Quantitative Elemental Analysis of a Single Cell by Using Inductively Coupled Plasma-Mass Spectrometry in Fast Time-Resolved Analysis Mode.

The elemental composition of a single yeast, green alga, or red blood cell (RBC) was precisely determined by using inductively coupled plasma-mass spectrometry (ICP-MS) operating in fast time-resolved analysis (TRA) mode. The technique is known as single-cell (SC)-ICP-MS. Phosphorus, sulfur, magnesium, zinc, and iron were detected in the three types of cell. The elemental composition of yeast and green alga obtained by SC-ICP-MS was consistent with results obtained from conventional ICP-MS measurements following acid digestion of the cells. Slight differences were found in the measured values between SC-ICP-MS and the conventional ICP-MS results for RBC. However, the SC-ICP-MS results for S and Fe in RBC were closer to the estimated values for these elements that were calculated from the level of hemoglobin in RBCs. The data suggest that SC-ICP-MS is suitable for the analysis of various cell types, namely, fungus, plant, and animal cells.

Enhanced Universal Quantification of Biomolecules Using Element MS and Generic Standards: Application to Intact Protein and Phosphoprotein Determination.

Tools that provide absolute quantification of biomolecules, particularly of proteins and their post-translational modifications, without needing suitable specific standards, are urgently demanded nowadays. To this end, we have significantly improved the recently introduced strategy based on CH4 addition to the plasma for absolute quantification of biomolecules using HPLC-ICP-MS. Addition of CO2 has been optimized and finally selected as a safer, more efficient quantitative strategy that is able to provide constant (<6% error) signal response factor for the six elements assayed (S, P, As, Se, Br, I) under compromised conditions. In the particular case of absolute protein quantification, accuracy and precision attainable for S-based absolute determination of intact proteins using internal and external S-generic standards were compared. Potential for real sample analysis was demonstrated by the high-sensitivity analysis of toxins present in snake venoms. Finally, multielemental speciation capabilities of the approach have been also demonstrated through P and S simultaneous analysis in phosphoproteomics. Simultaneous accurate determination of both absolute protein amount and corresponding phosphorylation degree for intact ß-casein, and even impurity traces of κ and α-s1 isoforms present, has been successfully achieved using a simple mixture of inorganic P and S standards. The lowest detection limits (<1 fmol protein) ever published for S- and P-based intact protein quantification with ICP-MS are reported.

A phenotypic drug discovery study on thienodiazepine derivatives as inhibitors of T cell proliferation induced by CD28 co-stimulation leads to the discovery of a first bromodomain inhibitor.

A phenotypic screening of thienodiazepines derived from a hit compound found through a binding assay targeting co-stimulatory molecules on T cells and antigen presenting cells successfully led to the discovery of a thienotriazolodiazepine compound (7f) possessing potent immunosuppressive activity. A chemical biology approach has succeeded in revealing that 7f is a first inhibitor of epigenetic bromodomain-containing proteins. 7f is expected to become an anti-cancer agent as well as an immunosuppressive agent.

Agreement in the Postural Assessment of Older Adults by Physical Therapists Using Clinical and Imaging Methods.

Postural assessment is one of the indicators of health status in older adults. Since the number of older adults is on the rise, it is essential to assess simpler methods and automated ones in the future. Therefore, we focused on a visual method (imaging method). The purpose of this study is to determine the degree of agreement between the imaging method and the palpation and visual methods (clinical method). In addition, the influence of differences in the information content of the sagittal plane images on the assessment was also investigated. In this experiment, 28 sagittal photographs of older adults whose posture had already been assessed using the clinical method were used. Furthermore, based on these photographs, 28 gray and silhouette images (G and S images) were generated, respectively. The G and S images were assessed by 28 physical therapists (PTs) using the imaging method. The assessment was based on the Kendall classification, with one of four categories selected for each image: ideal, kyphosis lordosis, sway back, and flat back. Cross-tabulation matrices of the assessments using the clinical method and imaging method were created. In this table, four categories and two categories of ideal and non-ideal (KL, SB, and FB) were created. The agreement was evaluated using the prevalence-adjusted bias-adjusted kappa (PABAK). In addition, sensitivity and specificity were calculated to confirm the reliability. When comparing the clinical and imaging methods in the four posture categories, the PABAK values were -0.14 and -0.29 for the S and G images, respectively. In the case of the two categories, the PABAK values were 0.57 and 0.5 for the S and G images, respectively. The sensitivity and specificity were 86% and 57% for the S images and 76% and 71% for the G images, respectively. The four categories show that the imaging method is difficult to assess regardless of the image processing. However, in the case of the two categories, the same assessment of the clinical method applied to the imaging method for both the S and G images. Therefore, no differences in image processing were observed, suggesting that PTs can identify posture using the visual method.

Rim Enhancement on Contrast-Enhanced CT as a Predictor of Prognosis in Patients with Pancreatic Ductal Adenocarcinoma.

This study investigated the utility of imaging features, such as rim enhancement on contrast-enhanced CT (CECT), in predicting the prognosis of pancreatic ductal adenocarcinoma (PDAC). This retrospective study included 158 patients (84 men; mean age, 68 years) with pathologically confirmed PDAC. The following imaging features were evaluated on CECT by two radiologists: tumor size, tumor attenuation, and the presence of rim enhancement. Cox proportional hazards analysis was performed to identify the imaging and clinicopathological features for predicting disease-free survival (DFS) and overall survival (OS). Pathological features were compared with the presence of rim enhancement. Among the 158 patients, 106 (67%) underwent curative surgery (surgery group) and 52 (33%) received conservative treatment (non-surgery group). Rim enhancement was observed more frequently in the non-surgery group than in the surgery group (44% vs. 20%; p < 0.001). Rim enhancement showed significant associations with shorter DFS and OS in the surgery group (hazard ratios (HRs), 3.03 and 2.99; p < 0.001 and p = 0.003, respectively), whereas tumor size showed significant associations with shorter OS (HR per 1 mm increase, 1.08; p < 0.001). PDACs with rim enhancement showed significant associations with higher histological tumor grades (p < 0.001). PDAC with rim enhancement on CECT could predict poorer prognosis and more aggressive tumor grades.

In vivo selection of high-metastatic subline of bladder cancer cell and its characterization.

The majority of deaths associated with solid tumors are caused by tumor metastasis. To prevent metastasis, it is vital to understand its detailed process. In hematogenous metastasis of bladder cancer, some cancer cells disseminating into blood circulation extravasate into the lung tissues to form metastases. To study the molecular basis of the lung metastasis of bladder cancer, we employed an in vivo selection system that mimics hematogenous metastasis of bladder cancer on a low-metastatic bladder cancer cell line (KK-47). We have successfully isolated a high-metastatic bladder cancer subline, KK-47HM4, from KK-47 cells. We characterized KK-47HM4 in in vitro experimental systems. No significant difference in growth rate and susceptibility to NK cell attack between KK-47 and KK-47HM4 cells was observed. However, KK-47HM4 exhibited the higher capacities of Matrigel Matrix invasion and transendothelial invasion than KK-47. These results suggest that the extravasation of KK-47HM4 cells was enhanced among the multiple steps of the lung metastasis of bladder cancer. Our cDNA microarray analysis identified 67 genes whose expression was up- or downregulated in KK-47HM4 cells compared with KK-47 cells. This analysis data implied that one possible cause for enhanced extravasation of KK-47HM4 is its higher adhesion to extracellular matrix proteins. KK-47HM4 is the first bladder cancer subline with enhanced extravasation potential using the in vivo selection system. The information provided by our cDNA microarray analysis using KK-47HM4 will be useful for further investigation into the molecular basis of extravasation of cancer cells.

[Adult mature teratoma of the testis: a case report].

A 33-year-old man visited our hospital complaining of painless left scrotal swelling. Serum alphafetoprotein (AFP) and human chorionic gonadotropin-beta (HCG-ß) were within normal range. Computed tomography revealed a heterogeneous tumor in the left scrotum and para-aortic lymph node swelling. We diagnosed the tumor as stage IIA testicular cancer and performed left high inguinal orchiectomy. Histopathological diagnosis was mature teratoma with no associated malignant germ cell tumor. The patient was followed-up without adjuvant chemotherapy because the size of para-aortic lymph nodes was remarkably reduced one month after the orchiectomy. The patient must be followed up carefully for possible metastasis.

A Case of Rectal Dissemination of Gastric Cancer Diagnosed by Simultaneous 18 F-FDG PET/MRI.

ABSTRACT: Metastatic rectal cancer is rare and difficult to differentiate from primary rectal cancer. A 79-year-old man with a rectal mass detected by CT during postoperative follow-up of gastric cancer underwent 18 F-FDG PET/MRI. Fused PET/MRI images revealed a lower FDG uptake within the mass, which appeared to surround the outside of the rectum, than in the rectal wall, suggesting rectal dissemination of gastric cancer. PET/MRI was useful for differentiating between mass and rectal wall uptake, because of the high contrast resolution of MRI and precise image fusion made possible by simultaneous image acquisition.

[Adrenal pheochromocytoma with multiple neurofibromatosis on the trunk].

We report a case of adrenal pheochromocytoma in a patient with neurofibromatosis type 1 (NF1). A 65-year-old female patient was admitted to our hospital for examination of a right adrenal mass. The adrenal tumor was incidentally discovered by abdominal computed tomography during examination for hypertension in another hospital. She had large multiple neurofibromatous lesions and café-au-lait spots on the trunk. We thought that it was difficult to make a skin incision on normal skin. Serum and urinary catecholamines were markedly increased. Magnetic resonance imaging revealed a solid round tumor 3 cm in diameter, located in the right adrenal gland. Laparoscopic right adrenalectomy was performed. Serum and urinary catecholamines returned to the normal range on post-operative day 10. Laparoscopic surgery may be a good option for NF1 patients with pheochromocytoma, especially those who had multiple neurofibromatosis on the trunk.

Core 2 N-acetylglucosaminyltransferase-1 expression induces aggressive potential of testicular germ cell tumor.

We studied orchiectomy specimens from 130 patients immuhistochemically with testicular germ cell tumor (TGCT) using anti-core 2 N-acetylglucosaminyltransferase-1 (C2GnT-1) antibody. The incidence of C2GnT-1 positivity in stage I disease (29.5%, 21/71) was significantly lower than that in higher stages (84.7%, 50/59) (P < 0.001, chi(2) test). This significant difference was also found when the cases were divided into seminoma and NSGCT according to histopathological classification. Kaplan-Meier plots and the log rank test showed that in the patients with stage I seminoma, C2GnT-1-positive cases had a higher risk for recurrence (P < 0.001). This was also the case with the patients with stage I NSGCT (P < 0.001). To determine whether C2GnT-1 promotes aggressive behavior of cancer cells, a C2GnT-1-negative human TGCT cell line, JKT-1, was stably transfected with a mammalian expression vector containing C2GnT-1 cDNA. In vitro assays revealed that JKT-1-C2 cells are more invasive than mock transfectants, although there are no differences in proliferation activity. When orthotopically inoculated into athymic nude mice, JKT-1-C2 cells produced larger testicular tumors extending to the retroperitoneum with mesenteric metastasis, while mock transfectants produced small tumors without metastasis (P < 0.01, Mann-Whitney's U-test). When injected via the tail vein, JKT-1-C2 cells produced a number of metastatic lung foci. In contrast, mock transfectants produced a small number of nodules (p < 0.01, Mann-Whitney's U-test). These results strongly suggest that C2GnT-1 enhances the metastatic potential of TGCT and may be a reliable biomarker for aggressive potential of TGCT.

[The giant schwannoma in the pelvic cavity: a case report].

A 62-year-old man presented with a giant tumor in the pelvic cavity that was incidentally revealed by abdominal ultrasonography. Abdominal magnetic resonance imaging showed the heterogenous tumor in the pelvis with cystic components. The tumor was 10.8 × 10.5 × 11.7 cm in diameter and adhered to the sacral wall. The tumor was extirpated following diagnosis as a benign neurogenic tumor by needle biopsy. The pelvic cavity was occupied by the tumor rigidly adhered to the sacrum. The histopathological diagnosis of the specimen was benign schwannoma, type Antoni A.

[Ganglioneuroma with calcification mimicking adrenal tumor: a case report].

A 28-year-old woman was referred to our hospital complaining of upper abdominal discomfort. The patient had been receiving medical treatment for hypertension. Computed tomography revealed a 30 mm solid tumor with calcification in the left adrenal gland and a 8 mm nodule in the right adrenal gland. Endocrinological examinations revealed no activity of either adrenal mass. The left adrenal tumor was extirpated, because malignancy of the tumor was not ruled out. Histopathological examination proved that the tumor was ganglioneuroma arising from the extra-adrenal retroperitoneum.

[Experience of laparoscopic surgery of pheochromocytoma found on induction for hemodialysis].

A 64-year-old female patient had been receiving hemodialysis since June 2008. A left adrenal mass, 2.5 cm in diameter, was incidentally found by screening computed tomography at the initiation of hemodialysis. Plasma epinephrine and norepinephrine were increased to 1.21 ng/ml and 4.71 ng/ml, respectively. In the scintiscan using ¹²³I-metaiodobenzylguanidine (MIBG), accumulation of the radionuclide in the left adrenal tumor region was confirmed. Laparoscopic left adrenalectomy was performed without peri-operative complications under the diagnosis of left pheochromocytoma. The elevated catecholamines and the blood pressure were restored after surgery.

[Multiple inflammatory pseudotumor of the paranephric fat].

A 57-year-old man was referred to our hospital because of a paranephric tumor detected by preoperative computed tomography (CT) for the ascending colon carcinoma. Since the paranephric tumor was small, surgical treatment was performed only for the colon carcinoma. The follow-up CT showed increase in size and number of paranephric tumor masses. It was suspected to be a soft tissue malignant tumor. Left radical nephrectomy was performed. Pathological findings revealed inflammatory pseudotumor of the paranephric fat. To our knowledge, this is the 4th case with inflammatory pseudotumor of the paranephric fat.

Integration of temporal subtraction and nodule detection system for digital chest radiographs into picture archiving and communication system (PACS): four-year experience.

Since May 2002, temporal subtraction and nodule detection systems for digital chest radiographs have been integrated into our hospital's picture archiving and communication systems (PACS). Image data of digital chest radiographs were stored in PACS with the digital image and communication in medicine (DICOM) protocol. Temporal subtraction and nodule detection images were produced automatically in an exclusive server and delivered with current and previous images to the work stations. The problems that we faced and the solutions that we arrived at were analyzed. We encountered four major problems. The first problem, as a result of the storage of the original images' data with the upside-down, reverse, or lying-down positioning on portable chest radiographs, was solved by postponing the original data storage for 30 min. The second problem, the variable matrix sizes of chest radiographs obtained with flat-panel detectors (FPDs), was solved by improving the computer algorithm to produce consistent temporal subtraction images. The third problem, the production of temporal subtraction images of low quality, could not be solved fundamentally when the original images were obtained with different modalities. The fourth problem, an excessive false-positive rate on the nodule detection system, was solved by adjusting this system to chest radiographs obtained in our hospital. Integration of the temporal subtraction and nodule detection system into our hospital's PACS was customized successfully; this experience may be helpful to other hospitals.

Synthesis and evaluation of (11)C-labeled (S)-N-[[1-(2-phenylethyl) pyrrolidin-2-yl]methyl]-3-methylthiobenzamide as a PET 5-HT(1A) receptor ligand.

We prepared 5-HT(1A) receptor ligands (S)-N-[[1-(2-phenylethyl)pyrrolidin-2-yl]methyl]-3-[11C]methylthiobenzamide ([11C](S)-PPMMB) (Ki = 4.3 nM) and the less active [(11)C](R)-PPMMB (Ki = 160 nM) by reduction of the disulfide dimer and subsequent [(11)C]methylation of demethyl (S)- and (R)-PPMMB, respectively. Both radioligands showed similar brain distribution in mice with relatively higher affinity for the hippocampus being rich in 5-HT(1A) receptors than for other brain regions. Uptake of [(11)C](S)-PPMMB was not reduced by carrier-loading nor by pretreatment with 5-HT(1A) receptor ligands. [(11)C](S)-PPMMB is therefore not a suitable radioligand for mapping 5-HT(1A) receptors using positron emission tomography.

Blood group antigen-targeting peptide suppresses anti-blood group antibody binding to antigen in renal glomerular capillaries after ABO-incompatible blood reperfusion.

BACKGROUND: Antibody-mediated rejection after ABO-incompatible kidney transplantation (ABO-I KTx) is a major barrier to transplantation success. The advent of immunosuppressive therapy has markedly improved graft survival in ABO-I KTx. However, compared with normal KTx, clinical conditions during ABO-I KTx are difficult to control because of overimmunosuppression. To reduce the need for immunosuppression, we aimed to develop a novel blood group antigen-neutralizing therapy. METHODS: We screened for an ABO blood group antigen-targeting peptide (BATP) by screening of T7 phage-displayed peptide library. After screening, hemagglutination inhibition assays, enzyme-linked immunosorbent assay, and cytotoxicity assay were used to analyze the blood group antigen-blocking effect and toxicity of BATP. We also tested the inhibitory effects on anti-blood group antibody binding in normal human kidney tissues blocked with BATP and excised kidneys perfused ex vivo with BATP. RESULTS: We identified six peptide sequences that efficiently suppressed hemagglutination of red blood cells by anti-ABO blood group antibodies and binding of these antibodies to ABO histo-blood group antigens in kidney tissues. Surprisingly, ex vivo perfusion of BATP in kidneys excised from renal cell carcinoma patients caused significant suppression of anti-blood group antibody binding to antigen and IgG and IgM deposition in renal glomerular capillaries after ABO-I blood reperfusion. CONCLUSIONS: These data indicate that A/B blood group antigens on red blood cells and in kidney tissues may be neutralized by BATP. This approach may enable the development of a novel blood group antigen-neutralizing therapy to overcome the challenges of ABO-I KTx.

Skin perfusion pressure is a prognostic factor in hemodialysis patients.

Peripheral arterial disease (PAD) is common in hemodialysis patients and predicts a poor prognosis. We conducted a prospective cohort study to identify risk factors for PAD including skin perfusion pressure (SPP) in hemodialysis patients. The cohort included 373 hemodialysis patients among 548 patients who received hemodialysis at Oyokyo Kidney Research Institute, Hirosaki, Japan from August 2008 to December 2010. The endpoints were lower limb survival (peripheral angioplasty or amputation events) and overall survival of 2 years. Our results showed that <70 mmHg SPP was a poor prognosis for the lower limb survival and overall survival. We also identified age, history of cardiovascular disease, presence of diabetes mellitus, smoking history, and SPP < 70 mmHg as independent risk factors for lower limb survival and overall survival. Then, we constructed risk criteria using the significantly independent risk factors. We can clearly stratify lower limb survival and overall survival of the hemodialysis patients into 3 groups. Although the observation period is short, we conclude that SPP value has the potential to be a risk factor that predicts both lower limb survival and the prognosis of hemodialysis patients.

Efficacy of SMART Stent Placement for Salvage Angioplasty in Hemodialysis Patients with Recurrent Vascular Access Stenosis.

Vascular access stenosis is a major complication in hemodialysis patients. We prospectively observed 50 patients in whom 50 nitinol shape-memory alloy-recoverable technology (SMART) stents were used as salvage therapy for recurrent peripheral venous stenosis. Twenty-five stents each were deployed in native arteriovenous fistula (AVF) and synthetic arteriovenous polyurethane graft (AVG) cases. Vascular access patency rates were calculated by Kaplan-Meier analysis. The primary patency rates in AVF versus AVG at 3, 6, and 12 months were 80.3% versus 75.6%, 64.9% versus 28.3%, and 32.3% versus 18.9%, respectively. The secondary patency rates in AVF versus AVG at 3, 6, and 12 months were 88.5% versus 75.5%, 82.6% versus 61.8%, and 74.4% versus 61.8%, respectively. Although there were no statistically significant difference in patency between AVF and AVG, AVG showed poor tendency in primary and secondary patency. The usefulness of SMART stents was limited in a short period of time in hemodialysis patients with recurrent vascular access stenosis.