Dynamics of the suprapatellar bursa during knee joint extension.

PURPOSE: The suprapatellar bursa is located in the proximal deep layer of the patella and is thought to reduce tissue friction by changing from a single-membrane structure to a double-membrane structure during knee joint motion. However, the dynamics of the suprapatellar bursa have only been inferred from positional relationships, and the actual dynamics have not been confirmed. METHODS: Dynamics of the suprapatellar bursa during knee joint motion were observed in eight knees of four Thiel-fixed cadavers and the angle at which the bursa begins to show a double membrane was revealed. The flexion angles of knee joints were measured when the double-membrane structure of the suprapatellar bursa began to appear during knee joint extension. RESULTS: The suprapatellar bursa changes from a single membrane to a double-membrane structure at 91 ± 4° of flexion, when the knee joint is moved from a flexed position to an extended position. CONCLUSION: The suprapatellar bursa may be involved in limitations to knee joint range of motion and pain at an angle of approximately 90°. Further studies are needed to verify whether the same dynamics are observed in living subjects.

A New Boron-Rhodamine-Containing Carboxylic Acid as a Sugar Chemosensor.

We propose a boron-rhodamine-containing carboxylic acid (BRhoC) substance as a new sugar chemosensor. BRhoC was obtained by the Friedel-Crafts reaction of 4-formylbenzoic acid and N,N-dimethylphenylboronic acid, followed by chloranil oxidation. In an aqueous buffer solution at pH 7.4, BRhoC exhibited an absorption maximum (Absmax) at 621 nm. Its molar absorption coefficient at Absmax was calculated to be 1.4 × 105 M-1 cm-1, and it exhibited an emission maximum (Emmax) at 644 nm for the excitation at 621 nm. The quantum yield of BRhoC in CH3OH was calculated to be 0.16. The borinate group of BRhoC reacted with a diol moiety of sugar to form a cyclic ester, which induced a change in the absorbance and fluorescence spectra. An increase in the D-fructose (Fru) concentration resulted in the red shift of the Absmax (621 nm without sugar and 637 nm with 100 mM Fru) and Emmax (644 nm without sugar and 658 nm with 100 mM Fru) peaks. From the curve fitting of the plots of the fluorescence intensity ratio at 644 nm and 658 nm, the binding constants (K) were determined to be 2.3 × 102 M-1 and 3.1 M-1 for Fru and D-glucose, respectively. The sugar-binding ability and presence of a carboxyl group render BRhoC a suitable building block for the fabrication of highly advanced chemosensors.

A positive genetic selection for transmembrane domain mutations in HRD1 underscores the importance of Hrd1 complex integrity during ERAD.

Misfolded proteins in the endoplasmic reticulum (ER) are retrotranslocated to the cytosol for ubiquitination and degradation by the proteasome. During this process, known as ER-associated degradation (ERAD), the ER-embedded Hrd1 ubiquitin ligase plays a central role in recognizing, ubiquitinating, and retrotranslocating scores of lumenal and integral membrane proteins. To better define the mechanisms underlying Hrd1 function in Saccharomyces cerevisiae, several model substrates have been developed. One substrate is Sec61-2, a temperature sensitive allele of the Sec61 translocation channel. Cells expressing Sec61-2 grow at 25 °C because the protein is stable, but sec61-2 yeast are inviable at 38 °C because the mutated protein is degraded in a Hrd1-dependent manner. Therefore, deleting HRD1 stabilizes Sec61-2 and hence sec61-2hrd1∆ double mutants are viable at 38 °C. This unique phenotype allowed us to perform a non-biased screen for loss-of-function alleles in HRD1. Based on its importance in mediating substrate retrotranslocation, the screen was also developed to focus on mutations in sequences encoding Hrd1's transmembrane-rich domain. Ultimately, a group of recessive mutations was identified in HRD1, including an ensemble of destabilizing mutations that resulted in the delivery of Hrd1 to the ERAD pathway. A more stable mutant resided in a buried transmembrane domain, yet the Hrd1 complex was disrupted in yeast expressing this mutant. Together, these data confirm the importance of Hrd1 complex integrity during ERAD, suggest that allosteric interactions between transmembrane domains regulate Hrd1 complex formation, and provide the field with new tools to define the dynamic interactions between ERAD components during substrate retrotranslocation.

Prognostic value of immune factors in the tumor microenvironment of patients with pancreatic ductal adenocarcinoma.

BACKGROUND: Both activated tumor-infiltrating lymphocytes (TILs) and immune-suppressive cells, such as regulatory T cells (Tregs), in the tumor microenvironment (TME) play an important role in the prognosis of patients with pancreatic ductal adenocarcinoma (PDAC). METHODS: The densities of TILs, programmed death receptor 1 (PD-1) + T cells, and forkhead box P3 (Foxp3) + T cells were analyzed by immunohistochemical staining. The associations of the immunological status of the PDAC microenvironment with overall survival (OS) time and disease-free survival (DFS) time were evaluated. RESULTS: PDAC patients with a high density of TILs in the TME or PD-1-positive T cells in tertiary lymphoid aggregates (TLAs) demonstrated a significantly better prognosis than those with a low density of TILs or PD-1-negativity, respectively. Moreover, PDAC patients with high levels of Foxp3-expressing T cells showed a worse prognosis than those with low levels of Foxp3-expressing T cells. Importantly, even with a high density of the TILs in TME or PD-1-positive T cells in TLAs, PDAC patients with high levels of Foxp3-expressing T cells showed a worse prognosis than patients with low levels of Foxp3-expressing T cells. A PDAC TME with a high density of TILs/high PD-1 positivity/low Foxp3 expression was an independent predictive marker associated with superior prognosis. CONCLUSION: Combined assessment of TILs, PD-1+ cells, and Foxp3+ T cells in the TME may predict the prognosis of PDAC patients following surgical resection.

Postoperative outcomes of gastric carcinoma with lymphoid stroma.

BACKGROUND: Gastric carcinoma with lymphoid stroma (GCLS) is a rare subtype of gastric cancer. There have been several reports demonstrating the favorable prognosis of early GCLS without lymph node metastasis (LNM) compared with gastric adenocarcinomas. However, it remains unknown whether advanced GCLS (AGCLS) with LNM has a similar prognosis and clinicopathological features. This study aimed to assess the clinicopathological features of GCLS of all stages. METHODS: We retrospectively assessed 375 patients who were pathologically diagnosed with gastric cancer and underwent curative surgical resection at Tokyo Medical University, Japan, between September 2013 and October 2019. Of these patients, 357 (95.2%) patients were pathologically diagnosed with gastric adenocarcinomas, and 18 (4.8%) patients were diagnosed with GCLS. The GCLS patients (n = 18) were compared with the gastric adenocarcinoma patients (non-GCLS patients, control) (n = 357) in terms of their clinicopathological features and clinical outcome. RESULTS: The GCLS patients showed significantly predominant upper gastric locations (P = 0.003), lower number of LNM (P = 0.01), and better overall survival rate than the non-GCLS patients (P = 0.029). The predominant upper gastric locations (P = 0.0002), lower number of LNM (P = 0.003), and better overall survival rate (P = 0.04) were significantly correlated in the AGCLS with LNM patients compared with the advanced non-GCLS with LNM patients. For survival analyses, surgical procedure, tumor location, and numbers of positive LNM were adjusted by 1:1 propensity score matching. After adjustment, the overall survival rate was significantly higher in the AGCLS group than in the advanced non-GCLS group (P = 0.03). CONCLUSION: AGCLS has distinct clinicopathological features and clinical behavior that are similar to those of early GCLS. AGCLS with LNM patients showed a significantly lower number of LNM and a better survival rate than advanced non-GCLS with LNM patients. To our knowledge, this study is the first report to describe the clinicopathological features of AGCLS.

The segment IV approach: a useful method for achieving the critical view of safety during laparoscopic cholecystectomy in patients with anomalous bile duct.

BACKGROUND: The critical view of safety (CVS) method can be achieved by avoiding vasculo-biliary injury resulting from misidentification during laparoscopic cholecystectomy (LC). Although achieving the CVS has become popular worldwide, there is no established standardized technique to achieve the CVS in patients with an anomalous bile duct (ABD). We recently reported our original approach for securing the CVS using a new landmark, the diagonal line of the segment IV of the liver (D-line). The D-line is an imaginary line that lies on the right border of the hilar plate. The cystic structure can be securely isolated along the D-line without any misidentification, regardless of the existence of an ABD. We named this approach the segment IV approach in LC. METHODS: In this study, we adopted the segment IV approach in patients with an ABD. RESULTS: From October 2015 to June 2020, 209 patients underwent LC using the segment IV approach. Among them, three (1.4%) were preoperatively diagnosed with an ABD. The branching point of the cystic duct was the posterior sectional duct, anterior sectional duct, or left hepatic duct in each patient. The CVS was achieved in all cases without any complications. CONCLUSION: It is a promising technique, especially even for patients with an ABD during LC.

[Long-Term Recurrence-Free Survival in a Patient Who Received Chemotherapy for Multiple Metastases of Rectal Cancer Including the Lesser-Curvature Lymph Node Metastasis-A Case Report].

A 49-year-old man was preoperatively diagnosed with rectosigmoid carcinoma, c-T4a, N3, M1b, Stage Ⅳb. On CT, lymph node swelling outside that area, including lesser-curvature lymph nodes(LNS), and liver metastases were seen. Laparoscopic high anterior resection was performed with the aim of local control. Additionally, D3 dissection and LNS sampling were performed. The tumor had invaded the bladder wall, and removed LNS were positive for metastasis. The final diagnosis was f-T4b, N3, M1b, Stage Ⅳb. One month after surgery, a CV port was implanted, and chemotherapy was initiated for unresectable cancer. The regimen was capecitabine and oxaliplatin(CAPOX)plus bevacizumab(BEV). After 5 courses, the patient was hospitalized for a CV thrombus that had occurred, and his chemotherapy was withdrawn for approximately 1 month while he was receiving antithrombotic therapy. After discharge, BEV was discontinued, and he received CAPOX alone. Bleeding from a pituitary adenoma was seen after a total of 19 courses. He was hospitalized for 2 months for the treatment, including surgery. A clinical complete response was determined based on CT and PET-CT performed after chemotherapy had been withdrawn for approximately 3 months. For approximately 1 year since the chemotherapy was discontinued, progression-free survival has been maintained.

[Examination of Treatment Results and Risk Factors for Recurrence of Advanced Lower Rectal Cancer].

BACKGROUND: The standard treatment in Japan for advanced lower rectal cancer is total mesorectal excision(TME)plus lateral lymph node dissection(LLND). However, the standard treatment in Western countries is preoperative treatment plus TME. There have been some discussions on preoperative chemotherapy and chemoradiation therapy. This study was aimed at identifying the prognostic factors of recurrence after curative surgery for advanced lower rectal cancer. METHODS: A total of 54 patients with advanced lower rectal cancer who had undergone curative operation at our department from 2010 to 2015 were retrospectively analyzed, excluding patients with both LLND and preoperative therapy. The primary endpoint of this study was the 5-year recurrence-free survival(5RFS). RESULTS: The overall 5RFS was 57.6%. The univariate analysis demonstrated that lymph node metastasis(p=0.038)and radial margin(RM, p=0.015)were significant risk factors, with a 5RFS of 39.7% and 0%, respectively. The multivariate analysis revealed that only RM significantly affected 5RFS(p= 0.009). CONCLUSION: Our results suggest that securing an adequate circumferential resection margin together with proper surgical technique and preoperative therapy are important for decreasing postoperative recurrence rates of advanced lower rectal cancer.

[Preoperative Therapy for Advanced Lower Rectal Cancer in Our Department].

PURPOSE: This study was aimed at evaluating the oncologic outcomes of our preoperative treatment strategies for cStage Ⅱ/Ⅲ lower rectal cancer. At our hospital, neoadjuvant chemotherapy is administered for patients with bulky mesenteric lymph nodes on pretreatment imaging, and neoadjuvant chemoradiotherapy is administered for patients whose circumferential radial or distal margin cannot be secured because of strong local extension. METHODS: Thirty patients who underwent preoperative therapy followed by total mesorectal excision for cStage Ⅱ/Ⅲ lower rectal cancer were retrospectively analyzed from October 2010 to October 2015. RESULTS: Twenty-five patients underwent neoadjuvant chemotherapy, and 5 patients underwent neoadjuvant chemotherapy. Tumor recurrence occurred in 10 patients at local(5 patients)and distant(5 patients)sites. The 5-year recurrence-free survival(5RFS)was 63.9%. CONCLUSION: We performed preoperative therapy in poor-risk locally advanced lower rectal cancer and obtained good results.

Effects of physical therapy on blood pressure in daily clinical practice-a pilot study.

[Purpose] Most exercise therapy procedures induce hemodynamic changes and could be a cardiovascular risk. This pilot study investigated factors that induce an exaggerated increase in blood pressure during exercise therapy. [Participants and Methods] We measured the blood pressure and pulse rate before and after exercise therapy for ambulation on days 1, 2, and 7 of the exercise therapy in patients (n=23; age, 69 ± 11 years) who were hospitalized for a stroke or an orthopedic surgery. [Results] Each participant's blood pressure and pulse rate were significantly increased after the exercise therapy. Regression analysis demonstrated that the increase in systolic blood pressure was independently predicted by body weight and pulse rate before the exercise therapy. In the logistic regression analysis, age and body weight were independent predictors of the exaggerated increase in systolic blood pressure (fourth quartile). [Conclusion] A significant increase in blood pressure was induced by exercise therapy. There was a correlation between systolic blood pressure increase and pulse rate before the exercise therapy. Old age or increased body weight predicts exaggerated increase in blood pressure during exercise therapy.

[A Case of Contralateral Oculomotor Nerve Palsy due to Internal Carotid Artery-Anterior Choroidal Artery Ruptured Aneurysm].

Sudden oculomotor palsy with severe headache is known to suggest a ruptured ipsilateral internal carotid artery aneurysm. We encountered a case of contralateral oculomotor nerve palsy due to internal carotid artery-anterior choroidal artery ruptured aneurysm. A 63-year-old woman presented with severe headache and sudden right oculomotor palsy. Computed tomography(CT)showed subarachnoid hemorrhage, and three-dimensional CT showed a left internal carotid artery-anterior choroidal artery aneurysm. We performed neck clipping via a left pterional approach. After the surgery, right oculomotor palsy was not observed. We think the causes of oculomotor nerve palsy in this case were hematoma and elevated intracranial pressure. Once these factors were removed, we think that oculomotor nerve palsy was not observed.

[Availability of Early Balloon Kyphoplasty for the Treatment of Osteoporotic Compression Fractures].

INTRODUCTION: Japan has many patients with osteoporosis; however, only about one-fifth of these patients receive treatment. Although some treatment guidelines exist for osteoporosis, the number of newly diagnosed patients with osteoporotic compression fractures is increasing and protocols for treatment of osteoporotic compression fractures vary from one hospital to another. This study aims to investigate the availability of early balloon kyphoplasty(BKP)in relation to our treatment strategy for osteoporotic compression fractures. METHODS: In our hospital, patients diagnosed with osteoporotic compression fractures were treated conservatively with a corset and rehabilitation. In cases where pain was prolonged and computed tomography(CT)imaging revealed formation of a cavity, we performed BKP. We divided the patients admitted between April 2016 and December 2016 with osteoporotic compression fractures into 2 groups, based on whether they received conservative treatment or BKP. We assessed the patients' age, fracture site, CT and MRI findings, bone density, Numerical Rating Scale(NRS), duration of hospital stay, and outcomes. RESULTS: In the BKP group, the number of Th12 and L1 compression fractures was higher than fractures to other vertebral bodies. No difference was observed in bone density, improvement of NRS, and outcomes between groups. CT cavity signs were more frequently observed in the BKP group than in the conservative group. CONCLUSIONS: This study establishes a correlation between the appearance of CT cavity sign and prolonged pain, which increases the likelihood of a patient undergoing BKP. The CT cavity sign and prolonged pain could be indicators of pre-stage pseudoarthrosis. BKP performed in the early stages of a fracture is safe and does not result in complications. However, BKP should be performed according to appropriate indications, including delayed neurological deficit, pain, and reduced bone adhesion.

Pallidotomy for Writer's Cramp after Failed Thalamotomy.

BACKGROUND/AIMS: Although many reports have confirmed the effects of stereotactic thalamotomy for writer's cramp, pallidotomy for writer's cramp is yet to be investigated. METHODS: After a 22-year-old woman with writer's cramp had undergone stereotactic thalamotomy twice, symptomatic relief was only temporary. Because her dystonic symptoms spread around the proximal part of the upper limb, she underwent unilateral pallidotomy 21 months after the second thalamotomy. RESULTS: Unilateral pallidotomy improved her dystonic symptoms without any adverse effects immediately after the surgery. During a follow-up observation of 1 year, no recurrent writer's cramp was observed. CONCLUSION: For writer's cramp, when symptoms cannot be improved by thalamotomy, pallidotomy may achieve an effective outcome.

The miR-506-Induced Epithelial-Mesenchymal Transition is Involved in Poor Prognosis for Patients with Gastric Cancer.

BACKGROUND: MicroRNAs have roles in the regulation of the epithelial-mesenchymal transition (EMT). Findings have shown that miR-506 inhibits the expression of SNAI2 and that low expression of miR-506 is associated with poor prognoses in ovarian and breast cancers. This study investigated the role of miR-506 in survival and the EMT in patients with gastric cancer. METHODS: In this study, miR-506 and SNAI2 mRNA levels were measured in 141 cases of gastric cancer by quantitative reverse transcription polymerase chain reaction, and the protein expressions of SNAI2 and E-cadherin in 39 cases were validated by immunohistochemical analysis. Next, the associations between their expression levels and clinicopathologic factors were evaluated. In addition, cell proliferation, migration, and luciferase activity of the 3' untranslated region (UTR) of SNAI2 were analyzed using pre-miR-506 precursor in two human gastric cancer cell lines. RESULTS: Low expression of miR-506 was significantly correlated with poor overall survival in both the univariate analysis (P = 0.016) and the multivariate analysis (P < 0.05). Low miR-506 expression was significantly correlated with high SNAI2 expression (P = 0.009) and poorly differentiated type (P = 0.015). In vitro, miR-506 suppressed SNAI2 expression by binding to its 3'UTR, resulting in increased expression of E-cadherin (P < 0.05), verified by immunohistochemical analysis. Pre-miR-506 transfected cells showed significantly suppressed cell proliferation and migration (P < 0.05) compared with the control cells. CONCLUSIONS: The EMT was directly suppressed by miR-506, and its low expression was an independent prognostic factor in gastric cancer patients. The data indicated that miR-506 may act as a tumor suppressor and could be a novel therapeutic agent.

Overexpression of Transcription Termination Factor 1 is Associated with a Poor Prognosis in Patients with Colorectal Cancer.

BACKGROUND: RNA polymerase 1 transcription termination factor (TTF1) mediates the transcription of ribosomal RNA (rRNA). In the current study, we investigated the clinical and biological significance of the TTF1 gene in colorectal cancer (CRC). METHODS: The expression of TTF1 messenger RNA (mRNA) in tumor and normal tissues from 136 patients with CRC was examined by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). We also performed in vitro cell proliferation and migration assays in TTF1-expressing CRC cells. The biological role of TTF1 in CRC was further elucidated using gene set enrichment analysis (GSEA) with CRC samples. RESULTS: TTF1 expression was significantly higher in tumor tissues than in corresponding normal tissues (p = 0.016). In clinicopathological analysis, the high-TTF1 expression group showed a higher incidence of liver metastasis and lymphatic invasion than the low-TTF1 expression group (p < 0.05), and tended to have more frequent venous invasion than the low-TTF1 expression group. Furthermore, the high-TTF1 expression group had a significantly poorer prognosis than the low-TTF1 expression group (p = 0.011). Moreover, overexpression of TTF1 enhanced the proliferation and migration capacity of CRC cells in vitro. GSEA revealed that TTF1 was significantly associated with the RAS and MYC pathways, and this observation was confirmed in samples from 136 patients with CRC. CONCLUSION: TTF1 was involved in cancer progression via the RAS and MYC pathways in CRC, suggesting that TTF1 may be a prognostic indicator and therapeutic target in CRC.

A Long Non-coding RNA Activated by Transforming Growth Factor-ß is an Independent Prognostic Marker of Gastric Cancer.

BACKGROUND: A recent study reported that long non-coding RNA activated by TGF-ß (lncRNA-ATB) induced epithelial-mesenchymal transition (EMT) through the transforming growth factor-ß (TGF-ß)/miR-200s/ZEB axis in hepatocellular carcinoma. Herein, we focused on the clinical significance of lncRNA-ATB in gastric cancer (GC) patients. MATERIALS AND METHODS: Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed to examine expression of lncRNA-ATB, miR-200b, and miR-200c in GC tissues (n = 183). Patients were divided into high and low lncRNA-ATB expression groups using a cutoff of lncRNA-ATB/GAPDH ≥0.60 or <0.60 to determine the clinicopathological significance of lncRNA-ATB in GC. Moreover, we evaluated the expression of TGF-ß, lncRNA-ATB, miR-200s, and ZEB1 in GC cell lines by qRT-PCR. GC cell lines were treated by recombinant TGF-ß1 or TGF-ß receptor inhibitor to examine morphologic changes and genetic alterations, such as lncRNA-ATB, miR-200s, and ZEB1 levels, with respect to the EMT phenotype. RESULTS: The high lncRNA-ATB group experienced a lower overall survival rate compared with the low lncRNA-ATB group, and multivariate analysis indicated that lncRNA-ATB was an independent prognostic factor (hazard ratio 3.50; 95 % CI 1.73-7.44; p = 0.0004). miR-200c levels were lower and ZEB1 levels were higher in the high lncRNA-ATB group than in the low lncRNA-ATB group. Treatment with TGF-ß in GC cell lines resulted in morphological EMT changes, upregulation of lncRNA-ATB and ZEB1, and downregulation of miR-200c and CDH1. SB431542 reduced lncRNA-ATB expression. CONCLUSION: LncRNA-ATB plays an important role in EMT to promote invasion and metastasis through the TGF-ß/miR-200s/ZEB axis, resulting in a poor prognosis in GC. LncRNA-ATB is a novel biomarker of lncRNA, indicative of a poor prognosis in GC patients.

Downregulation of PRRX1 Confers Cancer Stem Cell-Like Properties and Predicts Poor Prognosis in Hepatocellular Carcinoma.

BACKGROUND: Downregulation of paired related homeobox 1 (PRRX1) is associated with the acquisition of cancer stem cell (CSC)-like properties and poor prognosis in cancers. The purpose of this study is to clarify the role of PRRX1 expression in predicting prognosis and mediating CSC-like properties in hepatocellular carcinoma (HCC). METHODS: The association between PRRX1 expression and overall survival (OS) of patients with HCC was analyzed in three independent datasets: 62 resected primary cases, 242 cases from GSE14520, and 162 cases from The Cancer Genome Atlas (TCGA). A cell line expressing PRRX1 (HuH7) was established for the functional analyses. The ability to form spheres, the expression levels of the hepatic CSC surface markers (CD13, CD133, and EpCAM), in vitro chemosensitivity to 5-fluorouracil (FU), and radiosensitivity were evaluated. RESULTS: Univariate and multivariate analyses showed that the 5-year OS of the low PRRX1 expression group was significantly poorer than that of the high PRRX1 expression group (P = 0.024 and P = 0.045, respectively). Consistent with this, the low PRRX1 expression group in GSE14520 and TCGA datasets showed significantly shorter OS (P = 0.027 and P = 0.010, respectively). Gene set enrichment analysis on GSE14520 and TCGA datasets indicated that downregulation of PRRX1 was correlated with the stemness signature. The number of spheres and the expression levels of CSC markers were significantly decreased when PRRX1 was expressed. Moreover, PRRX1 impaired resistance to 5-FU and radiation. CONCLUSIONS: Downregulation of PRRX1 expression contributes to the poor prognosis of patients with HCC through acquisition of CSC-like properties.

Facilitation of adipocyte differentiation of 3T3-L1 cells by debrominated tetrabromobisphenol A compounds detected in Japanese breast milk.

Tetrabromobisphenol A (TeBBPA) is widely used type of brominated flame retardant. In this study, we newly synthesized materials for the debrominated congeners, 2,2',6-tribromobisphenol A (TriBBPA), 2,2'-dibromobisphenol A (2,2'-DiBBPA), 2,6-dibromobisphenol A (2,6-DiBBPA), and 2-monobromobisphenol A (MoBBPA) and evaluated the actual extent of contamination with bisphenol A (BPA), TeBBPA and debrominated congeners in Japanese breast milk samples. TriBBPA was detected at higher levels than that of TeBBPA, while DiBBPA and MoBBPA were detected at lower levels than that of TeBBPA. This observation suggested that humans are exposed to debrominated congeners, which might cause adverse effects. Contamination of the congeners in breast milk was concern about risk infant health, having vulnerable defense system. As pilot study by in vitro experiment, we assessed the toxic potency of debrominated congeners by studying their effect on adipocyte differentiation in 3T3-L1 cells. We observed 2,6-DiBBPA, TriBBPA and TeBBPA elevated the lipid accumulation and adipocyte-specific protein 2 expression in a manner dependent on the number of substituted bromines. Moreover, PPARγ transcriptional activities increased in a dose-dependent manner in the presence of 2,6-DiBBPA and TriBBPA as well as TeBBPA. Our study clarified that TeBBPA and its debrominated congeners accumulated in breast milk and the debrominated congeners promoted adipocyte differentiation, showing that a comprehensive evaluation of the influences of these compounds including the debrominated congeners of TeBBPA on health in infants is necessary.

Immunization method for multi-pass membrane proteins using highly metastatic cell lines.

A novel method using metastatic breast cancer cell lines was established for producing monoclonal antibodies (mAbs) against multi-span membrane proteins. Grafting of metastatic cells (MCF7-14) into the mammary gland of BALB/cJ/nu/nu mice induced splenic hypertrophy (1.6-3.0×10(8)cells/spleen [n=6]). More than half of the mAbs against MCF7-14 cells reacted with the cell membrane. Inducing production of antibodies against the extracellular domain of multi-pass membrane proteins is difficult. Because the protein structure becomes more complex as the number of transmembrane domains increases, preparing antigens for immunization in which the original structure is maintained is challenging. Using highly metastatic MDA-MB231 cells as the host cell line, we produced mAbs against a 12 transmembrane protein, solute carrier family 6 member 6 (SLC6A6), as a model antigen. When SLC6A6-overexpressing MDA-MB231 cells were grafted into nude mice, the number of splenocytes increased to 2.7-11.4×10(8)cells/spleen (n=10). Seven mAb-producing clones that not only recognized the extracellular domain of SLC6A6 but also were of the IgG subclass were obtained. Immunocytochemistry and flow cytometry analyses revealed that these mAbs recognized the native form of the extracellular domain of SLC6A6 on the cell surface. Our novel immunization method involving highly metastatic cells could be used to develop therapeutic mAbs against other multi-pass membrane proteins.

Up-regulation of NEK2 by microRNA-128 methylation is associated with poor prognosis in colorectal cancer.

BACKGROUND: NIMA-related kinase 2 (NEK2), an enzyme involved in the development and progression of cancer, is abnormally expressed in a wide variety of human cancers, including colorectal cancer (CRC), and is known to have roles in cell division and mitotic regulation through centrosome splitting. We investigated the clinical significance of NEK2 in CRC. In particular, we examined miR-128 expression, which is thought to target NEK2. METHODS: We measured NEK2 mRNA and miR-128 levels in clinical samples by quantitative reverse transcription real-time PCR and analyzed the associations between NEK2 levels, miR-128 levels, clinicopathological factors, and prognoses. Furthermore, we performed in vitro assays using a pre-miR-128 precursor and conducted miR-128 methylation analyses. RESULTS: MiR-128 inhibited NEK2 expression and cancer cell proliferation via cell cycle arrest. Moreover, miR-128 was silenced by DNA methylation. Increased NEK2 expression was associated with serosal invasion, lymphatic invasion, and peritoneal dissemination. Patients with high NEK2 expression also had significantly poorer prognoses. Multivariate analysis indicated that high NEK2 expression was an independent prognostic factor for survival. Patients with high miR-128 expression had significantly lower NEK2 expression and lower recurrence rates than those with low miR-128 expression. CONCLUSIONS: NEK2 may be an independent prognostic factor for CRC and was regulated by miR-128, a microRNA that was subjected to epigenetic regulation. Thus, this miR-128/NEK2 pathway may be a prospective therapeutic target for patients with CRC.