In the decapod crustacean brain, neurogenesis persists throughout the animal's life. After embryogenesis, the central olfactory pathway integrates newborn olfactory local and projection interneurons that replace old neurons or expand the existing population. In crayfish, these neurons are the descendants of precursor cells residing in a neurogenic niche. In this paper, the development of the niche was documented by monitoring proliferating cells with S-phase-specific markers combined with immunohistochemical, dye-injection and pulse-chase experiments.Between the end of embryogenesis and throughout the first post-embryonic stage (POI), a defined transverse band of mitotically active cells  appears. Just prior to hatching and in parallel with the formation of the DPS, the anlagen of the niche appears, closely associated with the vasculature. When the hatchling molts to the second post-embryonic stage (POII), the DPS differentiates into the lateral (LPZ) and medial (MPZ) proliferative zones. The LPZ and MPZ are characterized by a high number of mitotically active cells from the beginning of post-embryonic life; in contrast, the developing niche contains only very few dividing cells, a characteristic that persists in the adult organism.Our data suggest that the LPZ and MPZ are largely responsible for the production of new neurons in the early post-embryonic stages, and that the neurogenic niche in the beginning plays a subordinate role. However, as the neuroblasts in the proliferation zones disappear during early post-embryonic life, the neuronal precursors in the niche gradually become the dominant and only mechanism for the generation of new neurons in the adult brain. During embryonic development in the emerging ventral nerve cord of malacostracan Crustacea , most neurons are generated by neuronal stem cells, the neuroblasts (NBs), by a division pattern called the stem cell mode -7. NBs rHyas araneus  by labeling with the S-phase cell cycle marker 5-bromo-2'-deoxyuridine (BrdU) [Carcinus maenas [Cherax destructor [Homarus americanus, and the long-term survival of the newborn cells was documented using pulse-chase experiments with BrdU [Cancer pagurus, C. maenas, C. destructor, H. americanus, H. araneus, Libinia emarginata, Pagurus bernhardus, Panulirus argus, and Sicyonia brevirostris, suggesting that this is a common phenomenon in this group of organisms [Post-embryonic neurogenesis in a decapod crustacean was first shown in the spider crab e (BrdU) . Subsequs maenas , and crastructor , where istructor . Furtherith BrdU . Later sith BrdU . To daterganisms -18,20-29P. argus), an evolutionarily less derived species than crayfish, contrasting mechanisms underlying adult neurogenesis have been proposed [Adult neurogenesis in crayfish is driven by a neurogenic niche on the ventral surface of the brain in which first-generation progenitor cells reside ,28. Thisproposed ,21,29.Procambarus clarkii [Despite the growing body of literature concerning adult neurogenesis in the crustacean brain, the embryonic origin of the niche is still unknown. It has been reported that the niche is already present in the second post-embryonic (POII) stage in  clarkii . QuestioP. clarkii, in which additional experiments injecting fluorescently labeled dextran into the brain vasculature of embryos just prior to hatching explored the relationship between the developing niche and vasculature. The gradually changing appearance and function of the niche through development are discussed in the light of the current literature related to the adult crayfish brain.In this study, the development of the niche was monitored during embryonic and post-embryonic development of the brain of the procambarid parthenogenetic marbled crayfish (Marmorkrebs) ,38. The P. clarkii.This work describes the appearance and development of the neurogenic niche in the brain of the marbled crayfish from early stages  to late embryonic and post-embryonic development . Images in vivo labeling of embryos (E40% to E50%) with the S-phase-specific proliferation marker BrdU for 4 h followed by immunohistochemical processing revealed a high rate of mitotic activity in the entire germ band and especially in the anlagen of the appendages  [P. clarkii) [These studies suggest that during the 48-h chase period, at least some of the progeny of NB-X and the cap migrate to or become displaced towards cell cluster 9. Because we do not know the BrdU clearing time, it is possible that some of the BrdU+ cells in trail 9 were labeled only shortly before the EdU pulse. In fact, experiments in brains of embryonic lobsters (ricanus)  and the clarkii)  suggest Pulse-chase experiments  demonstrated that, in the POI stage, most cells in the cap are in S-phase Figure ; both laThese experiments also revealed intense mitotic activity in cells in the LPZ that extends into cell cluster 10 Figures ; many ofPulse-chase experiments with a 7-day interval between the two pulses  revealed that after one week the mitotic cells that incorporated the first label had completely moved out into cell clusters 9 and 10 Figure . Stream While the animals undergo a molt to the POII stage, the DPS is transformed as the cap becomes more and more distinct from the protoniche and starts to detach from it Figure . At thisP. clarkii [C. destructor in [P. clarkii [P. clarkii just prior to hatching  or into clarkii ). Therefg Figure . These irespects . In adulDye injections into the dorsal sinus of embryos revealed the cerebral artery and its lateral branches serving the accessory and olfactory lobes. Primary branches of the cerebral artery were as previously described for the adult crab brain , as wereP. clarkii. BrdU labeling identifies the DPS and cap, while the immunolocalization of tyrosinated tubulin delineates the protoniche  [To our knowledge the only available study on the development of a crustacean neurogenic niche is that of Song et al.  on the c adults' . However thread) . TherefoIt is well known that asymmetrically dividing stem cells, the NBs, drive embryonic neurogenesis not only in the ventral nerve cord  but alsoDrosophila melanogaster to contribute to post-embryonic protocerebral structures [Schistocerca gregaria [The DPS comprises an array of NBs, ganglion mother cells and/or other intermediate precursors. The term NB in arthropods classically refers to neuronal stem cells that divide in a geometrical and molecularly asymmetrical pattern and give rise to ganglion mother cells . Recructures ,54-56 angregaria -60.The mitotic cells of the DPS not only comprise NBs but also a heterogeneous assortment of intermediate-sized cells that are positive to S-phase markers and seem to be involved in the post-embryonic rapid massive production of cells in the deutocerebrum. They may be functionally equivalent to the insect intermediate or amplifying progenitors and the presence of such a cell type has already been suggested in the literature on crustacean post-embryonic neurogenesis in the brain ,30,61. HDuring late embryogenesis and early post-embryogenesis, the DPS divides into two parts, laterally into the LPZ and medially into the cap. Both the LPZ and the cap contain numerous cells in S-phase at this stage. At the same time, an additional structure, the protoniche, which will become the neurogenic niche of the adult crayfish ,28, emerThe structure that in adults is known as the MPZ derives from the cap during POII/ADI, when the cap and protoniche are displaced away from each other. Our data demonstrate that while the migratory stream 10 between the protoniche and LPZ is present at hatching and gets thinner and more defined while the brain matures, the stream between the protoniche and MPZ emerges only after the detachment of the cap from the protoniche (POII-juvenile). Therefore, the components of this neurogenic system do not form simultaneously and may not derive from the same cell source , but the development of the system embraces a period of time that lasts about two weeks (from hatching until ADI).P. clarkii undergo geometrically symmetrical divisions and both daughter cells migrate away [in vitro studies have shown that cells circulating in the blood (hemocytes) are attracted to and incorporated into the niche, suggesting that the hematopoietic system may play a role in maintaining the supply of neuronal precursors in the niche [Our study suggests that persistent neurogenesis in the early post-embryonic stages is mostly driven by local amplification in the LPZ and MPZ, while the protoniche in the beginning is characterized by a very low level of mitotic activity and plays a subordinate role. Hence it would appear that during early post-embryogenesis the proliferation zones operate relatively independently from progeny that reach them from the protoniche via the migratory streams. The proliferative characteristics of the protoniche (low level of mitotic activity) closely resemble those in the adult crayfish niche ,30. The ate away , suggestP. clarkii extended this understanding by confirming features of the DPS and protoniche just prior to hatching and in early post-embryonic stages. Further, these studies explored the relationship between the vascular system and the protoniche. The vascular system appears to be a common feature of all stem cell niches, including those in the nervous system [Our studies in the marbled crayfish identified the timing of appearance of the niche anlagen and followed the differentiation of the neurogenic system during post-embryonic stages. Additional work in the crayfish s system ,63. In bs system . Endothes system -67. In as system . The vass system .The literature on 'neurovascular' relationships is rapidly expanding, particularly as these relate to mechanisms of adult neurogenesis, although the role the vascular system may play in the development of neurogenic niches is not known. It is interesting, however, that the subgranular zone was first defined in mammals as the co-association of newborn neurons with dividing endothelial cells . FurtherCommunication between the blood vasculature and the crustacean neurogenic niche has been demonstrated previously ,30,44, aThis study defines the appearance of proliferative areas during early post-embryonic stages that will persist and become the functional elements producing neurons throughout the life of the organism. The protoniche, which originates in close association with vascular elements, is first identifiable at around the time of hatching. Although protoniche cells are actively in the cell cycle, they proceed very slowly compared with other proliferative structures in the post-embryonic brain, a feature that is also indicative of niche function in the mature brain. In contrast, the LPZ, which arises in the POI stage from the DPS, contains cells that are rapidly proliferating. The MPZ does not become recognizable in its adult form until POII, when the post-embryonic cap, derived from the DPS, separates from the protoniche. As the brain expands during the early juvenile stages (ADI), the migratory streams linking the protoniche to both the MPZ and LPZ extend. During POI and POII, the streams do not readily label with S-phase markers, suggesting that they do not yet contain cells migrating between the niche and the proliferation zones (LPZ and MPZ); this is consistent with the slow cycling activity of cells in the protoniche. Our data therefore suggest that the LPZ and MPZ are largely responsible for the production of new neurons in the early post-embryonic stages, and that the neurogenic niche in the beginning plays a subordinate role. However, as the NBs in the proliferation zones disappear during early post-embryonic life, the neuronal precursors in the niche gradually become the dominant and only mechanism for the generation of new neurons in the adult brain.Procambarus fallax [The marbled crayfish is a recently discovered 'parthenogenetic form' of the freshwater decapod s fallax . Once ths fallax . Embryoss fallax ,48 and hs fallax . Althougs fallax ,41,72-84et al. [In the present study, marbled crayfish embryos and post-embryonic stages were taken from a laboratory culture in which single adult individuals (or one adult together with the juvenile progeny) were kept in small aquaria filled with oxygenated tap water (approximately 5 liters at 20°C) and a 10-h light/14-h dark regime. Aquaria were provided with pottery shelters, dried leaves and sand. Water was changed twice a week, and animals were normally fed three times a week with crustacean food pellets (TetraMin) and vegetables (carrots and peas) and checked for mortality, molts or freshly laid eggs. For the experiments, embryos were staged according to the developmental staging system established by Seitz et al. , which q2, 1.2 MgCl2, 2.4 NaHCO3, pH 7.4). Brains were removed from the animals and either fixed for 4 h at room temperature (RT) or overnight at 4°C in 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB). Subsequently, brains were rinsed for 2 h in 0.1 M phosphate-buffered saline , and incubated for half an hour in PBS plus 0.3% Triton X-100 (PBS-TX), before exposure to primary antibodies (4 h or overnight at 4°C). Specimens were then rinsed for 2 h in PBS, followed by 30 minutes in PBS-TX and then incubated overnight in the appropriate secondary antibodies. Some brains were also counterstained with the nuclear stain YOYO (0.25 μl/ml) for 15 minutes. Afterwards, samples were rinsed for 2 h in PBS and mounted either in Gelmount  or Mowiol .Crayfish embryos of different developmental stages were dissected in crayfish saline . Glutamine synthetase is an octamer of 45 kDa subunits and catalyzes the amination of glutamic acid to form glutamine. The presence of two isoforms of glutamine synthetase have been found in the nervous system of P. clarkii, where enzymatic activity has been demonstrated [P. argus, it was confirmed that glutamine synthetase immunostaining in the nervous system is specific for glia [Monoclonal mouse anti-glutamine synthetase antibody (1:100) was from BD Biosciences Pharmingen . Microtubules are mainly formed by tubulin, which is a heterodimer consisting of α-tubulin and ß-tubulin, subject to specific post-translational modifications at the carboxyl termini of both units [Monoclonal anti-tyrosinated tubulin  was from Sigma  was used. EdU, another modified nucleoside, was detected with a cell proliferation kit . EdU was detected only in fluorescently labeled preparations and always with the secondary Alexa 488 antibody, included in the kit. Details of the protocol are reported below.BrdU and EdU, synthetic thymidine analogs that are incorporated into single-stranded DNA during S-phase, can be used to identify cells actively in the cell cycle. Single and multiple labelings were performed and thus different cocktails of secondary antibodies were used. In single peroxidase BrdU labeling, the Cell Proliferation Kit RPN 20  was employed. The primary antibody was a mouse anti-BrdU included in the kit ; details of the protocol are described below. For fluorescent staining, both the primary mouse anti-BrdU (Amersham kit) and the rat anti-BrdU (Sigma) were utilized. In this case, according to the goals of the experiment, goat anti-mouse Alexa 488 or 546, or goat anti-rat Alexa 488 or 546  were incubated directly in a BrdU/saline solution  for 4 h at RT. The 4-h time span has been demonstrated to provide an optimal labeling of cycling cells while the number of labeled cells is still low enough to be analyzed efficiently [Subsequently, animals were fixed in PFA for 30 minutes at RT before dissection. Brains were removed and incubated in 4% PFA for at least 4 h at room temperature or overnight at 4°C. Specimens were washed in at least three changes of PBS buffer for 30 minutes and then pre-incubated in PBS-TX for 30 minutes. To denature the DNA and hence improve binding of the antibody, the tissue was exposed to an acid treatment (2N HCl) for 20 minutes. After another cycle of PBS rinses, brains were incubated in mouse anti-BrdU antiserum  for 2.5 h at RT. Afterwards, specimens were washed in PBS overnight at 4°C and incubated in goat anti-mouse antiserum conjugated with peroxidase for 60 to 90 minutes at RT. Specimens were washed in PBS overnight at 4°C. To detect the bound antibody, the enzymatic diaminobenzidine reaction was used according to the manufacturer's instructions: in the presence of cobalt and nickel, diaminobenzidine gives a blue-black precipitation where the secondary antibody is bound and hence in those nuclei that incorporated BrdU. After dehydration in a graded EtOH series, specimens were mounted in methyl salicylate.® EdU Cell Proliferation Assay (Molecular Probes) is an alternative to the BrdU method. It is not antibody-based and does not require DNA denaturation for detection of the incorporated nucleoside. Animals  were incubated for 4 h at RT and afterwards fixed in PFA for 30 minutes. Brains were dissected and fixed for an additional 4 h. Specimens were then washed in at least three changes of PBS buffer and pre-treated in PBS-TX for 30 minutes. Incubation in Alexa 488 Click-iT reaction cocktail was performed for 2 h at RT. The solution was prepared fresh and contained 1X reaction buffer . Several washing steps followed this application before the incubation in primary antibodies used for the double labeling.The Click-iTIn order to clarify the activity over time of cells residing in the niche, a series of pulse-chase experiments was performed. Animals, placed in a small container for liquid (100 ml volume), were exposed first to BrdU for 4 h (first pulse). Then, the labeling solution was quickly rinsed off the animals and these were kept undisturbed in fresh water for 24 h, 48 h or 7 days, depending on the experiment. During this time, animals were in similar conditions as before the treatment . After this survival period, animals were exposed to the second labeling reagent (EdU) for 4 h (second pulse) and immediately fixed and dissected. Protocols followed the directions described above and used rat anti-BrdU (1:100 in PBS-TX) and mouse anti-tyrosin-tubulin  antibodies overnight. The application of the Alexa 488 Click-iT reaction cocktail was performed for 2 h at RT. Tissues were then incubated for 4 h (or overnight at 4°C) in a cocktail of secondary antibodies (anti-mouse Alexa 630 and anti-rat Alexa 546). After final washings, brains were mounted in Mowiol mounting medium.Actin and nuclear counterstaining provided more details of brain structure. Phalloidin-labeled phallo-toxins such as Alexa 488-phalloidin  and Alexa 546 phalloidin  are convenient probes for labeling and identifying F-actin in tissue. Using phalloidin, emerging structures of the brain during embryogenesis were highlighted, which allowed us to determine the exact developmental stages of specimens. As a nucleic acid stain, the green fluorescent YOYO  was used in order to distinguish nuclei (hence cells) from acellular structures in the brain .Digital images of light microscopic specimens were obtained with a Zeiss Axioskop fitted with a CCD-1300B digital camera  and processed with the Lucia Measurement 5.0 software package ). Fluorescent samples were screened with a Zeiss LSM 510 Meta confocal microscope. Images are based on stacks of 15 to 20 optical sections (single images are averages of 4 to 8 laser sweeps) of a z-series. Measurement of cells residing in the niche were obtained using the software ImageJ. The size of images in two dimensions was 1,024 × 1,024 × 1 pixel; sizes of images in three dimensions (z stack) varied according to the sample. Images from stacks represent projection of the z sections. Some images were adjusted for brightness and contrast with Adobe Photoshop 7.0 software  and arranged into plates by using the illustration program Illustrator 11.0 (Adobe Systems).P. clarkii embryos just prior to hatching  [ n = 80)  were remADI: first immature adult stage; BrdU: 5-bromo-2'-deoxyuridine; DPS: deutocerebral proliferative system; EdU: 5-ethynyl-2'-deoxyuridine; GMC: ganglion mother cell; LPZ: lateral proliferative zone; MPZ: medial proliferative zone; NB: neuroblast; PBS: phosphate buffer; PBS-TX: PBS plus 0.3% Triton X-100; PFA: paraformaldehyde; POI: first post-embryonic stage; POII: second post-embryonic stage; RT: room temperature.The authors declare that they have no competing interests related to this study.P. clarkii. BSB and SH contributed to the planning and analysis of experiments, as well as to the composition of the final manuscript. BSH assisted in drafting the manuscript. All authors read and approved the final manuscript.SS performed the primary experimental work and analysis for this paper, which contributed to her PhD thesis. JLB repeated aspects of this work in marbled crayfish, confirming the major findings of the study, and did the pulse-chase experiments with 1 week survival time. She also did the dextran injections of the vasculature in A video taken from a confocal stack. The video from a confocal stack  was imaged from the ventral surface through to the mid-brain of a P. clarkii brain at hatching (approximately 26 μm into the brain tissue). There are three labels: (1) dextran dye (green) micro-injected into the cerebral artery via the dorsal sinus combined with (2) the S-phase marker BrdU (red) identifying the cap and the immunolocalization of (3) tyrosinated tubulin (white) delineating the protoniche and its pore. The video shows that the cerebral artery bifurcates and then pushes ventrally toward the two hemispheres, each branch dividing laterally and then many times over into the neuropile regions as continuous tubes. The protoniche has blood vessels confluent with its structure and connects to blood vessels slightly deeper into the tissue. The central pore, considered as an early stage of the cavity, is confluent with the blood vasculature.Click here for file


In the treatment of hepatocellular carcinoma (HCC), hepatic resection has the advantage over radiofrequency ablation (RFA) in terms of systematic removal of a hepatic segment.We enrolled 303 consecutive patients of a single naïve HCC that had been treated by RFA at The University of Tokyo Hospital from 1999 to 2004. Recurrence was categorized as either intra- or extra-subsegmental as according to the Couinaud's segment of the original nodule. To assess the relationship between the subsegments of the original and recurrent nodules, we calculated the kappa coefficient. We assessed the risk factors for intra- and extra-subsegmental recurrence independently using univariate and multivariate Cox proportional hazard regression. We also assessed the impact of the mode of recurrence on the survival outcome.During the follow-up period, 201 patients in our cohort showed tumor recurrence distributed in a total of 340 subsegments. Recurrence was categorized as exclusively intra-subsegmental, exclusively extra-subsegmental, and simultaneously intra- and extra-subsegmental in 40 (20%), 110 (55%), and 51 (25%) patients, respectively. The kappa coefficient was measured at 0.135 . Multivariate analysis revealed that of the tumor size, AFP value and platelet count were all risk factors for both intra- and extra-subsegmental recurrence. Of the patients in whom recurrent HCC was found to be exclusively intra-subsegmental, extra-subsegmental, and simultaneously intra- and extra-subsegmental, 37 (92.5%), 99 (90.8%) and 42 (82.3%), respectively, were treated using RFA. The survival outcomes after recurrence were similar between patients with an exclusively intra- or extra-subsegmental recurrence.The effectiveness of systematic subsegmentectomy may be limited in the patients with both HCC and chronic liver disease who frequently undergo multi-focal tumor recurrence. Hepatic resection is regarded as the most appropriate first-line treatment for patients with solitary hepatocellular carcinoma (HCC) who are non-cirrhotic or cirrhotic without portal hypertension Percutaneous tumor ablation methods, such as ethanol injection and microwave coagulation, have played an important role as nonsurgical treatments that can achieve high local cure rates without reducing background liver function Hepatic resection is supposed to have the advantage over RFA as an effective intervention as it involves the systematic removal of a hepatic segment containing the tumor. Indeed, microscopic satellite nodules, not detected by radiological examination prior to resection, are often observed in the resected specimen Whereas RFA can reliably eliminate target nodules together with some of the surrounding tissue, most of the liver parenchyma of the tumor-bearing segment is left unablated. In contrast to anatomical resection, it is possible to observe and analyze intra- and extra-subsegmental recurrence by following up patients after ablation. The aim of our present study was to assess the frequency, risk factors and survival outcomes associated with intra-subsegmental HCC recurrence after RFA in comparison with extra-subsegmental recurrence.http://gastro.m.u-tokyo.ac.jp/med/0602A.htm) and participants who do not agree to the use of their clinical data can claim deletion of them.This retrospective study was conducted according to the ethical guidelines for epidemiological research designed by the Japanese Ministry of Education, Culture, Sports, Science and Technology and Ministry of Health, Labour, and Welfare. The study design was included in a comprehensive protocol of retrospective study at the Department of Gastroenterology, The University of Tokyo Hospital approved by The University of Tokyo Medical Research Center Ethics Committee . The following statements were posted at a website  and long-term  outcomes. Obtained data were stored in an encrypted hard disk separated from outside of the hospital. When reporting analyzed data, we protect the anonymity of participants for the sake of privacy protection. If you do not wish the utilization of your data for the clinical study or have any question on the research content, please do not hesitate to make contact with us.3/mm3 and prothrombin activity levels of no less than 50%. Patients with a portal vein tumor thrombosis, refractory ascites, or extrahepatic metastasis were excluded. In general, we performed RFA on patients with three or fewer lesions of 3 cm or less in diameter. However, we also performed ablation on patients beyond these criteria if it was predicted to be clinically effective From 1999 to 2004 a total of 569 patients with HCC underwent RFA as the initial treatment for naïve HCC. Of them, 304 patents had a single nodule. We enrolled 303 of these patients in our current study excluding one patient who could not achieve complete ablation. The inclusion criteria for RFA had been as follows: a total bilirubin level of less than 3 mg/dL, a platelet count of no less than 50×10HCC was diagnosed using dynamic computed tomography (CT), with a consideration of hyperattenuation in the arterial phase with washout in the late phase as a definite sign of this disease All patients received dynamic CT with a slice thickness of 5 mm within one month prior to ablation for comparison. The interval between the initiation of contrast material infusion and CT image recording was 30 and 120 sec for single detector-row spiral CT  and 25, 40 and 120 sec for multidetector-row CT . The images were presented after axial reconstruction with a slice thickness of 5 mm. RFA was performed on an in-patient basis using a cooled-tip electrode  under real-time ultrasound guidance. After 1 to 2 sessions of RFA, dynamic CT was performed to evaluate the treatment efficacy. During the treatment evaluation, we compared the CT findings for early and late phase before ablation and late phase after ablation. A lesion was judged to be completely ablated when the non-enhanced area shown in the late phase of CT post-ablation covered the entire lesion shown in both early and late phase of CT pre-ablation with a safety margin in the surrounding liver parenchyma. We confirmed complete ablation in all slices on which the target nodule was visualized. Patients received additional sessions until complete ablation was confirmed in each nodule. Finally, 303 of the 304 patients enrolled in this study were judged to be completely ablated.The General Rules for the Clinical and Pathological Study of Primary Liver Cancer, Second English EditionThe follow-up regimen consisted of blood tests and monitoring of tumor markers in an outpatient setting. Ultrasonography and dynamic CT were also performed every four months. Tumor recurrence was defined as a newly developed lesion on a dynamic CT that showed hyperattenuation in the arterial phase with washout in the late phase. The nomenclature used for the hepatic segments conformed to When HCC recurrence was identified, patients who met the same criteria used for primary HCC underwent RFA. Survival analysis was performed on a per patient basis. Patients without an indication for RFA due to a multiplicity of recurrent nodules underwent TACE if liver function was categorized as Child-Pugh class B or better. Patients with localized portal tumor invasion were treated by radiotherapy Data were expressed as the mean ± standard deviation (SD) unless otherwise indicated. To assess whether the location of recurrent nodules was independent of the subsegment of the original nodule, we calculated the kappa coefficient and its 95% confidence interval (CI) To assess the exclusively intra-subsegmental recurrence rate separately from all kinds of recurrence, we used cumulative incidence estimation with competing risk methods We assessed the risk factors for intra- and extra-subsegmental recurrence independently using univariate and multivariate Cox proportional hazard regression. In assessing the risk factor for intra-subsegmental recurrence, patients with exclusively extra-subsegmental recurrence were treated as censored data and vice versa. The following factors were used for these analyses: age, gender, hepatitis B surface antigen positivity, hepatitis C antibody positivity, platelet count, alanine aminotransferase (ALT), tumor size, alpha-fetoprotein (AFP), des-gamma-carboxyprothrombin (DCP) and lens culinaris agglutinin-reactive fraction of AFP (AFP-L3). Factors showing statistical significance as a predictor in univariate analysis were further analyzed using a multivariate Cox proportional hazard regression model with stepwise selection of variables based on the Akaike information criterion (AIC).P value of less than 0.05 were considered statistically significant. All statistical analyses were performed with S-Plus Ver. 7  and R 2.13.0 (http://www.R-project.org).We plotted survival curves according to the mode of recurrence  using the Kaplan-Meier method. Statistical significance among these three groups was assessed using the log-rank test. We also calculated adjusted hazard ratios for survival according to the mode of recurrence using multivariate Cox proportional hazard regression with factors that showed statistical significance in a univariate analysis of survival. Differences with a The enrolled HCC patient cohort in this study consisted of 191 males and 112 females with a mean age of 67.5 years . The meaDuring the follow-up period , tumor recurrence in the HCC patient cohort was identified in 201 cases. The recurrent nodules were distributed in a total of 340 subsegments. Recurrent nodules were exclusively intra-subsegmetal in 40 patients (20%), and exclusively extra-subsegmental in 110 patients 55%, . SimultaUnivariate Cox proportional regressions revealed that the following factors were significantly associated with intra-subsegmental recurrence: tumor size, AFP, DCP, AFP-L3, platelet count and anti-HCV antibody positivity. The final model for predicting intra-subsegmental recurrence with stepwise variable selection included tumor size, AFP, platelet count and anti-HCV antibody positivity . FactorsAmong the 40, 110 and 51 patients in whom recurrent HCC was found to be exclusively intra-subsegmental, exclusively extra-subsegmental, and simultaneously intra- and extra-subsegmental, 37 (92.5%), 99 (90.8%) and 42 (82.3%), respectively, were treated using RFA. Of the three patients with an exclusively intra-segmental recurrence, one individual was treated by hepatic resection and one patient was treated by TACE. The remaining patient received best supportive care because of deterioration in liver function. During the follow up period up to December 31, 2010, 130 patients died and 9 patients were lost to follow-up. The median survival time (95% CI) was 5.72 (3.51-NA) years in patients with exclusively intra-subsegmental recurrence, 4.95 (4.19–5.76) years in patients with exclusively extra-subsegmental recurrence, and 2.43 (1.90–4.26) years n patients with simultaneously intra- and extra-subsegmental recurrence, respectively  were risk factors for intra-subsegmental recurrence as well as extra-subsegmental recurrence. The risk of recurrence due to de novo carcinogenesis might be reduced by a subsegmentectomy according to the resected liver volume. However, as most patients with HCC have chronic liver disease, removal of non-cancerous liver parenchyma may have a negative impact on long-term survival, especially for those individuals with impaired liver regeneration. Therefore, the key issue is to what extent the liver parenchyma should be removed to sufficiently treat the patient on a case by case basis. It may be speculated that extensive resection could be tolerable and beneficial to those who have a well-preserved capacity for liver regeneration There is no doubt that tumor recurrence deteriorates the long-term prognosis for HCC patients. However it is also true that there are effective, sometimes potentially curative treatments for recurrent HCC. The re-resection after recurrence of HCC is indicated in 10–30% of patients We would touch upon the possibility that TACE prior RFA might affect the results. Approximately one third of patients underwent TACE prior RFA. TACE prior RFA can increase the ablated area through the blockade of arterial flow and may decrease the risk of local tumor progression. However the influence would be minimal because local tumor progression was found in only 10 patients and the location of recurrent nodules was weakly but significantly related to that of the original tumor, even after the exclusion of local tumor progression.In conclusion, the vast majority of recurrent nodules in HCC patients were found to be independent of the subsegment of the original tumor. In addition, whether these recurrences were intra-subsegmental or extrasubsegmental had no impact on the survival outcomes.


Musicians and musically untrained individuals have been shown to differ in a variety of functional brain processes such as auditory analysis and sensorimotor interaction. At the same time, internally operating forward models are assumed to enable the organism to discriminate the sensory outcomes of self-initiated actions from other sensory events by deriving predictions from efference copies of motor commands about forthcoming sensory consequences. As a consequence, sensory responses to stimuli that are triggered by a self-initiated motor act are suppressed relative to the same but externally initiated stimuli, a phenomenon referred to as motor-induced suppression (MIS) of sensory cortical feedback. Moreover, MIS in the auditory domain has been shown to be modulated by the predictability of certain properties such as frequency or stimulus onset. The present study compares auditory processing of predictable and unpredictable self-initiated 0-delay speech sounds and piano tones between musicians and musical laymen by means of an event-related potential (ERP) and topographic pattern analysis (TPA) [microstate analysis or evoked potential (EP) mapping] approach. As in previous research on the topic of MIS, the amplitudes of the auditory event-related potential (AEP) N1 component were significantly attenuated for predictable and unpredictable speech sounds in both experimental groups to a comparable extent. On the other hand, AEP N1 amplitudes were enhanced for unpredictable self-initiated piano tones in both experimental groups similarly and MIS did not develop for predictable self-initiated piano tones at all. The more refined EP mapping revealed that the microstate exhibiting a typical auditory N1-like topography was significantly shorter in musicians when speech sounds and piano tones were self-initiated and predictable. In contrast, non-musicians only exhibited shorter auditory N1-like microstate durations in response to self-initiated and predictable piano tones. Taken together, our findings suggest that besides the known effect of MIS, internally operating forward models also facilitate early acoustic analysis of complex tones by means of faster processing time as indicated by shorter auditory N1-like microstate durations in the first ~200 ms after stimulus onset. In addition, musicians seem to profit from this facilitation also during the analysis of speech sounds as indicated by comparable auditory N1-like microstate duration patterns between speech and piano conditions. In contrast, non-musicians did not show such an effect. In the past 15 years, highly trained musicians have become an important model in neuroscience to study the effects of learning-induced cortical plasticity. It is now widely known and accepted that the intensive practice and training needed to achieve high musical skills leads to structural and functional short- and long-term alterations in the brain .Grand-averaged waveforms evoked by CV-syllables recorded at Cz and the respective mean N1 peak amplitudes are illustrated in Figure post-hoc t-tests indicated smaller amplitudes in the MA  and MAU  tasks compared to the A task. The difference between the MA and MAU tasks failed to reach significance . Thus, a distinct attenuation to self-relative to externally initiated speech sounds was observed in general, replicating the results of various other studies addressing MIS and SIS, respectively  = 40.362, p < 0.001], pointing to smaller amplitudes evoked by the unvoiced CV-syllable /ta/ than by the voiced CV-syllable /da/. Again, this finding is in line with the pre-existing literature covering the topic of sub-segmental speech processing  = 4.408, p < 0.05] and pitch . In addition, a significant interaction task by pitch  was revealed. With respect to group-related differences regarding N1 peak amplitudes elicited by piano sounds, no significant effects were found.Figure post-hoc analyses were applied for low and high piano tones separately, using Bonferroni–Holm adjusted t-tests. Low piano tones-evoked stronger N1 potentials in the MAU task relative to the A task , whereas high piano tones-evoked stronger potentials in the MAU task relative to the MA task . All other comparisons failed to reach significance. Thus, no significant attenuation of mean N1 peak amplitudes to self-initiated piano sounds was observed.Subsequent https://sites.google.com/site/cartoolcommunity/user-s-guide), we first visually inspected the landscape and template maps resulting from the segmentation process to determine the particular time period and maps for the single-subject fitting. In order to provide consistency with the examination of the classical N1 component, we focused our analyses to the one template map that expressed a typical N1-like auditory topography (map 1) and therefore chose the time period for the single-subject fitting according to the first on- and last offset of this particular map. This resulted in a time window for the fitting procedure of 0–192 ms. Duration values of this map were then extracted from the fitted single-subject data in terms of timeframes and compared by means of a repeated measure ANOVA.Figure group and within-subjects factors task and voicing yielded a significant main effect for the factor task  and a significant task by group interaction .Results of the 2 × 3 × 2 repeated measure ANOVA with between-subjects factor post-hoc analyses were conducted for the two experimental groups, using Bonferroni–Holm adjusted t-tests. Since no significant effect for the factor voicing was found, the respective auditory N1-like map (map 1) durations for the two CV-syllables (/da/ vs. /ta/) were averaged and analyzed together. Post-hoc t-tests revealed significantly longer durations for map 1 in the A task relative to the MA task in the pianist group . Moreover, map 1 durations were longer in the MAU task compared to the MA task in the same group , though this particular difference failed to reach significance after correction for multiple comparisons. On the other hand, no significant differences between tasks regarding auditory N1-like map durations were found in the non-musician group at all.Separate Piano-tone related results of the topographical EP mapping are illustrated in Figure task  and group . Post-hoc t-tests revealed significantly longer durations for map 1 in the MAU relative to the MA task  and in the A task relative to the MA task . Though, the latter comparison failed to reach significance after correction for multiple comparisons. In addition, musicians exhibited longer auditory N1-like map durations in the MA and MAU tasks compared to non-musicians . Again, the latter comparison did not reach significance after correcting for multiple comparisons. Other than that, neither further main effects nor interactions were found.Here, the 2 × 3 × 2 repeated measures ANOVA yielded significant main effects for the factors MIS  and low- and high-pitched piano tones . Furthermore, we introduced a task in which the particular sensory consequence of a certain motor-action was unpredictable in terms of voicing (speech condition) or pitch (piano condition). To control for mere motor activity possibly being responsible for any observed suppression effects involving auditory processing, a M task was also applied and corresponding difference waves between MA and M tasks were calculated for further analysis. High-density EEG recordings were derived during the early acoustic processing stages and analyzed in two different ways: Firstly we calculated and analyzed conventional AEP components and focused on the N1 component, which is particularly susceptible for MIS-effects  sounds mainly corresponds to the existing literature addressing this topic . In opposition to this, the tasks used in our current study emphasize a rather passive/implicit processing of auditory speech stimuli. While the participants attention was mainly focused on maintaining the interval between button presses of ~3 s, active/explicit processing of the auditory stimuli was not required to accomplish the task. At the same time, there are a variety of studies showing that auditory processing can be affected by specific task properties such as cognitive load, difficulty, repetition, and active vs. passive conditions . High-pitched piano tones on the other hand substantially enhance N1 amplitudes when self-triggered and unpredictable relative to self-triggered but predictable  ones. Furthermore, we found that durations of the auditory N1-like template map are significantly longer during early auditory processing stages  in the pianist group in the MA task while in both groups the respective microstate durations are generally shorter when piano tones are self-triggered and predictable.Our results at hand regarding self- vs. externally triggered piano tones seem somewhat contradictory to begin with, considering that most studies examining auditory processing of self- versus externally initiated sounds consistently showed motor-induced suppression of early AEP components (e.g., Schafer and Marcus, Anyhow, this still does not explain why AEP N1 amplitudes in response to predictable self-initiated piano tones do not exhibit significant suppression relative to predictable externally initiated ones in our current study. Though, a plausible explanation can be derived from the results of a recent MEG study: Aliu et al.  reportedDespite the absence of significant suppression effects of AEP N1 amplitudes, the results of the corresponding TPA reveal alterations of auditory processing of self-initiated piano tones in both experimental groups. Auditory N1-like map durations are significantly shorter in pianists as well as in musical laymen when the piano tones are self-initiated and predictable. Again, this indicates a facilitation effect of accurate sensory predictions by the forward model such as less processing time is needed for early auditory analysis. Moreover, pianists show enhanced N1-like map durations relative to non-musicians in the MA task, suggesting a more refined auditory analysis and increased neuronal representation of self-initiated and predictable piano tones in pianists. This finding fits in well with the results of various other studies showing enhanced auditory processing of complex tones in musicians (e.g., Pantev et al., However, it is also of importance to briefly address one additional issue at this point: the auditory N1 has been shown to vary as a function of attention. Thus, it might be the case that possible differences in MIS between the groups are corrupted by mere attention effects. Though, the question whether attention influences the N1-suppression effect for self-initiated sounds has recently been addressed by Timm et al. . Their rOur findings indicate that intense musical training facilitates the processing of predictable self-initiated speech sounds in terms of faster early auditory analysis in the first ~200 ms. In particular, pianists show a comparable auditory N1-like template map duration pattern in response to predictable self-initated speech and piano tones, whereas a substantial reduction of processing time in non-musicians is only present in response to predictable self-initiated piano tones. Moreover, pianists and non-musicians did not significantly differ with respect to suppression of AEP N1 amplitudes neither in response to self-initiated speech sounds nor piano tones. Taken together, our results suggest that pianists rely on their forward model's predictions of sensory outcomes to the same extent when processing speech sounds and piano tones. In contrast, non-musicians seem to analyze predictable self-initiated speech sounds in a more elaborate manner even in the case of a positive match between their forward model's predictions and the actual auditory input. In addition, internal forward mechanisms do not mandatorily lead to suppressed cortical feedback to self-initiated complex sounds but can take beneficial effect with respect to processing time, as indicated by the absence of significant suppression of AEP N1 amplitudes and nonetheless shortened auditory N1-like map durations in response to predictable self-initiated piano tones.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Genes duplicated by polyploidy (homoeologs) may be differentially expressed in plant tissues. Recent research using DNA microarrays and RNAseq data have described a cacophony of complex expression patterns during development of cotton fibers, petals, and leaves. Because of its highly canalized development, petal tissue has been used as a model tissue for gene expression in cotton. Recent advances in cotton genome annotation and assembly now permit an enhanced analysis of duplicate gene deployment in petals from allopolyploid cotton.Gossypium using the Gossypium raimondii genome sequence as a reference. In the polyploid, most homoeologous genes were expressed at equal levels, though a subset had an expression bias of AT and DT copies. The direction of gene expression bias was conserved in natural and recent polyploids of cotton. Conservation of direction of bias and additional comparisons between the diploids and tetraploids suggested different regulation mechanisms of gene expression. We described three phases in the evolution of cotton genomes that contribute to gene expression in the polyploid nucleus.Homoeologous gene expression levels were quantified in diploid and tetraploid flower petals of cis-acting modifications that occurred prior to polyploidization. Some duplicated genes were intriguing exceptions to general trends. Mechanisms of gene regulation for these and other genes in the cotton genome warrants further investigation.Compared to previous studies, a surprising level of expression homeostasis was observed in the expression patterns of polyploid genomes. Conserved expression bias in polyploid petals may have resulted from  Gossypium currently consists of approximately 45 diploid and six polyploid species [G. hirsutum and G. barbadense, produce spinnable fiber used by the textile industry and represent the majority of world-wide cotton production. An investigation of the effects of polyploidization on gene expression could further our understanding of the basis of superior cotton fiber qualities and fiber yields in tetraploid cotton cultivars.The genus  species ,2. The s species ,4. Whilei.e. inter-allelic) in diploid Homo sapiens[Polyploidization causes a simultaneous duplication of all nuclear DNA, and some of the genomic consequences of polyploidization can be dramatic -7. One co sapiens-17. Regao sapiens,18,19. Oo sapiens. The expo sapiens,21-26.Gossypium tetraploids to those in related diploids for a given gene. When the tetraploid gene expression level was statistically indistinguishable from one of the diploids, it was assumed that the diploid with the expression level matching that found in the polyploid was dominant. When many genes throughout the genome exhibited expression dominance, the generalized trend was considered expression level dominance of the genome if one of the two genomes was more frequently dominant than the other genome in the tetraploid nucleus. An expression dominance of one of the two genomes was found in leaf [Gossypium polyploids. Expression level dominance has also been observed in other polyploid species such as Coffea[Spartina[cis- and trans- interactions of the regulatory machinery in the two distinct genomes are one explanation [Coffea species [Another consequence of polyploidization is expression level dominance. Expression level dominance has been characterized by the abundance of transcript rather than the transcript origin . It was  in leaf ,27 and p in leaf ,20 tissuas Coffea, Spartin[Spartina and whealanation . Externa species . DiffereG. raimondii to measure gene expression in several polyploid accessions of cotton within its phylogenetic framework.Previously, the transcript contributions of the two co-resident cotton genomes were quantified by custom microarrays ,10,18 orG. arboreum , G. raimondii , G. tomentosum , G. hirsutum cv. Acala Maxxa , G. hirsutum cv. T×2094  and a sterile diploid F1-hybrid between A2 and D5  (Table 1-hybrid was created by a hand pollination between reduced gametes of diploids G. arboreum (A2) and G. raimondii (D5), and its somatic cells only contain 13 chromosomes from each extant diploid genome . Diploid and tetraploid sequencing reads were individually mapped using GSNAP [G. raimondii[T and DT, using PolyCat with an index of 24 M homoeo-SNPs identified between several A1/A2 and D5 accessions [Reads were filtered and trimmed using sickle with a phred quality threshold of 20  uses a mixed-model approach to quantify the probability of gene expression in a sample . UPC det1-hybrid, G. tomentosum, G. hirsutum Tx2094, G. hirsutum Maxxa) and ‘genome’ with two levels (A-genome or D-genome). A single, nested interaction design was used to determine genes significantly differentially expressed between accessions. A separate, simple single factor experiment with 8 levels was used to detect genes differentially expressed between two genomes for each accession. EdgeR performs exact test for the negative binomial distribution coefficients to provide p-values and false discovery rates  for all the genes. Genes with <0.05 FDR were considered differentially expressed.EdgeR was used to normalize expression data and perform differential expression analysis . Two facA phylogeny based on expression levels of the genes from all the accessions was built using the neighbor-joining algorithm, with sum of squared differences for all genes between accessions within a distance matrix. We built another phylogeny using the neighbor-joining algorithm based on the difference between the two homoeologous gene expression levels.2 and D5 expression to the total expression of the polyploid. A matrix was constructed with the number of genes from the two comparisons and these numbers were used to calculate the total number of genes in each category of expression level dominance.To analyze expression level dominance, every gene of each polyploid accession was separately analyzed and characterized according to the relationships between the RPKM values of the different genomes. Genes without expression in petals as determined by UPC were excluded from analysis. Each gene was categorized after comparison of AG. tomentosum had the most RNA-seq reads (> 40 M each) and diploid D5 had the fewest RNA-seq reads (~37 M). Each of these reads was mapped  to the 13 large pseudo-molecules of the D-genome reference sequence  containT and DT). If the mapped reads overlapped a homoeo-SNP position (SNPs between the A- and D-genomes), they were categorized as belonging to one of the two genomes or as a chimeric read because it had A- and D-genome nucleotides at different loci in close proximity  . If a requences .Depending on the accession, approximately 45-50% of the genome is expressed in petals based on the UPC analysis. This total is lower than the number of cotton genes found to be expressed in fiber tissue (75-90%) at any developmental stages ,19. ThisDianthus[i.e. many genes were members of large gene families). These 654 enzymes were found to be part of 93 different enzymatic pathways in petal tissue. The enzymatic pathways can be divided into four general categories: Metabolic pathways, Biosynthetic pathways, Degradation pathways and signaling pathways  is referred to as ‘genome bias’ [i.e. bias ratio of AT/DT where AT > DT or vice versa) when the genes with 99% UPC expression likelihood were considered. Approximately 20% of genes expressed in petals had a significant bias towards the AT- or DT-genome  than other polyploids. However, we found only subtle differences in the distribution ratios of gene expression among any of the accessions using RNA-seq  was found to have an expression bias in each polyploid petal transcriptome. When the degree of bias of these genes was compared to the remaining biased gene pairs for each accession, these 478 biased pairs had a significantly larger degree of bias than the remaining gene pairs . For example, the degree of bias for the 478 ‘conserved’ genes was significantly higher than the degree of bias of the remaining 814 genes in the diploid F1-hybrid.Many commonly expressed genes with homoeologous expression bias were found in more than one accession Figure A. For exT direction  between the number of total genes/chromosome and number of biased genes/chromosome, the homoeologous gene expression bias was probably not related to their chromosomal location. Interestingly, 19 gene pairs in the diploid F1-hybrid had consistent, yet contrary biases compared to the natural polyploids . In addition, each of the polyploids had a unique set of a small number of gene pairs with a contrarian directional bias than the remaining accessions .The direction of bias for these 478 gene pairs was conserved for most petal transcriptomes with a few exceptions. Of the 478 gene pairs, 246 were consistently biased in the An Figure B and 2021-hybrid but not the natural polyploids, while 770 genes were biased in the natural polyploids but not the hybrid  was conserved for all but 1–2 pairs of genes . This conserved direction of homoeologous bias suggested a difference in cis-regulation of gene expression between genomes. Inspection of all of the other biases shared between two or more accessions found strong, consistent directions of homoeologous expression bias suggesting that the bias has roots in the independent evolution of the two progenitor species prior to reuniting within the common tetraploid nucleus.Zooming out to reconsider accession totals of biased genes, 339 genes were biased in the diploid Fi.e. expression level differences of AT - DT) between AT and DT homoeologous gene copies for each of the commonly expressed genes were also visualized within the phylogenetic framework  exhibited a consistent expression pattern . The diploid F1-hybrid frequently differed from the natural polyploids in gene expression levels and the categories of expression level dominance.Considering expression patterns for duplicated genes in a polyploid, relative to their orthologs in related diploids, twelve possible categories have been described, where each category corresponds to a combination of relative equalities or inequalities between two diploids and between the total expression in the tetraploid ,19,27. T1-hybrid, Maxxa, and G. tomentosum, respectively). This disagreement between the microarray and RNA-seq results could have been due to an artifact of microarray probe design where more EST templates were available for the D-genome than the A-genome . In addT- and DT-genomes compared to diploids) are ~10-fold more abundant than categories IX and XI  coincided with the RNA-seq read composition of genes with significant homoeolog expression bias. Perhaps, this also indicated cis-acting regulation of genes where a higher expression of one of the two diploid genomes was conserved in the polyploid nucleus and potential overexpression was controlled by and at the same levels as in the diploids.Because we could discern the As Figure . Thus, wTDT). It had equivalent contributions from each genome. In contrast to other categories, more genes were placed in this category on a percentage basis by our RNA-seq analysis than by the previous microarray experiment. There were also many fewer of these genes in the diploid F1-hybrid than in the natural polyploids. Perhaps this transgressive expression represent a different type of ‘additive’ gene expression where each diploid contributes an additive amount of transcript (such that two copies in the nucleus results in twice as much gene product), except that these loci have not individually evolved a mechanism for feedback control of expression. Such an interpretation would agree with a form of pseudo-overdominance (i.e. heterosis) between genomes and may have been due to complementary combinations of cis- and trans-acting factors resulting in more expression together than either genome alone.Interestingly, category VIII is described as exceptional or transgressive expression in the polyploid . In genes that were differentially expressed in both diploids and polyploids, it was rare for the direction of bias to change from one direction in the diploids to the opposite direction in the polyploid . Inspection of 1,000 bp upstream of the annotated genes did not reveal any significant differences in the number of SNPs between these two categories of genes . Perhaps the causative differences of expression were further upstream [T-genome than the DT-genome. Such a simplified perspective overlooks some complexities of cotton polyploid formation; however, it emphasizes the putative predominance of trans-acting regulation of gene expression in polyploid cotton if the ‘different’ categories are generally interpreted as cis-acting regulation.We generated a new perspective of gene expression in polyploid cotton by cross-listing two separate gene lists. The first list consisted of the genes that were differentially expressed between the diploids, Ads Table . It was 1-hybrid to the average degree of bias detected in the natural polyploids. When there was a significant DT bias detected in the diploid F1-hybrid, there was also a significant difference in the amount of bias between the diploid F1-hybrid and the other polyploids. When there was a significant AT bias detected in the diploid F1-hybrid, there was not a significant difference in the amount of bias between the F1 and the other polyploids.In addition, we compared the average degree of bias detected in the diploid FT-genome to the diploid A-genome and the DT-genome to the D-genome . The AT-genome in the diploid F1-hybrid had more expression level differences with the diploid A-genome than the DT-genome did with the diploid D-genome (390 vs. 115), but the natural polyploids all had more expression level differences with the diploids in the DT-genome than in the AT-genome. When the polyploid had a different level of expression than its respective diploid, the AT-genome expression levels were more frequently higher than the A-genome diploid expression levels and the DT-expression levels were more frequently lower than the D-genome diploid expression levels.To complement that previous perspective, we also compared the expression of the Ame Table . The dipGossypium species because of its economic importance [G. raimondii reference genome [Gossypium genome was expressed in the petal tissue of open flowers prior to pollination. This number was noticeably smaller than the number of genes expressed during the development of cotton fiber [Cotton fiber tissue has been the main focus of many transcriptome studies of portance -12,48,49portance ,20. In oe genome , the genon fiber , but we on fiber , the expDistributing transcripts in GO categories developed a molecular snap shot of the petal tissue. The cellular component ontology that includes multi-subunit enzymes and other protein complexes was most abundant GO category (88%). Petal cells undergo rapid elongation to reach full petal expansion stage. Actin cytoskeleton helps with cell elongation by transporting vesicles and organelles to the site of growth from cytoplasm. The cytoplasm (28%) and cytoplasmic parts (27%) were most represented under cellular component GO category. About 17% of transcripts fell under biological processes GO category and under this category cellular protein metabolic processes (31%) were most prominent. Petal tissue is an energy sink tissue for plant reproduction where starch and sucrose are mobilized from photosynthetic organs and broken down to sugars that function as precursors to essential primary and secondary metabolites . This waIn addition to a comprehensive assessment of gene expression in petals, the improvements in our analytical methodology resulted in three refinements of our understanding of the cotton transcriptome. First, both Yoo et al.  and thise.g., A is equal to P; D is equal to P, but A is not equal to D). Elimination of low-level expressed genes emphasized two clear trends of the polyploid transcriptome. The first trend was that we found negligible ‘down-regulation’ in the polyploid. When the low-level expressed genes (instead of a fold-change threshold) were eliminated, the frequency of categories of ‘down-regulation’ in the polyploid  were negligible compared to the frequency of ‘up-regulation’ categories . The second trend was that we found no ELD in the cotton petal transcriptome. Previously, a small D-genome ELD was found in petals, but it could be potentially explained by the origin of the microarray probes  as the A-genome diploid than the D-genome sequences, particularly when an EST assembly of AT and DT is used a reference. This potential inherent bias is fully considered when SNP-tolerant mapping is used since SNP positions are masked during the read alignment resulting in normalized read mapping efficiencies. This study was the first study to use SNP-tolerant mapping in RNA-seq analysis of a polyploid transcriptome.Second, UPC was used to assign an expression probability to each gene. Genes with low expression levels have less accuracy and cause inflated comparisons of expression level differences due to overlapping standard deviations of expression , it was previously found that the expression level of the polyploid was explained by an increased expression of the ‘recessive’ parent [Third, when the polyploid had equal to the higher diploid parent  of the diploid A- and D-genomes can be traced to a common ancestry 5–10 mya [Gossypium polyploids and a diploid F1-hybrid [Our results contribute to a growing understanding of the evolution of gene expression in cotton. Adams et al., first re1-hybrid ,20. Thes1-hybrid , our rescis-acting regulatory regions of genes prior to genome hybridization. In cotton, speciation of the two diploid genomes followed distinct evolutionary trajectories as evidenced by their differential accumulation of retrotransposons [cis-acting promoter regions of protein coding genes also likely changed for a modest number of genes. Evidence of changes to cis-acting regulatory regions was observed by the conservation of homoeologous gene expression bias. If the observed expression biases were the result of a stochastic process during polyploid formation, then the direction of homoeologous expression bias would also be stochastic. When we inspected all genes with an expression bias in more than one accession  could only be ascribed to the small number of genes where the polyploid expression patterns were different that the diploids  due to either accelerated evolution of one of the two genomes or a mechanism of epigenetic control of expression, did not agree with our current findings [The second phase of duplicate gene evolution is hybridization. When two genomes are combined into a single nucleus, they share gene regulatory factors, which can lead to novel regulatory processes and changes in regulatory networks. These novel interactions have been considered as a genome shock that accompanies polyploidization and may provide a new source of genetic variation , see 4545 for refindings ,20. Perh1-hybrid and natural Gossypium allopolyploids in previous microarray-based and RNA-seq studies [cis-acting transcriptional regulators. For example, suppose that the A- or D-genome has a cis-acting regulating sequence that is more efficient than the other genome’s homoeologous sequence due to mutation during the first phase of evolution. When combined into a polyploid nucleus, a more efficient cis-acting regulator would continue to induce one of the two homoeologous genes at the requisite level for expression in the polyploid nucleus just as it had in the diploid nucleus (i.e. polyploid expression was the same as the higher expressing diploid). The less common events where the polyploid had lower expression levels could be due to trans-acting transcription repressors where the active repressor of one genome reduces expression levels in both genomes.The second phase of gene evolution can also be interpreted through the lens of expression level dominance ,18,27. N studies ,27. We dT and DT transcripts. For example, if the polyploid matched or exceeded the expression level of the A-genome (Category IV and VI), the polyploid transcripts would largely consist of AT transcripts and not DT transcripts. The reverse was true for categories II and V where the polyploid matched the expression level of the D-genome diploid. Furthermore, most (50-70%) of the genes in the ‘expression level dominance’ categories actually showed no change in the AT- relative to the A-genome and in DT- relative to D-genome suggesting similar controls of expression between the diploid and the respective polyploid.In the second evolutionary phase, we can also consider the constitution of the polyploid expression levels in the categories that match or exceed the expression levels of the diploids because we can distinguish between A1-hybrid in agreement with Flagel et al. [T genomes of the natural polyploids than it was in the genome of the diploid F1-hybrid (but not the AT biased genes). Perhaps, this observation represents a higher rate of mutation accumulation in the AT-genome cis-acting regulatory regions than the homoeologous DT-genome regions. It also agreed with our previous finding of general nucleotide diversity between the two genomes [e.g. G. kirkii).The third phase of gene expression evolution occurs between hybridization and adaptation to the species current biological niche. We found a larger number of biased homoeologous genes in the natural polyploids when compared to the diploid Fl et al. . Since w genomes , our gen genomes . Althougcis- and trans-acting regulation of polyploid gene expression. We recognize that the simple explanations of increased and decreased levels of expression genes represent only a few of many possible combinations of cis- and trans-acting factors on gene expression within discrete and interconnected biochemical pathways. Indeed, the modest number of changes to polyploid gene expression corresponded to the limited number of genomic changes that were previously found to accompany polyploidization in cotton [Here we used RNA-seq to characterize gene expression in polyploid cotton petals with an unprecedented level of resolution within each genome. Future comparisons of these findings to similar results in other polyploids will perhaps enable an extrapolation of general trends to unifying concepts of polyploid gene expression in plants. Collectively, these results suggest that after polyploidization 1) most homoeologous gene pairs are expressed at approximately equal amounts, 2) ~20% of expressed genes have biased expression, 3) a portion of these genes have expression biases in more than one polyploid and the direction of their bias is largely conserved, 4) for any set of genes with expression biases in more than one accession, only a very a small number of genes have a direction of expression bias that was unique or contrary to the observed direction in other accessions, and 5) that general trends of gene expression can be interpreted as n cotton . PerhapsAR handled the plant material, prepared the libraries for sequencing, and helped write the manuscript. JTP performed the computational and statistical analyses and helped write the manuscript. JAU performed additional summaries of the results and wrote the manuscript. All authors read and approved the final manuscript.Distribution of gene ontology (GO) terms in 11,469 commonly expressed petal genes. a) Biological process; b) Cellular component; and c) Molecular function.Click here for fileThe enzymatic pathways can be divided into four general KEGG pathways: Metabolic pathways, Biosynthetic pathways, Degradation pathways and signaling pathways.Click here for fileT and DT genomes of each cotton species. Each histogram shows the number of genes  with each magnitude of homoeologous bias for Maxxa, G. tomentosum, the F1 diploid hybrid, or T×2094. Positive values indicate an At bias, while negative values indicate a Dt bias.Differences in log base 2 RPKM between the AClick here for fileCounts of duplicated genes from this RNA-seq study were placed into the 12 expression levels categories (identical to Figure Click here for file


Special Concern conservation status in Atlantic Canada. We developed maximum entropy distribution models for the lower BoF and the northeast Gulf of Maine (GoM) to describe spatiotemporal variation in habitat use of basking sharks. Under the Maxent framework, we assessed model responses and distribution shifts in relation to known migratory behavior and local prey dynamics. We used 10 years (2002-2011) of basking shark surface sightings from July-October acquired during boat-based surveys in relation to chlorophyll-a concentration, sea surface temperature, bathymetric features, and distance to seafloor contours to assess habitat suitability. Maximum entropy estimations were selected based on AICc criterion and used to predict habitat utilizing three model-fitting routines as well as converted to binary suitable/non-suitable habitat using the maximum sensitivity and specificity threshold. All models predicted habitat better than random . From July-September, a majority of habitat was in the BoF, in waters >100 m deep, and in the Grand Manan Basin. In October, a majority of the habitat shifted southward into the GoM and to areas >200 m deep. Model responses suggest that suitable habitat from July - October is dependent on a mix of distance to the 0, 100, 150, and 200 m contours but in some models on sea surface temperature (July) and chlorophyll-a (August and September). Our results reveal temporally dynamic habitat use of basking sharks within the BoF and GoM. The relative importance of predictor variables suggests that prey dynamics constrained the species distribution in the BoF. Also, suitable habitat shifted minimally from July-September providing opportunities to conserve the species during peak abundance in the region.The local distribution of basking sharks in the Bay of Fundy (BoF) is unknown despite frequent occurrences in the area from May to November. Defining this species’ spatial habitat use is critical for accurately assessing its  Cetorhinus maximus, Gunnerus 1765) are obligate filter feeders of zooplankton, primarily copepods, and are found circumglobally in temperate and tropical seas . Environmental variables representing the bathymetry, distance to bathymetric contours, chlorophyll- Toolbox  from theAverage Nearest Neighbor routine. Our attempts at correcting for autocorrelation by decimating presence locations by the autocorrelation as detected in a semivariogram and a Moran’s I-distance plot are summarized in the Material. Neither correction was applicable due to the autocorrelation structure detected in the semivariogram  is a machine learning species distribution modeling technique that seeks the probability distribution that maximizes entropy (i.e. closest to uniform) subject to constraints ; in our Following previous maximum entropy distribution models , we chosThe most often reported measure of Maxent outputs is the threshold-independent assessment using the Area Under the Curve (AUC) metric of the ROC curve . The ROCWe assessed variable importance using the percent contribution metric , which dMaxent provides three methods to assess model fit: cross-validation, bootstrapping, and subsampling. The number of sightings available in our database in July, August, and September allowed us to perform empirical comparisons of these model-fitting techniques. We performed model runs for those time periods where these techniques were estimated to be more appropriate  as well as in the October time period to compare a small dataset using all available variables. The random test percentage, used in subsampling, was set to 25% test and 75% training .The AICc model selection procedure selected models with different regularization values for each month; July: β = 5; August: β = 7; September: β = 3; October: β = 7. AICc did not follow any discernible pattern for any of the monthly Maxent models as regularization values were increased from 1 - 17. We evaluated the three model-fitting approaches and found that bootstrapping had the lowest false-positive rate as assessed by AUC values, followed by cross-validation, and then by subsampling . Howevera in August and September, and distance to the 200 m contour in October , ) Figure. July wast ranked - July, < 15 km; 3rd ranked - August, < 23 km; 2nd ranked - September, < 24 km)  . In July< 24 km) . Chlorop2 mg m-3 . In Sept2 mg m-3 . In July2 mg m-3 . Distanc2 mg m-3 . Based on the ROC curve for each model, the maximum sensitivity and specificity cumulative suitability score threshold varied  . These tpecial Concern in this area [Basking sharks in the western North Atlantic inhabit coastal waters  but thishis area . The results of our Maxent models reveal the consequences of using different model-fitting techniques of bootstrapping, subsampling, and cross-validation. We chose to use cross-validation because our primary focus was assessing spatial differences in basking shark distributions, not overall model performance. We show that when using other model-fitting techniques within Maxent with large presence-only data sets, a trade-off between model performance and variation in habitat suitability predictions may occur. The trade-off is relatively simple when models perform well. However, as model performance deteriorates, sacrifices in habitat suitability confidence may be necessary to maintain adequate model function. As we demonstrated, using the bootstrapping model-fitting technique improved model performance both in mean and standard deviation of AUC values but at the sacrifice of increasing uncertainty in habitat suitability maps. Importantly, the change from cross-validation to either subsampling or bootstrapping did not change variable importance.Models for July-October denoted variation in the distribution of basking sharks  & 3 and C. finmarchicus C4 and C5, presumably feeding on phytoplankton prior to the C5 diapause [Calanus helgolandicus, to the continental edge where they consumed primarily deep-water C. finmarchicus [Owen (1984) also observed evidence for habitat shifts by documenting basking shark distributions in the GoM changing from shallow water to deep water in September. Our model predicted an earlier shift to deep-water habitats in the BoF (August) that may reflect differences in the prey field between these habitats. Owen (1984) documented copepod swarms in shallow (<80 m) waters of the GoM throughout the summer and observed surface feeding sharks primarily along the western edge of Jordan Basin from June to August. These patches were likely diapause ,56. Habiarchicus . Interestingly, the narrow bands of suitable habitat along the Murr Ledges in the July suitability map overlap with the outflow of the cyclonic Bay of Fundy Gyre  Figure . This suThe appearance of suitable habitat further south in September  likely ra and SST, are limited to describing surface changes and cannot assess C5 C. finmarchicus dynamics or thermal stratification, we can only speculate on the mechanism behind distribution shifts. Owen (1984) proposed that basking sharks in the wNA may follow warm surface water as it undergoes vertical mixing because animals are thermally constrained, allowing October (or later) access to deeper-water habitats, thus eliminating basking sharks from surface sighting records. The shift to deep-water habitats in the October distribution in this study likely reflects such a movement as the model shifted from 180 m in depth in July-September to > 200 m in October-December. Additionally, response to the distance to the 200 m contour decreased from 20 km from the contour in September to less than 7 km in October. However, SST did not strongly influence the October distribution which implies that temperature is less important than prey requirements or that SST does not capture the thermal environment basking sharks are responding to [Southerly movement of the basking shark distribution, as predicted in our October model , does nonding to ,58,61. AThe model predictions have shown that sharks regularly use select areas within the BoF. These select areas may offer opportunities for management strategies, such as shifting traffic patterns and creating protection zones for basking sharks in the northern Grand Manan Basin ,62. HabiWe have developed the first maximum entropy model for basking sharks and made predictions of their distribution in the BoF. Suitable habitat was shown to vary between July-October over 2002-2011, indicating heterogeneous habitat utilization. We hypothesized these distributional shifts are related to prey dynamics but further understanding of the ecological drivers is necessary to elucidate the specific mechanisms of basking shark movements in the western North Atlantic. Also, we have compared model-fitting routines and reasoned that their choice must be weighed against study objectives. Defining the spatial distribution of basking sharks, as we have done, represents a critical step in future efforts towards conserving and understanding this large but understudied species, especially in data deficient areas such as Atlantic Canada. Figure S1Map for July-October of the distribution of sampling effort throughout the study area.(DOCX)Click here for additional data file.Figure S2Semi-variograms generated from sighting locations and environmental variables showing the spatial autocorrelation in the dataset used for Maxent estimations.(DOCX)Click here for additional data file.Figure S3Moran’s I of sightings locations and environmental variables as a function of distance used in an attempt to decimate sampling locations in order to correct for spatial autocorrelation.(DOCX)Click here for additional data file.Figure S4Model responses to distance to shore for July-September against a histogram of distance to shore in the study area.(DOCX)Click here for additional data file.Figure S5Model responses to distance to the 150 m contour for July and August against a histogram of distance to the 150 m contour in the study area.(DOCX)Click here for additional data file.Figure S6a for August and September against a histogram of chlorophyll-a in August and September in the study area.Model responses to chlorophyll-(DOCX)Click here for additional data file.Figure S7Model responses to distance to 200 m contour for September and October against a histogram of distance to 200 m contour in the study area.(DOCX)Click here for additional data file.Figure S8Model responses to sea surface temperature for July against a histogram of sea surface temperature in July in the study area.(DOCX)Click here for additional data file.Figure S9Model responses to distance to the 100 m contour for October against a histogram of distance to the 100 m contour in the study area.(DOCX)Click here for additional data file.Figure S10Map of major currents affecting the Bay of Fundy Gyre circulation.(DOCX)Click here for additional data file.