Early life exposures may modify risk of inflammatory bowel diseases , ulcerative colitis (UC)). However, the relationship between early life exposures and natural history of IBD has not been previously examined.This single center study included patients with CD or UC recruited in a prospective IBD registry. Enrolled patients completed a detailed environmental questionnaire that assessed various early life environmental exposures. Our primary outcome was requirement for disease-related surgery in CD and UC. Logistic regression models defined independent effect of early life exposures, adjusting for potential confounders.Our study included 333 CD and 270 UC patients. Just over half were female with a median age at diagnosis of 25 years. One-third of the cohort had history of bowel surgery (31%) and nearly half had used at least one biologic agent (47%). Among those with CD, being breastfed was associated with reduced risk of CD-related surgery (34% vs. 55%), while childhood cigarette smoke exposure was associated with increased risk. On multivariate analysis, history of being breastfed (odds ratio (OR) 0.21, 95% confidence interval [CI] 0.09–0.46) and cigarette smoke exposure as a child  remained independently associated with surgery. None of the early life variables influenced disease phenotype or outcome in UC.A history of being breastfed was associated with a decreased risk while childhood cigarette smoke exposure was associated with an increased risk of surgery in patients with CD. Further investigation to examine biological mechanisms is warranted.The online version of this article (doi:10.1186/s12876-014-0216-8) contains supplementary material, which is available to authorized users. Faecalibacterium prausnitzii [Inflammatory bowel disease (IBD), which comprises Crohn’s disease (CD) and ulcerative colitis (UC), are chronic immune mediated diseases characterized by a dysregulated immune response to commensal flora in a genetically susceptible host . The expusnitzii  and incrusnitzii  suggestsGiven the peak age of incidence of IBD in childhood and early adulthood, the role of early life environmental factors is of considerable interest. The spectrum of risk factors examined range from gestational age and mode of delivery to breastfeeding, antibiotic use, exposure to farm animals or pets, and cigarette smoke, all of which have variably been demonstrated to modify risk of CD or UC -19. It iExamining the influence of these early life environmental factors on disease outcome could shed light on its interplay with microbiome, potentially leading to novel therapies targeting intestinal flora. Consequently, we performed this study using a large, well-phenotyped cohort of IBD patients to investigate the effects of early environmental exposures on disease course in both CD and UC.The study population consisted of adult patients enrolled in the Prospective Registry in IBD Study at Massachusetts General Hospital (PRISM). This is a single center ongoing patient registry that aims to understand the causes of IBD and factors affecting disease progression. Patients were approached during their regularly scheduled clinic visits at the MGH Crohn’s and Colitis Center. Study research coordinators obtained consent and medical history was obtained and confirmed by review of the electronic medical record.Environmental questionnaires including ten questions on early life exposures were distributed to patients during regularly scheduled clinic visits subsequent to enrollment. The first wave of questionnaires was distributed in 2010 (n = 348) and the second wave to an additional set of patients was distributed during January-June 2014 (n = 255). Early life exposures included questions on gestational age, history of breastfeeding, exposure to cigarette smoke in childhood, treatment with antibiotics, hospitalization, and exposure to pets or farm animals , 17 to 40 years (A2), >40 years (A3) according to the Montreal classification . DiseaseOur primary outcome was IBD-related bowel resection for both CD and UC while our secondary outcome included need for anti-tumor necrosis factor biologic therapy . IBD-related surgery was selected as our primary outcome as this is a more robust and hard endpoint that is less influenced by physician practice variation. Disease characteristics and outcomes were confirmed by the treating physician.The data was analyzed using Stata 13.0 . Separate analyses were conducted for CD and UC. Patients with indeterminate colitis were grouped with UC owing to small numbers of the former. Continuous variables were summarized using means and standard deviations while categorical variables were expressed as proportions and compared using the chi-square test. We first performed univariate analysis comparing the environmental risk factors to our primary and secondary outcomes of interest. Multivariate regression models adjusting for disease specific covariates including phenotype and duration since diagnosis were used to identify the independent association of the exposures with our outcome of interest. This also allowed for exploration of whether the early life environmental influences exerted their effect through modification of disease phenotype. A p-value of <0.05 was considered statistically significant. Ethical approval for this study was obtained from the Institutional Review Board of Partners Healthcare (2004P001067). All patients enrolling in PRISM provided informed consent for participation in the registry.A total of 603 patients completed questionnaires on early life exposures . Just over half the cohort was female (54%), and 23% had family history of IBD in a first-degree relative. The median age at diagnosis of IBD was 25 years (interquartile range (IQR) 18 – 35 years) with median disease duration of 9 years at the time of enrollment (IQR 4 – 16 years). Nearly half of those with CD had inflammatory disease (44%), a third had penetrating disease (36%) while the remainder had stricturing phenotype. The most common location was ileocolonic (51%) followed by colon alone (24%) or ileal alone (18%). Over half of those with UC (57%) had pancolitis. In the entire cohort, 186 patients (31%) had at least one IBD-related surgery, two-thirds had used immunomodulators (65%) and half required biologics (46%). Table On univariate analysis, a history of being breastfed was associated with reduced risk of surgery in CD (odds ratio (OR) 0.42, 95% confidence interval (CI) 0.25 – 0.68) while childhood cigarette smoke exposure was associated with increased risk of surgery   in UC, the statistical power to detect associations may be lower. However, most associations except for daycare exposure did not demonstrate even a trend towards an association. We also did not have information on other factors that have been associated with aggressive disease course including steroid use at diagnosis.In conclusion, this study identified a history of being breastfed and exposure to cigarette smoke during childhood as environmental factors that may impact the natural course of CD. Further studies that examine these factors in conjunction with changes in the microbiome, epigenetic changes, and gene expression could offer insight into how the natural history of IBD is influenced by such influences, and offer avenues for improving disease outcomes.


The importance of soil humus in soil fertility has been well established many years ago. However, the knowledge about the whole mechanisms by which humic molecules in the rhizosphere improve plant growth remains partial and rather fragmentary. In this review we discuss the relationships between two main signaling pathway families that are affected by humic substances within the plant: one directly related to hormonal action and the other related to reactive oxygen species (ROS). In this sense, our aims are to try the integration of all these events in a more comprehensive model and underline some points in the model that remain unclear and deserve further research. Numerous studies have reported the beneficial effects of dissolved organic matter (DOM) in both mineral nutrition and development of plants cultivated in many different soil types –3. TheseIn terms of soil organic matter and more generally due to the complexity and heterogeneity of the humic supramolecules, their definition and classification are rather operational and are based on the different water solubility of HS components as a function of pH and ionic strength : humic a(i) Artificial HS (AHS). This indicates organic substances extracted by IHSS-method  from organic materials modified or transformed by using diverse alternative or complementary process different from composting, such as controlled pyrolysis (biochar), chemical oxidation, and anaerobic digestion (digestates). These HS may be named Artificial HS (AHS), and their fractions are as follows: artificial humic acids (AHA) and artificial fulvic acids (AFA). (ii) Fresh HS (FHS). This indicates organic substances extracted by IHSS-method from intact, non-biologically or chemically modified, fresh (living) organic materials, such as plant or animal fresh residues . These HS may be named Fresh HS (FHS) and their fractions are as follows: fresh humic acids (FHA) and fresh fulvic acids (FFA). (iii) Compost HS (CHS). This indicates organic substances extracted by IHSS-method from composted organic materials. These HS may be named Compost HS (CHS) and their fractions are as follows: compost humic acids (CHA) and compost fulvic acids (CFA). (iv) Sedimentary HS (SHS). This indicates organic substances extracted by IHSS-method from naturally humified organic matter with sedimentary origin present in terrestrial  and aquatic  environments. These HS may be named Sedimentary HS (SHS), and their fractions are as follows: sedimentary humic acids (SHA) and sedimentary fulvic acids (SFA). In principle, only SHS and, by analogy, CHS should properly be considered as HS since they are produced from fresh organic materials and processes both present in natural environments.A number of studies have reported that HS in solution, mainly SHS and CHS, can affect plant development –3, 7. Th−1) since their effects on plant growth are normally linked to their ability to provide complexed micronutrients to plants grown in soils prone to micronutrient deficiency [−1) [In general, the HS concentrations in the rhizosphere that is necessary to affect plant development and mineral nutrition through indirect effects and/or direct effects mediated mechanisms are different to each other . Thus, Hficiency –10. Howency [−1) . ConsequIt is well known that HS have beneficial effects on plant development when applied to both the rhizosphere and leaves (foliar sprays) , 8. In tAlthough the mechanism of action of HS has extensively been studied for many years, the knowledge about them is rather fragmentary and it has not been integrated in a comprehensive model yet . In geneIn the case of rhizospheric HS, direct effects mediated actions have to derive from the interaction of HS with plant roots. Although the possible penetration of some HS fraction into root apoplast and epidermal cell areas has been proposed, results are not very conclusive yet . However+-ATPase activity and nitrate root to shoot translocation [Regarding shoot growth promotion, studies carried out in cucumber with a sedimentary humic acid (SHA) obtained from leonardite reported the important roles of cytokinins (CKs) and mineral nutrient root to shoot translocation in the shoot growth promoting effect of SHA . This eflocation , 14. In location . These plocation .On the other hand, the shoot growth promoting action of SHA in cucumber was dependent on the increase in indoleacetic acid (IAA) and nitric oxide (NO) concentrations in roots caused by SHA root application . RecentlOverall, these results indicate that several interconnected hormone-dependent signaling pathways as well as physiological events are involved in the shoot promotion caused by HA with sedimentary origin .Regarding HS direct effects on root development, two main types of phenotypic effects might be distinguished: micromorphological effects  and macromorphological effects  –23.+-ATPase activity and expressed through auxin  and probably NO dependent pathways [Arabidopsis), as well as other genes encoding metabolic pathways belonging to the taking of nutrients [Several research groups working with HA obtained from vermicomposts of various vegetal residues (CHA) have reported their ability to promote lateral root proliferation and modify root architecture –23. Thespathways –25. Furtpathways . These eutrients –25  also showed the involvement of relevant phytoregulators in SHA-mediated effects on root development , 12–15. bidopsis   mutant recovery occur in Arabidopsis with the application of low-molecular-weight HS. This study also showed that Arabidopsis strains transformed with the reporter gene uidA (GUS) fused to auxin-responsive promoters (DR5:uidA and BA3:uidA) showed no change or a low response to HS application, concluding that the effects of low-molecular-weight HS may alter root morphology, by increasing the root surface through auxin-independent signaling pathways. In this line, Mora et al. [Root morphological auxin-like effects and their relationships with other mechanisms of action were investigated by Schmidt et al. , who dema et al.  demonstr2∙− levels [−1 increased the levels of H2O2 and decreased lipid peroxidation, possibly via the transformation and control exercised by POX enzymes [In parallel with these studies, other studies reported metabolic changes of reactive oxygen species (ROS) in rice plants upon vermicompost (VC) HA root application. Studies conducted by García et al. , 16, 19 2O2 levels and POX enzymatic activity were found in young rice plants  with known hormonal signaling pathways. In this sense, studies have reported regulatory relationships between hormone and redox metabolism, specifically in signaling pathways related to both auxin metabolism and ROS , 35 Fig.+-ATPase enzymes has already been demonstrated, studies showing the involvement of auxin transporters in HS-mediated signaling pathways have not been published so far.Point 2 in  in vitro O2∙− production [ Arabidopsis have been shown to correspond to stress response metabolism using large-scale gene expression methods (microarray and cDNA-AFLP) [ Brassica napus, wherein HA application stimulated 5.8% and 6.6% of stress response-related genes in shoots and roots, respectively [Point 3 in oduction . In thisoduction . ROS prooduction . In thisNA-AFLP) . The samectively .2+ channel activation is currently known [2+ channels is a key step in the regulation of other important processes, including antistress regulation and hormone signaling [2+gradient in the apical root region, leading to secondary root growth [ OsCPK7 and OsCPK17 gene expression upon CHA application have been reported [Most studies have shown a beneficial morphological effect on root system growth, albeit with different metabolic pathways involved in the HS mechanism of action in plants , 21. Thely known . ROS actignaling . ROS prot growth . Stimulareported .∙ species resulting from an increase in apoplast H2O2 may increase membrane permeability and therefore cellular Ca2+ influx [2∙− and H2O2 are apparently directly related to secondary root growth and emission acting on different regions. Thus, O2∙− predominantly accumulates in root elongation regions, whereas H2O2 accumulates in root differentiation regions [Although studies on the role of ROS as signaling molecules involved in root growth regulation remain ongoing, it has been reported that OH+ influx . O2∙− a regions .2∙− anions are detected at higher concentrations in the root elongation region of plants, whereas H2O2 is more concentrated in the root differentiation region (Root CHA application has shown that several components of the redox regulation system are simultaneously stimulated during the root growth promoted by CHA. Root CHA application to rice plants generates increases of ROS located in different root regions depending on the species. The On region .2∙− and H2O2 species, respectively, also responded to CHA application (Figures The CHA was also able to increase membrane permeability, as assessed by the release of electrolytes . Simulta Figures .The roles of ROS in metabolic processes related to plant cell differentiation, specifically those related to redox regulation and cell signaling pathways, are currently an open field of study. However, their involvement in secondary root growth and development is more evident today , 47. Sim Brachiaria upon HA application at high concentrations, and a redox imbalance caused by the high concentrations applied may lead to impaired root growth (Therefore, the role of ROS as signaling molecules or intermediate species in the HS mode of action in plants seems to respond to a fine adjustment action of redox homeostasis, where the final result is in beneficial effects, including root growth. It is known that this mechanism may be highly dependent on the HS concentration applied to plants. Adverse effects on the root system have been observed int growth .2O2 production and Ca2+ channel signaling for stomata opening and closing [ABA is a hormone that plays a key role in plant signaling pathways. ABA regulates and participates in H closing , 47; at  closing . Others  closing , where t closing .Both ABA and TIP-PIP subfamilies are involved in the mechanisms of action of HS in plants. Thus, the root application of SHA to cucumber plants caused a differential increase in ABA levels in roots and shoots –14. Conv OsTIP1;2 isoform in rice roots ( OsTIP1;2 isoform in water flux through vacuoles [In this sense, studies performed using CHA applied to roots of rice plants under normal and water stress growth conditions have shown that CHA regulates the gene expression of these isoforms both in leaves and in roots , 19 Fig. García ce roots  in line vacuoles .Conversely, under normal growth conditions, high CHA concentrations caused increased expression of this isoform, suggesting that CHA application at high concentrations may induce physiological stress  Figure . This re2+ [Overall, all results discussed above show that the whole mechanism responsible for the beneficial direct effects of HS on plant growth probably involves several complementary and interconnected signaling pathways related to both relevant hormonal networks and secondary messengers such as ROS and Ca2+ , 21, 54.Regarding the relevance of the origin of HS, it was noteworthy that both SHS and CHS presented many common hormone-mediated mechanisms regardless of their potential differences in structure , 4. As fThus, it becomes also clear that much further research is needed in order to integrate all signaling pathways affected by HS with different origins in a more comprehensive, holistic, model.


To investigate the redirection of lymphatic drainage post-lymphadenectomy using non-invasive near-infrared fluorescence (NIRF) imaging, and to subsequently assess impact on metastasis.Cancer-acquired lymphedema arises from dysfunctional fluid transport after lymphadenectomy performed for staging and to disrupt drainage pathways for regional control of disease. However, little is known about the normal regenerative processes of the lymphatics in response to lymphadenectomy and how these responses can be accelerated, delayed, or can impact metastasis.Changes in lymphatic “pumping” function and drainage patterns were non-invasively and longitudinally imaged using NIRF lymphatic imaging after popliteal lymphadenectomy in mice. In a cohort of mice, B16F10 melanoma was inoculated on the dorsal aspect of the paw 27 days after lymphadenectomy to assess how drainage patterns affect metastasis.NIRF imaging demonstrates that, although lymphatic function and drainage patterns change significantly in early response to popliteal lymph node (PLN) removal in mice, these changes are transient and regress dramatically due to a high regenerative capacity of the lymphatics and co-opting of collateral lymphatic pathways around the site of obstruction. Metastases followed the pattern of collateral pathways and could be detected proximal to the site of lymphadenectomy.Both lymphatic vessel regeneration and co-opting of contralateral vessels occur following lymphadenectomy, with contractile function restored within 13 days, providing a basis for preclinical and clinical investigations to hasten lymphatic repair and restore contractile lymphatic function after surgery to prevent cancer-acquired lymphedema. Patterns of cancer metastasis after lymphadenectomy were altered, consistent with patterns of re-directed lymphatic drainage. Lymphedema is a disfiguring and debilitating condition of impaired lymph uptake and/or transport manifested by chronic swelling, tissue fibrosis, and reduced immune response Vegf-c gene therapy and/or other pharmacological treatments have been proposed for amelioration of acquired lymphedema In order to evaluate the etiology of lymphedema, several animal models of acquired lymphedema in the limb of dogs Animals were maintained in a pathogen-free mouse facility accredited by the American Association for Laboratory Animal Care (AALAC). All experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC). Animal experiments were approved by University of Texas Health Science Center Animal Welfare Committee (AWC).Eight to ten week-old female C57BL/6 mice  were housed and fed sterilized pelleted food and sterilized water. Mice were anesthetized with isoflurane (2% oxygen) and maintained on a warming pad at 37°C. Two µl of 1% Evans blue dye (EBD) solution  was injected into the dorsal aspect of the left paw and taken up by the lymphatics See . Bupivac5 cells per mouse in 10 µl sterile PBS) were then implanted intradermally in the dorsal aspect of the left paw of mice (n = 4) which had undergone surgical removal of the left PLN 27 days prior. Mice (n = 4) without lymphadenectomy were also injected with B16F10 cells. Three weeks after inoculation, mice were euthanized, and intravital imaging using a stereoscope was performed to image LN metastasis. After intravital imaging, tissues were collected for histology.B16F10 murine melanoma cells were obtained from American Type Culture Collection (ATCC) and cultured in DMEM-F12 (GIBCO) supplemented with 10% FBS. B16F10 cells  24 hrs before imaging. Mice were imaged for baseline characteristics prior to PLN removal and for up to 4 weeks after surgery. Anesthetized mice with isoflurane were maintained on a warming pad. A volume of 2 µl of 645 µM of indocyanine green  dissolved in a mixture of distilled water and 0.9% sodium chloride in a volume ratio of 1∶9 was injected intradermally to the dorsal aspect of the left foot using 34-gauge needles . Fluorescence images were acquired immediately after and for up to 10 min after i.d. injection using a custom-built NIRF imaging system as described previously Lymphatic drainage through thoracic duct from the lumbar LNs (LLNs) was imaged in another two groups of mice: (i) mice without lymphadenectomy and (ii) two days following lymphadenectomy. Invasive, far-red and NIRF imaging was conducted 5 mins after i.d. injection of 2 µl of Alexa680-bovine serum albumin (BSA) and 2 µl of ICG to the dorsal aspect of the right and left hind paw, respectively. The peritoneal and thoracic cavities were exposed and imaged immediately after animals were euthanized.In vivo small animal CT imaging was performed to measure arm area from transverse CT slices Matlab  and ImageJ  were used to analyze the fluorescence imaging data in the following manner. To assess lymphatic contractility, fixed regions of interest (ROIs) in fluorescent afferent and efferent lymph channels were defined on fluorescence images. The mean of the fluorescence intensity within each ROI in each fluorescence image was then calculated and plotted as a function of imaging time as previously described Skin samples were fixed in 10% formalin overnight before transfer into 70% ethanol and embedded in paraffin. 4 µm sections were used in all staining procedures which included hematoxylin-eosin (H&E). For LYVE-1 detection, following paraffin removal and antigen retrieval using citrate buffer pH 6, tissues were blocked with 5% BSA and stained with rabbit anti-mouse LYVE-1 antibody  and biotin-anti rabbit secondary antibody . VectaStain Elite ABC peroxidase detection system and DAB chromagen were used before tissues were counter-stained with hematoxylin (Vector Labs). LYVE-1 expression in three different fields in each section was examined at x40 magnification (Leica Microsystems Inc.). Image-Pro Plus (Media Cybernetics) software was used to analyze the number of lymphatic vessels, and the percentage of positively stained lymphatic vessels per area was determined.For whole mount staining Data were presented as average values ± standard error. Statistical analysis was performed with Prism 5 . The D'Agostino and Pearson omnibus tests were used to assess data normality. Changes of ankle diameter, leg area, and lymphatic contraction frequency were analyzed with one-way repeated-measures analysis of variance (ANOVA) with a Bonferroni posttest (parametric) or a Friedman test with a Dunn posttest (nonparametric) for multiple comparisons over time. A one-way ANOVA with Bonferroni post hoc was used when three or more groups were compared. The student t-test was also used for comparisons made between two groups. The differences were considered significant at p<0.05.After i.d. injection of EBD on the dorsal aspect of the paw  was obsein vivo. Therefore, we performed longitudinal NIRF imaging to investigate changes of lymphatic drainage patterns in mice after PLN removal. As shown in Although EBD is suitable for intravital imaging of lymphatic vessels and LNs in dissected tissues, it is difficult to longitudinally image the distribution of EBD in mice Longitudinal NIRF lymphatic imaging demonstrated transient changes of lymphatic drainage patterns after PLN removal . At 2 daFluorescent lymphatic vessels draining both to the IsLN through the wound site and to the ILN were also observed in mice that underwent lymphadenectomy. As shown in another example in We also observed fluorescent collecting lymphatic vessels draining from the injection site to the IsLN, but not to the ILN, within 27 days after the procedure. As shown in As described previously, 3 out of 19 mice showed altered lymphatic drainage pathways at baseline. We detected fluorescent lymphatic vessels draining from the injection site to the IsLN through the PLN as well as to the ILN in these 3 mice . Our lonWe observed more fluorescent lymphatic vessels and extravascular ICG around the ankle in the operated hindlimb of mice at day 2 post-surgery. However, it is unknown whether the greater extent of staining of fluorescent lymphatic capillaries is due to distant lymphangiogenesis or is due to backflow in response to resistant fluid dynamics resulting from loss of normal lymph flow pathways. Therefore, we collected tissues from the wound area (red rectangle c in Tumor cells metastasize via lymphatic vessels to the sentinel LN (SLN). It has been shown that murine B16F10 cells inoculated on the paw of mice spread to the tumor-draining PLN. Thus, we sought to investigate where B16F10 cells metastasized to in mice which had previously undergone surgical removal of PLNs. We observed lymphatic drainage to the IsLN and ILN in 3 of 4 mice and to the ILN only in one mouse at 27 days post-surgery. At 3 weeks after B16F10 cell inoculation, intravital imaging data demonstrated that all 4 mice showed LN metastases. Three mice with lymphatic drainage to both the ILN and IsLN through the popliteal wound site showed B16F10 metastases to the ILN and the LLN through the IsLN as shown in As demonstrated above, we longitudinally imaged changes of lymphatic drainage pathways after PLN removal. Because afferent popliteal lymphatic vessels  and efferent (lower pressure) vessels, it is expected that removal of a LN can cause an increase in intralymphatic pressure. Our data shows a two-step process of recovery from PLN removal. Although minimally invasive PLN removal resulted in less traumatic lymphatic injury and thus mild swelling of the operated hind leg, we observed a complete blockage of normal lymphatic drainage to the popliteal region and thus triggering alternate, pre-existing collateral lymphatic vessels to drain stagnant lymph to the ILN in mice 2 days after lymphadenectomy. We also observed retrograde flow and more fluorescent lymphatic capillaries resulting from a greater extent of dermal backflow on the paw distal from the wound area at day 2. This lymphatic phenotype and alternate drainage pathways may be due to increased resistance to flow as a result of obstructed lymphatics, but not lymphangiogenesis, since there was no significant difference in the number of lymphatic vessels and the percentage of vessel area between tissues collected distally from operated and non-operated limbs . Once lyCollateral lymphatic circulation is a common manifestation of lymphatic obstruction and the lymphatic system can use it to overcome pathways rendered nonfunctional due to damage to the lymphatics in vivo with injection of ICG on the dorsal aspect of the paw. Thus, lymphatic function and drainage patterns can be longitudinally characterized in response to minimally invasive PLN dissection, which would affect all lymph flow from the lower hind limb and thus make this model relevant to post-surgical lymphedema. Previously, Ikomi et al. The popliteal afferent and efferent collecting lymphatic vessels are well defined and easily visualized et al. In situ imaging after injection of FITC dextran or other visibly excited fluorescent dyes has also been used to image functional maturation of newly formed lymphatic vessels in mice after LN removal and LN transplantation together with Vegf-c/d gene therapy Changes of lymphatic drainage patterns have been reported in mice after LN removal, alone or with the associated fat pad containing pre- and post-nodal collecting lymphatic vessels Since most cancers metastasize through lymphatic routes before spreading to distant organs, regional LN staging is important for prognosis estimation and therapy stratification. ALN dissection (ALND) and pelvic LND (PLND) have been employed for regional control of disease in order to achieve potential cure of breast, prostate, bladder, and other cancers. Our results, employing the metastatic B16F10 model, suggest that co-opting of collateral lymphatic vasculatures and re-direction of lymphatic drainage may negate the use of prophylactic lymphadenectomies to disrupt metastatic pathways.In breast cancer survivors, ALND is associated with a significant morbidity, with up to 60% of patients who undergo both ALND and radiation therapy suffering at some time from lymphedema In conclusion, we have non-invasively imaged dynamic lymphatic response to surgical removal of a PLN using NIRF imaging and provided assessment of its impact on metastatic spread. Changes in lymphatic function and drainage patterns via collateral lymphatic vessels were observed for a period of approximately 4 weeks after surgery. We observed dermal backflow, diffused dye patterns, and retrograde flow by day 2 post-surgery, during which lymphatic function was also significantly reduced. However, when lymphatic drainage capability was restored, as evidenced by lymphatic contractility, abnormal lymphatic drainage patterns regressed. Collateral lymph flow patterns to facilitate obstructed lymphatics disappeared in some mice, but were still observed in a majority of mice. These collateral draining pathways, while responsible for surgical repair, can also provide a route for metastatic dissemination of disease.


Many viruses express factors that reduce host gene expression through widespread degradation of cellular mRNA. An example of this class of proteins is the mRNA-targeting endoribonuclease SOX from the gamma-herpesvirus Kaposi’s sarcoma-associated herpesvirus (KSHV). Previous studies indicated that cleavage of messenger RNAs (mRNA) by SOX occurs at specific locations defined by the sequence of the target RNA, which is at odds with the down-regulation of a large portion of cellular transcripts. In this study, we address this paradox by using high-throughput sequencing of cleavage intermediates combined with a custom bioinformatics-based analysis pipeline to identify SOX cleavage sites across the mRNA transcriptome. These data, coupled with targeted mutagenesis, reveal that while cleavage sites are specific and reproducible, they are defined by a degenerate sequence motif containing a small number of conserved residues rather than a strong consensus sequence. This degenerate element is well represented in both human and KSHV mRNA, and its presence correlates with RNA destabilization by SOX. This represents a new endonuclease targeting strategy, in which use of a degenerate targeting element enables RNA cleavage at specific locations without restricting the range of targets. Furthermore, it shows that strong target selectivity can be achieved without a high degree of sequence specificity. The human tumor virus Kaposi’s sarcoma-associated herpesvirus uses a messenger RNA (mRNA)-targeting nuclease called SOX to cut cellular RNAs, thereby controlling gene expression in infected cells. How SOX causes destruction of the majority of mRNAs while at the same time achieving target and cut site specificity has remained unknown. To resolve this paradox, we mapped the location of SOX cut sites across the human RNA transcriptome, and confirmed that SOX recognizes specific RNA sequences. These sequences have some conserved features but are otherwise quite distinct. Thus, SOX uses a degenerate motif to achieve broad targeting capability while preserving cut site specificity within the RNA. Little is currently known about the targeting mechanisms of many host and viral ribonucleases, and thus these data provide a framework for understanding how these proteins may operate. Triggering wide-spread RNA degradation is a common strategy that viruses use to decrease host gene expression, also known as host shutoff ,2. ViralThe SOX family of proteins from gamma-herpesviruses is an example of a viral RNase that displays both broad targeting of RNAs and a poorly understood level of selectivity. Gamma-herpesviruses include the human pathogens Kaposi’s sarcoma-associated herpesvirus (KSHV), which causes Kaposi’s sarcoma as well as lymphomas in immunocompromised individuals and remains a leading cause of cancer-linked death in sub-Saharan Africa. The SOX ORF37) protein is expressed early during the lytic cycle of KSHV infection and its expression triggers RNA degradation, which is recapitulated by expression of the protein alone . Homolog protein in vitro studies . Howevea et al.  or proxia et al. ,21 or tha et al. . The SOXa et al. .To address how SOX specificity is mediated, we applied a degradome sequencing technique called parallel analysis of RNA ends (PARE)  to map cPrior analyses of individual mRNAs indicated that the KSHV RNase SOX cuts at specific locations within the RNA, in a manner dependent on the sequence surrounding the cleavage site . By perfTo dissect the specificity of SOX cleavage across the mRNA transcriptome, we designed an approach to map SOX cleavage sites in endogenous mRNAs using PARE ,24. PAREa posteriori, but initial attempts at detecting and validating cut sites indicated that this approach did not provide sufficient discriminatory power to identify SOX-specific cleavage sites. In previous studies using PARE or similar approaches [Conventional analysis of PARE and similar degradome datasets relies on detecting cut sites in each condition and then comparing conditions to each other proaches ,27, addip value < 0.0001), further demonstrating that these peaks correspond to specific SOX-mediated cleavages. For downstream analyses, we focused on cut sites that were detected in both the replicates using the 99.99% confidence level and were 0–5 nt apart  changed the efficiency of SOX cleavage, measured by the intensity of the degradation intermediate Figs , althougWe previously found that mutating a TGAAGT sequence 5 nt before the GFP cut site to TGAGTG could abolish the cleavage site in GFP . The LIMLastly, our analysis indicated that the nucleotide G is under-represented at the position immediately following the SOX cut site  or expressing shRNAs against Xrn1 (repeat 2) were transfected either with pCDNA3.1-GFP-SOX or pd2eGFP-N1. In both cases >75% transfection efficiency was observed. One day after transfection total RNA was harvested and purified using RNABee (Teltest). RNA was then treated as described in Zhai et al.  to generReads flagged by the CASAVA 1.8 program were eliminated and cutadapt  was usedA Bayesian probability framework was used to find peaks that were specific to test samples compared to control samples, which takes into account random variations in the observed number of reads. At a given location and a given experiment, we assume that there is an underlying rate at which reads are produced, and the observed count follows a Poisson distribution with mean equal to this rate.In both the control and test data sets, we find that the frequency of reads per location follows a power-law distribution, as is typical for gene expression and deep sequencing data ,41, and In practice, for a given control read count, we can compute a threshold for the test read count, beyond which the difference in underlying rates is significant. The software builds a table of the thresholds using bicubic splines so that many locations can be tested efficiently. The peak finding python scripts are attached as Parameters were empirically optimized for the analysis so that a scanning window of 4 nt, a multiplicative factor between test/control read counts of 4 and a confidence level of 99.99% or 99.999% were used to output specific peaks. Parameters used in the different analyses are specified in the figure legends. After identifying peaks in single test/control comparisons, the peaks found in the biological repeats were compared. For subsequent bioinformatics analysis of sequences only peaks that were found in both biological repeats were used Figs , 2B, S2CSequences surrounding cleavage sites as defined by chromosomal positions were extracted, using the human genome assembly GRch37 build as a reference. As many sequences as possible were used for each analysis. However, because the short reads do not provide information about mRNA isoform and splicing, for all sequence analyses only cut sites that had sufficient flanking sequences within the same annotated exon were used. For motif analysis Figs  and S7A Custom Python2.7 scripts  were use32P-labeled DNA probes made using Decaprime II (Ambion), against the 3’ UTR of the GFP reporters. Blots were imaged using a Fujifilm scanner FLA-9000. Quantification of the blots was carried out using ImageJ [Total cellular RNA was isolated for Northern blotting using Trizol (Life Technologies). RNA was separated on formaldehyde gels  in MOPS buffer (40 mM MOPS (Sigma), 10 mM sodium acetate, 1 mM EDTA, pH 7.0) and transferred by capillary blotting onto nitrocellulose membrane (Bio-rad) using 10x SSC buffer . Northern blots were probed with g ImageJ . 5’ rapiProtein was isolated for western blots in protein lysis buffer  containing cOmplete EDTA-free protease inhibitors (Roche), separated on SDS-PAGE gels run in Tris-glycine buffer and transferred onto PVDF membranes (EMD Millipore). Western blots were performed with mouse anti-Xrn1 antibodies  or mouse anti-tubulin antibodies . Secondary antibodies were used at 1:5000 dilution and purchased from Southern Biotech.S1 Fig5’ RACE analysis of GFP degradation fragment was carried out in KSHV-infected iSLK-219 cells also expressing shRNAs against Xrn1. Reactivation of the KSHV lytic cycle was induced by addition of sodium butyrate (NaBu) and doxycycline (dox). Doxycycline addition also induced expression of an shRNA against Xrn1. A) PCR amplification product of the 5’ RACE protocol detected in the lytically reactivated cells B) Mapping of the 5’ end of the GFP RNA fragment mapped to the GFP coding region. Arrows indicate the 5’ ends detected by sequencing of the cloned 5’ RACE product, with the number of individual clones that mapped to each nucleotide. Empty arrowheads indicate positions of GFP fragment 5’ ends in transfected 293T cells from Covarrubias, Gaglia et al. [(EPS)Click here for additional data file.S2 FigA) Western blots showing levels of Xrn1 proteins in the samples used for PARE. In repeat 1, inducible shRNAs were used, whereas in repeat 2 siRNAs were used for the knockdown. Tubulin is included as a loading control. B) The number of cut sites detected in the indicated comparisons is plotted against the parameters varied. C) Number of peaks that were identified in both replicates of SOX or GFP samples, as a function of the maximum distance allowed between the peaks. .(EPS)Click here for additional data file.S3 FigA-F) Plots of read counts (5’ ends only) for 200 nt surrounding the chromosomal position of selected SOX cut sites in the four samples. All plots represent a section of the marked exon. Note that y-axis has a logarithmic scale. G) 5’ RACE was carried out on RNA isolated from cells overexpressing SOX (“+”) or an empty vector (“-”) and treated with shRNA against Xrn1. Primers for four human RNAs detected fragments consistent with the cut sites predicted by the PARE analysis. Primers for PGAM1 did not replicate the deep sequencing data. Red asterix = expected position of RACE fragment from cleaved mRNA.(EPS)Click here for additional data file.S4 FigThe percentage of aligned reads (A) or of the filtered reads (B) that started with each of the four nucleotides was computed.(EPS)Click here for additional data file.S5 FigA) Predicted structure of the 50 nt surrounding the SOX cut site in the validated targets, highlighting the A trimer (asterisk) and the cut site (arrow). For SRSF3, the end of the 33 nt insertion used in B-C is marked. B-C) 200–33 nt surrounding the SOX cleavage site in the LIMD1 mRNA were inserted into the GFP reporter at nt 715. The GFP reporters were co-expressed with SOX (“+”) or an empty vector control (“-”) in shXrn1-treated cells. The GFP mRNA was detected using Northern blotting. The empty arrowheads point to the additional cleavage fragment resulting from insertions, whereas the filled arrowheads point to the normal GFP cleavage fragment. A representative blot is shown (B), as well as the quantified intensity of the signal from the different RNA species (C), plotted relative to the intensity of the bands from the 200 nt insertion construct. Error bars = standard deviation.(EPS)Click here for additional data file.S6 FigA) The TGAAGT sequence at positions -6 to -1 relative to the cut site in the GFP RNA see  was eith(EPS)Click here for additional data file.S7 Figp value (Kolgorov-Smirnoff test) < 0.001. C) The fold change in mRNA levels in SOX-expressing vs. control cells from Clyde and Glaunsinger [p < 0.001.A) Weblogo3  represenunsinger  is plott(EPS)Click here for additional data file.S1 Table% mapping (no restrictions) indicates the percentage of reads that map to the human genome if the requirement for unique mapping to a previously annotated region of the genome is removed.(DOCX)Click here for additional data file.S2 TableThe number of peaks detected by using each of the samples as test or control in the PyDegradome program (and plotted in (DOCX)Click here for additional data file.S3 TableThis table lists SOX cut sites identified in both replicates with confidence level of 99.99% or in one with confidence level 99.99% and in the second with confidence level 99.9%, and that were ≤ 5 nt apart in the two replicates. The table includes the chromosomal position of the cut site, the read count at the cut site in each replicate, the gene name and the confidence level setting used for the identification. It also indicates whether the cut site could be a transcriptional start site (TSS) and whether the cut site was used for the analyses in Figs (XLSX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S1 Files“Readme.txt” file with instructions on how to run the analysis, as well as the two scripts required for the analysis are included in the archive.(ZIP)Click here for additional data file.S2 Files“Readme.txt” file with instructions on how to run the analyses, and several scripts used to analyze the data.(ZIP)Click here for additional data file.


Endometrial carcinoma is the most common neoplasia of female genital tract. The prognosis of early stage disease (FIGO I and FIGO II) is excellent: recurrence after surgery is less than 15%, most of which are reported within 3 years after primary treatment. Herein we report a case of late rectal recurrence from FIGO Ib endometrial adenocarcinoma. Patient had also familiar and personal history of colonic adenocarcinoma and previous findings of microsatellite instability (MSI); molecular analysis evidenced heterozygotic somatic mutation in MLH1 gene. Twenty-eight years after hysterectomy and bilateral salpingoovariectomy, a rectal wall mass was detected during routine colonoscopy. Patients underwent CT scan, pelvic MRI, and rectal EUS with FNA: histopathological and immunohistochemical analysis revealed differentiated carcinoma cells of endometrial origin. No neoadjuvant treatment was planned and low rectal anterior resection with protective colostomy was performed; histology confirmed rectal lesion as metastasis from endometrial carcinoma. Recurrence of early stage endometrial carcinoma after a long period from primary surgery is possible. It is important to keep in mind this possibility in order to set a correct diagnostic and therapeutic algorithm, including preoperative immunohistochemical staining, and to plan a prolonged follow-up program. Endometrial carcinoma (EC) is the most common neoplasia of female genital tract: the highest incidence occurs in postmenopausal women (70%), whereas it is uncommon under 40 years of age (<5%) . The pro131 adjuvant radiotherapy, in 2001 to endoscopic resection of bladder papillary carcinoma, and in 2003 to right hemicolectomy for pT3 N1 (1/23) colonic adenocarcinoma followed by adjuvant chemotherapy. Due to positive familiar and personal history of oncologic diseases, oncologists referred patient to genetic evaluation: microsatellite instability (MSI) by heterozygotic somatic mutation in MLH1 gene was detected.A 72-year-old woman was admitted to our department for the findings of rectal mass, in the absence of abdominal pain and alteration of the intestinal canalization, after a routinely endoscopic investigation in the context of oncological follow-up. The patient was previously submitted in 1985 to hysterectomy and bilateral salpingoovariectomy for early stage low-grade endometrioid EC (FIGO Ib) associated with Intravaginal Brachytherapy (IVBT), in 1989 to left thyroid lobectomy for papillary carcinoma followed by IIn 2013 routine colonoscopy was performed and a right-sided compression of rectal wall with an unscathed mucosa was found; the patient was submitted to pelvic Magnetic Resonance Imaging (MRI) demonstrating a mass of 4.7 × 3.5 cm at 6.5 cm from the anocutaneous plane and about 3 cm from the anorectal junction.The mass had intramural development with compression of the gut lumen and without any pelviperineal muscular involvement. T1 and T2 weighted phases evidenced a small oval area (11 mm) in perivisceral adipose tissue with similar characteristics: a mesorectal lymphadenopathy with maximum diameter of 8 mm was also identified .Multidetector Computed Tomography (MDCT) confirmed a rectal hypodense lesion with a slightly eccentric hypodense area and medial component intensely enhanced in the arterial phase, with a single mesorectal lymph node: no liver or pulmonary metastasis was found .Endoscopic ultrasound (EUS) showed once more a subepithelial hypoechogenic mass in the right paramedian side of rectal wall , with abFine needle aspiration biopsy (FNAb) was performed on rectal wall mass (multiple aspiration with Procore 22-gauge needle).At cytohistology poorly differentiated carcinoma cells were found with immunocytochemical profile of endometrial origin in CEA (+), CDX2 (−), CA125 (−), and estrogen receptors (+). No endometriosis foci were found in all the specimens analyzed. Sieric neoplastic markers were negative. After multidisciplinary discussion, no neoadjuvant treatment (radiotherapy (RT), chemotherapy (CT), or CT + RT) was proposed, and indication for rectal resection was given. At laparotomy, a single hepatic metastasis in the segment 3 of 8 mm was found. After liver Intraoperative Ultrasound (IOUS), which confirmed absence of other focal lesions, hepatic wedge resection and rectal anterior resection associated with protective colostomy were performed. Postoperative course was uneventful and patient was discharged on 6th p.o. day. Histological examination confirmed rectal mass as a metastatic adenocarcinoma, not a primary rectal cancer, with immunohistochemical features of endometrial carcinoma CK7+, ER+, CDX2−, CK20−, antivimentin Ab+, and PAX 8+  .All antibodies used were rabbit monoclonal antibody . CK7 (clone SP52), CK20 (clone SP33), vimentin (clone V9), CA125 (clone 8C125), and estrogen receptors (clone SP1) were prediluted antibody; CEA (clone TF3H8-1) and CDX2 (clone EPR2764Y) were diluted 1 : 1. The slides were stained with these antibodies using the Ventana automated immunostainer . The hepatic and perirectal lesions evidenced the same immunohistochemical pattern. Invasion from endometrial adenocarcinoma in a single perirectal lymph node out of twelve was documented. After discharge, the revision of histological slides of patient's initial intervention was done: endometrial tumor removed 28 years before was confirmed to be a primary low-grade endometrioid EC with the same histopathological features. No indications for adjuvant treatment were done and clinical/radiologic follow-up was planned. After eight months the colostomy was closed and 23 months later the patient was disease-free.Among histological types of uterus body cancers, endometrial adenocarcinoma is the most frequent; it is found in 75% of cases and it is the most common gynaecological neoplasia in Western countries. Large majority of endometrial cancers are sporadic; genetic mutations cause endometrial cancer in about 5% of patients. Screening for genetic mutations should be considered in all patients with endometrial cancer, but especially in those younger than 50 years of age . OverallUntil 2014, 14 cases of solitary splenic metastasis from endometrial adenocarcinoma after a long period of clinical latency were also reported , and sevIn our patient, already from the early diagnostic stages, the lesion appeared with unusual endoscopic and radiological features. It was a subepithelial tumor with normal mucosa; in addition, MRI and MDCT scan showed an intramural well-defined lesion giving evidence for stromal tumor and especially for a rectal gastrointestinal stromal tumor (GIST). However, it was important to rule out the primary rectal origin of the lesion because the patient already underwent colectomy for colonic adenocarcinoma and she was affected by MSI . AsDifferential diagnosis was considered especially towards intestinal endometriosis, a rare but well-known identity; it often presents with cyclic intestinal symptoms of many years' duration . In addition, patients with suspected intestinal endometriosis have undergone one or more surgical procedures aimed at treating endometriosis previously. Absence of endometriosis does not exclude completely the possibility of a primary endometrial carcinoma arising from an endometriotic focus but, in this case, it would be at least probable to find other endometriotic foci in the surrounding tissue.Malignant transformation of endometriosis (EMT) is extremely rare and occurs in 1% of case; the association between development of malignancy and exogenous hormonal therapy is well known. EMT occurs electively in ovaries in more than 80% of patients, but gastrointestinal tract involvement has already been reported and classified as endometriosis-associated intestinal tumors (EAITs) . SampsonIn preoperative period, whenever possible, it is also important to use immunohistochemistry (IHC) to support difficult pathological differential diagnosis and to reduce the rate of false negative and false positive. Therefore it would be beneficial to integrate all of the current knowledge on IHC markers for the differential diagnosis of pelvic organs neoplasia. Carcinoma of pelvic organs presents distinct patterns of immunoreactivity for keratins (CK or KRTs) and it also expresses fundamental markers for differential diagnosis of different pelvic masses. From 2003, many reports document CDX2 and CK20 positivity in almost all rectal adenocarcinomas, but negativity in endometrial carcinoma . In the In conclusion, metastatic involvement of the GI tract from endometrial adenocarcinoma after a long period of clinical latency is a rare but possible occurrence. Consequently, it is important to keep in mind this possibility in order to set a correct diagnostic and therapeutic algorithm, including preoperative immunohistochemical staining. A prolonged clinical and instrumental follow-up program for high-risk cancer patients should always be performed.


Sleep disturbances represent important predictors of poor quality of life (QoL) in Parkinson's disease (PD). This open-label pilot study aimed to objectively assess, by means of actigraphic recording, effect of rotigotine on sleep in PD patients with self-reported sleep complaints. 15 PD patients underwent one-week actigraphic recording before (T0) and during (T1) rotigotine treatment, which was titrated to the dose subjectively improving motor symptoms (4–8 mg/24 h). Sleep disturbances, daytime sleepiness, cognitive performance, QoL, and depression were also evaluated with questionnaires. Actigraphic recordings showed a significant reduction in nocturnal motor activity and mean duration of wake episodes after sleep onset during rotigotine treatment compared to baseline. In 10 patients presenting objective evidence of poor sleep quality at T0 (sleep efficiency ≤ 85%), rotigotine also significantly improved other sleep parameters and further reduced nocturnal motor activity and mean duration of wake episodes. A significant decrease in number and duration of daytime sleep episodes was also observed at T1. Finally we confirmed that rotigotine significantly improves perceived sleep quality and QoL. Our study showed for the first time that rotigotine is associated with an objective improvement of nocturnal and diurnal sleep disturbances in PD patients with self-reported sleep complaints. This study is registered with AIFA-observational study registry number 12021. Sleep disturbances represent independent and important predictors of poor quality of life (QoL) in Parkinson's disease (PD) , 2.Reduced sleep efficiency, arousal, and sleep fragmentation may be caused by PD pathology, motor symptoms , autonomic symptoms (nocturia), or a coexisting sleep disorder . In addition patients could complain of excessive daytime sleepiness (EDS), increased diurnal sleep episodes, or sudden sleep attacks that have been related to nocturnal sleep disturbances, PD pathology, or use of dopaminergic agents . The latActigraphy is routinely used to evaluate the sleep-wake cycle for long periods, through a portable device usually worn on the wrist using an accelerometer to detect movement, which is sampled several times a second. The accelerometer generates a voltage during each movement, which is amplified and band-pass filtered (2-3 Hz). The resulting signal is compared with a reference signal to determine if it exceeds a threshold for quantification and storage. Computerized scoring algorithms then qualified epoch of time  as wake or sleep by comparing the activity score expressed in counts for that epoch to a threshold of activity set by the users. The motor activity is also quantified and reported as mean or median value of activity per epoch (or per minute for epoch of 60 seconds) . In PD pThis open-label pilot study aimed to evaluate rotigotine's effect on sleep in PD patients with self-reported sleep complaints, providing also objective measurements by means of actigraphic recording.Between April 2013 and June 2014 we recruited from the outpatient Movement Disorders Clinic of our department 15 consecutive PD patients . Only paThe study was approved by the local ethics committee (AUSL of Bologna) and performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and subsequent amendments. Patients gave written informed consent before entering the study.Within 1 week after the screening, patients underwent the baseline visit (T0) including the following: (1) history taking, neurological examination, and blood pressure measurement to exclude orthostatic hypotension ; (2) quaAt the end of the actigraphic recording patients received rotigotine 24 h transdermal patch, which was titrated over 1–4 weeks to the optimal dose to subjectively improve motor symptoms, starting at 2 mg/24 h with weekly increments of 2 mg/24 h up to a maximum of 8 mg/24 h. The final dose was maintained for 4 weeks. The occurrence of rotigotine related adverse events and the emergence of impulse control disorders were monitored with weekly phone calls and additional visits when required.When adverse events occurred during the titration period, rotigotine dose was back-titrated to the previous tolerated dose for the maintenance period. At the end of the maintenance period patients underwent a new visit (T1) including the same assessment performed at T0.Rotigotine's effect on sleep disturbances was evaluated by comparing actigraphic data, sleep diary, and questionnaire results obtained at T1 and T0. Mean values of 7 days were calculated for each parameter for statistical analysis.Questionnaires and scales on motor performance, QoL, depression, and global cognitive profile were also compared between T1 and T0.p ≤ 0.05.As deviation from a normal distribution was found data were reported as medians and 25th–75th percentiles and comparisons performed by the Wilcoxon signed-rank test. Statistical significance was set at http://dx.doi.org/10.1155/2016/3724148).All the 15 enrolled PD patients  completed the study. At T0, 1 patient was free from PD medication, 1 was treated with selegiline, and 13 were treated with LD (mean daily dose ± SD = 333 ± 162 mg), in combination with rasagiline in 4 patients  and 4 mg/24 h (patient 15) as they experienced adverse events at higher dose .At T1 compared to T0 significant improvements of motor performance (UPDRS-III) and self-reported sleep complaints (PDSS-2) were observed . Single Four patients  reported RLS at T0 and a significant decrease in RLS severity while on rotigotine treatment . Ten patients had a positive questionnaire for RBD. Four out of the 6 patients reporting at least one RBD episode/week at T0 referred a RBD frequency decrease at T1.ESS score was globally unchanged after rotigotine treatment .Actigraphic and sleep diary data are reported in Tables Nights of good sleep per week were significantly increased and number and duration of nocturnal awakenings significantly decreased under rotigotine according to patients' diaries.During the diurnal period actigraphic recording showed a significant reduction in number and duration of sleep episodes at T1, while only reduction in the number of sleep episodes emerged from patients' diaries.Finally MMSE score, H&Y Score, and BDI score were not significantly different at T1 while a significant improvement of QoL was observed .In this open-label pilot study we demonstrated, by means of actigraphic recordings, that the introduction of rotigotine was objectively associated with a significant reduction in motor activity and mean duration of wake episodes during nocturnal sleep in PD patients with self-reported sleep complaints. In patients with pathological SE at baseline (≤85%) , 22, rotIn agreement with previous studies we also confirmed that rotigotine improves perceived sleep quality in PD patients –6 and isAccording to our actigraphic and patients' reported results, rotigotine's effect on sleep disturbances may depend on several factors: (1) reduction in motor activity during sleep, which could be related to the need to move limbs due to restlessness or to painful posturing, which may cause awakening or arousal from sleep; (2) improvement of a coexisting sleep disorder like RLS; 3) improvement of nocturia and painful posturing as what emerged from PDSS-2. Rotigotine may also contribute to the reduction of the number of spontaneous or movement related arousal times during sleep , which c improvemRotigotine was well tolerated in this study. Sleep attacks or worsening of daytime sleepiness was not observed under treatment, during which on the contrary we found a decrease in number and duration of daytime sleep episodes. This result is clinically significant only for the duration of sleep episodes while their number was already low before treatment with rotigotine.The main limitation of the study is the lack of a placebo group which does not allow us to evaluate the presence and magnitude of a placebo effect. However our subjective data are in agreement with the previous randomized placebo-controlled study showing a subjective sleep improvement with rotigotine .We demonstrated for the first time by means of objective measurements a positive effect of rotigotine on nocturnal and diurnal sleep disturbances in PD patients with self-reported sleep complaints. Larger randomized placebo-controlled studies with actigraphic or polysomnographic recordings are warranted to confirm these data and define the effect of rotigotine on sleep fragmentation and arousal from sleep.Supplementary Table 1 shows the demographic and clinical features of the 15 PD patients enrolled in the study.Supplementary Table 2 compares single items' score of the PDSS-2 questionnaire during rotigotine treatment (T1) and baseline (T0). A significant improvement of 9 out of 15 items was observed during rotigotine.


Evidence from human psychophysical and animal electrophysiological studies suggests that sensitivity to interaural time delay (ITD) in the modulating envelope of a high-frequency carrier can be enhanced using half-wave rectified stimuli. Recent evidence has shown potential benefits of equivalent electrical stimuli to deaf individuals with bilateral cochlear implants (CIs). In the current study we assessed the effects of envelope shape on ITD sensitivity in the primary auditory cortex of normal-hearing ferrets, and profoundly-deaf animals with bilateral CIs. In normal-hearing animals, cortical sensitivity to ITDs (±1 ms in 0.1-ms steps) was assessed in response to dichotically-presented i) sinusoidal amplitude-modulated (SAM) and ii) half-wave rectified (HWR) tones  presented at the best-frequency of the unit over a range of modulation frequencies. In separate experiments, adult ferrets were deafened with neomycin administration and bilaterally-implanted with intra-cochlear electrode arrays. Electrically-evoked auditory brainstem responses (EABRs) were recorded in response to bipolar electrical stimulation of the apical pair of electrodes with singe biphasic current pulses (40 µs per phase) over a range of current levels to measure hearing thresholds. Subsequently, we recorded cortical sensitivity to ITDs (±800 µs in 80-µs steps) within the envelope of SAM and HWR biphasic-pulse trains  over a range of modulation frequencies. In normal-hearing animals, nearly a third of cortical neurons were sensitive to envelope-ITDs in response to SAM tones. In deaf animals with bilateral CI, the proportion of ITD-sensitive cortical neurons was approximately a fifth in response to SAM pulse trains. In normal-hearing and deaf animals with bilateral CI the proportion of ITD sensitive units and neural sensitivity to ITDs increased in response to HWR, compared with SAM stimuli. Consequently, novel stimulation strategies based on envelope enhancement may prove beneficial to individuals with bilateral cochlear implants. A delay in the time of arrival of a sound between the ears, termed interaural time delay (ITD), contributes to our ability to localize sounds, detect speech in background noise and to segregate multiple sound sources This is of potential clinical interest to cochlear implant (CI) recipients, since this may provide a method to enhance binaural sensitivity. Until recently, CIs have been inserted in one ear only. Since recipients of unilateral CIs have particular difficultly localizing sounds and detecting speech in background noise, CIs in both ears have been trialled worldwide and, in some countries, have become the standard of care for children with severe to profound hearing loss. However, results suggest that ITD sensitivity is generally poor among CI recipients that may significantly limit potential benefits of bilateral CIs A recent study by Laback and colleagues Mustela putorius) were used in this study: 7 normal-hearing animals and 5 deaf animals with bilateral cochlear implants. This study was carried out in strict accordance with local ethics guidelines , the UK Home Office Animals (Scientific Procedures) Act 1986 and under a personal Home Office license.Twelve adult pigmented ferrets . On induction, an intramuscular injection of atropine sulphate  was given to minimize airway secretions. A 24-gauge cannula was inserted into a peripheral vein and anesthesia was maintained with a continuous infusion of a mixture of medetomidine hydrochloride (0.02 mg/kg/h) and ketamine  in 5% glucose/saline solution. The infusion was supplemented with 0.5 mg/kg/h dexamethasone  and 0.06 mg/kg/h atropine sulfate to minimize cerebral edema and bronchial secretions, respectively. The animal was intubated with an endotracheal tube through which the animal was ventilated. Core body temperature, end-tidal COFor normal-hearing animals (n = 7), acoustic stimuli were generated using Tucker-Davis Technologies  System III hardware and TDT Brainware and Real Time Processor Visual Design Studio (RPvdsEx) software (50-kHz sampling rate). Subsequently, they were presented binaurally over earphones  coupled to otoscope specula that were inserted into both ear canals. The transfer function of the earphones was canceled from the stimulus using an inverse filter to ensure the frequency response of the drivers was flat from 0.5 to 25 kHz (±2-dB SPL). Closed-field calibration was performed using a 1/8 inch condenser microphone .2 rise/fall time) presented to both ears was used as a search stimulus in order to establish whether a unit was acoustically responsive. Subsequently, the frequency tuning of each acoustically-responsive unit was characterized with tones  presented to both ears over a range of frequencies (0.4 to 22 kHz in 12 logarithmic steps) and levels (60- to 80-dB SPL in 10-dB steps). The best frequency (BF) of a unit was defined as the tone frequency that elicited the largest number of responses within a 500-ms recording window after the onset of the stimulus. Envelope ITD sensitivity was assessed in all driven units using stimuli that are described in detail below.Broadband noise , there were 84 stimulus parameter combinations presented 15 times each in a pseudo-random order. Stimuli were presented at a rate of one every second. The basic protocol required approximately 40 minutes. For most units the protocol could be completed in its entirety without losing any responsiveness.Our methods for deafening and cochlear implantation have been described in more detail elsewhere After deafening, a custom-made intra-cochlear electrode array was inserted through the round window of each ear to an approximate depth of 7.5 mm into the scala tympani. The electrode array, fabricated using injection molding techniques, consisted of three platinum ring electrodes at its tip (∼0.43 mm in diameter with an inter-electrode separation of ∼0.5 mm). All three electrodes were implanted inside the cochlea, followed by two markers just proximal to the last electrode spaced by 0.2 mm. Using these markers to judge the depth of implantation ensured place-matched intracochlear electrodes for each bilateral CI procedure.st-order finite impulse response bandpass filter (0.3–3 kHz pass band). Electrical stimuli were generated using Tucker-Davis Technologies  System III hardware and Real Time Processor Visual Design Studio (RPvdsEx) software (25-kHz sampling rate). Specifically, biphasic current pulses  with alternating polarity were delivered to the intra-cochlear electrode array in a wide-bipolar configuration . Pulses were presented over a range of levels  in a pseudorandom order and repeated five hundred times. The stimulus artifact alternates in polarity with the stimulus, whereas the neural response does not. Thus, the artifact was largely averaged out from the summed neural response Hearing thresholds can vary depending upon the position of the intra-cochlear electrode array In deaf cats, envelope ITD sensitivity of inferior colliculus (IC) neurons has been demonstrated using SAM pulse trains with a carrier frequency of 5000 pulses per second Extracellular single- and small multi-unit cluster activity was recorded within a sound-attenuated chamber  using single-shank silicon probes with 16 recording sites . All recordings were band-pass filtered (0.5 to 5 kHz), amplified and digitized at 25 kHz. Stimulus generation and data acquisition were synchronized using Brainware software (Tucker-Davis Technologies). Data were acquired in 1 second sweeps triggered by the onset of the stimulus. Any recorded event with a magnitude of 2.5 times the mean of the recorded amplitude of the raw waveforms was considered to be a potential spike. The latencies and shapes of all potential spikes were stored for offline analysis. For each animal, we recorded activity from approximately 10–40 evenly-spaced recording sites within the primary auditory cortex. Auditory cortex was targeted using sulcal landmarks according to the criteria of Bizley and colleagues In animals with CIs, cortical electrophysiological recordings were complicated by artifacts derived from bilateral intra-cochlear electrical stimulation . Pilot sSpike sorting algorithms were based on those used in previous studies within our laboratory Initially for each recording, a dot raster and peri-stimulus time histogram (PSTH) with a 1-ms bin width was constructed by counting the number of spikes in response to each trial . Similarth order fitted the data significantly better than a null model that assumed no effect of the stimulus. A likelihood ratio test was performed to determine whether the Poisson regression model fitted the data significantly better than the null model at P<0.05. A generalized linear model  was used to express the observed spike count (y) as a function of stimulus parameter (x) in the form0, …, b4b are free parameters. These polynomials can approximate the non-linearity associated with physiological recordings such as saturation, non-monotonicity and thresholding. Furthermore, they make few assumptions about the shape of the ITD response function to capture the diversity of observed binaural responses in cortical neurons In units with a significant onset and/or offset response to both modulation conditions (SAM and HWR), ITD sensitivity was assessed using methods previously described in detail in an earlier publication from our laboratory 0b, independent of the stimulus. Subsequently, a likelihood ratio test was used to determine whether the Poisson regression model fitted the data significantly better than the null model at P<0.05, by computing the deviance for the two models. The deviance is equivalent to minus twice the difference between the log-likelihoods of the fits of the two models to the data and is approximately a 2χ-distribution with degrees of freedom equal to the order of the regression model.This null model assumes that the spike count (y) randomly varies around a spontaneous rate of In the same units, an index of ITD sensitivity (d’) was also calculated across all recording sweeps using the formula:r max and r min represent the maximum and minimum firing rate averaged across all repeat presentations of the same stimulus, respectively, and ii) σ min and σ max represent the corresponding standard deviations where i) These results are based on 592 single and small multi-unit clusters recorded from the left auditory cortex of 12 adult ferrets. Specifically, 448 units were recorded from 7 normal-hearing ferrets and 144 units were recorded from 5 profoundly-deaf ferrets with bilateral CIs.Initially, for each recording a dot raster and PSTH with binwidth of 1 ms was constructed by counting the number of spikes in response to each trial . During To compare the time-course of on and off responses , mean reConsistent with previous results from our laboratory th order fitted the data significantly better than the null model at P<0.05. Of the units that had an on response (416 units) more units were ITD sensitive, as defined by the significance level test, in response to HWR (160 units = 38%), compared with SAM (122 units = 29%) stimuli  and response period  and ii) a significant interaction between stimulus condition and response period .Consistent with previous data from our laboratory Initially, a dot raster and PSTH were constructed for each recording . Subsequth order. Of the units that had a significant off response (112 units), more units were ITD sensitive in response to HWR (26 units = 23.2%), compared with SAM pulse trains (22 units = 19.6%).ITD response functions to SAM and HWR pulse-trains were generated from mean spike rates averaged across the offset recording window . ITD senThis paper demonstrates for the first time that envelope enhancement can increase neural sensitivity to ITDs in ferrets with bilateral CIs. Without envelope enhancement i) fewer cortical neurons were sensitive to ITDs and ii) those that did respond demonstrated weaker binaural interactions. Consistent with recordings from guinea-pig IC Likewise, animal electrophysiology and human psychophysics should be compared with caution due to species differences, anesthesia and questions concerning the contribution of single cortical neurons to behavioral responses Envelope ITD sensitivity of IC neurons has been previously demonstrated in deaf cats Although the present study is the first to investigate the effects of envelope shape on neural sensitivity to interaural envelope delays with bilateral CIs, neural sensitivity to ITDs in the envelope and fine structure of an acoustic signal has previously been demonstrated at various levels of the auditory pathway, including the medial superior olive (MSO) Whilst it has been shown that cortical response properties differ between awake and anaesthetized preparations Sensitivity to ITDs is generally worse for high-, compared with low-frequency stimuli cf) and decrease at frequencies to either side of that maximum mf) of the SAM or HWR stimulus, compared with SAM tones, HWR tones have additional side-band components. Specifically, SAM tones are characterized by three frequency components: the carrier frequency (cf) and two spectral side-bands (cf+mf and cf−mf). HWR tones have additional side bands spaced at multiples of twice the mf. In psychophysical studies it is necessary to limit the spectra of HWR tones to prevent the use of additional frequency components that would fall outside the effective filter width of auditory nerve fibers The acoustic spectra of SAM and HWR tones have most energy at the tonal carrier frequency (The data from auditory cortex of ferrets presented here is consistent with studies that showed enhanced ITD sensitivity using trapezoid carriers with longer offset times in humans with bilateral CIs and normal-hearing listeners PSTHs associated with HWR and SAM stimuli were broadly similar  and 14. Laback and colleagues Despite limitations in the temporal coding of neurons to high frequency sounds, the results presented here suggest sensitivity of cortical neurons to ITDs increases with envelope enhancement. These findings are relevant to individuals with bilateral cochlear implants because most current commercially-available devices partially restore ILDs and envelope ITDs alone. Envelope enhancement could be used in a CI processing strategy to improve binaural perception. Laback and colleagues


Aberrant activation of DNA repair is frequently associated with tumor progression and response to therapy in hepatocellular carcinoma (HCC). Bioinformatics analyses of HCC data in the Cancer Genome Atlas (TCGA) were performed to define DNA repair based molecular classification that could predict the prognosis of patients with HCC. Furthermore, we tested its predictive performance in 120 independent cases. Four molecular subgroups were identified on the basis of coordinate DNA repair cluster (CDRC) comprising 15 genes in TCGA dataset. Increasing expression of CDRC genes were significantly associated with TP53 mutation. High CDRC was significantly correlated with advanced tumor grades, advanced pathological stage and increased vascular invasion rate. Multivariate Cox regression analysis indicated that the molecular subgrouping was an independent prognostic parameter for both overall survival : 2.989) and tumor-free survival  in TCGA dataset. Similar results were also obtained by analyzing the independent cohort. These data suggest that distinct dysregulation of DNA repair constituents based molecular classes in HCC would be useful for predicting prognosis and designing clinical trials for targeted therapy. Hepatocellular carcinoma (HCC) is the leading cause of cancer death worldwid with dismal prognosisThe application of microarray or high-throughput technologies which rely on thousands of pieces of bio-information and provide an accurate landscape of HCC genetic alterations have enabled researchers to measure the expression of a large number of genes in HCC for identifying tumor subtypes78911DNA repair process is constantly active as it responds to damage in the DNA structure and proceed by several mechanisms, including base excision repair, mismatch excision repair, nucleotide excision repair, homologous recombinationA thorough understanding of the DNA repair genes expression pattern in tumors would be of utmost importance in HCC prognostic prediction and improving therapy and in achieving the best therapeutic responseBased on previous studies on molecular networks incorporating DNA repair genes linked with hepatocarcinogenesis or progression in HCC, 59 initial genes were included in the preliminary analyses1318Unsupervised two-way hierarchical clustering analysis was performed to assess the extent to which gene expression was coordinate or independent across the patient cohort. A 15-gene subset was identified by the visual analysis of the clustering, expression of which was coordinately regulated and formed a clear gene grouping . The valEach tumor accumulates numerous damaging mutations, therefore, three major groups of associated alterations, including the CTNNB1 cluster , the AXIN1 cluster  and the tumour protein p53 (TP53) cluster  were identified by exome sequencing of HCCTo determine whether any other molecular characteristics were associated with CDRC expression, we analyzed the correlation of the methylation and copy number status of the CDRC with the gene expression. Interestingly, most CDRC genes had a significant negative correlation between methylation beta-values and mRNA expression Z score . FurtherAge distribution was closely associated with four DNA repair based molecular subgroups of HCC described herein. Patients in group 2 had lower mean age compared to other CDRC clusters (p < 0.05). The gender distribution of all subtypes was presented in Survival analyses for all patients with available outcome data (n = 239) showed remarkable differences in overall survival (OS) and tumor-free survival (TFS) between the molecular subgroups. Patients in group 3 showed a favorable outcome, with 5-year OS rates of ~45%  and PFS 21Examining other clinical variables for subgroup-specific prognostic value showed that OS and TFS rate in HCC patients between group 2 and group 3 were independent of gender, age, and HCC history risk factors  and S7. According to the results above, a multivariate Cox regression analysis was performed to assess whether CDRC molecular subgrouping was independent prognostic parameters for HCC in group 2 and group 3. Results showed that in group 2 and group 3, CDRC molecular subgrouping was the independent prognostic parameter for both OS (hazard ratio (HR): 2.989, 95% confidence interval (CI) 1.431–6.243, P = 0.004) and TFS  . PatholoMSH2 protein expression was evaluated by immunohistochemical staining. According to the MSH2 expression pattern by immunohistochemical staining, HCC tissues was divided into low (n = 41), moderate (n = 48) and high (n = 31) expression groups . The chaMany genetic and epigenetic disruptions in HCC indicate that it is characterized by remarkable molecular heterogeneity252627The relationship between CDRC expression pattern and clinicopathologic parameters was evaluated. The results indicated that increased DNA repair capacity was correlated with poor tumor differentiation and poor survival293133DNA repair genes affect cell proliferation or survival indirectly by influencing the ability of the organism to repair nonlethal damage in other genes, including protooncogenes, tumor suppressor genes, and genes that regulate apoptosis38In addition, we investigated the mutational status of three major groups  associated with CDRC expression in HCC. TP53 mutant tumors which had a high mutational burden were associated with high CDRC expression, with only 6.7% of cancers in the group exhibiting very low expression (group 3) being TP53 mutant. Genomic instability could be enhanced by TP53 mutant, which might lead to an increase DNA repair capacity. Furthermore, convincing evidence show that TP53 mutant proteins play an important gain-of-function role in promoting invasion and metastasis of tumors. Recent studies have indicated that genome-wide and cell type-specific alterations in miRNA expression during DNA damage response could be regulated by TP534221Molecular characterization of HCC based on DNA repair gene expression would improve the prediction of the clinical outcome of HCC patients and selection of treatments for specific molecular subtypes of HCC. Nevertheless, our findings needed to be validated and further refined in a large prospective patient cohort. Furthermore, the efficacy of adjuvant therapies for HCC, such as chemotherapy, and/or molecular targeted therapies could be assessed in the context of specific molecular subgroups, as the response to different therapy modalities would likely differ.Human DNA repair genes or genes in DNA repair pathways were extracted as initial genes1318http://www.cbioportal.org/)http://www.cbioportal.org/). GISTIC2-generated copy-number estimates  were downloaded from the UCSC Cancer Browser (https://genome-cancer.ucsc.edu). Methylation data was generated using Illumina Infinium Human DNA Methylation 450 platform and downloaded as beta-values from the cBioPortal https://tcga-data.nci.nih.gov/tcga/) and tabulated with genetic data.HCC TCGA dataset containing Normalized Agilent microarray and RNAseq z-score data, copy-number status generated by GISTIC, log2 copy-number value, and methylation (HM450) beta-values with the most anti-correlated with expression for each initial gene, and mutation data in three major groups: the CTNNB1 cluster , the AXIN1 cluster  and the TP53 cluster  identified by exome sequencing of HCC were extractedA prospective cohort of 120 patients with HCC was recruited from the First Affiliated Hospital of Wenzhou Medical University. These patients were diagnosed with HCC between January, 2013 and December, 2014. Patients were excluded if they had a diagnosis of a concurrent cancer or cancers metastatic to the liver. Clinical information of each patient including details of pathology and outcomes with a regularly follow up was collected. The histology and clinical stages were classified according to the seventh edition of the American Joint Committee on Cancer (AJCC) staging system. The tumor grade 1–4 of HCC in pathology diagnosis is equivalent to well-differentiated, moderately-differentiated, poorly differentiated, or undifferentiated, respectively, under microscope . Grade 1Immunohistochemical staining of MSH2 was performed on the 120 HCC sections. The sections were blocked in 3% hydrogen peroxide solution for 10 min at room temperature and then incubated with the MSH2 primary antibody  overnight at 4 °C. A negative control was performed by replacing the primary antibody with PBS. The sections were then incubated with a horseradish peroxidase labeled secondary antibody  at room temperature for 120 min. Finally, the signal was developed for visualization with diaminobenzidine and the sections were counterstained with hematoxylin. MSH2 expression score was conducted according to the percent of positive cells: 0–5% scored 0; 6–35% scored 1; 36–70% scored 2; more than 70% scored 3 and staining intensity: no staining scored 0, weakly staining scored 1, moderately staining scored 2 and strongly staining scored 3. The final score was determined using the ratio of positive cell score × staining intensity score as follows: “−” for a score of 0–1, “+” for a score of 2–3, “++” for a score of 4–6 and “+++” for a score of >6. Low expression was defined as a total score <4, moderate expression with a total score ≥ 4 and ≤6, and high expression with a total score > 6.−∆∆Ct method and normalized to β-actin.Total RNA was extracted from a section of fresh frozen tumor tissue using the RNeasy Mini Kit . Reverse transcription and PCR were executed using the RevertAid First Strand cDNA Synthesis kit  and Power SYBR Green PCR kit  according to the manufacturer’s instructions. Quantitative real-time PCR was performed using a 7500 Real-time PCR system . Primer sequences for MSH2 detection were as follows, forward: 5′-AGAGACAGGTTGGAGTTGG-3′; reverse: 5′-CGGGTAAAACACATTCCTT-3′. The relative expression level of MSH2 was determined using the 2Total protein was extracted using a RIPA lysis buffer . The proteins were then separated by SDS-PAGE and transferred onto polyvinylidene fluoride membrane. The membrane was blocked with 5% nonfat dried milk and incubated with MSH2 primary antibody  and goat anti-rabbit secondary antibody . Proteins were detected by using enhanced chemiluminescence method and imaged by GelDoc™ XR+ system . GAPDH  was chosen as an internal control and the expression level of MSH2 protein was normalized by GAPDH.2) values using R software (v3.1.2). Multivariate linear regression models were used to analyze associations between genes expression with the mutations in three major groups: the CTNNB1 cluster , the AXIN1 cluster  and the TP53 cluster . An independent two-sample t-test was used for comparison of continuous variables for normally distributed data, and Mann–Whitney U tests for non-normally distributed data. The χ2-test was used for comparison of categorical variables, if any groups contained less than 5, the Fisher’s Exact test was used in preference. Survival curves were evaluated using the Kaplan-Meier method, and differences between survival curves were tested by the log-rank test in the different groups. Cox proportional hazards regression model was used to examine univariate and multivariate hazard ratios for the prognostic parameters, including molecular groups and clinicopathological variables that were dichotomized. Only significantly different variables in univariate analysis were entered into the next multivariate analysis. Furthermore, the performance of DNA repair genes clusters for identifying the HCC subgroups was validated in our own cohort. The deceased patients were excluded in the survival analysis due to overall survival more than 5 years; patients who died or were lost to follow-up within 30 days were also excluded. The analyses were carried out using the SPSS Statistics 21.0  and MedCalc version 14.8 . A 2-tailed P value < 0.05 was considered to be statistically significant.Unsupervised two-way hierarchical clustering of HCC samples and initial genes expression data was performed with Multiexperiment Viewer (MeV) 4.9.0 , designed to allow the analysis of microarray data to identify patterns of gene expression and differentially expressed genes. Heatmap view of a diagram will come up that looks like lots of tiny blue and yellow boxes. Each row represents a specific gene, whereas each column represents each patient. Pearson correlation was used as the distance metric for hierarchical clustering. The number of patient and gene clusters can be adjusted by varying sample-tree or gene-tree distance-thresholds and visual analysis. The lower the distance range, the more clusters there will be since a shorter distance between similar genes means more groups. Gene cluster which could identify similar patients would be established. Normality of distributions was confirmed with the Anderson–Darling test. The correlations in gene expression within the main clusters and correlations of genes expression with their copy-number or methylation status were investigated by Pearson Coefficient of determination .