We found that SpGlpO interacts with a wide range of

We found that SpGlpO interacts with a wide range of host glycans with the strongest binding to those that are commonly found as O-linked glycans of protein glycosylation and as lactoceramides/gangliosides. In addition, asialo-GM1 contains the core GalNAcβ1-4Gal suggesting that SpGlpO is an alternative binding partner for these gangliosides, with this interaction proving important in mediating pneumococcal adherence to nasopharyngeal cells. Given these findings, we propose that SpGlpO enhances pneumococcal colonization of the nasopharynx through its direct binding to host glycoconjugates. In this context, the use of oligosaccharides to block pneumococcal adherence to mucosal surfaces presents an attractive prospect for therapy and disease prevention, as demonstrated for LNnT using a rabbit model of pneumonia and bacteremia and an infant rat model of colonization (Idanpaan-Heikkila et al., 1997).
GlcNAcβ1-3Gal is a core structure of gangliosides, which are predominantly found on neuronal surfaces. Previous work has demonstrated a ganglioside-mediated colonization of the nasopharynx and purchase EZ Cap Reagent GG by pneumococci (van Ginkel et al., 2003). We previously reported that an SpGlpO mutant showed decreased adherence to human brain microvascular endothelial cells (Mahdi et al., 2012), although a detailed mechanism for this was not determined. This study suggests that the binding of SpGlpO to different gangliosides, like asialo-GM1, could facilitate attachment to brain cells, providing an alternative mechanism for pneumococcal attachment to brain tissue. Interestingly, this would suggest an additional role for SpGlpO, along with its hydrogen peroxide producing cytotoxic effects, in the pathogenesis of pneumococcal meningitis. Moreover, the binding of SpGlpO to gangliosides opens up the likelihood that such interactions play a major role in the pathogenesis of otitis media, as was previously shown in chinchilla tracheal epithelia (Tong et al., 1999), and in gerbils where CNS infection occurred after pneumococcal otitis media (Muffat-Joly et al., 1994).
Immune-mediated clearance of pneumococci from the nasopharynx involves both antibody-dependent and antibody-independent mechanisms (Goldblatt et al., 2005; Malley et al., 2007; van Rossum et al., 2005). Antibody-independent clearance is thought to involve an IL-17A-mediated T-cell response, resulting in the recruitment of neutrophils to the site of infection and subsequent clearance of colonization (Lu et al., 2008; Malley et al., 2006). Consistent with these reports, intranasal immunization of mice with SpGlpO or SpGlpO-adjunct elicited significant total serum IgG, negligible salivary IgA, and more IFN-γ and IL-17A responses compared with LTB controls. The finding that immunization with SpGlpO did not completely block colonization could be advantageous in the context of reducing colonization to levels that significantly impacts on overall pneumococcal translocation to deeper host tissues, while intra- and inter-species competition in the nasopharynx is maintained at asymptomatic levels. This is also likely to result in reduction in transmission from carriers to new hosts. Our previous work with WCH16 and WCH43 showed that stable colonization of the nasopharynx of CD1 (Swiss) mice was firmly established over a 48- to 96-h period, with optimal and uniform colonization at 72h post-infection (Mahdi et al., 2012). This finding led us to choose 72h after challenge as the ideal time-point for mouse sacrifice to determine the level of nasopharyngeal colonization.
In summary, we provide evidence for a direct contribution of SpGlpO to colonization of the nasopharynx through its binding to host glycoconjugates, and show that intranasal immunization of mice with SpGlpO elicited significant protection against subsequent nasal colonization. We conclude that SpGlpO warrants consideration for inclusion in an optimal, multi-component protein vaccine formulation that can provide robust, serotype-independent protection against nasopharyngeal colonization and all forms of invasive pneumococcal disease.

The initial Trinity de novo assembly of D japonica RNAseq

The initial Trinity de novo assembly of D. japonica RNAseq data produced a dataset with 195,271 sequences and an N50 of 1,587bp. This number of contigs exceeds the number of predicted gene models in published flatworm genomes [5–9], likely due to a high number of redundant or incorrectly/partially assembled transcripts in the D. japonica assembly. Therefore, this preliminary dataset was filtered to retain only (i) sequences with predicted open reading frames (ORFs) ≥100 clemastine that contain an assignment to a known PFAM structural domain, or (ii) sequences with predicted open reading frames (ORFs) ≥100 amino acids that were evidenced by read mapping (FPKM value ≥1). The resulting filtered assembly retained 44,857 sequences with an N50 of 2444bp. Sequences from the unfiltered assembly are provided as Dataset 1. The filtered ORF assembly of 44,857 sequences is provided Dataset 2.
Sequences belonging to the voltage-gated like ion channel superfamily were then curated by searching the translated D. japonica transcriptome for Pfam protein family hits corresponding to domains such as ion transport (PF00520, PF07885, PF08412), Cav (PF08763), Nav (PF06512), PKD (PF08016), BK (PF03493), SK (PF035630) or cyclic nucleotide gated channels (PF08412, PF00027). Sequences were inspected to confirm the presence of the appropriate number of transmembrane helixes and pore forming domains and expected architecture/topology for each family of ion channels. This analysis resulted in the prediction of 114 unique pore-containing channel sequences that could be assigned to VGL ion channel families.
The appended Table 1 details the contig identifier and assignment of each of the D. japonica sequences based upon our assembly and current filtering methods. Within each class, assignments are ordered by FPKM values (fragments per kilobase of transcript per million mapped reads) to convey which transcripts predominate within each class of channels.

Acknowledgements
Data collection supported by NSF, United States (MCB1615538, JSM) and a Stem Cell Biology Training Grant (T32 GM113846, JDC). We thank the Parasite Genomics Group at the Wellcome Trust Sanger Institute for their assistance with sequencing samples.

Data
Here, we present data on the melanin contents in B16F1 murine melanoma cells per total protein content in the cells, in culture medium containing 0, 0.001, 0.005, and 0.01% emu oil (Fig. 1). The data show significant reduction of melanin production in the presence of emu oil. However, Fig. 2 indicates that the melanin content in the presence of 1µM α-melanocyte-stimulating hormone (α-MSH) was not significantly altered despite supplementation with emu oil.

Experimental design
Melanin, which is the major pigment of skin, plays an important role in protection against UV light under normal physiological conditions. However, overproduction of the melanin causes cosmetic problems, such as staining and freckles on the skin. Here, we examined the melanin production in murine B16F1 melanoma cells in the presence of emu oil, which is widely utilized in cosmetics for its moisturizing and transdermal penetration enhancing properties [1]. In this study, we measured the melanin contents in B16F1 cells treated with various concentrations of emu oil. The melanin contents were measured in the cells in both the presence and absence of the α-MSH, which is one of the endogenous factors that regulate melanogenesis. The measured melanin contents were divided by the cellular protein amount in each sample to compare the cellular melanin production.

Materials and methods

Acknowledgements

Data
Data include all sperm quality parameters recorded in fresh and cryopreserved chicken semen (Table 1) and the recovery rates of viable and motile sperm after freezing–thawing (Table 2). The most effective cryoprotectant combination includes both trehalose and DMA; in contrast, the absence of DMA (DMA-0) is responsible for more severe loss in sperm quality.

While previous research has primarily focused

While previous research has primarily focused on how the developing chemokine receptor antagonist predisposes adolescents to health-compromising behaviors, there is an emerging literature exploring how adolescent brain development facilitates healthy outcomes. In particular, scholars suggest that hyperactive reward and affective processing, coupled with heightened social cognitive processing, also underlie adaptive and beneficial behaviors (see Telzer, 2016). For instance, the ventral affective system, which is often implicated in risk taking behaviors, is also responsive to healthy rewards, such as helping others (Telzer et al., 2015; Telzer et al., 2013a, 2014). Interactions between reward-related circuitry and prefrontal systems can even bolster inhibitory control (Teslovich et al., 2014; Pfeifer et al., 2011; Telzer et al., 2015). Overall, these findings highlight that normative changes in the adolescent brain are associated with both positive and negative outcomes. In subsequent sections, we will integrate these perspectives (i.e., that heightened reward sensitivity engenders negative risk taking and positive other-oriented behaviors) and propose that these neural systems may also facilitate behavior that is helpful towards others, even when there are inherent risks to oneself.

Risk-taking behaviors during adolescence
Risk taking, or engaging in a behavior with an uncertain outcome that may lead to detriments in a given domain (e.g. health, social, etc.), is one of the most widely studied topics in developmental science. This is due, in part, to its real-world impact: morbidity and mortality rates increase 200–300% from childhood to adolescence, with 70% of annual adolescent deaths in the United States stemming from risky behaviors such as reckless driving or unsafe sexual practices (Centers for Disease Control & Prevention, 2012; Victor and Hariri, 2016). In the following section, we briefly discuss the developmental trajectories of risk taking behaviors, the supporting brain systems involved, and how social factors influence risk-taking.

Prosocial behaviors during adolescence
The ability to become less self-oriented and more helpful to others has been considered one of the hallmarks of adulthood (Arnett, 2003). Yet, surprisingly less attention has been devoted to understanding the development of prosocial behaviors relative to risk taking behaviors. Prosociality describes voluntary actions intended to benefit another, which range from cooperating with others to making donations. Longitudinal work in humans has shown that children and adolescents who exhibit greater prosocial behaviors have better relationships with peers (Eisenberg et al., 2006), less internalizing and externalizing problems (Bandura, 1999), and better academic performance (Caprara et al., 2000; Wentzel et al., 2004). Prosocial youth are also more likely to have better cognitive and emotion regulation abilities (Rothbart and Rueda, 2005; Eisenberg et al., 2006), which may enable them to adapt to environmental stressors better than their less prosocial counterparts. Together, these findings underscore the adaptive benefits of prosocial behaviors.

Prosocial Risk Taking
Given key socialization processes and a rapid reorganization of motivational, social, and cognitive neural circuitry during adolescence (summarized in Table 1), we propose a new area of study, Prosocial Risk Taking (PSRT), to explore the possibility that adolescents engage in risk-taking behaviors to benefit others. In this section, we discuss the proposed behavioral and neural mechanisms of PSRT and describe four behavioral types that may emerge when considering how prosocial and risk-taking tendencies interact. We discuss evidence that suggests adolescence could be a sensitive period for PSRT, with a focus on how individual differences in several psychosocial factors contribute to PSRT. Finally, we consider the best methodological practices for investigating this phenomenon and discuss its implications on adolescent adjustment and neurodevelopment.

br Experimental design materials and methods

Experimental design, materials and methods

Funding

Specifications Table

Value of the data

Data
The data in this article provides information on how to develop an experimental system to determine specific activities of enzyme/mutants in cell lysates in HTP mode based on ITA of enzyme/mutants as protein and HTP assay of activities (Figs. 1–7 and Tables 1–25). Data were provided for the validity of the proposed strategy, the performance to recognize the positive mutant in each random pair of PAAS/mutants during HTP screening, and the efficacy to elucidate sequence-activity relationships of both BFU and PAAS in HTP mode (Fig. 8, Fig. 9 and Table 26).

Experimental design, materials and methods

Data
In this Data in Brief article we provide detailed information on blood protein profiling as an extension of the results reported in Ref. [1], 24 blood specimens were collected from 6 healthy and young volunteers in different sample collection tubes for serum and plasma. Tubes characteristics and the subsequent sample preparation are presented in Table 1. For the blood protein profiling a nanoLC-UDMSE method and standard search parameters were employed. Detailed description of methods can be found in Ref. [1] and its supplementary methods. 6 highly abundant blood proteins, namely serum albumin, immunoglobulin gamma, immunoglobulin alpha, serotransferrin, haptoglobin, and alpha-1-antitrypsin, were depleted by using a commercially available immunoaffinity depletion column. A detailed overview on depletion efficiency based on protein abundances for all sample collection methods is presented in Table 2. The distribution of the high abundant proteins before and after depletion is presented in Fig. 1 and more specific, fibrinogen coverage is shown in Fig. 2. Data regarding number of identified gallic acid and relatively quantified proteins for all sample types after depletion is shown in Fig. 3. Also, a top 10 list of the most abundant unique proteins for each of the EDTA-, heparin-, citrate plasma and serum samples is given in Table 3. The complete list of all relatively quantified proteins over all samples including their occurrence in the protein core set or as unique proteins interpreted in detail previously [1], can be found in the Supplementary material with data on individual sample abundance, mean abundance for each sample collection method and the abundance based coefficient of variation after depletion.

Experimental design, materials and methods
Experimental design and the materials and methods have been reported previously [1].

Acknowledgements
We would like to thank to the European Social Found, Human Resources Development Operational Programme 2007–2013 [Project no. POSDRU/159/1.5/136893], the Deutscher Akademischer Austauschdienst (German Academic Exchange Service) [Programme ID 57130104, Personal number: 91558112], the ERASMUS + Traineeship [Contract no. 06/24/08/2016] and the Iuliu Hațieganu University of Medicine and Pharmacy, Cluj-Napoca Romania [Grant no. 7690/57/2016] for the research grants awarded to Maria Ilies.

Data
Psychosocial risk factors identified in 20 cases (10 fatal, 10 near-fatal) of physical child abuse are presented in Table 1. An X indicates the presence of a given risk factor among one or more caregivers in the child׳s environment.

Experimental design, materials and methods
The Institutional Review Boards at the University of Louisville and the Kentucky Cabinet for Health and Family Services (KY CHFS) approved Pseudogenes research. Our retrospective record review included 20 children younger than four years of age who had been victims of fatal (n=10) or near-fatal (n=10) child abuse in the Commonwealth of Kentucky [1]. We utilized the Kentucky Revised Statues [KRS 600.020 (37)] definition of near-fatality: an injury that places a child in serious or critical condition as certified by a physician. The following documents were reviewed when available: medical records associated with all medical visits prior to and including the fatal/near-fatal event, social service evaluations prior to and including the fatal/near-fatal event, legal proceedings, criminal histories of caregivers, and autopsy findings (when applicable). Three independent investigators reviewed and abstracted data from each de-identified case simultaneously, noting the presence or absence of previously identified psychosocial risk factors in each case.

The malignant tumors are mostly circumscribed multinodular lesions which

The malignant tumors are mostly circumscribed, multinodular lesions which tend to recur as bulky nodules and metastasize after years or decades. However, factors affecting the risk and onset of malignant NCH transformation are still unknown. The time period for malignant tumor development has been estimated to be 1–6 years for acquired NCH and 15–67 years for congenital NCH. No guidelines are available for NCH management because of the overall small number of cases. It has been suggested that wide local excision should be considered to lessen malignant potential. Patients should be given clear guidance about possible risks and monitored with regular follow-up examinations.

Drug rash with eosinophilia and systemic symptoms (DRESS) syndrome is a severe adverse drug reaction characterized by a cutaneous eruption and multi-organ involvement. The characteristic features of DRESS include eosinophilia, visceral involvement, fever, atypical lymphocytes, mild mucosal involvement, lymphadenopathy, reactivation of the herpes virus (HHV-6), a relatively long latency from drug initiation, and protracted reactions. A prompt diagnosis of DRESS syndrome is crucial because of its life-threatening potential. Treatment of DRESS syndrome includes the prompt recognition and withdrawal of the causative drug, the salinomycin of systemic corticosteroids, and supportive care.
We report here the case of a patient with persistent transaminitis, hyperthyroidism, photosensitivity, rosacea, anhidrosis, and polyarticular juvenile idiopathic arthritis (JIA) following DRESS syndrome. A 13-year-old boy with no known drug allergies and a significant past medical history developed facial edema, fever, severe transaminitis with features of liver decompensation (coagulopathy and low albumin), and a generalized erythematous morbilliform rash (A). These symptoms persisted (A and B) for 6 months after his diagnosis of DRESS syndrome, which had begun after initiating treatment with salinomycin sulfamethoxazole/trimethoprim (Bactrim) for acne vulgaris. A skin biopsy sample taken during the acute stage of the disease showed basal vacuolar alteration with extensive interface dermatitis and the presence of eosinophils on hematoxylin and eosin stain (B). He was diagnosed with DRESS syndrome (RegiSCAR score 6, definite case) and his treatment was managed with pulsed intravenous methylprednisolone followed by intravenous hydrocortisone and prednisolone by mouth. The diagnosis of DRESS syndrome was made in view of the temporal association following sulfamethoxazole/trimethoprim administration, the presence of fever with drug-induced liver injury and severe transaminitis, and a positive response to treatment with steroid drugs. His serum HHV-6 polymerase chain reaction was positive. As a result of intermittent relapses of his skin rash and mild transaminitis, he remained on tapering steroid treatment for 8 months after discharge.
One month after the onset of DRESS syndrome, he was noted to have hand tremors and was subsequently diagnosed with hyperthyroidism and treated with carbimazole. He was noted to have photosensitivity to UVB light on phototesting with a minimal erythema dose of 40 mJ/cm (no photosensitivity noted to visible light and UVA) 2 months after his diagnosis of DRESS syndrome, which persisted for approximately 10 months. At the same time, he was noted to have steroid-induced rosacea (C) and persistent anhidrosis of his face and forehead, which made him unable to resume his active outdoor lifestyle. This persisted for about 10 months. Furthermore, 1 year after his initial diagnosis, he developed polyarticular JIA of the temporo-mandibular, shoulder, and wrist joints, which was proved by magnetic resonance imaging. He was subsequently treated with adalimumab (Humira, AbbVie, North Chicago, Illinois, USA) and methotrexate by a pediatric rheumatologist.
The long-term sequelae of DRESS syndrome include type I diabetes mellitus, autoimmune thyroid disease, sclerodermoid lesions, and systemic lupus erythematous. Impaired regulatory T cell function and viral infection or reactivation have been proposed to result in the development of autoimmune diseases in DRESS syndrome. Elevated liver transaminases are commonly found in patients with DRESS syndrome and can take up to several months to completely resolve. Endocrine abnormalities, including autoimmune thyroid diseases, are more frequently seen as long-term sequelae of DRESS syndrome rather than as acute reactions. Immunological dysregulation has been considered as a possible mechanism, resulting in carrier protein photon absorption in the UVB and visible light range in adverse drug reaction-induced photosensitivity. Our patient\’s rosacea is most likely to be induced by prolonged treatment with steroid drugs by mouth. Rebound vasodilation and proinflammatory cytokine release have been proposed as the mechanism for steroid-induced rosacea. Our patient\’s anhidrosis most likely occurred as a result of damage to the sweat glands and adnexal structure secondary to perivascular lymphocytic infiltrate in the papillary dermis, the presence of eosinophils, and dermal edema in DRESS syndrome. The exact pathogenesis of JIA is still poorly understood; however, it is assumed to result from a combination of genetic susceptibility and environmental triggers. Regulatory T cells have been hypothesized to play a role in the regulation of inflammation and the maintenance of immune tolerance, in which their reduction or dysfunction are responsible for the development of autoimmune diseases such as JIA. Our patient\’s persistent transaminitis and late presentation of JIA indicates that these are long-term sequelae instead of acute complications of DRESS syndrome. Our patient was well and in good health before the episode of DRESS syndrome and subsequently developed all the sequelae sequentially over the course of more than 1 year. Recovery was delayed due to persistent transaminitis and the patient had to tail off his steroid drugs slowly. This suggests that, because of the multisystem involvement of DRESS syndrome, the skin, appendages, endocrine organs, and joints can all be affected. This case report also suggests that the long-term sequelae of severe DRESS syndrome follow a chronological order and sequence. Further study is needed to determine the association of autoimmune disease as one of the long-term sequelae of DRESS syndrome. Close and regular monitoring for autoimmune disease is therefore important for patients diagnosed with DRESS syndrome.

The cause of the serious decline is therefore attributed

The cause of the serious decline is therefore attributed to a general lack of maintenance. Most of all, the decline resulted from poorly thought changes that worsened ventilation, such as the closure of spaces initially conceived of as partly open as the Peristyle, the replacement of the shade-providing louvers with non-opening transparent elements and iron-frame hopper windows with maintenance difficulties. The removal of the too noisy ventilation fans which, without replacing them with new air extractors, and the installation of security doors of solid glass panes that mostly remained obstructed. In addition, the natural oxidation of door and window frames and metal structures became evident with the recent removal of the suspended ceilings containing the warm air that penetrated underneath the sloping roof (Figure 7). The elimination of this interspace worsened a situation that had already been repaired by opening special ventilation slits on the façades to increase the circulation of the air.
Perhaps the physical, technical, and technological improvements of the structural glass might have solved the environmental issues without giving precedence to one beta adrenergic receptors or another, such as saving both the mosaics and their insertion into a museum with natural lighting, which was acknowledged by Guido Meli as a “milestone in archaeological restoration” (Meli, op. cit). Experimenting with the automatism of new shadings with “shape memory” materials was possible. These materials could guarantee optimum climatic conditions. The image of the coverings with larger windows could be lightened without frames, or perhaps with ventilated edges. However, the decision was to ignore the challenge posed by transparency, which was intended as an application of Cesare Brandi׳s critical theory, and thus, adopted a “type replanning” criterion.
The new proposal of the Regional Centre for Planning and Restoration called for the demolition of the Minissi׳s sheds and, although the visiting paths along the raised walls have been kept, casts the rooms into the shade. The museological structures have been darkened to concentrate the attention of visitorson the archaeological remains, lit by spotlights placed under walkways. New volumes have been built in honeycomb plasterboard. Wooden roofs rest on wooden trusses that are coated with copper with a rubber sheath in-between them. Therefore, the new system will require much care with due attention to the effects of rainwater when the leaching of copper oxides start to cover the surfaces with its inevitable green patina.
Although improvable, the works of Minissi aimed only at evoking the past, not at restoring it. On the contrary, the focus now is on reproducing a perception more similar to that of ancient Roman spaces, without considering that the loss of transparency breaks the ties acquired down through the centuries between the archaeology and the surrounding landscape, and accentuates precisely the extraneousness to the place of which Minissi׳s abstract coverings were accused (Figures 8 and 9).

Church of S. Nicolò Regale at Mazara del Vallo, Trapani (Sicily)
Minissi repeated the experience achieved at Piazza Armerina in the church of S. Nicolò Regale in Mazara del Vallo (Trapani, 1960–1966), where the marine environment had caused deep damage to the stone, like in the transparent covering of the cavea of the Greek Theatre at Heraclea Minoa.
When Minissi was called to restore this church, only the perimetral walls and some traces of the superior contour remained of the original building. Between 1946 and 1949, some urgent works had been carried out, such as removal of unstable masonry from the roof, ceiling, baroque dome, and stuccoes. Minissi suggested a cautious reconstruction that would outline the Arabic-Norman vaults, as this situation had already happened in other contemporary churches, without using stone. According to the critical approach of the time, reconstructions in stone blocks had to be avoided to not “take for granted all that could be arbitrary”. He refused to propose a simple shed as the previous wooden one, as Topoisomerase would not have considered the architectural features of the monument and would not have allowed the recovery of the functional aspects of the original existing elements. Such reconstruction would have prevented a rigorous and enhancing solution, despite the use of innovative techniques and modern materials.

The patient was discharged uneventfully days

The patient was discharged uneventfully 10 days after surgery. She was then treated by chemotherapy using cisplatin and etoposide, and radiotherapy with a total dose of 60Gy in 30 fractions over the resection area.
The patient supported the dapt secretase treatment well and led an independent day-to-day life thereafter. Unfortunately, about 10 months later a generalized seizure was noted. In addition, thereafter, there were also frequent headaches, vomiting, general weakness and memory impairment. The condition was complicated by progressive upper back pain. Imaging studies revealed no local recurrence of the tumor in the previous operative field, but did show enhancing masses involving the subdural spaces of fronto-parietal areas on both sides with perifocal edema (Fig. 2). T1, T4 and T6 vertebral metastases were also observed, with stenosis caused at the T4 level. The patient underwent craniotomies with bilateral subdural tumor removal and T4 laminectomy. A histopathological study confirmed the diagnosis of metastatic esthesioneuroblastoma. Chemotherapy and radiotherapy were continued, but the patient died about 2 years after her first diagnosis.

Discussion
Although esthesioneuroblastoma can show leptomeningeal dapt secretase and intracranial invasion in advanced disease, such a pure subdural extension without local manifestations is rare. Murakami et al in 2005 and Capelle et al in 2008 have each reported cases of recurrent esthesioneuroblastoma shown as diffuse subdural extension. While Tamase et al reported a case of recurrent esthesioneuroblastoma outside the initial field of radiation with progressive dural invasion. Our case resembled the latter in that no recurrent tumor was noted within the previous resection field. A review of the cases reported suggested that they all belonged to Kadish stage C disease during initial presentation, and Hyam’s grades II to III under histological examinations. The patients died of the original disease 8 years, 2 years, and 2 years after the first diagnosis in the cases of Murakami et al, Capelle et al and our patient, respectively. Therefore, close post-operative MRI or CT scan follow-up is mandatory in advanced cases, because such a pattern of disease appears to carry a poor prognosis.
There are an increasing number of reports supporting the effectiveness of combined surgery and radiotherapy for treating esthesioneuroblastoma. In Dulguerov’s 2001 meta-analysis, the 5-year overall survival for patients treated with surgery plus adjuvant radiotherapy was 65%, in contrast to 48% for those treated by surgery alone. A series from the University of Virginia gave a more promising result, with an 8-year median overall survival of 80.4% achieved in patients undergoing craniofacial resection with adjuvant therapy (radiotherapy ± chemotherapy). Chao et al reported the 5-year local control rate of 87.4% for the combination of surgery and radiotherapy compared to 51.2% for irradiation alone. In that series, the author even suggested that with adjuvant radiation therapy, the surgical margin did not influence local tumor control, showing that esthesioneuroblastoma is radiosensitive in nature. In both our case and that reported by Tamase et al, the tumor did not recur in the previous irradiation field. This could indicate the radiosensitivity of the tumor and the effectiveness of adjuvant radiotherapy in treating the disease, and highlight the importance of the setting of the irradiation field.
Since the introduction of the craniofacial approach in 1970s, the 5-year survival at the University of Virginia increased from 37.5% to 82%. There is an increasing interest in endoscopic surgery, and its role is currently being evaluated. As seen in our case, a trans-basal approach can also achieve a promising result in removing the tumor. With any approach, the arachnoid membrane should be preserved carefully and as much as possible to prevent cerebrospinal fluid (CSF) dissemination. As the tumor is friable, the tumor cells can spread diffusely along the subdural space during the first operation, and cause subdural extension when it recurs. Therefore, we suggest that unnecessary irrigation of the resection field should be avoided in order to prevent such a complication. In addition, post-operative subdural hematoma could also promote such a recurrence. Thus meticulous hemostasis is mandatory. During the operation of our case, the patient was placed in a supine position, with the neck extended. This made the fronto-parietal areas the dependent sites during tumor excision, and possibly contributed to tumor seeding. Therefore we suggest that avoidance of neck hyperextension might be helpful in this respect.

br Case report A year old

Case report
A 43-year-old man presented with odynophagia, progressive swallowing difficulty, and weight loss of 4 kg in 3 months. The patient had been smoking one pack of cigarettes per day for more than two decades and had a peptic ulcer, which had been under medical control for 5 years. Esophagography disclosed a huge tumor with severe mucosal destruction about 10 cm in length in the upper esophagus (Fig. 1). Endoscopic examination of the esophagus showed a huge tumor with irregular mucosa with ulceration 15 cm from the incisors, and the endoscope could not pass through this region (Fig. 2). Bronchoscopy demonstrated external compression on the posterior wall in the upper to middle trachea with obvious narrowing of up to 50% of the lumen. A computed tomography (CT) scan of the chest and upper abdomen showed a huge mass in the upper third of the esophagus with luminal widening (Fig. 3), but no direct invasion of adjacent structures or regional lymphadenopathy. No evidence of metastasis was found from the position emission tomography (PET) and whole-body bone scans. The tumor markers α-fetoprotein (AFP), squamous cell carcinoma antigen (SCC), and carcinoembryonic antigen (CEA) were all within normal limits. Histopathological examination via endoscopic biopsy showed high-grade pleomorphic spindle cell proliferation with an intersecting fascicular growing pattern and increased mitotic activities. These tumor buy Tedizolid HCl showed no immunoreaction to AE1/AE3, EMA, desmin, CD117, S100, or CD34. A high-grade pleomorphic sarcoma was diagnosed.
The patient first received neo-adjuvant chemotherapy with the MAID [mesna (700 mg), doxorubicin (15 mg/m2), ifosfamide (2.5 mg/m2), and dacarbazine (250 mg/m2)] regimen. After completion of six courses of chemotherapy, a CT scan of the chest demonstrated that the tumor had mildly shrunk in size. Although smaller, it still caused obvious symptoms such as odynophagia and dysphagia. In this period, the patient could tolerate a liquid diet. His body weight was within an acceptable range. The patient underwent radical surgery 1 year after diagnosis. Right-sided video-assisted minithoracotomy was carried out. The operation consisted of total esophagectomy, cardiectomy, and total laryngectomy with three-field radical lymph node dissection. Reconstruction was performed using a gastric tube transposed to the neck via the posterior mediastinal route. Pylomyotomy and feeding jejunostomy were also performed. During the operation, one huge tumor was noted in the upper esophagus just below the esophageal inlet. Enlarged lymph nodes were found in the para-esophgeal and parapancreatic areas.
The surgical specimen contained a huge intraluminal tumor with only a thin transverse pedicle stalk 3 cm in length connecting it to the esophageal mucosa. The gross size of the tumor was 7 × 5 × 3 cm. It was located near the esophageal inlet (Fig. 4) and had a firm and highly vascular nature. The tumor had only invaded the submucosal layer, and there were adequate peripheral margins of uninvolved tissue in all planes. The second histopathological examination revealed buy Tedizolid HCl the same tumor morphology as that of the initial examination, and no lymph node metastasis was found (Fig. 5). Immunohistochemical staining was AE1/AE3 (–), EMA (–), desmin (–), CD34 (–), CD-117 (–), S-100 (focal +), CD68 (focal +), CD163 (focal +), CD35 (–), and actin-M851 (focal +). The immunohistochemical staining showed complete absence of reactions with any lineage-selective markers. The diagnosis of high-grade undifferentiated pleomorphic sarcoma was confirmed.

Discussion
Primary sarcomas of the esophagus are rare neoplasms. In a previous study, sarcomas and carcinosarcomas accounted for approximately 0.1–1.5% of all esophageal tumors and comprised only 5% of all gastrointestinal sarcomas. Leiomyosarcoma is the most common cell type of the esophageal sarcomas. The rarity of esophageal sarcoma is illustrated by the small number of previous reports, consisting of single or small groups of patients. The clinical symptoms of esophageal sarcomas are nonspecific and different from those of esophageal carcinoma, and include progressive dysphagia, loss of weight, regurgitation, retrosternal pain, respiratory distress, odynophagia, sensation of a lump in the throat, hemorrhage, anemia, sudden death (due to asphyxia), vomiting (food or tumor fragments), fever, and cough. Our patient presented with odynophagia and dysphagia initially, which was consistent with symptoms in previously reported cases.

Loss of the ApcWT allele

Loss of the ApcWT allele in intestinal nicergoline in organoid culture induces Wnt target genes and high self-renewal potential in these cells upon which they give rise to neoplastic growth. This manifests in cyst-like organoids with aberrant tissue architecture, cell differentiation and proliferation. In vivo loss of ApcWT in ISC leads to immediate induction of Wnt signaling and rapid expansion of ISCs resulting in aberrant tissue architecture and secretory cell differentiation typical of early intestinal adenomas in mice (Barker et al., 2009; Sansom et al., 2004). However, these studies used mice with an induced simultaneous loss of both Apc alleles, thus circumventing events that influence loss of Apc function first hand. Therefore, the spontaneous formation of cyst-like organoids genuinely reflects the initiating step in intestinal adenoma formation and represents a unique window to investigate cellular and molecular determinants of this first step in CRC. Our data suggest that the cyst-like ApcMin/Min organoids arise from an expansion of cells positive for the ISC marker Lgr5, but negative for the ISC marker Olfm4, a detail we find reflected in ApcMin/Min adenomas. This is in contrast to Olfm4-positive adenomas formed through Apc loss in Lgr5-positive cells (Myant et al., 2013; Schepers et al., 2012), suggesting that spontaneous adenomas in Min mice do arise through a mechanism differing from induced ApcWT-loss in ISCs.
A benefit of the organoid in vitro system nicergoline is that it leaves out interactions of epithelial cells with stromal cells (Akcora et al., 2013; Barker et al., 2007), which can distort the detection of epithelial cell-specific signaling pathway interactions in vivo. Using this approach we wanted to know how the Notch and Myb pathways interact with Wnt signaling during the transition of ISC to tumor-initiating cells. We confirm findings of previous studies that loss-of-function of Notch (Pellegrinet et al., 2011; VanDussen et al., 2012) and Myb (Cheasley et al., 2011) strongly disturbs ISC self-renewal in vivo and in vitro. Surprisingly, we find that gain-of-function in these pathways does not increase self-renewal, suggesting that either both pathways alone are not sufficient to drive self-renewal, or their maximal capacity to do so, is limited and cannot be further increased in organoids.
Importantly we found that Notch activation increases the number of isolated Min crypts that process to cyst-like organoids, while Notch activation or inhibition does not affect growth or self-renewal once the cyst-like growth has been initiated. This is a striking parallel to the situation in vivo, where Notch activation increases the number of Wnt driven adenomas, but does not affect colon adenoma growth thereafter (Fre et al., 2009; Peignon et al., 2011). Intriguingly, we did not find any significant differences in SI adenoma formation across the genotypes on an Apcmin/+ background and we think this is because the activation of the Wnt pathway in the SI swamps out the effects of the other pathways. However, Notch activation and MybER had a significant effect in the colon in terms of adenoma formation and as colon cancer is approximately 10 times more common in humans we suggest that this was a more relevant matter to pursue. One explanation for this phenomenon is that Notch activation increases the number of tumor-initiating cells upon ApcWT-loss in the colon. This arguably can happen on three functional levels where we know aberrant Wnt activity affects initiation of intestinal adenomas: either by symmetric division of ISCs (Bellis et al., 2012), by the induction of a SC program in non-ISCs (Schwitalla et al., 2013) or by influencing the number of cells with ApcWT-loss able to clonally expand (Vermeulen et al., 2013). Notch activation leads to increased expression of the ISC markers Lgr5 and Olfm4, while the Lgr5-positive/Olfm4-negative phenotype of ApcMin/Min cyst-like organoids remains the same when Notch is activated and this is replicated in adenomas. This indicates that Notch-activation can affect the ISC phenotype, but does not affect the phenotype of tumor-initiating cells. Apparently the mechanism of how Notch influences adenoma initiation is complex and further studies will be necessary to elucidate it.

In light of the above findings we propose that

In light of the above findings, we propose that the β-gal+/NeuN+ cells and β-gal+/TH+ cells described by Shan et al. (2006) do not reflect neurogenesis and DA neurogenesis from Nestin+ NPCs, but rather expression of Nestin by mature midbrain neurons. Indeed, given Shan et al.\’s (2006) experimental design, their observations are possibly more consistent with this. They used transgenic mice (pNes-LacZ) that produce β-gal only whilst Nestin is being expressed (as opposed to our mice where Nestin expression triggers permanent expression of β-gal or eYFP). Thus they could not perform lineage tracing studies, and their β-gal+/NeuN+ and β-gal+/TH+ cells could only reflect neurogenesis from Nestin+ NPCs if the transition from Nestin expression to NeuN and TH expression (i.e. neuronal differentiation) occurs faster than degradation of the β-gal signal after Nestin expression ceases. To our knowledge the rate of β-gal degradation is not known but it is a foreign protein and we always observe it in what appear to be lysosomes (i.e. sites of protein degradation) in midbrain cells (e.g. bright spots in Fig. 4k). Presumably therefore it is degraded relatively quickly, leaving not much time for Shan et al.\’s (2006) β-gal+/NeuN+ and β-gal+/TH+ cells to arise via neurogenesis, particularly in adult midbrain where neurogenesis is generally considered to occur indolently or not at all.
Apart from the β-gal+ or eYFP+ neurons present at early time-points following tamoxifen, our findings suggest other β-gal+ or eYFP+ midbrain cells are indeed derived from Nestin+ NPCs and might reflect constitutive adult midbrain neurogenesis. In this context, our findings provide the following insights. First, there are many such cells lining the midbrain Aq, particularly the dorsal and ventral poles. These cells have a simple columnar morphology with multiple cilia extending into the ventricular space. In these respects they are typical ependymal cells, a type of glial cell that is important for production, circulation, and exchange of cerebrospinal between the ventricles and the ST 2825 manufacturer parenchyma. In addition, these ependymal cells might be activated in response to injury or withdrawal of canonical Notch signaling to produce new neurons or glia, as they do in the LV (Carlén et al., 2009). Note, however that LV ependymal cells appear to be a limited resource for repair because they do not self-renew at rates sufficient to maintain their own population; i.e. they are not true stem cells (Carlén et al., 2009). This is consistent with the fact that very few of our β-gal+ or eYFP+ ependymal cells were GFAP immunoreactive (Hermann et al., 2009), and none were Sox2 immunoreactive, both of which are considered markers of true stem cells. These findings are broadly consistent with those of Hermann et al. (2009), who described a preservation of Nestin+ cells along the entire rostral-to-caudal extent of the adult mouse paraventricular region, despite a gradual reduction of stem (Nestin+/GFAP+) cells and neuroblasts (Nestin+/PSA-NCAM), and a complete absence of transit amplifying cells caudal to LV. Hermann et al. (2009) also noted Nestin+ Aq cells have a distinct midbrain phenotype, and were therefore unlikely to have migrated in from the forebrain (LV and 3V). Also, despite being non-proliferative in vivo (evidenced by the absence of BrdU incorporation), they could be readily proliferated when provided EGF and FGF2 in vitro (Hermann et al., 2009).
There are also many β-gal+ or eYFP+ cells in the ventral midline region below Aq [see also Shan et al. (2006)]. These cells had a migrating morphology suggestive of migration from the Aq ventrally. Developmental DA neurogenesis follows a similar trajectory (Deierborg et al., 2008), which initially led us to think it might be recapitulated in the adult. However, β-gal+ or eYFP+ cells in the ventral midline were the same distance away from Aq along the dorso-ventral axis with time following tamoxifen, indicating they are not moving ventrally (or dorsally). A similar lack of temporal dispersion of proliferating Aq cells labelled with retrovirus was noted by Yoshimi et al. (2005a,b). It remains possible they are migrating rostally or caudally, however they did not express classical markers of new cell migration PSA-NCAM or DCX (see also (Aponso et al., 2008; Hermann et al., 2009); but see (Yoshimi et al., 2005a,b; Peng et al., 2008). β-gal+ or eYFP+ ventral midline cells were not Sox2, NeuN or GFAP immunoreactive either, suggesting they are some kind of intermediate between stem- or precursor cells and post-mitotic differentiated cells.