When applying genome-wide sequencing technologies to disease investigation, it is increasingly important to resolve sequence variation in regions of the genome that may have homologous sequences. The human mitochondrial genome challenges interpretation given the potential for heteroplasmy, somatic variation, and homologous nuclear mitochondrial sequences (numts). Identical twins share the same mitochondrial DNA (mtDNA) from early life, but whether the mitochondrial sequence remains similar is unclear. We compared an adult monozygotic twin pair using high-throughput sequencing and evaluated variants with primer extension and mitochondrial preenrichment. Thirty-seven variants were shared between the twin individuals, and the variants were verified on the original genomic DNA. These studies support highly identical genetic sequence in this case. Certain low-level variant calls were of high quality and homology to the mtDNA, and they were further evaluated. When we assessed calls in preenriched mtDNA templates, we found that these may represent numts, which can be differentiated from mtDNA variation. We conclude that twin identity extends to mtDNA, and it is critical to differentiate between numts and mtDNA in genome sequencing, particularly as significant heteroplasmy could influence genome interpretation. Further studies on mtDNA and numts will aid in understanding how variation occurs and persists. Mitochondria play important roles in cellular function and human disease . The twins grew up together and lived in a similar environment.Zygosity of the twins was determined by genotyping highly polymorphic DNA loci using the PowerPlex® short tandem repeat kit . Amplified fragments were detected using the ABI  3730xl DNA Analyzer. Data were analyzed with GeneMapper software (Life Technologies).DNA libraries were generated using Illumina Paired-End  library preparation kit according to the manufacturer's instructions. Libraries were quantified by qPCR using a KAPA  library quantification kit and assessed on an Agilent Technologies  2100 Bioanalyzer using a High Sensitivity DNA chip. Following an automatic cluster generation, paired-end sequencing was performed on Illumina HiSeq 2000 using the V1 flow cell Hiseq system version (Illumina Inc.). The libraries were subject to a second sequencing run using the V3 flow cell HiSeq system version.http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/) executed in an internal Galaxy server  numbers, while some variants were only found in Mitomap mtDNA Sequence Polymorphism database (http://www.mitomap.org/MITOMAP), and others were previously not reported.To check read quality, raw sequencing data were analyzed using the next generation tool Fastqc (Variants detected at low frequency (<0.01%) were examined using the fluorescent primer extension assay SnapShot . We designed custom multiplex reactions according to the manufacturer's instruction and pooled up to five templates per reaction for scalability. As a first step we designed template primers and one extension primer for each variant and performed primer extension on 33 candidates. In order to verify if the detected variant nucleotides were from the mitochondrial or the nuclear genome, we amplified the entire mtDNA in two large fragments of more than 8 kb with an overlap of 183 bp , the presence of the variant in at least one sequencing read, and variant surrounded by nonvariant sequence .We noted the presence of potential single-base sequence differences present at very low level, one to three reads per locus, excluding duplicate reads. As these differences could represent a number of possibilities including sequencing errors, read mapping ambiguity due to numts, or true mitochondrial variation, we investigated these further. In analyzing potential low-level variants in the mitochondrial genome, we used stringent filters: a strict nucleotide quality score ; the presence of more low-level potential variants was correlated with higher coverage. Importantly, we performed additional validation on the original genomic DNA to rule out error introduced by library preparation and complementary studies to evaluate their origin. We focused on 76 low-level variants that were shared between twins Fig. . We desiUsing the original genomic DNA, we found the major reference allele in 33/33 positions, which confirms the results from the high-throughput sequencing, and the low-level variant was seen in 9/33 (27.3%) Table . An examGiven these findings, for the seven variants confirmed by primer extension, we considered the possibility that nuclear mtDNA fragments (numts) could be generating apparent mitochondrial sequence variants. To test this possibility, we generated hemigenomes of mtDNA using high-fidelity long PCR and used these as templates for the primer extension assays, as this would eliminate numts , we directly compared variant-containing reads to the genome . We founTo detect numts that could be mapping to the mitochondrial genome and producing low-level apparent variants, we performed read mapping using whole genome reads from the twins to the mtDNA only as reference. Depth of coverage increased up to 600 reads higher than the initial mapping using the whole hg19 genomic reference, and almost all of these increased the only allele or major allele call. We examined the low-level variants previously analyzed using the initial mapping data and the primer extension assay in table These findings support mitochondrial similarity in twins and demonstrate the importance of distinguishing mitochondrial genome from homologous nuclear DNA using a combination of methods.In this study, we analyzed mitochondrial sequence variants in a pair of adult twins using high-throughput sequencing and validation. These data also allowed us to ask whether these two independent individuals harbored mitochondrial somatic variation in their blood. Our results revealed identical homoplasmic variants in mtDNA sequence from blood-derived genomic DNA, which lend support to the idea that monozygotic twins may be highly similar in their mitochondrial genomic sequence, as they seem to be in nuclear genomic sequence  these adult twins had highly similar mtDNA sequence from blood. (2) We did not find differential somatic heteroplasmy >1% to suggest accumulating mutation over time, although more twin pairs and tissues should be tested in the future. (3) Low-level variants, only some of which are numts, are detected by high-throughput sequencing and can be confirmed by primer extension.


Ecosystem service assessments have increasingly been used to support environmental management policies, mainly based on biophysical and economic indicators. However, few studies have coped with the social-cultural dimension of ecosystem services, despite being considered a research priority. We examined how ecosystem service bundles and trade-offs emerge from diverging social preferences toward ecosystem services delivered by various types of ecosystems in Spain. We conducted 3,379 direct face-to-face questionnaires in eight different case study sites from 2007 to 2011. Overall, 90.5% of the sampled population recognized the ecosystem’s capacity to deliver services. Formal studies, environmental behavior, and gender variables influenced the probability of people recognizing the ecosystem’s capacity to provide services. The ecosystem services most frequently perceived by people were regulating services; of those, air purification held the greatest importance. However, statistical analysis showed that socio-cultural factors and the conservation management strategy of ecosystems  have an effect on social preferences toward ecosystem services. Ecosystem service trade-offs and bundles were identified by analyzing social preferences through multivariate analysis . We found a clear trade-off among provisioning services  versus regulating services and almost all cultural services. We identified three ecosystem service bundles associated with the conservation management strategy and the rural-urban gradient. We conclude that socio-cultural preferences toward ecosystem services can serve as a tool to identify relevant services for people, the factors underlying these social preferences, and emerging ecosystem service bundles and trade-offs. The ecosystem services concept has been increasingly used by academics, researchers and policy-makers Because ecosystem service assessments are determined by analyzing the effect of ecosystems and biodiversity on human well-being, it is necessary to understand the ways society benefits from nature and, hence, the many reasons that societies value ecosystem services Based on socio-cultural preferences, the concept of ecosystem service bundles emerge as a useful tool for identifying ecosystem service synergies and trade-offs i) the probability that people recognize the capacity of ecosystems to deliver services to society and the factors influencing such recognition; (ii) the relative importance given by people to different categories of ecosystem services  and their underlying factors; (iii) the factors that affect the relative importance stakeholders give to particular ecosystem service and the potential trade-offs emerging; and (iv) the ecosystem service bundles that can emerge from diverging socio-cultural preferences.In this study, we analyzed socio-cultural preferences toward ecosystem services delivered by different types of Spanish ecosystems and how they can promote ecosystem service trade-offs and bundles. Here, the term “socio-cultural preferences” incorporates individual perceptions, knowledge, and associated values i) a representative sample of ecosystem diversity and (ii) different environmental management strategies . Thus, both biophysical and socio-cultural variability were considered to select case sites because they determine a different supply and demand of ecosystem services The research was conducted at eight sites in the Iberian Peninsula  to captuData sampling was conducted in each of the eight case sites by following a consistent survey design. The population sampled was randomly selected to cover a wide range of ecosystem service beneficiaries, such as local inhabitants, visitors, or environmental technical experts. The sampling population was restricted to individuals over 18 years old. A total of 3379 direct face-to-face questionnaires were conducted between 2007 and 2011. To test the suitability of the questionnaire design, we conducted a preliminary sampling in each case study. More details about the sampling characteristics are provided in i) the respondents’ previous knowledge about the studied ecosystem’s capacity to provide services to society; (ii) the respondents’ perception of the importance of ecosystem services for the well-being of people in the area, after pollsters provided information about the services potentially delivered by ecosystems; (iii) the individuals’ environmental behavior ; and (iv) socio-demographic characteristics of the surveyed individuals . While conducting the surveys, the term ecosystem service was always referred to as “the benefits that the ecosystems of the area provide for human well-being” to make the term more understandable and to avoid educational biases. In addition, to facilitate interpretation, a list of ecosystem services with related pictures was presented to respondents in the second portion (ii) of the questionnaire The questionnaire was structured in four sections, regarding the following topics:  was used to select the best model among all the possible combinations of independent variables U-test to analyze whether the relative importance of different service categories differed with gender, rural versus urban population, elderly versus younger people, and between respondents showing environmental behavior versus those who did not.We used the chi-squared test to analyze the relative importance of different service categories for respondents’ well-being, comparing the number of ecosystem services identified by the respondents in each of the service categories . Then, we used the non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparison test to analyze whether the relative importance given to each service category by respondents was affected by social  and management factors . We also performed a non-parametric Mann-Whitney A redundancy analysis (RDA) was used to identify socio-cultural factors associated with the relative importance of particular ecosystem services by relating ecosystem services to socio-demographic and environmental behavior variables, as well as the management strategy and ecosystem type. A Monte Carlo permutation test (1000 permutations) was performed to determine the significance of independent variables in determining the relative importance of ecosystem services. The factors with the highest inertia were used to identify ecosystem service bundles using a hierarchical cluster analysis. We used Ward’s linkage method with Euclidean distances to identify relatedness among ecosystem service preferences N = 250), we obtained that 92.8% of the answers were correctly classified.Overall, 90.5% of the sampled population recognized that ecosystems can deliver services to society. According to the logit model, factors affecting the probability that respondents recognized the ecosystem’s capacity to deliver services were the level of formal education, environmental behavior, and gender . Our resχ2 = 79.8, d.f. = 7, p<0.0001). While the scores regarding the recognition of an ecosystem’s capacity to supply services were high for coastal systems, forests, and wetlands, these scores were significantly lower for various ecosystems, including rivers and streams, drylands, and urban systems : regulating services showed the highest saliency , followed by cultural services (33%), and provisioning services (23%). When respondents were asked to identify the relative importance of particular services, more than 40% identified air purification, the existence value of biodiversity, and nature tourism, as the most important services . Few respondents seemed to perceive the role of ecosystems as providers of forests products (13% of respondents) or hunting as a recreational activity (11%). We found significant differences in the social perception about the relative importance of different ecosystem service categories , age, and reported level of environmental behavior . While rU = 919 499.0, p<0.0001), recreational hunting and local ecological knowledge obtained higher value scores from inhabitants of rural areas .Regarding cultural services, we found two groups of services depending on the respondents’ place of residence. While services such as nature tourism, aesthetic values, environmental education, and the existence value of biodiversity were mostly perceived by urban inhabitants .People who visited protected areas regularly largely recognized regulating services, while members of environmental or social organizations largely recognized provisioning services . RecreatRespondents recognized all categories of ecosystem services when they were interviewed in Natural Parks  . Howeverp<0.0001, from 1000 permutations). The first three axes explained 87.8% of the total variance  revealed a trade-off between provisioning  and all other ecosystem services . RDA1 also revealed different stakeholder perceptions regarding ecosystem services, mainly explained by a rural-urban dichotomy . Rural pThe second axis of the RDA (21.6% of the variance) revealed a gradient of ecosystem services perception related to the level of ecosystem protection, with non-protected areas having positive scores and ‘Natural Parks’ having negative scores. Positive scores were associated with services provided by ecosystems in non-protected areas . Negative scores were associated with services provided by ecosystems in Natural Parks  .In addition, the RDA highlights that the respondents’ acknowledgement of particular ecosystem services was associated with specific ecosystem types . For exaUsing the first three axes of the RDA in the hierarchical cluster analysis, we identified three well-defined bundles of ecosystem services . Group IPrevious studies have found that human preferences toward ecosystem services focus first on provisioning services, followed by regulating services, and finally, on cultural services e.g., . In contHowever, social preferences over specific ecosystem services may vary among respondents due to a complex set of factors, including individual needs, cultural traditions, access to ecosystem services, and sources of household income The fact that rural people recognized a highly diversified flow of services  may be bThe rural-urban gradient identified in this study in relation to the social perception of ecosystem services is a consequence of different lifestyles and socio-economic characteristics. Variables such as age, formal education level, gender, and income clearly vary along the rural-urban gradient, having an impact on the rural-urban pattern of ecosystem service perceptions Additionally, we found that gender roles are significant in defining preferences toward ecosystem services. In contrast to previous studies on the topic, which have shown that females perceived fewer ecosystem services than males Our results suggest that different types of knowledge may be required to capture the entire range of services that ecosystems provide, i.e., experiential or local knowledge  and technical or experimental knowledge  The fact that local ecological knowledge is bundled with regulating services related to water and soil suggests that most traditional land management practices in Spain focus on managing these ecosystem components to tackle soil erosion, aridity, drought, and flooding It is broadly recognized that over the past century most landscape management strategies have favored the delivery of provisioning services at the expense of regulating and cultural services To a large extent, the institutional response to accelerate land use change that promotes provisioning services has led to a parallel increase in biodiversity conservation policies through the establishment of protected areas Rather than supporting the revitalization of traditional rural practices, institutional responses to land-use change have favored the demands of an urban conservation movement through the creation of strict protected areas, i.e., National Parks i) different stakeholders hold different values and perceptions toward ecosystem services; (ii) there is an important rural-urban gradient in preferences toward ecosystem services based on gender, different lifestyles, and different sources of knowledge regarding ecosystem services  local ecological knowledge is bundled with key regulating services related to ecosystem functioning  (iv) a gender-differentiated role exists regarding perceptions of ecosystem services (v) the perception of ecosystem services may vary as a consequence of land management strategy  (vi) trade-offs can be identified from socio-cultural preferences as people’s willingness to trade-off conservation of one ecosystem service against another vii) ecosystem service bundles can be identified from people’s systemic representations of interrelationships between ecosystem services  ; (iv) a services  and 4; (d areas) ; (vi) trservices .Although different studies have recognized that ecosystem service assessments should incorporate ecological, socio-cultural, and monetary values Figure S1Characteristics of study sites in relation to climate data in Spain.(PDF)Click here for additional data file.Table S1Main characteristics of sites included in this study.(PDF)Click here for additional data file.Table S2Summary statistics of socio-economic characteristics of polled people for each case site.(PDF)Click here for additional data file.Table S3Summary of the variables obtained from the questionnaire and used for the different analysis performed in the study.(PDF)Click here for additional data file.


Cystoid macular edema (CME) is a condition that involves the macula, causing painless vision loss. In this paper, we report a case of niacin-induced bilateral cystoid macular edema (CME) in a middle-age woman taking low dose of niacin (18 mg of nicotinic acid). Optical coherence tomography (OCT) showed retinal thickening and cystoid spaces in both eyes, whereas fluorescein angiography  revealed the absence of fluorescein leakage also in later phases. Four weeks after discontinuation of therapy there were a complete disappearance of macular edema at funduscopic examination and an improvement of visual acuity in both eyes. Furthermore OCT showed a normal retinal profile in both eyes. In our opinion considering the wide availability of niacin, medical monitoring and periodical examination should be considered during niacin administration. To our knowledge, this is the first report in the literature that described the very low-dose niacin-induced bilateral niacin maculopathy. Cystoid macular edema (CME) is a disorder which involves the central retina, caused by an accumulation of extracellular fluid in the macular region with secondary formation of multiple cystic spaces .The edema affects outer plexiform and inner nuclear layers of the retina and is generally related to permeability changes in blood-retinal barrier that limits the intraretinal movements of plasmatic components and maintains a right homeostasis.Various conditions can cause cystoid maculopathy such as retinal vascular diseases, intraocular inflammatory diseases, ocular surgery especially cataract extraction, hereditary retinal dystrophies, and topical or systemic assumption of drugs , 3. In this paper we report the case of a healthy woman that, after a daily assumption of 18 mg of nicotinic acid, has developed a cystoid macular edema in both eyes.A 53-year-old female (P.P.) was admitted to our Department of Ophthalmology, Sapienza University of Rome, Polo Pontino, for a sudden decrease of visual acuity in both eyes, blurred vision, and metamorphopsia.She had a medical history of hypertension and type 2 diabetes mellitus well controlled with oral hypoglycemic agents. In the past she had never presented with any vision trouble, previous history of ophthalmological diseases, or ocular surgery. Plasma cholesterol levels slightly higher than the normal range were incidentally found on a routine blood testing. Therefore her general practitioner prescribed her dietary changes, lifestyle modifications, and oral nutritional supplement containing 18 mg of niacin per day .One month before the start of therapy she had undergone a complete ophthalmologic examination: the best-corrected visual acuity (BCVA) was 0.0 logMAR in both eyes; results of slit-lamp examination were unremarkable; no signs of diabetic retinopathy or hypertensive retinopathy were reported on funduscopic examination.μm in the right eye and 578 μm in the left eye  and 0.52 logMAR in the left eye (LE); the anterior segment examination was normal, pupil reflexes were evoked, crystalline lenses was clear, and in situ intraocular pressure (IOP) measured by Goldman applanation tonometry was 16 mmHg in both eyes. Dilated funduscopic evaluation confirmed the absence of diabetic or hypertensive retinopathy but revealed the presence of bilateral macular edema. In order to confirm and quantify macular edema optical coherence tomography  was performed. OCT showed an alteration of retinal profile with increasing of retinal thickness and the presence of hyporeflective cystoid spaces in the outer plexiform and inner nuclear layers in both eyes. Central macular thickness (CMT) was 646 left eye . Fluorescein angiography  revealed the absence of fluorescein leakage also in later phases Figures .We advised her to discontinue the assumption of dietary supplement and to make another followup after four weeks. Four weeks after discontinuation of therapy, she underwent a complete ophthalmologic examination. She, as ordered, stopped taking the dietary supplement, and she confirmed to have monitored her blood glucose levels and not changed her diet or lifestyle. BCVA was 0.04 logMAR in both eyes; at funduscopic examination the macular reflex was evoked with complete disappearance of macular edema; optic nerve head and vessels by size and course were normal in both eyes.μm in the right eye and 194 μm in the left eye  was reduced to 195 left eye .Niacin , one component of the dietary supplement taken by the patient, is a vitamin preparation usually used for the treatment of lipid disorders. The mechanism of action affects the plasma levels of the apolipoproteins (apo) A and B. The decrease of Apo B-100 causes a reduction of very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and lipoprotein(a) [Lp(a)], whereas the decrease of hepatic clearance of Apo A-I raises the level of plasma high-density lipoprotein (HDL) . NicotinPharmacological doses of niacin can cause different adverse effects. The most frequent is the prostaglandin-mediated vasodilatation of the small blood vessels resulting in cutaneous flushing, warmth, and pruritus, usually involving the face, neck, and upper trunk. The duration of the reaction is variable from a few minutes to 2–4 hours, and with continuation of therapy the symptoms become less intense until to disappear. Dry skin and cutaneous hyperpigmentation (acanthosis nigricans) are also commonly reported . ClinicaFraunfelder et al. reported that 3 g or more per day of nicotinic acid could cause many ocular side effects such as blurred vision, eyelid edema, toxic amblyopia, proptosis, loss of eyelashes or eyebrow, superficial punctate keratitis, and cystoid macular edema, which represents the most serious ocular complication. All these adverse effects are reversible with discontinuation of niacin therapy , 9.In 1973, Donald M. Gass reported the case of three patients (mean age 50 years) with hypercholesterolemia that, after high doses of niacin (3–6 g/day), developed a sudden decrease of central vision due to cystoid macular edema in both eyes .Nicotinic acid maculopathy affects more frequently men, with a male : female ratio of 10 : 1 . A retroIn our case a very low dose of niacin was administered (18 mg of nicotinic acid) and niacin maculopathy appears 4 weeks later with a complete resolution after discontinuation of drug, confirming the relationship between drug administration and macular edema appearance. Funduscopic examination showed bilateral macular edema, but no signs of diabetic or hypertensive retinopathy excluding the presence of other factors that may contribute to the capillary dysfunction associated with edema onset. At funduscopic examination the foveomacular area has a peculiar aspect: the foveola takes on a bright yellow hue, similar to an exudate. Cysts primarily involve the foveal area and they are smaller than those seen in postsurgical or inflammatory cystoid macular edema . FA and OCT are useful to confirm the diagnosis. FA showed the absence of fluorescein leakage or vascular alteration, whereas OCT confirms the presence of cystic hyporeflective spaces in macular region, as reported in the literature. Indeed previous studies reported that the fluorangiographic pattern is atypical. The petaloid appearance caused by fluorescein dye leakage typical of CME is absent. Instead OCT shows the presence of intraretinal cystoid spaces, that appear more numerous and larger in the outer plexiform layer that is in the inner nuclear one –15. Whether nicotinic acid induced cystoid macular edema is still unknown. A first hypothesis suggests that, in patients with vascular or inflammatory diseases, niacin induces the release of prostaglandins, causing the blood-retinal barrier compromise with extracellular accumulation of fluid in many cystic spaces. This theory is not fully accepted for the absence of fluorescein leakage . A seconTo our knowledge this is the first report in the literature of CME caused by very low dose of niacin (18 mg). In our opinion niacin should be used with caution and under medical monitoring or periodic examination. In particular, patients with ocular symptoms, such as blurred vision, decrease of visual acuity, and metamorphopsia, should immediately go to an ophthalmologist and stop the assumption of nicotinic acid. Further studies would be desirable to investigate the safety dose, pathophysiologic mechanisms or predisposing risk factors, and the possible interaction with other agents that may cause niacin maculopathy.


Hepatic metastases of neuroendocrine tumors (NETs) are considered a major prognostic factor associated with significantly reduced survival compared to patients without liver metastases. Several surgical and nonsurgical strategies are present to treat resectable and nonresectable liver metastases, some of which have the potential to cure liver mestatases.The aims of the four systematic reviews presented in the paper are to determine the effectiveness of liver resection versus nonsurgical treatment of patients with NET liver metastases, to investigate the impact of neoadjuvant and adjuvant treatment options on the tumor-free survival, to assess the role of liver transplantation in patients presenting with unresectable bilateral hepatic metastases, and to evaluate the role of primary tumor resection in presence of unresectable liver metastases.Literature search was performed on Medical Literature Analysis and Retrieval System Online, Excerpta Medica Database, and the Cochrane Library . No language restrictions were applied. Randomized controlled trials, prospective and retrospective comparative cohort studies, and case-control studies will be used for the qualitative and quantitative synthesis of the systematic reviews. Case series will be only included in a separate database for descriptive purposes.This study is ongoing and presents a protocol system of four systematic reviews that will assist in determining the effectiveness of liver resection versus nonsurgical treatment of patients with NET liver metastases. This study is also assumed to investigate the impact of neoadjuvant and adjuvant treatment options on the tumor-free survival, the role of liver transplantation, and the relevance of primary tumor resection in presence of unresectable liver metastasis.The systematic reviews will show the current evidence based on the effectiveness of surgical strategies in patients with NET liver metastases and serve as basis for clinical practice guidelines.The systematic reviews have been prospectively registered with the International Prospective Register of Systematic Reviews: liver resection (CRD42012002652); http://www.crd.york.ac.uk/prospero/display_record.asp?ID=CRD42012002652 . neoadjuvant and adjuvant treatment strategies (CRD42012002656); http://www.crd.york.ac.uk/prospero/display_record.asp?ID=CRD42012002656 (Archived by WebCite at http://www.webcitation.org/6LQVvEHuf). liver transplantation (CRD42012002655); http://www.crd.york.ac.uk/prospero/display_record.asp?ID=CRD42012002655 . resection of the locoregional primary NET (CRD42012002654); http://www.crd.york.ac.uk/prospero/display_record.asp?ID=CRD42012002654 (Archived by WebCite at http://www.webcitation.org/6LQWEIuGe). Neuroendocrine tumors (NETs) developing from neuroendocrine cells can originate almost everywhere in the body . PrimaryAccording to their functional behavior, NETs can be subdivided into two categories: functioning NETs and nonfunctioning NETs . FunctioDespite the slow growing nature of NETs, Pape et al reported liver metastases of gastroenteropancreatic NETs in 84.7% of cases at the initial diagnosis . Due to A wide array of options is available to treat liver metastases from NETs, which improves the 5-year OS in 60-80% patients who have undergone curative surgery compared to less than 40% untreated patients -17. SurgDisease recurrence after surgical treatment of liver metastases is often observed, even when resection is performed with curative intent . To incrFor liver metastases arising from a non-NET primary tumor, the benefit of neoadjuvant and adjuvant strategies combined with liver resection has already been investigated in more detail ,29. NordControversy concerning liver metastases from NETs as an indication for liver transplantation arises inter alia from the relatively low number of such patients being transplanted. Moreover, heterogeneous 5-year OS data have been published with ranges between 33% and 96% ,30. TherAnother important question is whether the primary tumor should be removed in presence of nonresectable liver metastases as the answer may improve the outcome. Potential benefits of resection are seen in providing relief from hormonal and local tumor mass-related symptoms . Since eThe purpose of these four systematic reviews is to assess the role of surgical strategies in the management of liver metastases of nets, to evaluate the use of adjuvant and neoadjuvant therapies, to define selection criteria for patients who benefit the most from liver transplantation, and to study the influence of resection of the primary tumor.These four systematic reviews dealing with surgical treatment options for NET liver metastases attempt to answer the questions with regard to liver resection in patients with hepatic metastases see , neoadjuWe will report our review findings in accordance with the standards of the Preferred Reporting Items for Systematic reviews and Meta-Analyses . Our revThe systematic review inclusion and exclusion criteria are listed in In patients with resectable NET liver metastases, does liver resection with a curative intent (R0/R1) improve outcome  when compared to non-surgical treatment ?In patients with NET liver metastases, does R2 liver resection (debulking) improve outcome  when compared to non-surgical treatment ?In patients with NET liver metastases, do locally ablative techniques as an adjunct to R2 liver resection improve outcome ?In patients with NET liver metastases, does neoadjuvant treatment improve outcome  after liver resection compared to no neoadjuvant treatment?In patients with NET liver metastases, does adjuvant treatment improve the outcome  of liver resection as opposed to no adjuvant treatment?In patients with NET liver metastases, do both neoadjuvant and adjuvant treatment strategies improve the outcome  of liver resection compared to no neoadjuvant and adjuvant treatment?In patients with non-resectable NET liver metastases, does liver transplantation improve outcome  as opposed to R2 liver resection (debulking) or non-surgical treatment ?In patients with NET liver metastases, which selection criteria should be used for liver transplantation in order to improve outcome ?In patients with NET liver metastases and consideration for liver transplantation, does a delay (≥6 months) to assess tumor progression before transplanting improve the selection of patients  as opposed to early transplantation (<6 months)?In patients with NET liver metastases listed for liver transplantation, does downstaging  improve outcome ?In patients with non-resectable NET liver metastases, does living donor liver transplantation improve outcome  as opposed to deceased-donor transplantation or non-surgical treatment ?Does the outcome of the recipient justify the risk of the donor in the setting of liver transplantation for NET liver metastases?In patients with a pancreatic primary NET and non-resectable liver metastases, does resecting the primary tumor improve outcome  when compared to non-surgical treatment ?In patients with an intestinal primary NET and non-resectable liver metastases, does resecting the loco-regional primary tumor improve outcome  when compared to non-surgical treatment ?In patients with a lung primary NET and non-resectable liver metastases, does resecting the primary tumor improve outcome  when compared to non-surgical treatment ?Librarians of the Medical Library Careum, University of Zurich, Switzerland, developed the electronic search strategy to query databases and to identify all potentially relevant articles see . The folThe following parameters will be chosen for data extraction: first author’s name, publication year, answering scientific questions, study design, total number of patients, number of patients in the study group, number of patients in the comparison group, type of nonsurgical treatment, age , male-to-female ratio, progression-free survival, OS, quality of life (tools), and hazard risk ratio. The Grading of Recommendations Assessment, Development, and Evaluation will be used to grade the quality (level) of evidence and the strength of recommendations .2<50%). It is anticipated that there will be a limited scope for meta-analysis despite a relatively large number of studies due to different outcome measurements of the existing trials as such tumors are rare. However, results from studies using the same type of intervention and comparator with the same outcome measurements will be pooled using a random-effects meta-analysis. In addition, risk ratios for binary outcomes, 95% CI, and two-sided P values will be calculated for each outcome.A narrative synthesis of the findings from the included studies will be provided. A quantitative synthesis will be used for studies that are sufficiently homogenous from a clinical  and from a statistical perspective  and surgical procedure they underwent (pancreatic resection prior to liver transplantation or simultaneous resection). The 5-year OS was found to be significantly different between patients who were less than 55 years of age and had pancreatic resection prior to transplantation compared to patients who were 55 years of age or older and underwent simultaneous resection (5-year OS 61% vs 0%) . ReachinSurgical resection of NETs is the treatment strategy whenever a curative intent is anticipated. However, it is not clear whether resection of the primary NET is still beneficial in advanced disease stage presenting with unresectable liver metastases. Bettini et al investigated the role of primary tumor resection in nonfunctioning pancreatic NETs with unresectable liver metastases . OS did The four systematic reviews described in this protocol will help to elucidate the role of surgical strategies and serve as a basis for developing clinical practice guidelines.


Experimental detection of nitrogen fixation in microbes requires species-specific conditions, making it difficult to obtain a comprehensive census of this trait. The recent and rapid increase in the availability of microbial genome sequences affords novel opportunities to re-examine the occurrence and distribution of nitrogen fixation genes. The current practice for computational prediction of nitrogen fixation is to use the presence of the minimum set of six genes coding for structural and biosynthetic components, namely NifHDK and NifENB. Using this criterion, we conducted a comprehensive search in fully sequenced genomes and identified 149 diazotrophic species, including 82 known diazotrophs and 67 species not known to fix nitrogen. The taxonomic distribution of nitrogen fixation in Archaea was limited to the Euryarchaeota phylum; within the Bacteria domain we predict that nitrogen fixation occurs in 13 different phyla. Of these, seven phyla had not hitherto been known to contain species capable of nitrogen fixation. Our analyses also identified protein sequences that are similar to nitrogenase in organisms that do not meet the minimum-gene-set criteria. The existence of nitrogenase-like proteins lacking conserved co-factor ligands in both diazotrophs and non-diazotrophs suggests their potential for performing other, as yet unidentified, metabolic functions.Based on a careful comparison of the repertoire of nitrogen fixation genes in known diazotroph species we propose a new criterion for computational prediction of nitrogen fixation: the presence of a Our predictions expand the known phylogenetic diversity of nitrogen fixation, and suggest that this trait may be much more common in nature than it is currently thought. The diverse phylogenetic distribution of nitrogenase-like proteins indicates potential new roles for anciently duplicated and divergent members of this group of enzymes. After r8. This PSyntrophobotulus glycolicus DSM 8271 contains nine protein pairs related to the alpha and beta subunits of nitrogenase. In two cases, these are organised as four linked genes  potentially located in operons, suggesting that some of these gene pairs may provide scaffolding functions for co-factor assembly into the structural subunits, analogous to the nifDKEN gene clusters encoding conventional nitrogenase.Representatives of these non-conventional enzymes cluster in distinct clades relative to the conventional NifDKEN, Vnf/AnfDK and the C-group DK proteins, which are coloured dark blue in Figure Clostridium botulinum strains and Alkaliphilus oremlandii encode NifEN-like sequences  in the NifD/E-related components (Mlab_1040 and Dhaf_1539) and only a single conserved Cys residue (corresponding to ßC95/ßC44) in the NifK/N related subunits (Mlab_1039 and Dhaf_1540). Representative species from the Human Microbiome project, including Coprococcus catus GD/7 and Dorea longicatena DSM 13814, also appear in these clades . In contrast, several representatives of the Archaea possess only a single gene encoding a homolog of the alpha and beta chains of nitrogenase   is commonly used as a search tool in many phylogenetic studies. However, when using a single-gene survey in the database of microbial sequenced genomes, we detected orphan false-positive hits in several non-diazotrophic genomes. For example, the Methanobrevibacter ruminantium M1 and Methanocaldococcus fervens AG86 genomes include only a sequence similar to NifH, while the Methanosphaera stadtmanae DSM 3091 genome contains only a NifD-like sequence. In this case orphan nifD-like sequences may be evolutionary relics of divergent enzymes in which the NifD/E component does not contain conserved FeMoco ligands (see below). Thus genome analysis of environmental samples based purely on BLAST hits to NifH or NifD may lead to false indications of diazotrophy. To eliminate hits from orphan sequences our initial approach was to search in silico for the co-occurrence of NifH and NifD and then subsequently filter these hits for the occurrence of other nitrogen fixation protein sequences.The universal presence of gene sequences coding for the nitrogenase catalytic components in diazotrophs  or a large substrate (protochlorophylide). Although none of the alpha subunit related sequences we have analysed contain the FeMoco ligand His442, it is not possible to distinguish whether the function of these sequences is likely to relate to catalysis (i.e. NifDK-like) or to biosynthesis (i.e. NifEN-like). Biochemical and structural studies of NifEN reveal its functional diversity, since it can catalyse cluster conversion, molybdenum incorporation into the cofactor in association with NifH, and potentially the incorporation of homocitrate into FeMoco . AlthougRhodospirillum rubrum ATCC 11170, Clostridium beijerinckii NCIMB 8052, Geobacter sp. FRC-32, Additional file Clostridium cellulovorans 743B, Methanocorpusculum labreanum Z, Clostridium botulinum A2 Kyoto-F). Possibly this might provide a mechanism for coupling metal transport to the assembly of a metal cofactor. In Coprococcus catus GD/7 and other representatives of the Firmicutes, NifHEN-like proteins are associated with hydrogenase maturation proteins and may possibly play a role in the assembly of the active site metallocluster. The NifD proteins present in methanogenic Archaea have been proposed to function in coenzyme F430 biosynthesis, and NflD has been shown to co-purify with a NifH-like gene, NflH [Nitrogenase-like sequences could potentially perform analogous roles in association with a NifH-like component. The genomic organisation of these proteins may provide some clues to their possible metabolic functions  and the nitrogenases (Nif/Vnf/AnfDK) ,27,31. Rin silico prediction of this biochemical pathway. The occurrence of nif-orphan sequences or incomplete gene sets in several species questions single-gene approaches used in phylogenetic studies of nitrogen fixation. Furthermore our analysis highlights the presence of nitrogenase-like sequences with potential to catalyze as-yet unidentified functions.This work led to the identification of 67 potential diazotrophic species included in twelve taxonomic phyla, indicating that this metabolic trait is more widespread than formerly predicted. The identification of a minimum gene set required for nitrogen fixation provides a more robust method for the th 2011. Only one representative of species containing more than one sequenced genome was manually selected resulting in 999 unique Bacterial species and 93 unique Archaeal species. BLAST [A. vinelandii nitrogen fixing protein sequences: NifH (Avin_01380), NifD (Avin_01390), NifK (Avin_01400), NifE (Avin_01450), NifN (Avin_01460), NifU (Avin_01620), NifS (Avin_01630), NifV (Avin_01640), NifB (Avin_51010), NifQ (Avin_51040), AnfG (Avin_48980), and VnfG (Avin_02600). Initially hits were selected based on a relatively weak threshold (≥ 20%amino acid identity over the query length); using the minimum gene set criterion, hits to anf/vnfG, and presence of synteny the initial list was refined, yielding the protein sequences listed in Additional file Nitrogen fixing genes present in species with completely sequences genomes were identified through the protein database of microbial genomes at the National Center for Biotechnology Information up to July 17s. BLAST  searcheshttp://img.jgi.doe.gov) and then used as queries in a BLAST [−20. This returned 1117 unique geneIDs, which were then filtered against known NifD/E and NifK/N sequences  of known diazotrophs. Table S3. Nitrogen fixation genes (locus tags) of potential diazotrophs. Table S4. Nitrogen fixation genes (locus tags) of Group-C species. Click here for fileFigure S1. Neighbor joining phylogenetic tree of the Nif/Vnf/AnfD and K sequences derived from the species shown in Figure Click here for fileFigure S2. Gene neighborhoods of selected nitrogenase-like proteins.Click here for file


Mirounga angustirostris) biannually travel thousands of kilometers to forage in coastal and open-ocean regions of the northeast Pacific Ocean and then return to land where they fast while breeding and molting. Our study examined potential effects of age, adipose percent, and the difference between the breeding and molting fasts on PCB concentrations and congener profiles in blubber and serum of northern elephant seal females. Between 2005 and 2007, we sampled blubber and blood from 58 seals before and after a foraging trip, which were then analyzed for PCBs. Age did not significantly affect total PCB concentrations; however, the proportion of PCB congeners with different numbers of chlorine atoms was significantly affected by age, especially in the outer blubber. Younger adult females had a significantly greater proportion of low-chlorinated PCBs  than older females, with the opposite trend observed for hepta-CBs, indicating that an age-associated process such as parity (birth) may significantly affect congener profiles. The percent of adipose tissue had a significant relationship with inner blubber PCB concentrations, with the highest mean concentrations observed at the end of the molting fast. These results highlight the importance of sampling across the entire blubber layer when assessing contaminant levels in phocid seals and taking into account the adipose stores and reproductive status of an animal when conducting contaminant research.Persistent organic pollutants, including polychlorinated biphenyls (PCBs), are widely distributed and detectable far from anthropogenic sources. Northern elephant seals ( Persistent organic pollutants such as polychlorinated biphenyls (PCBs) are harmful to wildlife because they can disrupt endocrine and immune function Phoca vitulina) Eumetopias jubatus)Pusa hispida)Balaenoptera physalus), pilot whales , Dalls porpoises , and killer whales (Orcinus orca) had declining PCBs in blubber with increasing age Demographic and physiological parameters such as age, sex, and adipose stores may affect persistent organic pollutant concentrations. Variability in concentrations within a population may cause certain individuals to be more at risk from the negative effects of persistent organic pollutants. The percent of adipose tissue often has a significant, negative correlation with contaminant concentrations, and fluctuations in adipose tissue as a result of prolonged fasting can increase contaminant concentrations in remaining tissues Tursiops truncatus) foraging adjacent to point-sources of PCBs showed higher concentrations than bottlenose dolphins foraging farther from point sources Phoebastria spp.), humpback whales (Megaptera novaeangliae), killer whales, and harbor seals displayed geographically-associated variability in total PCB concentrations The major source of contaminants to marine mammals is from their food Mirounga angustirostris) are unique in that all of these variables can be quantified when they arrive on land. They have been monitored at the Año Nuevo colony  since 1968, where flipper tags are attached to several hundred pups annually, providing a consistent subset of known-age animals within the population that can be used to investigate relationships between age and contaminant concentrations. Northern elephant seals are long-lived, mesopelagic, high-trophic level predators. Based on diving behavior Assessing contaminants in free-ranging, open-ocean marine predators is complex because demographic parameters, estimates of adipose percent, and indices of foraging behavior are difficult to concurrently obtain from wild and visibly healthy marine predator populations. Northern elephant seals  with no visual health impairments, were selected for tissue sampling at Año Nuevo State Reserve in San Mateo County, California, USA between 2005 and 2007. Most seals (N = 54) were known-age individuals between 4–17 years old, identified by a uniquely numbered, plastic tag  placed in a lobe of their hind flipper. Female elephant seals have two distinct periods of fasting on land (breeding/lactation and molting) between foraging trips . The annAll 58 adult females were included in our summary of PCB concentrations . Only knGirth and length measurements were taken at eight locations along the seal, including six locations where blubber thickness was measured dorsally, laterally and ventrally using a handheld ultrasound backfat meter  63Ni ECD , serum and each section of blubber biopsy were analyzed separately for 22 PCB congeners . Details of sample preparation, clean-up, and analysis, including quality assurance, are provided in Debier et al. −1 (ppb) of serum fresh weight, 2 ng g−1 (ppb) of serum lipid weight and 0.7 ng g−1 (ppb) of lipid weight of adipose tissue in our analytical conditions. The limit of quantification (LOQ) of PCB congeners was determined by means of spiked bovine serum and bovine fat samples used as quality control (QC) and was the lowest concentration which could be quantified and which showed a recovery range between 70% and 130% −1 of serum fresh weight, 10 ng g−1 of serum lipid weight and 2.5 ng g−1 of lipid weight in adipose tissue. Congeners falling below the LOQ for elephant seal samples were recorded as measured concentrations. When a congener concentration was recorded as zero, this means that the congener was not detected at all in the sample. Blubber concentrations were quantified as ng g−1 lipid, while serum samples were quantified both by unit of serum lipid (ng g−1 lipid) and unit of whole serum (ug L−1). Lipids in serum were quantified as described in Vanden Berghe et al. Blubber biopsies were cut into three equal parts. Inner (closest to the muscle) and outer (closer to the skin) blubber layers were analyzed in 2008 at the University of Liege. Using a Thermo Quest Trace 2000 gas chromatograph coupled with a Linear mixed effects (LME) models were run using the nlme package The subset of individuals with paired pre- and post-foraging trip samples were additionally analyzed using paired t-tests to examine if ∑PCB concentrations in tissues  changed between the start and end of the short or the long foraging trip. T-tests were also used to examine whether the magnitude and direction of change in tissue concentrations were the same for both foraging trips (short versus long). The level of statistical significance for all analyses was set at p≤0.05.PCB concentrations were further examined at the level of specific congeners and congener families, based on the degree of chlorination. Low-chlorinated biphenyls included all congeners with three or four chlorines (tri- and tetra-CBs). The percentage of the total contaminant concentration made up by each congener family was calculated for every sample . Octa-, nona-, and deca-CBs were below the detection limit for the majority of samples, therefore statistical analyses were focused on tri/tetra-CBs, penta-CBs, hexa-CBs, and hepta-CBs. LME models were run with the percentage of each congener family as the response variable, after an arcsin square root transformation was applied −1 lipid in blubber and 512–2,837 ng g−1 lipid in serum , -138, -153 (hexa-CBs), -180, -183, and -187 (hepta-CBs), all of which had a mean percent contribution of the total burden greater than 5% . These eSerum PCB profiles had seven congeners that contributed a mean percent of more than 5% of the total PCB concentration: PCB-101, -110, -118, -138, -153, -180, and -183. These seven congeners comprised 78.8±5.9% of the total serum concentrations. PCB-153 comprised between 14.0–26.4% of the total serum PCB concentrations.1,44 = 12.8, p<0.001) and a significant, positive relationship between age and the percent of hepta-CBs  in inner blubber. There was no detectable relationship between age and the percent of either tri/tetra-CBs or hexa-CBs in the inner blubber. In the outer blubber, there was also a significant, negative relationship between age and the percent of penta-CBs  and a significant positive relationship between age and the percent of hepta-CBs . Additionally, in the outer blubber, the percent of tri/tetra-CBs had a significant, negative relationship with age  and the percent of hexa-CBs had a significant, positive relationship with age . The same significant, negative relationship observed for both blubber layers between age and the percent of penta-CBs was also detectable for lipid-normalized serum  and serum concentrations using wet-weight. The percent of tri/tetra-CBs also had a significant, positive relationship with age in the serum , which was the opposite trend as observed in the outer blubber layer. The percent of hexa-CBs and hepta-CBs in serum did not change with age.There was no detectable relationship between age and total PCB concentrations in inner blubber, outer blubber or serum (p≥0.230). However, age had a significant effect on congener groups ; Table 2Elephant seals were fattest early in the breeding season fast  and leanest late in the breeding fast . Seals sampled early in the molting fast had a mean adipose percent of 30.8±2.7% (range: 23.4–33.8%) compared to 30.9±2.4% (range: 26.1–36.1%) late in the molting fast, indicating that adipose and lean tissue were lost in similar proportions over the course of the molt.1,20 = 19.7, p<0.001). Direct comparisons of late breeding and late molting, while accounting for adipose percent, revealed that seals had higher mean inner blubber ∑PCB concentrations at the end of the molt  , and there was no detectable difference between ∑PCB concentrations in late breeding and late molting outer blubber samples  . Convers1,21 = 6.1, p = 0.023); however, when the single highest serum concentration was removed from analysis (2837 ng g−1 lipid measured during the early molting fast) this relationship became non-significant . Serum ∑PCB concentrations measured per unit of wet weight had a significant relationship with adipose percent even with the highest concentration removed . There was a significant difference between mean late breeding and late molting serum ∑PCB concentrations per unit lipid  but there was no significant difference between the late breeding and late molting serum ∑PCB concentrations per unit of wet weight .The relationship between the percent of adipose tissue and total serum ∑PCB concentrations was dependent on whether the concentrations were analyzed using lipid-normalized or wet weight concentrations. Serum ∑PCBs measured per unit lipid initially appeared to have a significant relationship with adipose percent , while the opposite was true for adipose percent and the proportion of penta-CBs in the inner blubber . There was no detectable relationship between adipose percent and the proportion of hexa-CBs or hepta-CBs in the inner blubber. In the outer blubber, adipose percent also had a significant, positive relationship with the proportion of tri/tetra-CBs  and a significant, negative relationship with the proportion of penta-CBs . Additionally, adipose percent had a significant, negative relationship with the proportion of hepta-CBs in the outer blubber . There was no detectable relationship between adipose percent and the proportion of hexa-CBs in the outer blubber. In serum, there was a significant, positive relationship between adipose percent and the proportion of penta-CBs , the opposite trend to that observed in blubber. There were no significant relationships detected between adipose percent and the proportion of tri/tetra-CBs, hexa-CBs or hepta-CBs in serum.Adipose percent had significant relationships with some congener groups in blubber and serum . Fatter Paired tissue samples from the same individuals pre- and post-foraging revealed that changes in ∑PCB concentrations within individuals differed by tissue type and whether the change was measured across the short or long foraging trip . The conOur findings highlight that toxic contaminants are detectible in seals that forage in remote depths of the Pacific Ocean, thousands of kilometers from anthropogenic sources. Mesopelagic and open-ocean foraging behaviors do not leave northern elephant seals immune to the accumulation of PCBs, as PCBs were detected in blubber and blood of all 58 sampled northern elephant seal females. ∑PCB concentrations in elephant seal serum and blubber sampled during early and late breeding in the present study are similar to those previously observed in elephant seal females Zalophus californianus) Quantification of ∑PCBs in marine mammals varies somewhat based on the number of congeners that are quantified in each study, which makes direct comparison of ∑PCBs or the percent of individual congeners difficult between studies. Additionally, the reporting of individual congeners varies between study, making one to one comparisons difficult. Nevertheless, some broad comparisons can be made between our observations and other published studies, realizing that the absolute values may differ slightly based on study design. Female northern elephant seals have substantially lower ∑PCB concentrations than stranded adult California sea lions  Monachus schauinslandi) The most common PCB congener quantified in elephant seal blubber for our study is CB-153, which contributes the greatest proportion to ∑PCBs in a broad range of marine mammal species The absence of a significant age relationship with ∑PCB concentrations indicates that elephant seals likely offload contaminants during lactation and replenish contaminants while foraging. Age does not affect ∑PCB concentrations in the blubber of other female pinnipeds, including harbor seals Based on previous research on elephant seals from Año Nuevo quantifying average ∑PCB concentrations in milk ow). This decreases the transfer efficiency from maternal blubber to offspring through milk, potentially due to differences of mobilization efficiency from blubber ow comprised a greater proportion of PCBs in maternal harbor seals ow values, while selectively retaining congeners with higher Kow values. Another potential mechanism that can explain the age relationship observed in our study is the preferential metabolism of lower chlorinated congeners. It is possible that both selective excretion through milk and preferential metabolism could play a role in the age and congener trends observed in female northern elephant seals.Age significantly affects the proportion of PCB congener families in female elephant seal blubber and serum. This suggests a positive relationship between an age-associated process, such as the number of times a female has given birth, and the proportion of higher chlorinated congeners in elephant seal tissues. Congeners with increased chlorination are more lipophilic and have a higher octanol-water partitioning coefficient  have significant, negative relationships with the percent of adipose tissue. However, this trend is not observed in outer blubber or serum measured per unit lipid. This suggests that varying physiological and temporal mechanisms mediate fluctuations within the main reservoir (blubber) and the transport system (serum) of PCBs in northern elephant seal females. Asynchronous fluctuations between inner blubber, outer blubber, and serum are likely due to differential use of these tissue components during fasting and lactation, and the potential for transfer of contaminants to offspring. Trends observed here are supported by previous studies. In general, inner blubber ∑PCB concentrations may be more dynamic than outer blubber ∑PCB concentrations because inner blubber is the main energy reserve mobilized during fasting When animals are at their fattest, outer blubber concentrations may be higher than inner blubber concentrations Biannual fasting periods of seals on land are associated with breeding or molting and result in significant reductions in adipose tissue and body mass Ursus maritimus), that undergo significant fasting periods While concentrations of ∑PCBs in blubber and serum in our study are below the concentration threshold that corresponds with immune system suppression in harbor seals Animals may be especially vulnerable to the effects of contaminants if normal fasting causes increased tissue concentrations and is then followed by decreased post-fasting foraging opportunities. This was the possible explanation for elevated ∑PCB concentrations observed in a female from the present study. This female lost adipose tissue over the course of a short, post-breeding foraging trip, which corresponded to an increase in blubber and serum ∑PCB concentrations of 92% and 213%, respectively, over her late fasting concentrations. Her tissue concentrations, measured before and after the foraging trip, are among the highest observed during any time period and, based on our observations of other seals in this study, it is likely that her ∑PCB tissue concentrations would have been even higher at the end of the molting fast. Similar trends are observed in polar bears, where fluctuations in seasonal food availability outside of the hibernation period can influence body condition and thus affect PCB concentrations Examining paired tissue samples from the same individuals before and after a foraging trip provides insight into certain dynamics that are not observable in a larger population level analysis. These results are unique because tissue samples were paired between the start and end of a foraging period and not between the start and end of a fasting period, as is common for studies on the PCB dynamics in the blubber and blood of marine mammals. Inner blubber is the most metabolically active layer in northern elephant seals Overall, our findings demonstrate that PCBs accumulate in female northern elephant seals that forage across the mesopelagic north Pacific. While total accumulation of PCBs does not change with age, our findings reveal that more chlorinated PCB congeners comprise a higher proportion of the total PCB burden of older females while younger seals have a higher proportion of less-chlorinated PCBs. At this time, the health consequences of these observations remain unknown.Northern elephant seals exhibit a set of life history strategies that depend on two seasonal fasting periods. Our findings suggest that female northern elephant seals are most vulnerable to high ∑PCB concentrations at low adipose percentages, which occur at the end of their two seasonal fasts. Depuration to pups may mitigate high blubber ∑PCB concentrations in adult female tissues during the breeding fast and prevent overall ∑PCB concentrations from increasing with increasing age. However, no mitigating mechanism is known to occur during the molting fast, and therefore, our results suggest the female elephant seals are most vulnerable to increased concentrations of ∑PCBs during their molting fast. Free ranging adult male elephant seals may accumulate significantly more ∑PCBs than females since they have no ability to mitigate their contaminant load through reproductive transfer. More research is needed on males to verify this hypothesis.In addition, the inverse relationship between body condition and total PCB concentration in the inner blubber has implications for the development of bio-monitoring strategies. First, the presence of a significant, negative relationship between adipose percent and ∑PCB concentration in the inner blubber, but not in the outer blubber, indicates the complexity of blubber as a biological indicator. For elephant seals, our results suggest that ∑PCB concentrations of inner blubber represent recent trends in contaminant fluctuation, while ∑PCB concentrations of outer blubber, a tissue of slower turnover, may represent longer-term accumulation. Bio-monitoring of more elusive marine predators, where biopsy darting is the only option, may provide misleading results if only the outer-most tissue is sampled. It is imperative that adipose percent and the life history of an animal be fully considered for both the design of contaminant monitoring programs and appropriate interpretation of the results.


During the modified Bentall surgery (aortic root replacement), a cuff of native aorta is implanted, together with the coronary ostium, into the aortic graft. Multi-detector computed tomography (MDCT) imaging can accurately assess the coronary ostial anastomosis site post-surgery. In this study, we assessed the feasibility of imaging the coronary ostial anastomosis site using transthoracic echocardiography (TTE).Patients  with previous Bentall surgery underwent TTE study, with MDCT (64-slice) as the reference standard. TTE used conventional and novel acoustic windows to interrogate the coronary ostia.All coronary ostia (n = 28) were well-visualized with MDCT. The optimum TTE acoustic window for visualizing the coronary ostia was a superiorly positioned parasternal short-axis view with the probe tilted towards the left shoulder, medially angulated for the right coronary artery ostia (RCAos) and laterally angulated for the left main coronary artery (LMAos). In this off-axis position, 10 (71%) LMAos and 13 (93%) RCAos could be visualized. In the conventional parasternal views, only 5 (36%) RCAos and no LMAos could be visualized. TTE underestimated the diameter of the LMAos , but was similar to MDCT for the RCAos .We report a novel TTE acoustic window to image the coronary ostia of post-Bentall surgery patients. Although TTE underestimates the left coronary ostium size, recognition of the ostial dilation with TTE appears feasible in most patients. Those that cannot be imaged will require alternative imaging modality such as MDCT. The incidence of thoracic aortic aneurysm is approximately 4.5 per 100,000 people, of which 60% predominantly affects the supravalvular aorta (aortic root and/or ascending thoracic aorta) . The patThe Bentall procedure , coupledThe present study reports a novel TTE acoustic window that appears to be superior to conventional acoustic windows in assessing the coronary ostial dilation of patients who had undergone the modified Bentall operation, with dual-source 64-slice MDCT imaging as the reference standard.Patients who had prior aortic root replacement with the Bentall’s technique were recruited over a period of 15 months. The referrals for imaging were all based on clinical review and recommendation by patients’ treating physicians as part of their long-term follow-up. Detailed background history was obtained from each patient and further corroborated with the patient’s medical record. Clinical information collected included indications for surgery, original pathology, comorbidites, original operation report, and associated histopathology reports.The original Bentall procedure and its subsequent modifications have been published previously ,5. In brStandard two-dimensional and Doppler TTE was performed by an experienced sonographer using commercially available transducer and equipment . Echocardiograms were recorded and stored in a digital database and then analysed offline using commercially available software . Echocardiogram was performed with patients in the left lateral decubitus position. TTE used conventional and novel acoustic windows to interrogate the coronary ostia. Conventional acoustic windows concentrated on the parasternal short-axis views whilst several off-axis views were attempted to determine the optimum TTE acoustic window for visualizing the coronary ostia. The coronary ostial diameter was measured using electronic calliper at the coronary ostial-aortic graft junction from the optimum acoustic window.All patients also underwent MDCT imaging using a dual-source 64-slice scanner . The scanner parameters have been previously published . 100 ml The diameters of the coronary vessels on CT scan were assessed offline using commercially available software . Measurements were performed from the ‘best diastole’ data set. The coronary ostial diameter was measured using electronic calliper at the coronary ostial-aortic graft junction. 0.6 mm multi-planar reconstructions were manipulated to obtain an image perpendicular to the aortic root at the level of the coronary ostium that showed the greatest ostial dilation on visual assessment. A soft tissue window was used (600/200).The study complies with the Declaration of Helsinki and was approved by the institution  Ethics Committee on Human Research (CH62/6/2008-059).t tests. Analysis was performed using PRISM 4.03 . A two-tailed probability value <0.05 was considered statistically significant.All continuous variables are expressed as mean ± standard deviation. Mean coronary ostial diameters obtained from TTE versus MDCT were compared using paired A total of 14 patients  underwent both TTE and MDCT imaging studies to assess the thoracic aorta  . In the conventional parasternal views, only 5 (36%) RCAos and no LMAos could be visualized. Different off-axis views were then attempted to visualize the coronary anastomotic sites. The optimum TTE acoustic window for visualizing the coronary ostia was found to be in a superiorly positioned parasternal short-axis view with the probe tilted towards the left shoulder, medially angulated for the right coronary artery ostia (RCAos) and laterally angulated for the left main coronary artery (LMAos). In this off-axis position, 10 (71%) LMAos and 13 (93%) RCAos could be visualized (Figure We previously identified apparent dilation of the post-surgical coronary ostium in almost all patients who had undergone the modified Bentall’s operation some years earlier using a dual-source 64-slice MDCT . The lonThe normal diameter of native coronary vessels can be underestimated in post-mortem and conventional invasive angiographic studies -14. ThesVeira et al. reported a dilated appearance of the aortic-coronary artery anastomosis site of a patient immediately following composite aortic root and valve replacement using intraoperative transoesophageal echocardiogram , suggestHowever, if conventional TTE acoustic windows were used, none of the LMAos could be visualized and only a minority of the RCAos was seen. This was despite significant coronary ostia dilation in nearly all patients confirmed on MDCT. This would explain the lack of reported coronary ostia dilation using TTE in these patients. This is not unexpected given the altered anatomy of the coronary ostia-aortic junction post surgery. The button technique results in the explanted native coronary arteries being sutured onto side-holes of the prosthetic aorta in a position that is more superior to the original position of the native vessels Figure . Hence, Although this is a small cohort study, and will require validation, we combined the images derived from MDCT to corroborate our findings from TTE. Most of the TTE were performed several months later than the MDCT. Future studies should address the issue of whether the coronary ostia dilation of these patients can change or progress with long-term imaging assessments. Although majority of the coronary ostia could be visualized on TTE using the proposed novel acoustic window, not all were observed. The TTE acoustic window may be limited in some post-thoracic surgery patients irrespective of the position of the probe. Those that cannot be observed on TTE may require MDCT to monitor their coronary vessels and aorta. We did not have a TTE control group. Our purpose was to show that conventional TTE acoustic windows are inadequate in visualizing the coronary ostial dilation of post-Bentall patients that were easily seen on MDCT. While our proposed novel TTE acoustic window allowed visualization of most of these patients’ coronary ostia-aortic junction, it is not known if the measured coronary ostial diameters will be significantly different from normal coronary vessels diameters measured using TTE.The significance of coronary ostial dilation in patients after modified Bentall surgery remains unclear. Long-term follow-up assessments using echocardiography as well as MDCT may help assess the impact of this finding on patient course and management. The present study described a novel transthoracic echocardiographic acoustic window that visualized the coronary ostia of most patients after Bentall surgery and confirmed similar dilated coronary ostia seen on MDCT. Although transthoracic echocardiography underestimates the size of the left main coronary ostium, recognition of the ostial dilation with transthoracic echocardiogram appears feasible in many patients and avoids the radiation exposure associated with MDCT. In those patients with inadequate echocardiographic acoustic window, using MDCT to image the coronary ostia of these patients post-surgery can be considered.AscAo: Ascending thoracic aorta; AR: Aortic regurgitation; AV: Aortic valve; LMAos: Left main coronary artery ostia; MDCT: Multi-detector computed tomography; PS off-axis view: Parasternal off-axis view; RCAos: Right coronary artery ostia; TTE: Transthoracic echocardiography.We declare that we have no competing interest.ACN designed the study, performed the main data collection, performed all data analyses and wrote the first draft of the report and subsequent revision with assistance from LK and JY. DH reviewed the data analyses, reviewed and revised the manuscript. LK reviewed the data analyses, reviewed and revised the manuscript. JY designed the study, reviewed the data analyses, reviewed and revised the manuscript. All authors read and approved the final manuscript.


Cortico-basal ganglia-thalamocortical circuits are severely disrupted by the dopamine depletion of Parkinson's disease (PD), leading to pathologically exaggerated beta oscillations. Abnormal rhythms, found in several circuit nodes are correlated with movement impairments but their neural basis remains unclear. Here, we used dynamic causal modelling (DCM) and the 6-hydroxydopamine-lesioned rat model of PD to examine the effective connectivity underlying these spectral abnormalities. We acquired auto-spectral and cross-spectral measures of beta oscillations (10–35 Hz) from local field potential recordings made simultaneously in the frontal cortex, striatum, external globus pallidus (GPe) and subthalamic nucleus (STN), and used these data to optimise neurobiologically plausible models. Chronic dopamine depletion reorganised the cortico-basal ganglia-thalamocortical circuit, with increased effective connectivity in the pathway from cortex to STN and decreased connectivity from STN to GPe. Moreover, a contribution analysis of the Parkinsonian circuit distinguished between pathogenic and compensatory processes and revealed how effective connectivity along the indirect pathway acquired a strategic importance that underpins beta oscillations. In modelling excessive beta synchrony in PD, these findings provide a novel perspective on how altered connectivity in basal ganglia-thalamocortical circuits reflects a balance between pathogenesis and compensation, and predicts potential new therapeutic targets to overcome dysfunctional oscillations. Parkinson's disease is a progressive age-related neurodegenerative disorder that severely disrupts movement. The major pathology in Parkinson's disease is the degeneration of a group of neurons that contain a chemical known as dopamine. Treatment of Parkinsonism includes pharmacological interventions that aim to replace dopamine and more recently, implanted devices that aim to restore movement through electrical stimulation of the brain's movement circuits. Understanding the electrical properties that emerge as a result of depleted dopamine may reveal new avenues for developing these technologies. By combining a novel model-based approach with multi-site electrophysiological recordings from an animal model of Parkinson's disease we provide empirical evidence for a link between abnormal electrical activity in the Parkinsonian brain and its physiological basis. We have examined the connections along the brain's motor circuits, and found an abnormality in inter-area connections in a particular neural pathway, a pathway critically dependent on dopamine. The scheme makes strong and testable predictions about which neural pathways are significantly altered in the pathological state and so represent empirically motivated therapeutic targets. In Parkinson's disease (PD), degeneration of midbrain dopamine neurons severely disrupts neuronal activity in looping circuits formed by cortico-basal ganglia (BG)-thalamocortical connections . Studies have shown that excessive oscillations at beta frequencies (13–30 Hz) are a key pathophysiological feature of these Parkinsonian circuits, when recorded at the level of unit activity and/or local field potentials (LFPs) in several key circuit nodes. These nodes include the frontal cortex, subthalamic nucleus (STN), external globus pallidus (GPe) and internal globus pallidus (GPi) . Suppression of pathological beta-activity is achieved by dopamine replacement therapies [10] and surgical treatments e.g. high-frequency, deep brain stimulation (DBS) of the STN; where prolonged attenuation after stimulation is observed . Bradykinesia and rigidity are the primary motor impairments associated with beta activity and, following dopamine replacement therapies, improvements in these motor deficits correlate with reductions in beta power . Moreover, a recent report has shown that stimulating the STN at beta frequencies exacerbates motor impairments in Parkinsonian rodents [17], in line with similar findings in PD patients .Precisely how dopamine depletion leads to abnormal beta power is unknown. Recent work in rodents has revealed that excessive beta-activity emerges in cortex and STN after chronic dopamine loss but not after acute dopamine receptor blockade . Here, we examine whether changes in effective connectivity between the nodes of the cortico-basal ganglia-thalamocortical network can account for enhanced beta oscillations following chronic dopamine loss. To test this hypothesis we used dynamic causal modelling (DCM). This approach allows one to characterise the distributed neuronal architectures underlying spectral activity in LFPs. DCM is a framework for fitting differential equations to brain imaging data and making inferences about parameters and models using a Bayesian approach. A range of differential equation models have been developed for various imaging modalities and output data features. The current library of DCMs includes DCM for fMRI, DCM for event related potentials and DCM for steady state responses (DCM-SSR). The current paper is based on DCM-SSR, designed to fit spectral data features .a posteriori estimates of connectivity and synaptic parameters . Using these estimates, we characterised the sensitivity of beta oscillations to changes in particular connection strengths to identify candidate connections that may represent therapeutic targets in idiopathic PD.Using spectral data, recorded simultaneously from multiple basal ganglia nuclei and the somatic sensory-motor cortex, we asked whether systematic changes in re-entrant neural circuits produce the excessive beta oscillations observed in LFPs recorded from the 6-hydroxydopamine (6-OHDA)-lesioned rat model of PD . We inverted the models  using LFP data collected simultaneously from electrodes implanted in frontal cortex, striatum, GPe and STN. Specifically, we used neural mass models that characterise the main projection cell types at each circuit node as glutamatergic or GABAergic. Neural mass models describe neuronal dynamics in terms of the average neurophysiological states  over populations of neurons. Inference on effective connectivity differences observed between the Parkinsonian and control cases was based on in vivo, where we assume observed responses are caused by interactions among neuronal populations or sources, with known neurotransmitters and directed connections. The starting point for analyses of effective connectivity in this paper is the end point of classical functional connectivity analyses; namely, observed cross-spectral densities (and their associated cross-correlation and coherence functions). In other words, we place special emphasis on explaining how functional connectivity emerges in terms of directed connections that rest on a particular model of neuronal interactions. In what follows, we illustrate this approach when applied to the directed circuitry of a cortico-basal ganglia-thalamocortical system.Measures of functional connectivity have been applied previously to examine frequency-specific signal correlations between nodes in the cortico-basal ganglia-thalamocortical network. These measures have highlighted excessive coupling between the cortex and STN [22] and between STN and GPe  in animal models of PD. While functional connectivity and effective connectivity measures share some technical aspects e.g. likelihood models [23] or Bayesian estimators [24], the underlying concepts are fundamentally different [25]. The distinction between functional connectivity  and effective connectivity  emerged from the analysis of electrophysiological time series: Aertsen et al. [26], used the term effective connectivity to define the neuronal interactions that could explain observed spike trains using a “minimum simple neuronal model”. In what follows, we employ such a minimum model approach, using the key elements of known cortico-basal-ganglia-thalamocortical interactions. Our model predicts the output of this loop circuit Dynamic causal models for LFP data typically comprise connected cortical sources, where each source is described by a neural mass [27]. This neural mass ascribes point estimators to hidden neuronal states (ensemble depolarisation and firing rates), capturing the average activity of a population of neurons [28], i.e. a mean-field approximation. These dynamics depend on model parameters that encode, for example, inter-regional connectivity, the amplitude of postsynaptic responses and/or synaptic rate constants. Here, three (layered) populations were used to model the cortical source, while a single population of neurons, (either glutamatergic [excitatory] or GABAergic [inhibitory]) was used for distinct BG nuclei see . The difThe connections in our standard DCM were based on the well characterised re-entrant circuits linking the cortex, basal ganglia and thalamus in rodents and primates . The maiRecent work has emphasized the significance of the hyperdirect pathway for the functional organisation of cortico-basal ganglia-thalamocortical circuits [30] and, importantly, this pathway has been shown to be crucial for the expression of abnormal slow oscillations in the STN-GPe network in Parkinsonism [33]. In short, our standard model architecture incorporates the major glutamatergic and GABAergic connections between the six key components of the cortico-basal ganglia-thalamocortical circuit. In accommodating the core elements of the loop circuit, the model also adheres to the established organisational principles embodied in the direct, indirect and hyperdirect pathways. Note that our standard model does not include all known connections. However, the addition of more connections does not necessarily improve the ability of the loop circuit (and model) to sustain beta oscillations. To test this, we tried adding two less well-studied, but potentially important, pallidofugal connections, either from GPe to EPN or that from GPe to striatum [34]. The addition of either connection did not improve the performance of (evidence for) the standard model, which provided the optimum balance of accuracy and complexity for our given data set  estimates are plotted with 95% Bayesian confidence intervals. We considered connections changed when the probability of a difference was greater than 99.99%. Along the hyperdirect pathway, cortical output to the STN increased in the Parkinsonian animals compared to the control group while, conversely, efferent connections from the STN to GPe decreased. These changes in effective connectivity, occurring after dopamine cell lesions, characterise the circuit generating beta oscillations and the net balance between pathogenesis and any consequent compensatory changes. The DCMs of the grand averaged spectral responses (where one DCM was fit to the average control cross-spectra and one DCM was fit to the average lesioned cross-spectra) confirm the inference based on individual DCMs  using a z-transform [21]. This transform produces a transfer function, which summarises the model system's input-output spectral properties.In F  = 7.42, p<0.0001;Post hoc two way t-tests (to cover positive and negative derivatives), indicated that the beta contributions were significant for two connections when corrected for multiple comparisons. We found that variations in striatal connections to GPe (p<0.05 Bonferroni corrected) and GPe connections to STN (p<0.05 Bonferroni corrected) produced significant increases in beta power  beta activity. This entailed using the optimised DCM from the lesioned animals to predict spectral responses to changes in the model circuit architecture: To do this, we quantified the degree to which a change in each connection affected beta activity throughout the circuit (power summed over 16–18 Hz and channels). This provided a measure of ‘beta contribution’ per animal, per connection; in terms of the partial derivative of summed beta activity, with respect to each connection. Note that beta contributions (derivatives) could be either positive, whereby small changes exacerbate beta, or negative whereby small changes ameliorate beta  that profoundly influenced beta activity in our simulations did not differ in their mean strength between the control and Parkinsonian groups. This apparent paradox can be resolved by considering the extended network that incorporates all nine connections. The strength of a connection need not necessarily change between the healthy and diseased states in order for that connection to have a different effect on oscillatory strength within the new network; in other words, perturbations to a connection with a given strength will have a different functional effect, depending on the network in which it is embedded. Moreover since the conditional densities of the parameters of interest in the DCM are identifiable . This analysis showed that the frequency promotion in the Parkinsonian state was specific to the beta band and could not be generalised to higher frequencies .We have clear evidence that the extended network differs between the control and Parkinsonian states with increases in the effective connection strength of the hyperdirect cortex to STN pathway and reduction in that from STN to GPe. Above, we identified that several connections in the indirect pathway have the capacity to dynamically modulate beta. Thus we compared beta contribution between the control and Parkinsonian animals for the ‘beta critical’ indirect pathway connections. This showed that these connections engendered much higher beta activity when embedded in the Parkinsonian network  would normally predispose to ‘over fitting’ in a maximum likelihood setting. However, Bayesian inference finesses this problem because it optimises the marginal likelihood (model evidence), which includes a complexity term. This complexity rests on the use of priors or constraints on the underlying neurobiology . This effectively limits the degrees of freedom in the model to those parameters with relatively uninformative priors; i.e., the connectivity parameters of interest. Moreover, the standard model performed no better when two additional pallidofugal connections were incorporated , and supplemental doses of ketamine  and xylazine . The electrocorticogram (ECoG), a type of cortical local field potential, was recorded via a 1 mm diameter steel screw juxtaposed to the dura mater above the right frontal (somatic sensory-motor) cortex  and amplified  before acquisition. Extracellular recordings of LFPs in the striatum, GPe and STN were simultaneously made in each animal using ‘silicon probes’ . Each probe had one or two vertical arrays of recording contacts . The same probe was used throughout these experiments but it was cleaned after each experiment in a proteolytic enzyme solution to ensure that contact impedances and recording performance were not altered by probe use and re-use [33]. Monopolar probe signals were recorded using high-impedance unity-gain operational amplifiers  and were referenced against a screw implanted above the contralateral cerebellar hemisphere. After initial amplification, extracellular signals were further amplified (1000×) and low-pass filtered at 6000 Hz using programmable differential amplifiers . The ECoG and probe signals were each sampled at 17.9 kHz using a Power1401 Analog-Digital converter and a PC running Spike2 acquisition and analysis software . Neuronal activity was recorded during episodes of spontaneous ‘cortical activation’, which contain patterns of activity that are similar to those observed during the awake, behaving state [48]. Cortical activation was defined according to ECoG activity . Neuronal activity patterns present under this anaesthetic regime may only be qualitatively similar to those present in the unanesthetized brain. However, the urethane-anesthetized animal still serves as a useful model for assessing ensemble dynamics within the basal ganglia [46]. Indeed, in 6-OHDA-lesioned animals, exaggerated beta oscillations emerge in cortico-basal ganglia circuits during activated brain states  thus accurately mimicking the oscillatory activity recorded in awake, unmedicated PD patients [10].Experimental procedures were carried out on adult male Sprague-Dawley rats , and were conducted in accordance with the Animals (Scientific Procedures) Act, 1986 (UK). Recordings were made in eight dopamine-intact control rats (288–412 g) and nine 6-OHDA-lesioned rats (285–428 g at the time of recording), as described previously . Briefly, anaesthesia was induced with 4% v/v isoflurane  in OUnilateral 6-OHDA lesions were carried out on 200–250 g rats, as described previously . Twenty five minutes before the injection of 6-OHDA, all animals received a bolus of desipramine  to minimize the uptake of 6-OHDA by noradrenergic neurons [49]. Anaesthesia was induced and maintained with 4% v/v isoflurane (see above). The neurotoxin 6-OHDA  was dissolved immediately before use in ice-cold 0.9% w/v NaCl solution containing 0.02% w/v ascorbate to a final concentration of 4 mg/ml. Then 3 µl of 6-OHDA solution was injected into the region adjacent to the medial substantia nigra . The lesion was considered successful in those animals that made >80 net contraversive rotations in 20 min. Note that the emergence of exaggerated beta oscillations after 6-OHDA lesions is not dependent on apomorphine ). Electrophysiological recordings were carried out ipsilateral to 6-OHDA lesions in anesthetized rats 21–42 days after surgery, when pathophysiological changes in the basal ganglia are likely to have levelled out near their maxima [6].i and j  were evaluated from the channels' time-series, We restricted our analysis to the ECoG and LFP activities present during a spontaneous activated brain state, which mimics that accompanying alert behaviours in which beta oscillations are most prominent in PD patients and 6-OHDA-lesioned rats . Thirty second epochs of cortical activation were analysed from one contact in each BG nucleus and the contemporaneous ECoG from each animal. Cross-spectral density n,i and j can then be constructed from the (complex) transfer functions http://www.fil.ion.ucl.ac.uk/spm/). These cross-spectra were then used for dynamic causal modelling:Data from the four channels  were summarised using an autoregressive (AR) process [52] of order DCM is a model comparison framework for the inversion and comparison of generative (forward) models based on differential equations. DCM allows data from multiple recording sites to be analysed as a distributed system. Originally developed for fMRI [54], the framework uses a generative model of the neural processes   that cause observed data. Bayesian model inversion furnishes estimates of coupling or effective connectivity between regions and how this coupling is changed by experimental context [57]. For LFP and ECoG data, the generative model contains details about the structure and synaptic properties within neuronal sources, as well as the synaptic input that each source receives [21]. In what follows, we describe the precise form of the dynamic causal model we used in this application. The underlying neural mass model has been described and validated in a series of previous papers In DCM for steady-state responses, one is trying to predict or explain observed spectral activity. Effectively, this entails modelling the mapping between the spectral density of neuronal fluctuations or innovations and the resulting responses. This mapping is parameterised in terms of the (synaptic) parameters of a neural mass model. The neural mass model is used to determine how random neuronal fluctuations are filtered to produce observed cross-spectra. Model inversion involves optimising model parameters to explain empirical cross-spectra. In what follows, we will describe the generative model in terms of the kernels or transfer functions that couple the spectra of neuronal innovations to observed cross-spectra and then describe the neural mass model that determines how the transfer functions (kernels) are parameterised. k-th neuronal innovations  to each channel times the spectral density of each input: We require the model to predict cross-spectra corresponding to the data features described above. This prediction is based on the parameters k-th innovation  that constitute our model of hidden neuronal states. The effect of neuronal noise or fluctuations is modelled here in terms of their expression as cross spectra in channel space. The neural model is composed of subpopulations, where each subpopulation can have different synaptic rate constants and amplitudes. Subpopulations are grouped into sources and coupled with intrinsic connections, while sources are connected by extrinsic connections between specific subpopulations in different sources. Our model comprised a cortical source, four basal-ganglia nuclei and a thalamic source . The corj-th subpopulation in the k-th source:j-th subpopulation in the k-th source, while and l-th subpopulation , weighted by an intrinsic coupling parameter i-th source: The dynamics of hidden neuronal states and the ensuing observation are assumed to have the form, where i-th electrode, as described above.The architecture depicted in i-th parameter. A non-zero prior variance allows the parameter to be pushed from its prior mean. A relatively tight or informative prior obtains when Because the model parameters are non-negative they are treated as scale-parameters with Gaussian priors on the log of their values. These are specified in terms of a prior mean a posteriori (MAP) parameter estimates  and the conditional covariance. This scheme is known as Variational Laplace The priors, together with the likelihood model (Equation 3), constitute a generative model of observed cross spectral densities based on a neuronal state-space model formulated in continuous time. This generative model is summarised in −2. Note that Variational Laplace generalises previous variational schemes based upon expectation maximisation, which ignore condition uncertainty about the parameters optimised in the maximisation step: e.g., [69]. For dynamic models of the sort used in this paper, this approach has been evaluated in relation to Monte Carlo Markov chain (MCMC) techniques and has been found to be robust, providing accurate posterior estimates while being far superior to sampling schemes in terms of computational efficiency [70].Variational Laplace appeals to a mean field approximation and factorises an approximate joint posterior into two densities over the covariance and model parameters: Given the empirical data, either from each animal separately  contribute to beta frequencies more than others. This analysis assumes that the generative model of the spectra has been optimised and omits channel noise; i.e. using As outlined above, the free energy is a lower bound on the model evidence [71] and is used for model selection, when testing a series of possible neural architectures using Bayes Factors  model. We can hence perform additional system identification, where all spectral properties of the BG-cortical circuits, as approximated by our model can be summarised. The system's transfer function can be constructed from the state-space  formulation given above , renders the system a MIMO Click here for additional data file.