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Abstract Background The creation of minimally redundant tile paths (hereafter MTP) from contiguous sets of overlapping clones (hereafter contigs) in physical maps is a critical step for structural and functional genomics. Build 4 of the physical map of soybean (Glycine max L. Merr. cv. 'Forrest') showed the 1 Gbp haploid genome was composed of 0.7 Gbp diploid, 0.1 Gbp tetraploid and 0.2 Gbp octoploid regions. Therefore, the size of the unique genome was about 0.8 Gbp. The aim here was to create MTP sub-libraries from the soybean cv. Forrest physical map builds 2 to 4. Results The first MTP, named MTP2, was 14,208 clones (of mean insert size 140 kbp) picked from the 5,597 contigs of build 2. MTP2 was constructed from three BAC libraries (BamHI (B), HindIII (H) and EcoRI (E) inserts). MTP2 encompassed the contigs of build 3 that derived from build 2 by a series of contig merges. MTP2 encompassed 2 Gbp compared to the soybean haploid genome of 1 Gbp and does not distinguish regions by ploidy. The second and third MTPs, called MTP4BH and MTP4E, were each based on build 4. Each was semi-automatically selected from 2,854 contigs. MTP4BH was 4,608 B and H insert clones of mean size 173 kbp in the large (27.6 kbp) T-DNA vector pCLD04541. MTP4BH was suitable for plant transformation and functional genomics. MTP4E was 4,608 BAC clones with large inserts (mean 175 kbp) in the small (7.5 kbp) pECBAC1 vector. MTP4E was suitable for DNA sequencing. MTP4BH and MTP4E clones each encompassed about 0.8 Gbp, the 0.7 Gbp diploid regions and 0.05 Gbp each from the tetraploid and octoploid regions. MTP2 and MTP4BH were used for BAC-end sequencing, EST integration, micro-satellite integration into the physical map and high information content fingerprinting. MTP4E will be used for genome sequence by pooled genomic clone index. Conclusion Each MTP and associated BES will be useful to deconvolute and ultimately finish the whole genome shotgun sequence of soybean.

The black locust Robinia pseudoacacia (Robinieae, Fabaceae) is a common invasive riparian tree in Japan. There are less effective management strategies to remove the tree from the riparian area because of its quickly established high population. We investigated the expansion patterns of R. pseudoacacia through sympatric (i.e. between high- and low-water channel (HWC/LWC) within a study site) and allopatric (i.e. along river corridor) dispersal in the Tama River (Tokyo, Japan). Four microsatellites were used to examine the effects of gene flow on six populations in three sites. These subpopulations showed small genetic distance (i.e. no barrier or slightly limited) and genetically mixed population structure. It indicated that both sympatric and allopatric dispersals were active. Many migrants were younger individuals (i.e. <5 years old) and were found in the LWC area. Thus, the LWC could receive more migrants than the HWC through both types of dispersals. In addition, our age and genetic structure analyses reveal that recruited individuals likely settled immediately after the clearing project of R. pseudoacacia through sympatric dispersal. It appears that the migration by allopatric dispersal occurred following this. For the effective management of R. pseudoacacia , migrants should be removed regularly following initial removal of invaders during site restoration., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Annals of Botany Company.)

Adaptive divergence is a key mechanism shaping the genetic variation of natural populations. A central question linking ecology with evolutionary biology is how spatial environmental heterogeneity can lead to adaptive divergence among local populations within a species. In this study, using a genome scan approach to detect candidate loci under selection, we examined adaptive divergence of the stream mayfly Ephemera strigata in the Natori River Basin in northeastern Japan. We applied a new machine-learning method (i.e., random forest) besides traditional distance-based redundancy analysis (dbRDA) to examine relationships between environmental factors and adaptive divergence at non-neutral loci. Spatial autocorrelation analysis based on neutral loci was employed to examine the dispersal ability of this species. We conclude the following: (a) E. strigata show altitudinal adaptive divergence among the populations in the Natori River Basin; (b) random forest showed higher resolution for detecting adaptive divergence than traditional statistical analysis; and (c) separating all markers into neutral and non-neutral loci could provide full insight into parameters such as genetic diversity, local adaptation, and dispersal ability., Competing Interests: None declared., (© 2020 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Sediment bypass tunnels (SBTs) are guiding structures used to reduce sediment accumulation in reservoirs during high flows by transporting sediments to downstream reaches during operation. Previous studies monitoring the ecological effects of SBT operations on downstream reaches suggest a positive influence of SBTs on riverbed sediment conditions and macroinvertebrate communities based on traditional morphology-based surveys. Morphology-based macroinvertebrate assessments are costly and time-consuming, and the large number of morphologically cryptic, small-sized and undescribed species usually results in coarse taxonomic identification. Here, we used DNA metabarcoding analysis to assess the influence of SBT operations on macroinvertebrates downstream of SBT outlets by estimating species diversity and pairwise community dissimilarity between upstream and downstream locations in dam-fragmented rivers with operational SBTs in comparison to dam-fragmented (i.e., no SBTs) and free-flowing rivers (i.e., no dam). We found that macroinvertebrate community dissimilarity decreases with increasing operation time and frequency of SBTs. These factors of SBT operation influence changes in riverbed features, e.g. sediment relations, that subsequently effect the recovery of downstream macroinvertebrate communities to their respective upstream communities. Macroinvertebrate abundance using morphologically-identified specimens was positively correlated to read abundance using metabarcoding. This supports and reinforces the use of quantitative estimates for diversity analysis with metabarcoding data.

Atherosclerosis plays the major role in myocardial infarction and stroke and its pathophysiology is closely related to blood flow. Among clinical imaging modalities, ultrasound has the highest temporal resolution. Doppler ultrasound has been clinically applied for blood flow measurement and several parameters obtained with Doppler have been considered as essential for diagnosis. However, conventional Doppler method merely measures one-dimensional component of the blood flow along the ultrasonic beam. Based on previous approaches with multi-angle Doppler measurement for two-dimensional (2D) blood flow, this study aims to expand 2D flow measurement into three-dimensional (3D) flow estimation by applying continuity equation on multiplane 2D velocity mapping. The algorithm was validated by numerical simulation based on computational fluid dynamics and comparison with particle image velocimetry of carotid artery model. The method visualized 3D spiral flow in the carotid artery bifurcation model where 2D blood flow showed Iaminar flow. Clinical application of 3D blood flow visualization will provide important information on pathophysiology in common sites of atherosclerosis.

Human noroviruses are excreted in feces from infected individuals and included in wastewater. It is critical to remove/inactivate them in wastewater treatment processes, particularly in the disinfection step, before release to aquatic environments. However, the high mutation rates of human noroviruses raise concerns about the emergence of strains that are less susceptible to disinfectants and can survive even after wastewater treatment. This study aimed to demonstrate the strain-dependent susceptibility of norovirus to free chlorine. A population originated from the murine norovirus strain S7-PP3, a surrogate for human noroviruses in environmental testing, was exposed to free chlorine and then propagated in a host cell. This cycle of free chlorine exposure followed by propagation in cells was repeated 10 times, and populations with lower susceptibility to free chlorine were obtained from two independent trials of chlorine exposure cycles. Open reading frame 2 (ORF2) and ORF3 of the murine norovirus genome were analyzed by next-generation sequencing, and a unique nonsynonymous mutation (corresponding to a change from phenylalanine to serine) at nucleotide (nt) 7280 in ORF3, which encodes the minor capsid protein VP2, was found in chlorine-exposed populations from both trials. It was confirmed that all of the clones from the chlorine-treated population had lower susceptibility to free chlorine than those from the control population. These results indicate that exposure to free chlorine and dilution exert different driving forces to form murine norovirus (MNV) quasispecies, and that there is a selective force to form MNV quasispecies under free chlorine exposure. IMPORTANCE This study showed that free chlorine disinfection exerted a selection pressure for murine norovirus (MNV). The strain-dependent viral susceptibility to the disinfectant elucidated in this study highlights the importance of employing less susceptible strains as representative viruses in disinfection tests, because the disinfection rate values obtained from more susceptible strains would be less useful in predicting the virus inactivation efficiency of circulating strains under practical disinfection conditions., (Copyright © 2018 Rachmadi et al.)

This article documents the addition of 283 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Agalinis acuta; Ambrosia artemisiifolia; Berula erecta; Casuarius casuarius; Cercospora zeae-maydis; Chorthippus parallelus; Conyza canadensis; Cotesia sesamiae; Epinephelus acanthistius; Ficedula hypoleuca; Grindelia hirsutula; Guadua angustifolia; Leucadendron rubrum; Maritrema novaezealandensis; Meretrix meretrix; Nilaparvata lugens; Oxyeleotris marmoratus; Phoxinus neogaeus; Pristomyrmex punctatus; Pseudobagrus brevicorpus; Seiridium cardinale; Stenopsyche marmorata; Tetranychus evansi and Xerus inauris. These loci were cross-tested on the following species: Agalinis decemloba; Agalinis tenella; Agalinis obtusifolia; Agalinis setacea; Agalinis skinneriana; Cercospora zeina; Cercospora kikuchii; Cercospora sorghi; Mycosphaerella graminicola; Setosphaeria turcica; Magnaporthe oryzae; Cotesia flavipes; Cotesia marginiventris; Grindelia Xpaludosa; Grindelia chiloensis; Grindelia fastigiata; Grindelia lanceolata; Grindelia squarrosa; Leucadendron coniferum; Leucadendron salicifolium; Leucadendron tinctum; Leucadendron meridianum; Laodelphax striatellus; Sogatella furcifera; Phoxinus eos; Phoxinus rigidus; Phoxinus brevispinosus; Phoxinus bicolor; Tetranychus urticae; Tetranychus turkestani; Tetranychus ludeni; Tetranychus neocaledonicus; Tetranychus amicus; Amphitetranychus viennensis; Eotetranychus rubiphilus; Eotetranychus tiliarium; Oligonychus perseae; Panonychus citri; Bryobia rubrioculus; Schizonobia bundi; Petrobia harti; Xerus princeps; Spermophilus tridecemlineatus and Sciurus carolinensis., (© 2009 Blackwell Publishing Ltd.)

Genomes that have been highly conserved following increases in ploidy (by duplication or hybridization) like Glycine max (soybean) present challenges during genome analysis. At http://soybeangenome.siu.edu the Soybean Genome Database (SoyGD) genome browser has, since 2002, integrated and served the publicly available soybean physical map, bacterial artificial chromosome (BAC) fingerprint database and genetic map associated genomic data. The browser shows both build 3 and build 4 contiguous sets of clones (contigs) of the soybean physical map. Build 4 consisted of 2854 contigs that encompassed 1.05 Gb and 404 high-quality DNA markers that anchored 742 contigs. Many DNA markers anchored sets of 2-8 different contigs. Each contig in the set represented a homologous region of related sequences. GBrowse was adapted to show sets of homologous contigs at all potential anchor points, spread laterally and prevented from overlapping. About 8064 minimum tiling path (MTP2) clones provided 13,473 BAC end sequences (BES) to decorate the physical map. Analyses of BES placed 2111 gene models, 40 marker anchors and 1053 new microsatellite markers on the map. Estimated sequence tag probes from 201 low-copy gene families located 613 paralogs. The genome browser portal showed each data type as a separate track. Tetraploid, octoploid, diploid and homologous regions are shown clearly in relation to an integrated genetic and physical map.

Sudden death syndrome (SDS) of soybean is a complex of root rot disease caused by the semi-biotrophic fungus Fusarium solani f. sp. glycines (Fsg) and a leaf scorch disease caused by toxins produced by the pathogen in the roots. Development of partial rate-reducing resistance in roots to SDS was studied. The recombinant inbred line 23 (RIL23) that carried resistance conferred by six quantitative trait loci (QTL) derived from cultivars 'Essex' x 'Forrest' was compared to the susceptible cultivar Essex. Roots of RIL23 and its susceptible parent Essex were inoculated with Fsg. Transcript abundance (TA) of 191 ESTs was studied at five time points after inoculation. For most of the genes, there was an initial decrease in TA in the inoculated roots of both genotypes. By days 7 and 10 the inoculated roots of Essex failed to increase expression of the transcripts of defense-related genes. In RIL23 inoculated roots, the TA of 81 genes was increased by at least two-fold at day 3 (P=0.004), 88 genes at day 7 (P=0.0023) and 129 genes at day 10 (P=0.0026). A set of 35 genes maintained at least a two-fold higher abundance at all three time points. The increase in TA in RIL23 was in contrast to that observed in Essex where most of the ESTs showed either no change or a decreased TA. The ESTs with an increased TA had homology to the genes involved in resistance (analogs), signal transduction, plant defense, cell wall synthesis and transport of metabolites. Pathways that responded included the protein phosphorylation cascade, the phospholipase cascade and the phenolic natural products pathways, including isoflavone and cell wall synthesis.

We report use of a new bronchial blocker through a single-lumen endotracheal tube to achieve one-lung ventilation to perform thoracoscopic operation in patients in whom placement of the double-lumen tube failed and difficult intubation is predicted. The bronchial blocker tube was placed into the aimed bronchus under the bronchoscopic vision and the cuff of the blocker was inflated to achieve one-lung ventilation. In all of the 4 patients, the bronchial blocker could be inserted and placed safely, quickly, and exactly under the fiberoptic flexible bronchoscopic vision to perform thoracoscopic operation without any complications. The new bronchial blocker tube through the indwelling endotracheal tube may have advantages in situations where placement of double-lumen endotracheal tubes is technically impossible or inappropriate. The use of the new bronchial blocker tube will, however, require careful evaluation in larger series.

We have performed 5 thoracoscopic pericardial resections for malignant pericardial effusions. An initial trocar was placed in the seventh or eighth intercostal space posterior to the midaxillary line. Two additional trocars were placed, usually in the sixth intercostals spaces in the anterior axillary and posterior axillary lines. Using an endoscopic grasping instrument and scissors through the working ports, a pericardial resection was performed. All patients were successfully managed by thoracoscopic pericardial resections. Two of the 5 patients had associated malignant pleural effusions that were able to be managed at the same time by thoracoscopy. The average chest tube duration was 1.8 days. There has been no reaccumulation of pericardial effusions in all patients at an average follow-up of 5 months. The thoracoscopic approach could be minimally invasive and the procedure of choice in performing pericardial resections in selected patients with malignant pericardial effusions who are expected to have a reasonable life expectancy.

The injury of the lung occurred during thoracoscopic operation, especially, of the fragile, severely emphysematous lung is often difficult to treat. A fibrin glue spraying device in which an air compressor was connected to an intravenous catheter was constructed, and connected to a Dupuloject syringe. Fibrin glue was administered by spraying on the target lesion under a thoracoscopic vision. The procedure was done while the tip of the intravenous catheter was held and manipulated with forceps through a thoracoport. And then, the polyglycolic acid (PGA) mesh that had been cut into small pieces was put on the target lesion with forceps. Fibrin glue was sprayed on the lesion again in the above-mentioned manner. When necessary, the procedure was repeated. Our technique during thoracoscopic operation may be easy to manipulate and be able to apply the target lesion uniformly within a short time with a successful repair.

When a metastatic lung tumor is found to be deep to the visceral surface of the pleura, or when it is found to be a small lesion, it may important to help identify the lesion by preoperative methods in order to localize it at the time of thoracoscopic operation. We performed computed tomography-guided localization of metastatic lung tumors with use of a needle with a suture in 11 cases prior to thoracoscopic resection. Placement of the needle, immediately before moving to the operation room were successfully performed in all patients. It took from 16 to 25 minutes (20.3 minutes on average). Complications included minimal pneumothorax in 8 patients, mild airway bleeding in 2, and penetration through the middle lobe to the lower lobe of the right lung in 1. However, treatment was not necessary in any of the patients. Thoracoscopic resections were successfully achieved in all patients. Our technique may be a simpler technique and advantageous for identification of small lesions and lesions deep to the visceral surface of the pleura during the thoracoscopic operation for metastatic lung tumors.

We herein report an initial experience of thoracoscopic surgery for a solitary arterior-venous malformation (PAVM) with the AESOP 3000 HR voice-controlled robot to hold a thoracoscope. A 52-year-old woman was hospitalized due to a transient loss of consciousness. A brain magnetic resonance image and electroencephalogram (EEG) were normal. A chest computed tomogram indicated a suspicion of PAVM. The definite diagnosis of the PAVM was made by the 3-dimensional computed tomogram and pulmonary angiography. Thoracoscopic operation with the voice-controlled robot (AESOP 3000 HR) was conducted. The operating staffs were able to assemble the AESOP 3000 HR robotic system safely and quickly without difficulty. The robot functioned without problems throughout the procedure. No complications or events related or unrelated to the maneuvers of the robot during the operation were noted. The procedure of the pulmonary resection with the voice-controlled robot was successfully preformed by a single surgeon. The operating time was 110 minutes, and the volume of the intraoperative bleeding was 10 g. The postoperative course was uneventful. The use of the AESOP 3000 HR robot may be more convenient and friendly in thoracoscopic procedure.

We investigated aberrations of the neurofibromatosis type 2 (NF2) gene in breast tumors of 60 patients by single-strand conformation polymorphism analysis of polymerase chain reaction products followed by nucleotide sequencing. We detected a tumor-specific single-base substitution in codon 398 in exon 12 of the gene, resulting in an alteration of a single amino acid, in DNA from a breast cancer sample. The results indicated possible but infrequent involvement of mutations of the tumor suppressor NF2 gene in human breast cancers.

Mutations of the p53 gene have been found in a variety of human cancers and are implicated in the biologic functions of cancer. To investigate the clinical implications of p53 mutations in pancreatic adenocarcinoma, we examined the association of mutations of the p53 gene with patients' prognosis. Single-strand conformational polymorphism analysis and direct DNA sequencing were used to detect p53 gene mutations in 37 pancreatic adenocarcinomas. p53 gene mutations were detected in 16 (43%) of the 37 pancreatic adenocarcinomas. Direct sequencing did not reveal preferential clustering at any specific codon. There was no significant association of the presence of p53 gene mutations with histologic types, extent of tumor invasion, the presence of lymph node metastasis, or tumor stage. Univariate analysis showed that survival of patients with p53-gene-mutated tumors was significantly poorer than that of patients with p53-gene-nonmutated tumors (P = 0.02). Cox's multivariate analysis of ten clinicopathologic features including p53 gene mutations revealed that presence of p53 gene mutations (P = 0.026) and curativity of operation (P = 0.014) were independent predictors of survival. Furthermore, the survival of patients with p53-gene-mutated tumor was significantly poorer than that of patients with p53-gene-nonmutated tumors, both in patients who underwent curative operation (P = 0.04) and in patients who underwent non-curative operation (P = 0.01). These results suggested that mutations of the p53 gene might play an important role in cancer aggressiveness and could be a clinically useful predictor of prognosis in patients with pancreatic adenocarcinoma.