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Water temperature is the most important and critical factor for freshwater invertebrates and it fluctuates with an increase in air temperature. In this study, the effect of water temperature on egg development was clarified in Stavsolus japonicus, and the response to climate change in stoneflies with long egg periods were considered. Water temperatures prior to 43 days before hatching likely do not affect egg development in Stavsolus japonicus. Instead, they use egg diapause as an adaptive strategy to survive hot summer conditions. Increased water temperatures may cause migration to higher elevations for stoneflies that have lower adaptability in their egg development period, with populations eventually becoming stranded where no higher elevation or cooler habitat is available. Species extinction is expected to increase with increasing temperatures, leading to reduced biodiversity in many ecosystems. Indirect effects of water warming on maturation and reproduction may lead to substantial reductions in benthic invertebrate populations.

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INTRODUCTION: Locoregional recurrence after curative resection remains an important issue in the treatment of colorectal cancer (CRC). The aim of the present study was to investigate the clinical significance of quantitative detection of intraperitoneal free cancer cells by a PCR-based method for predicting locoregional recurrence after CRC resection. METHOD: A total of 114 patients with CRC were enrolled between March 2017 and December 2018, and 95 patients with Stage I-III CRC were analyzed. Peritoneal lavage fluid was collected before and after tumour resection and subjected to cytology and quantitative reverse transcription-PCR (qRT-PCR) with carcinoembryonic antigen (CEA) as a genetic marker. RESULTS: 2.1% of patients had positive cytology after resection, whereas 9.5% had positive CEA qRT-PCR (PCR+) after resection. Eight of nine PCR+ patients after resection had tumours in the rectum. Fifteen (15.8%) patients developed recurrence during the follow-up period, including three with locoregional recurrence. One of 86 (1.2%) PCR- patients and 2 of 9 (22.2%) PCR+ patients after resection developed locoregional recurrence. Overall and in rectal cancer patients, the 3-year cumulative risk of locoregional recurrence was 25.0% and 28.6% for PCR+ patients, which is significantly higher than PCR- patients (1.3% and 0%, P < 0.001 and P = 0.001, respectively). CONCLUSION: Intraperitoneal free cancer cells can serve as a sensitive predictor of locoregional recurrence after rectal cancer resection. qRT-PCR for CEA can be a suitable method for detecting intraperitoneal free cancer cells in peritoneal lavage fluid.

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The Fukushima accident emitted radioactive substances into the environment, contaminating litter, algae, sand substrate, aquatic invertebrates, and fish in freshwater streams. Because these substances have substantial effects on stream ecology over many years, it is necessary to clarify the diffusion and decay mechanisms of radiocesium. The transfer coefficient differed among aquatic invertebrate groups, likely due to the differences in habitat. The ecological half-life of cesium was longer where the air dose rate was lower. The transfer coefficient was also higher in areas with lower air dose rate. The radiocesium concentration in algae was inversely related to stream current velocity in the radiocesium-contaminated area. However, this relationship was not observed in the lower air dose rate area: the radiocesium concentration in algae in the rapid-velocity areas tended to be higher than that in the slow-velocity areas. This reverse trend would lead to a longer period of freshwater contamination. The radiocesium concentration would continue to decrease in highly contaminated areas, but it would be difficult to reduce the radiocesium concentration in less-contaminated areas because different contamination mechanisms are at work. Controlling the water flow is key to regulating radiocesium concentration in freshwater ecosystems.

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BACKGROUND/AIM: To examine the dynamics of circulating tumour cells (CTCs) in pancreatic cancer (PC), new mouse CTC models from human PC xenografts were developed. MATERIALS AND METHODS: Orthotopic (pancreas) and heterotopic (subcutaneous) transplantation models using GFP-tagged SUIT-2 PC cells were prepared. Using a cytology-based CTC detection platform, CTCs and metastasis were compared. RESULTS: The two types of orthotopic models, including the surgical transplantation model and the intraperitoneal injection model, showed a similar pattern of initial pancreatic tumour formation and subsequent development of peritoneal and hematogenous lung metastases. In the heterotopic model, only hematogenous lung metastasis was observed, and the number of CTCs and lung metastases was higher than that of the orthotopic model. Furthermore, KRAS mutation (G12D) was detected in CTCs. CONCLUSION: These orthotopic and heterotopic models clearly differ in terms of the pattern of metastasis and CTCs and therefore, would be useful PC models to investigate the effect of drug-therapy on CTCs and the role of KRAS mutation.

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Circulating tumor cells (CTCs) in tumor draining vein blood (DB) are potential sources for liquid biopsy. However, the identification of CTCs in DB of breast cancer has not been attempted. In this study, we investigated the feasibility of CTC detection in DB of breast cancer patients using a newly developed filtration-based microfluidic CTC detection device. Samples of peripheral vein blood (PB) and DB drawn from the lateral thoracic vein of the resected breast tissue were collected during the perioperative period. We investigated 41 breast cancer patients who underwent breast surgery with axillary lymph node dissection. DB was successfully collected in 36 patients (87.8%), with a mean amount of 0.85 ml. CTCs were detected in 58.3% of PB samples and 80.6% of DB samples. DB had significant higher number of CTCs compared with PB (p < 0.001). CTCs were detected in 75.0% of DB samples and 50.0% of PB samples from patients achieving pathological complete response after neoadjuvant chemotherapy. These results suggest that abundant CTCs are released into the DB of breast cancer patients, indicating that CTCs in DB would be alternative sources for liquid biopsy and potential indicators for monitoring of treatment response and prognosis in breast cancer patients.

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BACKGROUND/AIM: Dynamics of circulating tumor cells (CTCs) after molecular targeting therapy remain unclear. MATERIALS AND METHODS: We examined changes in CTC numbers and morphology early after targeting therapy in EGFR-mutated PC-9 human lung cancer and HER2-gene amplified GLM-1 gastric cancer mouse CTC models using a cytology-based semi-automated CTC detection platform. RESULTS: Erlotinib and T-DM1 inhibited cell growth mainly by induction of apoptosis in vitro. The number of CTCs detected 5-10 days after targeting therapy in mice was significantly increased compared to CTC numbers before therapy. The increased CTCs after therapy consisted of apoptotic CTCs and viable CTCs. This heterogeneous population of CTCs reflects well the cell population of the primary tumor disrupted by therapy. CONCLUSION: CTCs can be mobilized from the primary tumor due to tissue disruption in acute response to targeting therapy, suggesting potential usefulness of CTC monitoring as a predictor of therapeutic response in the clinical settings.

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Numerous technologies exist to detect circulating tumor cells (CTCs), although reports on cytological detection of CTCs remain limited. We recently developed a cytology-based CTC detection device using glass slides and light microscopy. In this study, we automated this previously manual device to improve its efficiency and cost effectiveness for clinical applications. We conducted a pilot study using this device to compare CTCs in peripheral blood (PB) and draining venous blood (DVB) from patients with colorectal cancer (CRC). The cytology-based automated CTC detection platform consisted of a disposable filtration device with a three-dimensional (3D) metal filter and multichannel automated CTC enrichment device. This platform allowed rapid and gentle filtration of CTCs and their efficient transfer from the filter to glass slides for subsequent Papanicolaou (Pap) and immunocytochemical (ICC) staining. Cytological diagnosis of CTCs was performed by observing permanent glass slide specimens by light microscopy. The current pilot clinical study enrolled CRC patients (n = 26) with stage I-IV tumors, who underwent surgery. PB was collected before surgery, and DVB was obtained from the mesenteric vein immediately after resection. Based on the CTC morphology obtained from PB and DVB samples, we proposed the following cytological criteria for the diagnosis of CTCs: pan-cytokeratin-positive, atypical cells with malignant morphological features identified by Pap staining. The numbers of CTCs defined by these criteria were significantly higher in DVB than PB from CRC patients (p<0.01), and the number of CTCs in DVB was increased significantly with stage progression (p<0.05). These results suggest that DVB may be another potential source of CTCs other than PB for liquid biopsies including downstream analysis. This automated cytology-based CTC detection device therefore provides a unique and powerful tool to investigate the significance of CTCs in CRC patients in a clinical setting.

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OBJECTIVES: The lung was recently re-discovered as a hematopoietic organ for platelet production in mice. However, evidence for the role of the lung in thrombopoiesis in humans is still limited. In this study, we examined megakaryocytes in the pulmonary and systemic circulation, specifically in pulmonary arterial blood (PAB), venous blood (PVB) and peripheral blood using a newly developed microfluidic platform for rare cell isolation. MATERIALS AND METHODS: We analyzed 23 lung cancer patients who underwent surgery in our institute. PAB and PVB were obtained from the resected lung immediately after surgery. Blood samples were size-selected using a filtration-based microfluidic device and enriched rare cells on glass slide specimens were stained with Papanicolaou (Pap), immunocytochemistry (ICC), and immunofluorescence (IF). Lung tissues were also analyzed by immunohistochemistry. RESULTS: Pap/ICC/IF showed the presence of abundant CD61+/cytokeratin- giant cells with a megakaryocyte lineage in PAB, but only a few in PVB. These megakaryocytes were found to consist of CD61+/CD41+ immature megakaryocytes and CD61+/CD41- mature megakaryocytes with the potential to produce platelets. These findings were confirmed by the conventional hematological analysis of blood smears stained with Giemsa. In analysis of lung cancer, CD61+ megakaryocytes were observed exclusively in the capillaries of non-cancerous tissue, whereas platelets were selectively observed in the tumor blood vessels of cancerous tissue. CONCLUSIONS: These results indicate that numerous megakaryocytes migrate from systemic bone marrows to accumulate in PAs and arrest of mature megakaryocytes in the capillaries of normal lung, suggesting the possibility that the lung plays a physiological role in the systemic thrombopoiesis in lung cancer patients.

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Food-web complexity often hinders disentangling functionally relevant aspects of food-web structure and its relationships to biodiversity. Here, we present a theoretical framework to evaluate food-web complexity in terms of biodiversity. Food network unfolding is a theoretical method to transform a complex food web into a linear food chain based on ecosystem processes. Based on this method, we can define three biodiversity indices, horizontal diversity (DH ), vertical diversity (DV ) and range diversity (DR ), which are associated with the species diversity within each trophic level, diversity of trophic levels, and diversity in resource use, respectively. These indices are related to Shannon's diversity index (H'), where H' = DH  + DV  - DR . Application of the framework to three riverine macroinvertebrate communities revealed that D indices, calculated from biomass and stable isotope features, captured well the anthropogenic, seasonal, or other within-site changes in food-web structures that could not be captured with H' alone.

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PURPOSE: Circulating tumor cells (CTCs) can provide a potentially minimal invasive source for monitoring chemotherapeutic effects. However, detailed in vivo dynamics of CTC after chemotherapy remain largely unknown. METHODS: We monitored CTC number and morphology early after chemotherapy using a newly developed cytology-based CTC detection device and triple-negative breast cancer mouse CTC models with spontaneous lung metastatic potential. RESULTS: Paclitaxel inhibited cell growth of breast cancer cells by mainly G2/M cell cycle arrest and partly apoptosis, whereas doxorubicin inhibited cell growth mainly by apoptosis and partly G2 cell cycle arrest in vitro. The number of CTCs was significantly increased 3-10 days after paclitaxel and doxorubicin chemotherapy and decreased thereafter in two mouse CTC models. The transiently increased CTCs early post-chemotherapy consisted of not only G2/M arrested cells (apoptotic cells), but also morphologically near-intact live cells. This heterogeneous cell population of CTCs was similar to that of primary tumor tissue after chemotherapy. CONCLUSIONS: These results indicate that CTCs can be mobilized from the primary tumor in rapid response to chemotherapy and suggest the possibility that CTC monitoring from both numerical and morphological viewpoints early after chemotherapy using a cytology-based CTC detection device would be a useful diagnostic tool for predicting drug sensitivity/resistance in preclinical and clinical setting.

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Long-term monitoring of ecosystem succession provides baseline data for conservation and management, as well as for understanding the dynamics of underlying biogeochemical processes. We examined the effects of deforestation and subsequent afforestation of a riparian forest of Japanese cedar (Cryptomeria japonica) on stable isotope ratios of carbon (δ¹³C) and nitrogen (δ¹5N) and natural abundances of radiocarbon (Δ¹4C) in stream biota in the Mt. Gomadan Experimental Forest and the Wakayama Forest Research Station, Kyoto University, central Japan. Macroinvertebrates, periphytic algae attached to rock surfaces (periphyton), and leaf litter of terrestrial plants were collected from six headwater streams with similar climate, topography, and bedrock geology, except for the stand ages of riparian forests (from 3 to 49 yr old in five stands and > 90 yr old in one reference stand). Light intensity and δ¹³C values of both periphyton and macroinvertebrates decreased synchronously with forest age in winter. A Bayesian mixing model indicates that periphyton contributions to the stream food webs are maximized in 23-yr-old forests. Except for grazers, most macroinvertebrates showed Δ¹4C values similar to those of terrestrial leaf litter, reflecting the influence of modern atmospheric CO2 Δ¹4C values. On the other hand, the Δ¹4C values of both periphyton and grazers (i.e., aquatic primary consumers) were significantly lower than that of modern atmospheric CO2, and were lowest in 23-yr-old forest stands. Previous studies show that root biomass of C. japonica peaks at 15-30 yr after planting. These evidences suggest that soil CO2 released by root respiration and dispersed by groundwater weathers carbonate substrata, and that dissolved inorganic carbon (DIC) with low Δ¹4C is incorporated into stream periphyton and some macroinvertebrates. The ecological response in the studied streams to clear-cutting and replanting of Japanese cedar is much slower (~20 yr) than the chemical response (< 5 yr). More than 50 yr is required for the food web structure to completely recover from clear-cutting. The ecological delay is attributed to several biogeochemical factors, the understanding of which is critical to integrated management of forest-stream continuum and the prediction of ecosystem resilience in response to environmental change.

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This study reported the 15,435 bp-long complete mitochondrial genome of the relict Epiophlebia superstes (Odonata, Epiophlebiidae), an enigmatic dragonfly of the paraphyletic 'Anisozygoptera' possessing characteristics similar to members of both extant odonate suborders, the Zygoptera and the Anisoptera. This mitogenome comprises the common set of 37 genes and an A + T-rich control region, and has a gene arrangement identical to those of all available odonates. The genome contains three non-coding inter-genic spacers (s1-s3), which occurs in all of other known odonates, but it lacks the inter-genic spacer s5 typically found in the Anisoptera. This result suggests that E. superstes possesses a mitogenmic organization more closely related to that of the Zygoptera than that of the Anizoptera.

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The stable N isotopic composition of individual amino acids (SIAA) has recently been used to estimate trophic positions (TPs) of animals in several simple food chain systems. However, it is unknown whether the SIAA is applicable to more complex food web analysis. In this study we measured the SIAA of stream macroinvertebrates, fishes, and their potential food sources (periphyton and leaf litter of terrestrial C3 plants) collected from upper and lower sites in two streams having contrasting riparian landscapes. The stable N isotope ratios of glutamic acid and phenylalanine confirmed that for primary producers (periphyton and C3 litter) the TP was 1, and for primary consumers (e.g., mayfly and caddisfly larvae) it was 2. We built a two-source mixing model to estimate the relative contributions of aquatic and terrestrial sources to secondary and higher consumers (e.g., stonefly larva and fishes) prior to the TP calculation. The estimated TPs (2.3-3.5) roughly corresponded to their omnivorous and carnivorous feeding habits, respectively. We found that the SIAA method offers substantial advantages over traditional bulk method for food web analysis because it defines the food web structure based on the metabolic pathway of amino groups, and can be used to estimate food web structure under conditions where the bulk method cannot be used. Our result provides evidence that the SIAA method is applicable to the analysis of complex food webs, where heterogeneous resources are mixed.

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The Fukushima Daiichi Nuclear Power Plant (FDNPP) accident in March 2011 emitted radioactive substances into the environment, contaminating a wide array of organisms including fishes. We found higher concentrations of radioactive cesium ((137)Cs) in brown trout (Salmo trutta) than in rainbow trout (Oncorhynchus nerka), and (137)Cs concentrations in brown trout were higher in a lake than in a stream. Our analyses indicated that these differences were primarily due to differences in diet, but that habitat also had an effect. Radiocesium concentrations ((137)Cs) in stream charr (Salvelinus leucomaenis) were higher in regions with more concentrated aerial activity and in older fish. These results were also attributed to dietary and habitat differences. Preserving uncontaminated areas by remediating soils and releasing uncontaminated fish would help restore this popular fishing area but would require a significant effort, followed by a waiting period to allow activity concentrations to fall below the threshold limits for consumption.

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Chondroitin sulfate (CS) is a linear polysaccharide composed of repeating disaccharide units of glucuronic acid (GlcUA) and N-acetyl-d-galactosamine (GalNAc) with sulfate groups at various positions. Baculovirus is an insect-pathogenic virus that infects Lepidoptera larvae. Recently, we found that the occlusion-derived virus envelope protein 66 (ODV-E66) from Autographa californica nucleopolyhedrovirus (AcMNPV) exhibits chondroitin (CH)-digesting activity with distinct substrate specificity. Here, we demonstrate that the ODV-E66 protein from Bombyx mori nucleopolyhedrovirus (BmNPV) exhibits 92% homology to the amino acid sequence and 83% of the CH lyase activity of ODV-E66 from AcMNPV. ODV-E66 cleaves glycosyl bonds at nonreducing sides of disaccharide units consisting of nonsulfated and 6-O-sulfated GalNAc residues. We then investigated CS in the silkworm, Bombyx mori, which is the host of BmNPV. CS was present in insect tissues such as the midgut, peritrophic membrane, silk gland and skin. The polysaccharide consisted of a nonsulfated disaccharide unit, mono-sulfated disaccharide at Position 4 of the GalNAc residue and mono-sulfated disaccharide at Position 6 of the GalNAc residue. With regard to immunohistochemical analysis, the staining patterns of the silkworm tissues were different among anti-CS antibodies. Chondroitn sulfate that is digestible by ODV-E66 exists sufficiently in the peritrophic membrane protecting the midgut epithelium from ingested pathogens. Our results suggest that ODV-E66 facilitates the primary infection of the virus by digestion of CS in the peritrophic membrane.

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Primary carcinoma of the vermiform appendix is a rare disease with few clinical symptoms. Accordingly, preoperative diagnosis of appendiceal cancer is challenging because of the lack of specific symptoms. We herein report a case of appendicular adenocarcinoma found unexpectedly during laparoscopic surgery in a 69-year-old Japanese female patient diagnosed with serous papillary adenocarcinoma, in order to determine whether optimal cytoreduction could successfully be achieved at the time of primary surgery. We performed diagnostic laparoscopic surgery in order to make a correct diagnosis based on the histological tissue. The vermiform appendix was found to contain a tumor measuring 1.5 cm wide and 4.5 cm long. Laparoscopic appendectomy, partial omentectomy, and partial resection of the lesion in the peritoneum were performed. The histological diagnosis was mucinous adenocarcinoma of the vermiform appendix, and the stage was T4NxM1. The patient received adjuvant chemotherapy with mFOLFOX 6 (5FU, leucovorin, and oxaliplatin). She achieved stable disease and was alive with disease eleven months after surgery. We therefore recommend that gynecologists should not rule out the possibility of appendiceal cancer, even in cases with preoperative findings similar to those of serous papillary adenocarcinoma of the peritoneum with peritoneal disseminated tumors.

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PURPOSE: To assess the feasibility of visualizing noninvasively the physiologic flow of pancreatic juice by using serial magnetic resonance cholangiopancreatography (MRCP) with a spatially selective inversion-recovery (IR) pulse in volunteers and patients with pancreatic disease. MATERIALS AND METHODS: The study was approved by the institutional review board, and informed consent was obtained. Twelve healthy volunteers and three patients with acute pancreatitis were included. MRCP with a spatially selective IR pulse was repeatedly performed every 15 seconds during a total of 10 minutes (total of 40 images). MRCP images were evaluated for the presence, frequency, and magnitude of pancreatic juice inflow within the tagged area. The two groups were compared by using the Mann-Whitney test. RESULTS: Pancreatic juice inflow was observed in all healthy volunteers and in two of three patients with acute pancreatitis. The pancreatic fluid inflow was observed 25-37 times (median, 32 times; mean, 31.4 times; range, 25-37 times) in a series of 40 images in 12 healthy volunteers, while it was seen 0-11 times (median, 2 times; mean, 4.3 times; range, 0-11 times) in a series of 40 images in the three patients with acute pancreatitis (P = .009). No regularity in the timing of the pancreatic fluid inflow was noted. The distance that the pancreatic fluid moved in the pancreatic duct within the tagged area was significantly longer in healthy volunteers (median grade, 2.46; mean grade, 2.41; range, 1.6-3.3) than in patients with acute pancreatitis (median grade, 0.05; mean grade, 0.13; range, 0-0.35) (P = .009). CONCLUSION: The physiologic flow of the pancreatic juice can be visualized noninvasively with serial MRCP by using a spatially selective IR pulse.

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Peak preovipositional flight season and peak preovipositional flight time of day were compared among 12 species of Nemouridae, Chloroperlidae, Perlodidae and Perlidae. Species with a later peak date of preovipositional flight were found to have a later peak preovipositional flight time of day than species with an earlier peak in preovipositional flight season. A later peak preovipositional flight season correlated with a lower light intensity. Similarly, a later peak preovipositional flight time of day correlated with a lower light intensity and a later sunset. Individuals of one species (Sweltsa sp.), whose preovipositional flight date was later, flew over the stream at a later time of day. Species differences in peak preovipositional flight season and peak preovipositional flight time of day may be driven by species specific sensitivity for different light intensities.

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The relation between emergence date and the factors concerned in reproduction were studied in the stoneflies Isoperla aizuana (Perlodidae) and Sweltsa sp. (Chloroperlidae) in the laboratory. Individuals emerging earlier had larger adult size and greater adult longevity. Fecundity was also related to emergence date, adult size, and adult longevity. Furthermore, individuals emerging earlier had more days until first effective mating and tended to mate more effectively than later-emerging individuals. The maturity of the reproductive tissue may differ with emergence date. While most of the later-emerging individuals had a lower rate of effective mating, longer-lived and larger size adults mated more effectively in females of Sweltsa sp. The differences in mating character might affect the effectiveness of mating.

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