prognosis and recurrence after hepatectomy. Based on gene expression analysis of 110 training HCC cases, 20 genes whose mRNA expression levels were significantly upregulated and 50 genes that were downregulated correlated with high serum AFP-associated HCC patients. Gene expression profiles of Villin1 (Vil1) were obtained in high serum AFP-associated HCC tumor tissues. In the present analysis, only VIL1 was significantly correlated with the recurrence of HCC. The results were validated independently using Taqman gene expression assays and immunostaining analysis. Results showed that the upregulation of VIL1 mRNA was also correlated with high serum PIVKAII, vascular invasion (P < 0.05), poor differentiation, an advanced cancer stage (P < 0.01) and recurrence-free survival (P = 0.017). The upregulation of VIL1 mRNA was observed more frequently in the early recurrence patients as compared to the late recurrence patients. Cox regression univariate and multivariate analyses indicated that high serum AFP levels (overall survival, HR 1.675, P = 0.002; FRS, HR 1.359, P = 0.039) and Vil1 protein expression (overall survival, HR 0.253, P = 0.009; FRS, HR 0.401, P = 0.041) were independent, unfavorable prognostic factors for overall and recurrence-free survival of patients. We demonstrated that the VIL1 gene is a potential candidate molecular marker for high serum AFP-associated HCC and a predictive candidate for the postoperative recurrence and poorer prognosis of HCC., (© 2012 Japanese Cancer Association.)

The prognosis of oral squamous cell carcinoma (OSCC) is significantly dependent on the existence of cervical lymph node metastasis (LNM), with the overall survival rate being much lower in patients with LNM. Primary causes and molecular mechanisms of LNM are still largely unclear. We hypothesized that factors related with cancer progress and/or prognosis in OSCC are revealed by genome-wide investigation of DNA copy number aberrations (CNAs). In order to find biomarkers for occult LNM of OSCC, we comprehensively investigated genomic DNAs from 60 OSCC patients using Affymetrix mapping arrays and statistically analyzed correlations between CNAs of genes and the presence of occult LNM in the patients. The genome-wide CNA study indicated significant correlations between the presence of occult LNM and CNAs of certain genes. Through a literature survey, we narrowed down the candidates and focused on loss of NKX3-1, which is a homeodomain-containing transcription factor. NKX3-1 is known as a tumor suppressor gene in prostate cancer but has never been reported in OSCC. Quantitative RT-PCR and immunohistochemistry (IHC) analyses also showed significantly lower expression of NKX3-1 in the cases with occult LNM, which was further validated by IHC analysis in independent cases. The survival analyses indicated that NKX3-1 loss is a significant risk factor to decrease the disease-free survival (DFS) and the overall survival (OS) rates. This is the first time that the significant association of NKX3-1 loss and occult LNM was indicated in OSCC. The present results suggest that loss of NKX3-1 may be a potential biomarker for occult LNM of OSCC.

We investigated the expression and promoter methylation of dbpA in human hepatocellular carcinoma (HCC) and examined their correlation with clinicopathological features. In 96 paired samples of HCC and adjacent non-tumorous liver, and 10 normal liver specimens, dbpA mRNA was quantified by real-time RT-PCR, and promoter methylation was examined by methylation-specific polymerase chain reaction and bisulfite sequencing. The results showed that dbpA mRNA expression levels were higher in HCC compared to corresponding non-tumor tissues (P<0.01) and higher in non-virus-associated HCC compared to virus-associated cases (P<0.01). dbpA promoter was methylated in 37.7% of HCC samples and the promoter methylation was significantly correlated with the low expression of dbpA in non-virus-associated HCC (P<0.01), but not in virus-associated HCC. Surprisingly, poor prognosis was more significantly associated with high dbpA expression in non-tumorous liver (P=0.018) but not with that in HCC. Non-tumorous tissues consist of chronic hepatitis or liver cirrhosis, and these conditions are the background of hepatocarcinogenesis, defined as the hypercarcinogenic state. Our results suggest that the high expression of dbpA in the hypercarcinogenic state is an indicator of poor prognosis.

Aim: Unearthing of silenced genes in colorectal cancer (CRC)., Materials and Methods: Oligonucleotide microarray was used in order to find changes in gene expression in five CRC cell lines before and after 5-aza-2'-Deoxycitidine treatment. Up-regulated genes were integrated with expression profile of matched colorectal tissue samples. Methylation-specific polymerase chain reaction and Real-time quantitative reverse transcription polymerase chain reaction were used to further analyze candidates using 15 CRC cell lines and 23 paired samples., Results: After applying study selection criteria for 68 genes obtained from integrated arrays, we identified 16 genes; apoptosis-stimulating of p53 protein 1(ASPP1) and Scavenger receptor class A, member 5 (SCARA5) were selected for further analysis. Methylation was only identified for SCARA5 in 20% of the cell lines and in 17% of tumor the samples. Down expression of SCARA5 was observed in CRC cell lines and in tumor samples compared to normal (p<0.001 and p=0.001, respectively)., Conclusion: Genome-wide screening identifies genes potentially affected by methylation in CRC. SCARA5 may have a role in tumorigenesis in CRC.

The purpose of this study was to find a methylation-related gene that could become a biomarker or therapeutic target in colorectal carcinoma (CRC). We screened candidate genes suspected to be silenced by DNA methylation using cDNA microarray analysis. To investigate the clinical significance of one candidate gene (UNC5B), we analyzed the correlation between mRNA expression and clinicopathological features using clinical tissue samples. Moreover, methylation specific PCR analysis was performed to reveal whether the promoter region was methylated in CRC cell lines. We found 75 candidate genes that were potentially suppressed by DNA methylation in CRC. We focused on UNC5B, a possible tumor suppressor gene and regulator of apoptosis known to be inactivated in CRC. The mRNA expression analysis using tissue samples revealed that UNC5B mRNA was down-expressed in about 20% of CRC patients, and the patients with low-UNC5B-expression tumors showed a significantly higher recurrence rate after curative surgery. According to the univariate and multivariate analysis, low UNC5B expression was an independent risk factor for postoperative recurrence in stage I, II and III CRC patients. Furthermore, patients with low expression of UNC5B in tumors had significantly poorer prognosis than those with high expression of UNC5B. Although UNC5B mRNA expression was restored by the demethylation treatment in CRC cell lines, the promoter region of UNC5B was not methylated. UNC5B is a potential biomarker for the selection of patients with high risk of postoperative recurrence and worse prognosis in CRC.

Tumor stroma drives the growth and progression of cancers. A heparin-binding epidermal growth factor-like growth factor, HB-EGF, is an EGF receptor ligand that stimulates cell growth in an autocrine or paracrine fashion. While elevated expression of HB-EGF in cancer cells and its contribution to tumor progression are well documented, the effects of HB-EGF expression in the tumor stroma have not been clarified. Here, we show that HB-EGF is expressed in stromal fibroblasts where it promotes cancer cell proliferation. In uterine cervical cancers, HB-EGF was detected immunohistochemically in the stroma proximal to the cancer epithelium. Proliferation of cervical cancer cells in vitro was enhanced by coculture with fibroblasts isolated from tumor tissues of patients with cervical cancer. Inhibition of HB-EGF function or treatment with platelet-derived growth factor (PDGF) inhibitors abrogated cancer cell growth enhanced by cervical cancer-associated fibroblast (CCF) coculture. Furthermore, tumor formation in a mouse xenograft model was enhanced by cotransplantation of CCF or mouse embryonic fibroblasts, but not with embryonic fibroblasts from HB-EGF-deficient mice. Conversely, conditioned medium from cancer cells induced HB-EGF expression in CCF. Mechanistic investigations established that PDGF was the primary factor responsible. Together, our findings indicate that HB-EGF and PDGF reciprocally mediate the interaction of cancer cells with cancer-associated fibroblasts, promoting cancer cell proliferation in a paracrine manner that has implications for novel combinatorial cancer therapies., (©2011 AACR.)

Colorectal cancer is one of the leading causes of cancer death worldwide. To identify candidates for biomarkers and therapeutic targets, we investigated the proteome of colorectal cancer tissues. Using 2D-DIGE in combination with our original large format electrophoresis apparatus, we compared surgically resected normal and tumor tissues from 53 patients with colorectal cancer. We focused on proteins with an alkaline pI using IPG gels for the alkaline range. We observed 1687 protein spots, and found 100 spots with statistical (p<0.01) and significant (>2-fold) differences between the normal and the tumor tissue groups. Among these 100 protein spots, five showed a different intensity between tumor tissues from the stage-II and the stage-III patients. MS experiments revealed that these 100 protein spots corresponded to 58 unique proteins. These included six proteins which had not been previously reported to be associated with colorectal cancer. Among these proteins, five were not reported in any type of malignancy. IEF/western blotting confirmed the differences in protein expression between the normal and the tumor tissues. These results may provide an insight for biomarker development and drug target discovery in colorectal cancer., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Purpose: This study aimed to identify a novel biomarker or a target of treatment for colorectal cancer (CRC)., Experimental Design: The expression profiles of cancer cells in 104 patients with CRC were examined using laser microdissection and oligonucleotide microarray analysis. Overexpression in CRC cells, especially in patients with distant metastases, was a prerequisite to select candidate genes. The mRNA expression of candidate genes was investigated by quantitative reverse transcriptase PCR (RT-PCR) in 77 patients as a validation study. We analyzed the protein expression and localization of the candidate gene by immunohistochemical study and investigated the relationship between protein expression and clinicopathologic features in 274 CRC patients., Results: Using microarray analysis, we identified 6 candidate genes related to distant metastases in CRC patients. Among these genes, osteoprotegerin (OPG) is known to be associated with aggressiveness in several cancers through inhibition of apoptosis via neutralization of the function of TNF-related apoptosis-inducing ligand. The mRNA expression of OPG in cancer tissues was significantly higher in patients with distant metastases than those without metastases. Overexpression of OPG protein was associated with significantly worse overall survival and relapse-free survival. Moreover, overexpression of the OPG protein was an independent risk factor for CRC recurrence., Conclusion: Overexpression of OPG may be a predictive biomarker of CRC recurrence and a target for treatment of this disease., (©2011 AACR.)

Increased expression of heparin-binding EGF-like growth factor (HB-EGF) and membrane-type matrix metalloproteinase-1 (MT1-MMP) is frequently associated with various types of malignant tumor. HB EGF-like growth factor has been reported to promote the malignant progression of ovarian carcinoma. Based on this finding, inhibition of HB-EGF activity with CRM197 is now under phase I clinical evaluation. On the other hand, MT1-MMP expressed in ovarian carcinoma cells is thought to promote invasion and growth of tumor cells by degrading the extracellular matrix. However, we recently demonstrated that co-expression of MT1-MMP and HB-EGF in gastric carcinoma cells leads to cleavage of HB-EGF within its N-terminal heparin-binding region, converting it into a potent heparin-independent growth factor. In this study, we evaluated the importance of regulation of HB-EGF by MT1-MMP in clinical samples of ovarian carcinoma. We detected co-expression of HB-EGF and MT1-MMP in clear cell ovarian carcinoma tissues, particularly at the invasion front and in tumor cells that had disseminated into the ascites, whereas HB-EGF alone was expressed in non-invasive borderline ovarian tumor tissue. Furthermore, a soluble HB-EGF fragment that corresponds to that processed by MT1-MMP was detected in malignant ascites obtained from patients with metastatic ovarian carcinoma. Ovarian carcinoma cells that express MT1-MMP and HB-EGF exhibited enhanced cell growth in a 3D-collagen matrix and anchorage-independent growth in suspension. These results indicate that MT1-MMP co-expressed with HB-EGF in ovarian carcinoma cells potentiates the activity of HB-EGF to promote invasive tumor growth and spreading in vivo., (© 2010 Japanese Cancer Association.)

Microarray analysis has been applied to comprehensively reveal the abnormalities of DNA copy number (CN) and gene expression in human cancer research during the last decade. These analyses have individually contributed to identify the genes associated with carcinogenesis, progression, metastasis of tumor cells and poor prognosis of cancer patients. However, it is known that the correlation between profiles of CN and gene expression does not highly correlate. Factors which determine the degree of correlation remain largely unexplained. To investigate one such factor, we performed trend analyses between the lengths of CN segments and corresponding gene expression profiles from microarray data in hepatocellular carcinoma (HCC) and colorectal carcinoma (CRC). Significant correlations were observed in CN gain of HCC and CRC (p<0.05). The trend of the CN loss showed a significant correlation in HCC although there was no correlation between the length of CN loss segments and gene expression in CRC. Our findings suggest that the influence of CN on gene expression highly depends on the length of CN region, especially in the case of CN gain. To the best of our knowledge, this is the first study describing the correlation between lengths of CNA segments and expression profiles of corresponding genes.

Background: Variety of information relating between genome and the pathological findings in disease will yield a wealth of clues to discover new function, the role of genes and pathways, and future medicine. In addition to molecular information such as gene expression and genome copy number, detailed clinical information is essential for such systematic omics analysis., Results: In order to provide a basic platform to realize a future medicine based on the integration of molecular and clinico-pathological information of disease, we have developed an integrated clinical omics database (iCOD) in which comprehensive disease information of the patients is collected, including not only molecular omics data such as CGH (Comparative Genomic Hybridization) and gene expression profiles but also comprehensive clinical information such as clinical manifestations, medical images (CT, X-ray, ultrasounds, etc), laboratory tests, drug histories, pathological findings and even life-style/environmental information. The iCOD is developed to combine the molecular and clinico-pathological information of the patients to provide the holistic understanding of the disease. Furthermore, we developed several kinds of integrated view maps of disease in the iCOD, which summarize the comprehensive patient data to provide the information for the interrelation between the molecular omics data and clinico-pathological findings as well as estimation for the disease pathways, such as three layer-linked disease map, disease pathway map, and pathome-genome map., Conclusions: With these utilities, our iCOD aims to contribute to provide the omics basis of the disease as well as to promote the pathway-directed disease view. The iCOD database is available online, containing 140 patient cases of hepatocellular carcinoma, with raw data of each case as supplemental data set to download. The iCOD and supplemental data can be accessed at http://omics.tmd.ac.jp/icod&#95;pub&#95;eng.