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TRF1 facilitates the replication of telomeric DNA in part by recruiting the BLM helicase, which can resolve G-quadruplexes on the lagging-strand template. Lagging-strand telomeres lacking TRF1 or BLM form fragile telomeres—structures that resemble common fragile sites (CFSs)—but how they are formed is not known. We report that analogous to CFSs, fragile telomeres in BLM-deficient cells involved double-strand break (DSB) formation, in this case by the SLX4/SLX1 nuclease. The DSBs were repaired by POLD3/POLD4-dependent break-induced replication (BIR), resulting in fragile telomeres containing conservatively replicated DNA. BIR also promoted fragile telomere formation in cells with FokI-induced telomeric DSBs and in alternative lengthening of telomeres (ALT) cells, which have spontaneous telomeric damage. BIR of telomeric DSBs competed with PARP1-, LIG3-, and XPF-dependent alternative nonhomologous end joining (alt-NHEJ), which did not generate fragile telomeres. Collectively, these findings indicate that fragile telomeres can arise from BIR-mediated repair of telomeric DSBs.

Telomere crisis contributes to cancer genome evolution, yet only a subset of cancers display breakage-fusion-bridge (BFB) cycles and chromothripsis, hallmarks of experimental telomere crisis identified in previous studies. We examine the spectrum of structural variants (SVs) instigated by natural telomere crisis. Eight spontaneous post-crisis clones did not show prominent patterns of BFB cycles or chromothripsis. Their crisis-induced genome rearrangements varied from infrequent simple SVs to more frequent and complex SVs. In contrast, BFB cycles and chromothripsis occurred in MRC5 fibroblast clones that escaped telomere crisis after CRISPR-controlled telomerase activation. This system revealed convergent evolutionary lineages altering one allele of chromosome 12p, where a short telomere likely predisposed to fusion. Remarkably, the 12p chromothripsis and BFB events were stabilized by independent fusions to chromosome 21. The data establish that telomere crisis can generate a wide spectrum of SVs implying that a lack of BFB patterns and chromothripsis in cancer genomes does not indicate absence of past telomere crisis., Telomere crisis has been shown to induce chromothripsis and breakage fusion bridge (BFB) cycles in vitro. Here, the authors show that telomere crisis generates a much broader spectrum of structural variations, implying that cancers without chromothripsis and BFB cycles could have emerged from telomere crisis.

Background Several studies showed that first-line bevacizumab plus chemotherapy for HER2-negative metastatic breast cancer improves progression-free survival and tumor response rate but not overall survival. MERiDiAN trial evaluated plasma vascular endothelial growth factor-A (VEGF-A) prospectively as a predictive biomarker for bevacizumab efficacy in metastatic breast cancer. However, results of this trial do not support using baseline plasma VEGF-A to identify patients benefitting most from bevacizumab. We measured baseline serum VEGF-A level from stored blood samples of metastatic breast cancer patient with treated bevacizumab plus paclitaxel as fist-line and later line therapy, and evaluated a correlation between serum VEGF-A level and efficacy of bevacizumab and prognosis of breast cancer patients tread with bevacizumab, retrospectively. Patients and methods We examined blood samples from 57 metastatic breast cancer patients treated with bevacizumab and paclitaxel, after obtaining written informed consent. And, we evaluated a correlation between baseline serum VEGF-A level and time to treatment failure (TTF) and overall survival (OS). We also compared the serum VEGF-A level of response group (CR and PR) and that of non-response group (SD and PD). Results Baseline serum level of VEGF-A ranged from 80 to 2079 pg/ml. Cases of treatment line were as follows: first-line, 22 cases (38.6%); second line, 11 cases (19.3%) and third-line and the later line, 24 cases (42.1%). The cutoff identified by ROC curve analysis that was able to differentiate response group and non-response group in first-line setting was 360pg/ml for serum VEGF-A. And, we separated high serum VGEF-A group and low serum VEGF-A group of patients treated with bevacizumab plus paclitaxel. In patients treated as first line therapy, median TTF was 4.0 months with high serum VGEF-A group versus 5.0 months with low serum VEGF-A group, and median OS was 12 months with high serum VGEF-A group versus 11months with low serum VEGF-A group. There were no significant differences in both TTF and OS in first line setting. In patients treated as second line and later line therapy, median TTF was 2.8 months with high serum VGEF-A group versus 7.1 months with low serum VEGF-A group, and median OS was 6.4 months with high serum VGEF-A group versus 12.7 months with low serum VEGF-A group. The prognosis of high serum VEGF-A group was significantly worse than that of low serum group in both TTF and OS. The serum VEGF-A level of response group was tend to be higher than that of non-response group in first line setting, and was lower in second and later line setting. However, there were no significant differences. Conclusion In this study, serum VEGF-A cannot be a predictor for efficacy of bevacizumab plus paclitaxel as first line therapy for metastatic breast cancer patients. On the other hand, there was a possibility that high serum level of VEGF-A can be a poor prognostic factor in late line therapy setting of bevacizumab. Citation Format: Tozuka K, Nagai SE, Matsumoto H, Hayashi Y, Kubo K, Tsuboi M, Sato A, Takai K, Wang X, Yamada Y, Inoue K. Prognostic and predictive value of serum level of vascular endothelial growth factor-A in metastatic breast cancer patients treated with bevacizumab plus paclitaxel [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-12-17.

Background Catheter ablation for atrial fibrillation (AF) with high contact force (CF) has been reported to lead to the gaps after pulmonary vein isolation (PVI), and the appearance of the gaps depends on the site in the left atrium (LA). Purpose The aim of this study is to clarify the relationship between the gaps appearance and high CF. Methods In the clinical study, 74 consecutive patients (39 males, age 71±9, 46 with paroxysmal AF) underwent conventional PVI with a point-by-point radiofrequency (RF) ablation using a CF sensing catheter (TactiCath SETM). RF energy (J), number of RF point, Lesion index (LSI), force-time integral (FTI), average CF (g), and the degree of LA depression with high CF were evaluated. In the experimental study (figure), the relationship between the ablation area and the degree of depression with high CF using bovine left ventricular wall. Results In the clinical study, the gaps were found in 175 of 1182 sites after first path PVI ablation. The CF in the gap sites was higher than without the gap sites in RPV roof, anterior RSPV and anterior RPV carina (18.9±6.8 vs 15.0±5.7g p=0.0262, 26.4±11.6 vs 19.5±6.0g p=0.0029, 22.7±6.4 vs 19.1±5.5g, p=0.0161). Meanwhile, there were no differences in RF energy, number of RF point, LSI, FTI. High CF (≥30g) showed a depression of 3 mm or more in only RPV roof and anterior RPV (figure). In the experimental study, the ablation range narrowed as the indentation deepened with more than 30g CF (r=0.6417, p=0.0625). Conclusion Catheter ablation for AF with high contact force might lead to the gaps in RPV roof and anterior RPV site by the reduction of the ablation area due to depression caused by the pressure. Extension of each contact force Funding Acknowledgement Type of funding source: None

Telomere shortening is a presumed tumor suppressor pathway that imposes a proliferative barrier (the Hayflick limit) during tumorigenesis. This model predicts that excessively long somatic telomeres predispose to cancer. Here, we describe cancer-prone families with two unique TINF2 mutations that truncate TIN2, a shelterin subunit that controls telomere length. Patient lymphocyte telomeres were unusually long. We show that the truncated TIN2 proteins do not localize to telomeres, suggesting that the mutations create loss-of-function alleles. Heterozygous knock-in of the mutations or deletion of one copy of TINF2 resulted in excessive telomere elongation in clonal lines, indicating that TINF2 is haploinsufficient for telomere length control. In contrast, telomere protection and genome stability were maintained in all heterozygous clones. The data establish that the TINF2 truncations predispose to a tumor syndrome. We conclude that TINF2 acts as a haploinsufficient tumor suppressor that limits telomere length to ensure a timely Hayflick limit.

In DNA repair, the resection of double-strand breaks dictates the choice between homology-directed repair—which requires a 3′ overhang—and classical non-homologous end joining, which can join unresected ends1,2. BRCA1-mutant cancers show minimal resection of double-strand breaks, which renders them deficient in homology-directed repair and sensitive to inhibitors of poly(ADP-ribose) polymerase 1 (PARP1)3–8. When BRCA1 is absent, the resection of double-strand breaks is thought to be prevented by 53BP1, RIF1 and the REV7–SHLD1–SHLD2–SHLD3 (shieldin) complex, and loss of these factors diminishes sensitivity to PARP1 inhibitors4,6–9. Here we address the mechanism by which 53BP1–RIF1–shieldin regulates the generation of recombinogenic 3′ overhangs. We report that CTC1–STN1–TEN1 (CST)10, a complex similar to replication protein A that functions as an accessory factor of polymerase-α (Polα)–primase11, is a downstream effector in the 53BP1 pathway. CST interacts with shieldin and localizes with Polα to sites of DNA damage in a 53BP1- and shieldin-dependent manner. As with loss of 53BP1, RIF1 or shieldin, the depletion of CST leads to increased resection. In BRCA1-deficient cells, CST blocks RAD51 loading and promotes the efficacy of PARP1 inhibitors. In addition, Polα inhibition diminishes the effect of PARP1 inhibitors. These data suggest that CST–Polα-mediated fill-in helps to control the repair of double-strand breaks by 53BP1, RIF1 and shieldin.

Background: To avoid tissue expander exposure following mastectomy flap necrosis, several methods for covering expander by autologous flap in cases of immediate breast reconstruction have been reported. These methods are classified into two groups, complete or partial expander coverage. Two methods have potential risks of postoperative complications following: insufficient lower pole expansion and cranial migration in complete coverage methods, and lateral migration in partial coverage methods. However, the comparisons of complication rates between these two methods have not been reported. This study aims to compare the incidence of expander exposure following mastectomy flap necrosis and expander migration between two methods. Methods: A retrospective review of 93 patients (99 breasts) who underwent immediate expander-based breast reconstruction was performed. Patients were divided into two groups, complete or partial expander coverage by autologous flaps. In both groups, expanders were placed into subpectral position. In partial coverage group, the lateral borders of pectralis major muscles were sutured to the mastectomy skin flaps. If the skin flap was too thin to be sutured, the serratus anterior muscro-fascial flap was dissected and sutured to the lateral border of pectralis major muscle to cover the expander completely. Allograft products were not used in both groups. Demographics, intraoperative findings, and postoperative complications were compared between two groups. Results: Of the 99 breasts, 56 underwent complete expander coverage and 43 underwent partial coverage. Mastectomy flap necrosis rate was higher in the complete coverage group (Complete 14.3% versus Partial 0%; p=0.0091), however, there was no incidence of expander exposure in both groups. Lateral migration rate was higher in the partial coverage group (Complete 0% versus Partial 9.3%; p=0.033). There was no difference in cranial migration rate between two groups (Complete 12.5% versus Partial 2.3%; p=0.133). Conclusions: The thinness of the mastectomy flaps was considered to provide the higher incidence of mastectomy flap necrosis in the complete coverage group. The complete expander coverage reduced lateral migration rate and prevented expander exposure in cases of mastectomy flap necrosis. Citation Format: Kubo K, Hamahata A, Tozuka K, Tsuboi M, Hayashi Y, Takai K, Saito T, Sakurai H, Matsumoto H. A complication analysis between complete and partial tissue expander coverage using autologous flaps in cases of immediate breast reconstruction [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-13-11.

Studies to date have not completely determined the factors influencing hydrogen embrittlement of ferrite/bainite X80 pipeline steel. Hydrogen embrittlement susceptibility was evaluated based on fracture strain in tensile testing. We conducted a thermal desorption analysis to measure the amount of tracer hydrogen corresponding to that of lattice defects. Hydrogen embrittlement susceptibility and the amount of tracer hydrogen significantly increased with decreasing crosshead speed. Additionally, a significant increase in the formation of hydrogen-enhanced strain-induced lattice defects was observed immediately before the final fracture. In contrast to hydrogen-free specimens, the fracture surface of the hydrogen-charged specimens exhibited shallower dimples without nuclei, such as secondary phase particles. These findings indicate that the presence of hydrogen enhanced the formation of lattice defects, particularly just prior to the occurrence of final fracture. This in turn enhanced the formation of shallower dimples, thereby potentially causing premature fracture of X80 pipeline steel at lower crosshead speeds.

Background: Neoadjuvant chemotherapy (NAC) with taxanes, followed by fluorouracil, epirubicin, and cyclophosphamide (FEC), with concurrent trastuzumab is known to achieve a high pCR rate of more than 60% for HER2-positive breast cancer (BC) as well as good prognoses in those obtaining pCR. On the other hand, the prognostic significance of tumor-infiltrating lymphocytes (TILs) has recently been described in triple-negative BC. However, the prognostic and predictive values of TILs in HER2-positive BC remain unclear. In the present study, we examined the grades of TILs in pre-treatment cancer tissues and residual tumors after NAC with trastuzumab, and also investigated its predictive utility for pCR and prognostic power for HER2-positive BC. Patients and Methods: A total of 128 Japanese women with HER2-positive BC received either paclitaxel or docetaxel followed by FEC, with concomitant trastuzumab. The proportional grades of stromal (Str)-TILs in pre-treatment biopsy specimens and residual tumors after NAC with trastuzumab were determined as follows: low grade (0-10%), intermediate grade (10-40%), and high grade (40-90%), using the criteria of the International Working Group for TILs in BC. Analysis 1: The relationship between the grades of Str-TILs in pre-treatment tumors and pCR rates was investigated. Relapse-free survival (RFS) and cancer-specific survival (CSS) were analyzed for a correlation with pre-treatment Str-TILs. Analysis 2: Alterations in the grade of Str-TILs were examined in the residual tumors of non-pCR patients, and RFS and CSS were analyzed for a correlation with residual Str-TILs. Results: pCR was achieved in 83 out of the 128 patients (pCR rate, 64.8%) who received NAC with trastuzumab, and RFS was significantly better in the pCR group than in the non-pCR group (p = 0.0071). Analysis 1: The patient distribution of the Str-TILs grade in pre-treatment tumors was as follows: high: 24 (18.8%); intermediate: 38 (29.7%); and low: 66 (51.6%). pCR rates correlated with the Str-TILs grade in pre-treatment tumors: 83.3% in the high group, 71.1% in the intermediate group, and 54.5% in the low group (p = 0.026); however, the Str-TILs grade in pre-treatment tumors did not correlate with survival. Analysis 2: In 45 non-pCR patients, the distribution of the Str-TILs grade in residual tumors was as follows: high: 9 (20.0%); intermediate: 8 (17.8%); and low: 28 (62.2%), respectively. In non-pCR patients, the rate of a high Str-TILs grade was greater in residual tumors than in pre-treatment tumors (residual, 20.0%, pre-treatment, 8.9%). RFS was significantly better with a high grade than with a low grade of residual Str-TILs (p = 0.033). Conclusions: The status of TILs in pre-treatment tumors predicted responses to NAC concomitant with trastuzumab in HER2-positive BC. The grade of TILs was higher in residual tumors than in pre-treatment tumors, and, among non-pCR patients, the prognosis of patients with a high residual-TILs grade was better prognosis than that of patients with a low residual-TILs grade. We speculate that an examination of TILs in residual tumors after NAC with trastuzumab may be necessary for selecting patients with a good prognosis from non-pCR patients. Citation Format: Kurozumi S, Inoue K, Matsumoto H, Hayashi Y, Tozuka K, Kubo K, Komatsu K, Takai K, Nagai SE, Oba H, Horiguchi J, Takeyoshi I, Kurosumi M. Prognostic value of tumor-infiltrating lymphocytes in residual tumors after neoadjuvant chemotherapy concomitant with trastuzumab for HER2-positive breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-14-17.

The power output of a wind turbine depends on both wind speed and turbine control. There is a fluctuation in the natural wind speed, which changes the turbine power output. In this paper blade pitch control based on detailed monitoring of the inflow wind, including vertical velocity distributions, has been experimentally evaluated in a field test wind turbine. The inflow wind speeds were observed by 11 sets of ultrasonic anemometers. In this first test, the blade pitch is changed to keep optimum angle of attack based on the local inflow wind speed in the rotor plane. The blade pitch is independently controlled as a reference radial position of 80% of rotor radius. As a result of the pitch controls, the wind turbine power output was increased and the power output fluctuation was decreased for the cyclic pitch control based on the local wind speed. With the cyclic pitch control, the fluctuation in rotor thrust and average rotor thrust were also reduced compere to collective pitch control.

To prevent ATR activation, telomeres deploy the single-stranded DNA binding activity of TPP1/POT1a. POT1a blocks the binding of RPA to telomeres, suggesting that ATR is repressed through RPA exclusion. However, comparison of the DNA binding affinities and abundance of TPP1/POT1a and RPA indicates that TPP1/POT1a by itself is unlikely to exclude RPA. We therefore analyzed the central shelterin protein TIN2, which links TPP1/POT1a (and POT1b) to TRF1 and TRF2 on the double-stranded telomeric DNA. Upon TIN2 deletion, telomeres lost TPP1/POT1a, accumulated RPA, elicited an ATR signal, and showed all other phenotypes of POT1a/b deletion. TIN2 also affected the TRF2-dependent repression of ATM kinase signaling but not to TRF2-mediated inhibition of telomere fusions. Thus, while TIN2 has a minor contribution to the repression of ATM by TRF2, its major role is to stabilize TPP1/POT1a on the ss telomeric DNA, thereby allowing effective exclusion of RPA and repression of ATR signaling.

Background The detection of circulating tumor cells (CTCs) in peripheral blood is an independent predictor of the efficacy of systemic therapy, and also a prognostic marker for patients with metastatic breast cancer. One of the main methods to detect CTCs is CellSearch system, which uses immune-magnetic separation followed by immunocytochemistry. A microdevice (CTChip from ClearCell system) can capture and enumerate CTCs based on distinctive physiological differences (size and deformability) between cancer cells and blood cells. CTChip thus obtains a larger CTC yield than affinity-based separation, which enriches a particular subgroup of cells expressing EpCAM. In this study, we enumerate CTCs in peripheral blood from early and metastatic breast cancer patients using a size-based method. Patients and methods We examined blood samples from a total of 18 early and metastatic breast cancer patients, after obtaining written informed consent. Blood samples were taken in sodium EDTA tubes after discarding the first 1ml of blood from the syringe. Two ml blood samples were applied to CTChip (ClearCell system), and CTCs were eventually trapped in the microwells of the CTChip. Trapped cells were analyzed by immunocytochemistry with monoclonal antibodies specific for leukocytes (CD45) and epithelial cells (CK8/18), along with 4',6-diamidino-2-phenylindole (DAPI) for nuclei: CK8/18-positive, DAPI-positive and CD45-negative cells more than 10 μm in diameter were defined as CTCs. Eight patients were examined using both the CTChip and CellSearch system to compare the yield of CTCs. Results Of 18 patients, 6 were de novo stage IV, 6 were recurrent and 6 were early stage breast cancer patients. Of primary tumors, 8 were HER2- and ER and/or PR +, 6 were HER2-and ER- and PR-, 3 were HER2+ and ER and/or PR +, and one was HER2+ and ER- and PR-. Using CTChip, detected CTCs ranged from 3 - 107 cells/2 ml in all cases: 3 - 83 for early stage, 19 - 156 for stage IV and 21 - 146 for recurrent. The number of CTCs found in recurrent patients tended to be higher than in early stage patients. Size-based method using CTChip clearly showed high sensitivity compared with the CellSearch system, which detected CTCs in only 2 cases out of 8. In analysis by immunochemistry, we found CK-negative, CD45-negative and DAPI positive cells with larger diameter (>16 μm) than CK-positive CTCs in most patients, and the numbers were higher in stage IV (8.5 cells of median value) and recurrent (13 cells) patients than in early stage patients (1.5 cells). Our study suggested that CK-negative large cells might be CTCs with epithelial–mesenchymal transition (EMT). Conclusion This size-based technology enables us to capture CTCs regardless of EpCAM expression. Enumerated CTCs varied in size and positivity of CK8/18, suggesting the heterogeneity of CTCs. Further research, especially focusing on EMT will be crucial to understand the key mechanism of metastasis and drug resistance. Citation Format: Tozuka K, Nagai SE, Kubo K, Komatsu K, Takai K, Inoue K, Matsumoto H, Hayashi Y, Tsuboi M, Yamada Y, Wang X, Suganuma M. Enumeration of heterogeneous circulating tumor cells (CTCs) using size-based method in early, and metastatic, breast cancer patients [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-01-08.

Thermal desorption spectroscopy (TDS) has been used to reveal the nature of defects acting as trapping sites of hydrogen. Hydrogen was charged to ferritic and eutectoid steels deformed to various degrees and then given annealing treatment. Desorption with a single peak appeared between room temperature and 600 K from ferritic steels. Under constant hydrogen charging conditions, the amount of desorption increased with strain. However, when the deformed samples were subjected to annealing at temperatures as low as 500 K, the increase of desorbed hydrogen no longer appeared. Vacancy clusters, which themselves annihilate in the course of TDS measurement, are the probable source of hydrogen desorption. When heavy deformation was given to ferritic steels, a two-step decrease of hydrogen desorption took place with increasing annealing temperature, corresponding to annihilation of vacancy clusters and decrease of dislocation density, respectively. The desorption with a single peak has two origins, one due to the annihilation of the trapping sites themselves and the other to desorption from stable sites. For heavily deformed eutectoid steel, an additional desorption peak centered at around 640 K appeared. The peak likely results from deformation-induced defects within the cementite phase or supersaturated carbon in ferrite. Two types of desorption, one due to the annihilation of trapping sites in the course of measurement and the other due to desorption from stable sites, should be discriminated. TDS using hydrogen as a tracer can be applied as a tool to investigate the various defects induced by plastic deformation.

We report a method of endoscopic retrograde sphincterotomy in patients in whom the optimal viewing of the papilla of Vater is hardly obtained. Percutaneous transhepatic cholangiodrainage (PTCD)-tube is placed under ultrasonographic guidance. PTCD-tube coming out of the papilla of Vater is observed by the endoscope and the guide wire inside the shpincterotome is inserted into the PTCD-tube. Sphincterotome is advanced into the common bile duct by the guidance of the guide wire and PTCD-tube. Sphincterotomy is performed in a usual fashion. Two patients with previous history of gastrectomy underwent this procedure with successful results. This method should be tried when usual method of EST is difficult and unsuccessful.

Behçet's disease (BD) is a multi-systemic inflammatory disease, characterised by recurrent oral aphthosis, genital ulcers, skin lesions and uveitis. We have reported excessive Th1 cell activity in patients with BD. More recently, Th17 cells were suggested to associate with several autoimmune diseases. This study was designed to investigate the role of Th17 related cytokines and signalling molecules in patients with BD.We examined mRNA expressions of Th1 and Th17 related cytokines and related signalling molecules in PBMC of 12 patients with BD and 14 normal controls (NC) using quantitative RT-PCR. We studied expressions of the Th17 related cytokines in other four BD patients' skin lesions by immunofluorescence.Major Th17 related cytokines were not detected in unstimulated PBMC in patients with BD. After stimulation, mRNA expressions of TGFβ receptor type 1, IL-12 receptor β2 and suppressor of cytokine signalling protein (SOCS) 1 on PBMC were significantly enhanced in patients with BD, as compared with NC (p0.05). mRNA expression of RORC, a key transcription factor for Th17 cell differentiation, was comparable between BD and NC. CD4+ T cells infiltrating into BD skin lesion expressed TGFβ1 much more than those infiltrating into non-Behçet's disease erythema nodosum.These findings suggest that TGFβ/Smad signalling pathway of T cells is overactive in patients with BD.

Human telomeres bind shelterin, the six-subunit protein complex that protects chromosome ends from the DNA damage response and regulates telomere length maintenance by telomerase. We used quantitative immunoblotting to determine the abundance and stoichiometry of the shelterin proteins in the chromatin-bound protein fraction of human cells. The abundance of shelterin components was similar in primary and transformed cells and was not correlated with telomere length. The duplex telomeric DNA binding factors in shelterin, TRF1 and TRF2, were sufficiently abundant to cover all telomeric DNA in cells with short telomeres. The TPP1.POT1 heterodimer was present 50-100 copies/telomere, which is in excess of its single-stranded telomeric DNA binding sites, indicating that some of the TPP1.POT1 in shelterin is not associated with the single-stranded telomeric DNA. TRF2 and Rap1 were present at 1:1 stoichiometry as were TPP1 and POT1. The abundance of TIN2 was sufficient to allow each TRF1 and TRF2 to bind to TIN2. Remarkably, TPP1 and POT1 were approximately 10-fold less abundant than their TIN2 partner in shelterin, raising the question of what limits the accumulation of TPP1 x POT1 at telomeres. Finally, we report that a 10-fold reduction in TRF2 affects the regulation of telomere length but not the protection of telomeres in tumor cell lines.

The shelterin complex at mammalian telomeres contains the single-stranded DNA–binding protein Pot1, which regulates telomere length and protects chromosome ends. Pot1 binds Tpp1, the shelterin component that connects Pot1 to the duplex telomeric DNA–binding proteins Trf1 and Trf2. Control of telomere length requires that Pot1 binds Tpp1 as well as the single-stranded telomeric DNA, but it is not known whether the protective function of Pot1 depends on Tpp1. Alternatively, Pot1 might function similarly to the Pot1-like proteins of budding and fission yeast, which have no known Tpp1-like connection to the duplex telomeric DNA. Using mutant mouse cells with diminished Tpp1 levels, RNA interference directed to mouse Tpp1 and Pot1, and complementation of mouse Pot1 knockout cells with human and mouse Pot1 variants, we show here that Tpp1 is required for the protective function of mammalian Pot1 proteins.

Basic device characteristics were investigated for a new high-speed and non-volatile nano electro-mechanical systems (NEMS) memory device with nanocrystalline silicon (nc-Si) dots embedded in its movable floating gate beam.Over 1 GHz operation is possible due to the size reduction of the NEMS. From a simulation of mechanical properties of the movable floating gate beam, advantage of using the nc-Si dots array was shown for low power operation. The mechanical bistability of the fabricated SiO2 beam was clearly observed in both experimental and simulation studies.

Bile duct injury can occur more frequently during laparoscopic cholecystectomy than in open cholecystectomy. Three cases of common bile or hepatic duct injuries occurred in a series of eighty laparoscopic cholecystectomies; In case 1, the common bile duct was misidentified as the cystic duct. In case 2, bile peritonitis occurred on the fourth postoperative day caused by necrosis of the common hepatic duct involving the cautery surrounding it. In case 3, a bile leak occurred due to an incision at the confluence of the cystic and common duct. Dissection of the cystic duct at the infundibulum of the gallbladder, blunt dissection of the Calot's triangle, the handling of clips with special attention for safety were thought to be necessary in order to lower the risk of bile duct injury. Preoperative endoscopic retrograde cholangiography (ERCP) is recommended to avoid bile duct injury.

Summary: We herein present a case of an internal carotid artery embolism associated with a hidden internal carotid artery aneurysm. The aneurysm was visualized during successful endovascular treatment of the carotid embolism. In retrospect, the aneurysm was at risk of rupture during the procedure. In the endovascular treatment of cerebral embolism, the possibility that aneurysms are hidden by emboli must be borne in mind. Care should be taken not to injure unidentified arterial walls while advancing a catheter blindly.

The large species difference in the pharmacokinetics/pharmacodynamics of 7-hydroxystaurosporine (UCN-01) can be partially explained by the high affinity binding of UCN-01 to human alpha1-acid glycoprotein (AGP) (Fuse et al, Cancer Res., 58: 3248-3253, 1998). To confirm whether its binding to human AGP actually changes the in vivo pharmacokinetics, we have studied the alteration in its pharmacokinetics after simultaneous administration of human AGP to rats: (a) the protein binding of UCN-01 was evaluated by chasing its dissociation from proteins using dextran-coated charcoal. The UCN-01 remaining 0.1 h after adding dextran-coated charcoal to human plasma or AGP was approximately 80%, although the values for other specimens, except monkey plasma (approximately 20%), were1%, indicating that the dissociation from human AGP was specifically slower than from other proteins; and (b) the pharmacokinetics of UCN-01 simultaneously administered with human AGP has been determined. The plasma concentrations after i.v. administration of UCN-O1 with equimolar human AGP were much higher than those after administration of UCN-01 alone. The steady-state distribution volume and the systemic clearance were reduced to about 1/100 and 1/200, respectively. Human AGP thus reduced the distribution and elimination of UCN-01 substantially. On the other hand, dog AGP, which has a low binding affinity for UCN-01, did not change the pharmacokinetics of UCN-01 so much. Furthermore, human AGP markedly reduced the hepatic extraction ratio of UCN-01 from 0.510 to 0.0326. Also, human AGP (10 microM) completely inhibited the initial uptake of UCN-01 (1 microM) into isolated rat hepatocytes, whereas the uptake of UCN-01 was unchanged in the presence of human serum albumin (10 microM). In conclusion, the high degree of binding of UCN-01 to human AGP causes a reduction in the distribution and clearance, resulting in high plasma concentrations in humans.

The expression of T-cell antigen receptor (TCR) alpha beta was investigated in rat CD4- CD8- thymocytes during thymic reconstitution after the exposure of animals to irradiation or glucocorticoid. The effect of the immunosuppressant FK506 on the expression of TCR alpha beta in rat CD4- CD8- thymocytes was also examined. The percentage of CD4- CD8- thymocytes constituted 2.6% of total thymocytes and that of CD4- CD8- TCR alpha beta high cells constituted 12.6% of CD4- CD8- thymocytes in normal adult Lewis rats. The percentage of CD4- CD8- TCR alpha beta high cells increased during thymic reconstitution after irradiation, and maximally constituted 28.6% of CD4- CD8- thymocytes on day 7. Similar results were obtained during thymic reconstitution after glucocorticoid treatment. In contrast, continuous treatment with FK506 for 7 days markedly decreased not only the percentages of CD4+ CD8- TCR alpha beta high and CD4- CD8+ TCR alpha beta high thymocytes, but also that of CD4- CD8- TCR alpha beta high thymocytes. These results indicate that rat CD4- CD8- thymocytes contain a subpopulation of mature (TCR alpha beta high) cells. The possible implications of the existence of this subpopulation with regard to thymocyte differentiation and maturation are discussed.

In an attempt to know the applicability of non-cultural detection technique for N. gonorrhoeae and C. trachomatis on pharyngeal and rectal swabs, the swabs taken from N. gonorrhoeae- and C. trachomatis-free subjects were tested by Gonozyme, Chlamydiazyme, Ideia Chlamydia, Gen-Probe and Gen Probe Pace 2. As N. gonorrhoeae- and C. trachomatis-free subjects, the castrated males older than age 70, with known prostate cancer treated at least for 6 months with anti-androgens who were seronegative for C. trachomatis and had a history of Ofloxacin treatment in the last 3 months and whose pharyngeal and rectal swabs were negative for N. gonorrhoeae, were subjected for study. The false negative rates were for N. gonorrhoeae from the pharynx by Gonozyme 57.7%, by Gen-Probe 0%, by Gen-Probe Pace 2 0%, from rectum by Gonozyme 0%, by Gen-Probe 0% by Gen-Probe Pace 2 0%, for C. trachomatis from pharynx by Chlamydiazyme 93.8%, by Ideia Chlamydia 8.3%, by Gen-Probe 0%, by Gen-Probe Pace 2 0%, from rectum by Chlamydiazyme 94.4%, by Ideia Chlamydia 70.9%, by Gen-Probe 20.0% and by Gen-Probe Pace 2 0%. To investigate the incidence of pharyngeal and rectal infection by N. gonorrhoeae among the patients with gonococcal genital infection, as well as pharyngeal and rectal infection by C. trachomatis among the patients with chlamydial genital infection, pharyngeal and recal swabs obtained from the patients with genital infection by N. gonorrhoeae or C. trachomatis were tested by Gen-Probe Pace 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Summary The effect of administration of FK506 at 1 mg/kg body weight for 14 days on rat lymphoid tissues, especially the thymus, and recovery after discontinuation of treatment, were investigated by the immunoperoxidase technique and flow cytofluorometry using monoclonal antibodies OX6, OX7, OX8, OX18 and W3/25, reactive with rat lymphocytes. Marked reduction of the thymic medulla upon treatment was clearly demonstrated by staining with OX18 and OX6. Changes produced by FK506 were also observed in the cortical area of the thymus, and were especially marked in the subcapsular area and around blood vessels. Eventually, the thymic cortex appeared patchy, this change being maximal 14 days after the start of administration. Obvious restitution of the thymic medulla was evident about 14 days after withdrawal of FK506. Flow cylometric analysis of the thymus showed that the percentages of cells labelled positively with OX7, OX8 and W3/25 were increased with FK506 treatment, and recovered to the normal levels soon after withdrawal. Furthermore, the peak of fluorescence intensity of OX7+, OX8+ and W3/25+ cells showed a temporary shift to the right during FK506 treatment; however, the peak of fluorescence intensity of OX18+ cells showed a temporary shift to the left. Treatment with FK506 also produced a significant change in 3H-thymidine uptake by thymocyte. These results suggest that FK506 may inhibit the proliferation, maturation and differentiation of thymocytes. However, thymocytes prepared from FK506-treated rats and labelled with FITC behaved similarly to rat thymocytes in normal recipient rats. This suggests that during FK506 treatment thymocytes may retain their potential for peripheral mobilization.