Ionizing irradiation is an important clinical approach to treat solid tumors. Modern radiation technologies aim to selectively kill tumor cells and protect the surrounding normal tissue. The standard paradigm for radiation effects in cellular systems involves damage of the DNA including DNA double-strand breaks, which are considered as most effective in destroying tumor cells. Due to their enhanced physical and radiobiological properties, high-linear energy transfer radiation qualities are of special interest in tumor therapy. Future radiation therapy strategies aim to utilize carbon ions to effectively treat highly aggressive tumors. More recently, evidence is emerging for non-DNA targeted effects of radiation, including mutations, chromosomal aberrations, and changes in gene expression, which can occur in cells that were not directly exposed to radiation. Radiation oncologists are only gradually beginning to appreciate the clinical relevance of radiation-induced bystander effects, genomic instability, and abscopal effects. Since these effects are sensed by the immune system, a combination of immunotherapy and irradiation presents a new therapeutic opportunity in the future. The long-standing conventional paradigm for radiobiology for radiation effects in cellular systems has involved DNA double-strand breaks (DSBs) as the triggering lesions leading to mutation, cell death, and transformation. Depending on the linear energy transfer (LET) and dose, ionizing radiation causes a variety of different DNA lesions, including single- and double-strand breaks, DNA–protein cross-links, and DNA base damages . IonizinIn recent years, high LET irradiation is gaining greater interest in tumor therapy, due to their improved physical and radiobiological properties. It is well-known that a spatial focused deposition of high energy by heavy ions can cause complex damage types . The oxyThe oxygen enhancement ratio (OER) decreases with increasing LET . This suRecently, non-targeted irradiation effects that are not a direct consequence of the initial lesions produced by damages of the cellular DNA have been reported . Since tFigure 1). While the mechanisms underlying these effects are still not completely understood, it is very apparent that their implications are much wider than the field of classical radiobiology. The major adverse consequences caused by irradiation, such as initiation of secondary malignancies, are attributed to an inadequate repair in DNA damage in normal and tumor tissues. However, new studies have shown damage in cells that were not exposed to irradiation. These findings are explained by a potential interplay of irradiated and non-irradiated cells.Bystander effects, abscopal effects, and genomic instability are three phenomena which will lead to a paradigm shift in radiation biology (Since the discovery of X-rays in 1895, it was assumed that the deleterious effects of ionizing radiation such as mutations and carcinogenesis are mainly due to a direct damage of the DNA. Radiation-induced bystander effects are defined as biological effects in cells that are in close proximity to cells that have been irradiated . In 1992in vivo chromosomal instability (in vitro (Previous studies implicate that pro-inflammatory cytokine signaling is associated with tability  and the in vitro . The stuin vitro . Since Nin vitro . Immune in vivo experiments have demonstrated that cells of the innate immune system can be activated by ionizing radiation to produce pro-inflammatory mediators of genomic instability (Importantly, tability . Mutou-Ytability . Bystandtability .The following conclusions can be drawn from experiments analyzing bystander effects: irradiation of the cytoplasm can induce genetic effects in the nucleus that was not directly exposed to radiation. It appears that the traversal of high-LET particles through the cytosol is more efficient than through the nucleus . PresumaThe term “abscopal” is derived from the Latin prefix “ab,” meaning “away from,” and the Greek word “scopos,” meaning “target.” An abscopal effect has been defined as a reaction of cells within an organism that had not been directly exposed to irradiation, but cause tumor regression of the non-irradiated tumors . These rIt is assumed that the abscopal effect is mainly mediated by an activation of the immune system via cytokines. The abscopal effect refers to distant effects observed after local radiation therapy . TherefoImmune-mediated abscopal effects have been observed in mice with 67NR tumors after radiotherapy by studying the maturation status of dendritic cells . RadiatiClinical reports of abscopal effects after radiotherapy have been shown in different tumor types, including lymphoma, melanoma, and a variety of carcinoma . AbscopaA better understanding of abscopal effects might improve the clinical outcome of radiotherapy.Although ionizing radiation is known to induce secondary malignancies in many tissues, the underlying mechanisms at the cellular and molecular level are not completely understood. An attractive hypothesis is that radiation induces “genomic instability” in a subpopulation of cells harboring multiple mutational events that are required for the transformation of a normal tissue into an invasive tumor . DNA damThe severity of genomic instability is influenced by the LET . While tAs a clinical implication, genomic instability can serve as a marker for an increased risk to develop secondary malignancies after radiation therapy. Of particular interest is the observation that transmissible instability can be induced in somatic cells from normal individuals by exposure to ionizing radiation, leading to a persistent enhancement in the rate at which chromosomal aberrations arise in non-irradiated cells after many generations of replication .Current anti-cancer modalities such as surgery, chemo-, and radiation therapies have only limited success in the cure of solid tumors in advanced stages. During the past decade progress has been made in the understanding of the fundamental mechanisms and biological significance of the immune system in the control of cancer. The major challenge in the field is to understand the various molecular mechanisms involved in non-DNA-targeted irradiation effects that counteract tumor-related signaling pathways. Irradiation-induced abscopal and bystander effects have been shown to stimulate the immune system of cancer patients and thus might exert beneficial effects. A better understanding of the immune-modulatory effects of heavy-ion beam treatment will help to develop innovative and more effective strategies for charged-particle therapy in clinical settings.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The two principal antibody classes present in saliva are secretory IgA (SIgA) and IgG; the former is produced as dimeric IgA by local plasma cells (PCs) in the stroma of salivary glands and is transported through secretory epithelia by the polymeric Ig receptor (pIgR), also named membrane secretory component (SC). Most IgG in saliva is derived from the blood circulation by passive leakage mainly via gingival crevicular epithelium, although some may be locally produced in the gingiva or salivary glands. Gut-associated lymphoid tissue (GALT) and nasopharynx-associated lymphoid tissue (NALT) do not contribute equally to the pool of memory/effector B cells differentiating to mucosal PCs throughout the body. Thus, enteric immunostimulation may not be the best way to activate the production of salivary IgA antibodies although the level of specific SIgA in saliva may still reflect an intestinal immune response after enteric immunization. It remains unknown whether the IgA response in submandibular/sublingual glands is better related to B-cell induction in GALT than the parotid response. Such disparity is suggested by the levels of IgA in submandibular secretions of AIDS patients, paralleling their highly upregulated intestinal IgA system, while the parotid IgA level is decreased. Parotid SIgA could more consistently be linked to immune induction in palatine tonsils/adenoids (human NALT) and cervical lymph nodes, as supported by the homing molecule profile observed after immune induction at these sites. Several other variables influence the levels of antibodies in salivary secretions. These include difficulties with reproducibility and standardization of immunoassays, the impact of flow rate, acute or chronic stress, protein loss during sample handling, and uncontrolled admixture of serum-derived IgG and monomeric IgA. Despite these problems, saliva is an easily accessible biological fluid with interesting scientific and clinical potentials. Ancient people recognized the antimicrobial properties of external body fluids and used topical application of saliva, colostrum, or urine as a cure or prophylactic . Many inBesredka proposed the existence of an external antibody system in 1919 when he showed that rabbits, after oral immunization with killed Shigella, were protected against fatal dysentery irrespective of the serum antibody titer . Over th+ PCs and their immediate precursors (plasmablasts) are approximately 20 times as numerous as IgG+ PCs.A molecular basis for secretory antibodies emerged in the 1960s when it was shown that saliva contains immunoglobulin (Ig) molecules . The dis+ PCs produce mainly dimers and perhaps some larger polymers  rather than monomers  has been considered a better alternative for salivary IgA , implying that total salivary IgA may be concurrently elevated because of induction of unknown specificities ; the intestinal immune response reflected in saliva as specific IgA could hence be partly masked by ‘normalizing’ the specific titer to total IgA in the same sample , whereas in others the actual protein concentration  vaccination as well as after various infectious diseases have been published, it may be questioned how reliable or meaningful some of the results are. Many of the early reports are summarized in a previous review .+ PCs occurs scattered among acini of histologically normal major salivary glands and are often seen in clusters adjacent to ducts , they may be relatively abundantly exposed to exogenous antigens and are probably important in the defense of the oropharynx. This possibility is supported by the observation of numerous IgAir ducts  and the ir ducts . In facty glands  has beeny glands . Becauses , has indicated that almost 90% of them are variably involved in production of polymers , such as the numerous isolated lymphoid follicles and the appendix , 79. FroVibrio cholerae vaccine, the specific IgA antibody detection sensitivity in whole saliva was not better than in serum and only approximately 50% of that in intestinal lavage , such as the adenoids and palatine tonsils in humans , may be + PCs, as mentioned previously  show a striking dichotomy between the two body regions  of infants have detectable IgD in their whole saliva during the first months of life , and IgDy glands . The locE. coli, and occasionally to poliovirus, have been reported to be present in Swedish infants during the first days of life  are of the IgA1 subclass and virtually all express J chain  and S. mitior (previously S. mitis) but also Porphyromonas/Prevotella (previously Bacteroides) and Capnocytophaga species which are involved in periodontal disease , B. fragilis and E. coli is carried mainly by in the IgA2 isotype  actinomycetemcomitans were seen in subjects whose subgingival plaque harbored this microorganism (A. actinomycetemcomitans and also to P. gingivalis patients with deep periodontal pockets (Several studies have shown a positive relationship between the concentration of salivary IgA  and periorganism . Other s pockets . A possi pockets . HoweverSecretory immunity may hence be involved in host resistance to periodontal disease; but SIgA antibodies probably have little or no impact on the growth of an established dental plaque. Thus, an effect of accumulated plaque in an experimental gingivitis group of students was neither found on the total parotid IgA level nor on the IgA titer to a large number of oral bacteria, whether the subjects were smoking or not . Also, aE. coli tended to be associated with relatively good resistance to infections of the respiratory tract (+ PCs mainly with cells producing IgG or IgD, whereas exclusive replacement with IgG+ and IgM+ PCs apparently confers satisfactory mucosal resistance (Most subjects with selective IgA deficiency in developed countries are clinically healthy, but some have increased tendency to allergy and infections, especially when compensatory production of SIgM is lacking . It has ry tract . Pronenesistance .In addition, several studies have been performed on subjects with no overt immunodeficiency to see if the level of salivary IgA shows a negative relationship to susceptibility to infection or allergy, mainly in the upper airways. Usually no clear-cut associations have appeared with infections , althougOther studies have reported that total salivary IgA tends to be reduced in infection-prone children with no overt immunodeficiency . This is+ lymphocytes in the samples (Saliva of HIV-infected subjects may variably carry this virus , probabl samples . Saliva  samples .Controversial results have been published concerning the effect of HIV infection on the salivary IgA level . Such inConversely, we showed that AIDS patients had reduced output of both parotid IgA1 and IgA2 . This ob+ PCs (The level of IgG antibodies to p24 in whole saliva correlates with that in serum , again s+ PCs  than the+ PCs , 73.Adequately performed measurements of salivary IgG antibodies to HIV do show high specificity and sensitivity , and comOral microorganisms and aerodigestive antigens are continuously influenced by the two major antibody classes in saliva: SIgA and IgG. The former is synthesized as pIgA by PCs in salivary glands and is exported by an epithelial receptor-mediated mechanism. Conversely, most IgG occurring in saliva represent systemic immunity because it is derived from serum by passive diffusion – preferentially through gingival crevices – although a minor fraction may originate from glandular, gingival or tonsillar PCs. Along with the paracellular leakage of monomeric IgA and IgG antibodies, other serum-derived or locally produced factors and mediators will also appear in whole saliva such as IL-6 in patients with ulcerative colitis .The secretory immune system is subject to complex regulation which influences distinctly the activity of the various cell types involved in SIgA formation. However, a number of immunological phenomena induced by mucosal antigen exposure are poorly defined in experimental animals and still more obscure in humans. There is evidence to suggest that GALT and NALT do not contribute equally to the induction of secretory immunity in various regions of the body. Because of such compartmentalization, it is unclear whether enteric immunization is the best way to stimulate salivary IgA responses. In fact, the various salivary glands may have different preferences.In addition to possible compartmentalization of the oral secretory immune system, there are several other variables influencing the levels of total IgA and specific antibodies in salivary fluids. These include the impact of flow rate, protein loss during sample handling, difficulties with reproducibility and standardization of immunoassays, and uncontrolled admixture of serum-derived monomeric IgA and IgG to the samples. Nevertheless, salivary secretions still have interesting scientific and clinical potentials.


There is growing evidence for exaggerated oscillatory neuronal synchronisation in patients with Parkinson's disease (PD). In particular, oscillations at around 20 Hz, in the so-called beta frequency band, relate to the cardinal symptoms of bradykinesia and rigidity. Deep brain stimulation (DBS) of the subthalamic nucleus (STN) can significantly improve these motor impairments. Recent evidence has demonstrated reduction of beta oscillations concurrent with alleviation of PD motor symptoms, raising the possibility that suppression of aberrant activity may mediate the effects of DBS. Here we review the evidence supporting suppression of pathological oscillations during stimulation and discuss how this might underlie the efficacy of DBS. We also consider how beta activity may provide a feedback signal suitable for next generation closed-loop and intelligent stimulators. Converging evidence from experimental and clinical studies indicates that deep brain stimulation (DBS) of the basal ganglia (BG) can be a very successful therapeutic approach in Parkinson's disease (PD). Accordingly, there has been a progressive shift from lesion techniques, which had been used for decades, to stimulation techniques, which have the major advantage of being—at least partly—reversible. Introduced in the early 1990's, DBS, particularly of the subthalamic nucleus (STN), is now a common therapeutic solution for advanced patients. However, the mechanisms underlying its remarkable efficacy in reducing parkinsonian symptoms remain largely unclear. Here, we will explore the hypothesis that DBS of the STN suppresses pathological synchronisation at low frequencies (13–35 Hz) and that this contributes to symptomatic benefit. To this end we will begin with a critical discussion of the evidence that such oscillatory synchronisation underlies motor impairment in PD and briefly discuss the efficacy of current high-frequency DBS regimes, before considering the evidence that DBS does suppress oscillatory synchronisation.There is extensive evidence indicating that abnormally synchronised activity patterns in the beta band (13–35 Hz) are a hallmark of untreated PD. These activities have principally been found in recordings from the STN and globus pallidus interna (GPi) of PD patients withdrawn from their medication. Activities synchronised in this band have been captured in single-unit recordings and cross-correlograms of neuron pairs  , and also over other functionally connected elements of the cortex—BG network (Hashimoto et al., in vitro recordings and computational studies to suggest that high-frequency DBS may modulate local currents and, in particular, potassium and sodium conductances (Beurrier et al., The mechanisms by which STN DBS might induce beta suppression are also unclear. There is evidence to suggest that dopamine attenuates beta oscillations through the damping of resonance phenomena in local circuits (Eusebio et al., Although STN DBS seems to suppress pathological beta activity, this need not imply that STN DBS-induced motor improvement is exclusively due to beta suppression. Certainly a minority of patients do not have spectral peaks in the beta band during the post-operative period (Eusebio et al., The potential local suppression of low gamma activity by STN DBS should be distinguished from its potential effects at even higher frequencies. Thus, it has been suggested that some of the effects of DBS may relate to the driving of neurons and axons near the point of stimulation (Garcia et al., Although current fixed stimulation DBS regimes afford good control of motor symptoms in PD, they are by no means perfect. In particular, some side-effects may relate to the indiscriminate suppression or over-riding of residual physiological functioning in BG-cortical circuits (Chen et al., The advantage of the above approach is that it relies only on a strong correlation between beta activity and motor impairment at any given time. However, the growing evidence that exaggerated beta synchrony may be mechanistically linked to motor impairment in PD suggests another and more sophisticated approach. Here, the temporal patterning of stimulation is organised so that it specifically targets the putative pathological activity. There are several approaches that can be taken (Tukhlina et al., In conclusion, we have reviewed evidence that high-frequency STN DBS suppresses excessive beta synchrony in PD and that this is—at least in part—responsible for the improvement in motor symptoms. Several issues remain unresolved and in particular the mechanisms by which STN DBS suppresses beta oscillations and the possibility that other consequences of high-frequency STN DBS—unrelated to beta suppression—may contribute to symptomatic improvement.Peter Brown is a consultant to Medtronic Ltd. Hayriye Cagnan and Alexandre Eusebio have no commercial or financial relationships that could be construed as a potential conflict of interest.


Emerging evidence suggests that vascular disease confers vulnerability to a late-onset of depressive illness and the impairment of specific cognitive functions, most notably in the domains of memory storage and retrieval. Lower limb athero-thrombosis and abdominal aortic aneurysm (AAA) have both been previously associated with neuropsychiatric symptoms possibly due to associated intracerebral vascular disease or systemic inflammation, hence suggesting that these illnesses may be regarded as models to investigate the vascular genesis of neuropsychiatric symptoms. The aim of this study was to compare neuropsychiatric symptoms such as depression, anxiety and a variety of cognitive domains in patients who had symptoms of peripheral athero-thrombosis (intermittent claudication) and those who had an asymptomatic abdominal aortic aneurysm AAA.In a cross-sectional study, 26 participants with either intermittent claudication or AAA were assessed using a detailed neuropsychiatric assessment battery for various cognitive domains and depression and anxiety symptoms . Student t test and linear regression analyses were applied to compare neuropsychiatric symptoms between patient groups. AAA participants showed greater levels of cognitive impairment in the domains of immediate and delayed memory as compared to patients who had intermittent claudication. Cognitive dysfunction was best predicted by increasing aortic diameter. CRP was positively related to AAA diameter, but not to cognitive function. AAA and aortic diameter in particular were associated with cognitive dysfunction in this study.AAA patients are at a higher risk for cognitive impairment than intermittent claudication patients. Validation of this finding is required in a larger study, but if confirmed could suggest that systemic factors peculiar to AAA may impact on cognitive function. New evidence is emerging suggesting that vascular disease confers vulnerability to a late-onset of depressive illness and the impairment of specific cognitive functions, most notably in the domains of memory storage and retrieval The term “Vascular Depression” has been used to describe the broad nature of late-life depression resulting from a multitude of potential vascular mechanisms. Consistent with the Vascular Depression Hypothesis, patients with a later onset of depression have greater neuropsychological and neuroradiological abnormalities, increased disability, medical morbidity and mortality Traditionally, psychological and psychiatric conditions such as stress and depression have been associated with poor immune system function and an increased susceptibility towards infectious disease and illness The same vascular mechanisms which are associated with vascular depression may also increase vulnerability towards cognitive decline and dementia The current investigation aims to explore the relationship between abdominal aortic aneurysms (AAA) and the symptoms of depression and cognitive functioning. Both local and systemic inflammation has been identified in patients with AAA, with increased circulating levels of a range of cytokines previously identified, such as resistin and osteopontin (OPN) It is hypothesised that patients with infrarenal AAA have an increased risk towards the symptoms of depression and cognitive impairment in comparison to patients with occlusive atherosclerosis (without AAAs). We chose these two groups of patients since the mechanisms underlying the pathogenesis of abdominal aortic aneurysm (AAA) and occlusive atherosclerosis appears to have a number of distinctions Participants in the current study were recruited from around the local Townsville area that had undergone, or were currently receiving treatment for a blood vessel disease in the Vascular Clinic at The Townsville Hospital. Prior to invitation, participants were screened in order to assess research suitability. Only participants with either symptoms of intermittent claudication or an AAA were invited to participate. Ethics approval was provided by the local ethics committees of the Townsville Hospital and James Cook University. Participants gave written informed consent to participate in the study prior to commencing any study procedures. Participants were either contacted at home or were directly approached during consultation hours at the Vascular Clinic. Collection and analysis of blood serum was processed by a commercial pathology laboratory (Sullivan Nicolaides Pathology). Pathology tests included full blood count, fasting lipids, electrolytes and urea. Physician based medical history confirmed that all patients were free of mental retardation or dementia history or other psychiatric disorders such as schizophrenia. Participation in the research was entirely voluntary and no incentives were offered for participation.Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) The cognitive status of participants was assessed using the Boston Naming Test and verbal fluency tests. Inter-correlation scores between indexes remained at a moderate range of .20–.40, suggesting that each RBANS index is measuring different and distinct cognitive constructs Normative data is based on a standardised U.S. sample of 540 adults aged 20–89 years. Factors such as age, sex, race/ethnicity, education level and geographic region were evenly weighed in accordance with 1995 Census data. Reliability coefficients for each index were calculated using spilt-half reliability and Spearman-Brown correction. Reliability coefficients calculated for each subtest and age group ranged from .77 to .95 Hamilton Depression and Anxiety Scales. The Hamilton scales is a semi-structured interview consisting of 17 items which assess depressed mood, suicide, work and loss of interest, retardation, agitation, gastro-intestinal symptoms, general somatic symptoms, hypochondriasis, insight and weight loss p<.0001) was found between depression, anxiety and control groups Depressive and anxiety symptoms were assessed using the 2. Specific blood tests for this analysis included C-reactive protein (CRP), Haemoglobin (HB g/l), Triglycerides (TAG) and Low Density Lipoprotein (LDL).Patients recruited to the study had either an AAA or intermittent claudication. Intermittent claudication was diagnosed by a consultant vascular physician based on an appropriate history along with clinical signs of lower limb ischemia and computed tomography angiography (CTA) evidence of occlusive or stenotic peripheral artery disease. AAA was defined as maximal infra-renal abdominal aortic diameter ≥30 mm from axial slices of the CTA. Patients with AAA included in the current study did not have symptoms of intermittent claudication or clinical evidence of impaired lower limb blood supply. Patients with intermittent claudication included in the current study had an AAA excluded by CTA imaging (aortic diameter <30 mm). Hypertension and diabetes were defined by previous history or treatment for these conditions. Cigarette smoking classification was based on smoking history (defined as ever or never smokers). CHD was defined by a history of myocardial infarction, angina or coronary revascularisation. Body mass index (BMI) was calculated as weight(kg)/height(m)Demographic and clinical data were compared between AAA and intermittent claudication groups using Chi-square test for categorical data and two sample t-test for continuous data . We emplM = 70.3 years, SD = 6.3 years) and 6 female  participants of Caucasian descent. Measure of education was total years of school completed, the sample ranged from 8–11 years . The AAA group consisted of 10 males and 1 female whilst the intermittent claudication group had 10 males and 5 females. Sociodemographic and clinical characteristics such as diabetes, hypertension, smoking, CHD, BMI and CRP showed no significant differences between the AAA and intermittent claudication groups  with AAA patients performing worse than intermittent claudication patients. AAA group participants generally scored worse on most individual and summary cognitive scores as compared to intermittent claudication participants. Significant differences were noted on tasks involving short and long-term memory  and delayed memory retention  and overall cognitive functioning   indicating a relationship between aortic development and systemic inflammation, which may indicate an indirect relationship between CRP and memory function in our study; however no direct evidence was found for this assumption.In this cross-sectional study of vascular patients, we examined if neuropsychiatric and neurocognitive symptoms were associated with AAA presence. We found that overall cognitive function and specifically short-term and delayed memory were associated with AAA. Furthermore aortic diameter was associated with worse cognitive scores in the memory domains. In contrast, no association between depressive or anxiety symptoms and AAA was observed.The findings of this small study suggest worse cognition function in patients with AAA compared to those with occlusive peripheral artery disease. There are numerous possible explanations for these findings. AAA has been associated with distinct circulating concentrations of biomarkers, including pro-inflammatory cytokines, proteolytic enzymes and thrombus products Cytokine Model of Cognitive Function’. The model proposes that pro-inflammatory cytokines play an intimate role in the molecular and cellular mechanisms sub-serving learning, memory and cognition Early perspectives on systemic inflammation suggested that the CNS was not susceptible to inflammation or immune activation, and was considered to be mostly unaffected by systemic inflammatory and immune response It is possible that cytokines, such as resistin and OPN, which are upregulated in AAA, may also exert their effects on pro-inflammatory cytokines  either directly in the CNS, or through a process of peripheral inflammation. It is not yet clear if resistin and OPN are capable of crossing the blood brain barrier; however evidence suggests that it may indeed be possible given the small physical profile of the molecules Another possible reason for the association between cognition and AAA identified in the current study could be associated intracerebral vascular disease. Intracerebral vascular disease is one of the commonest causes of dementia. It is possible that the patients with AAA we studied could have more severe vascular pathology, including intracerebral disease, than those with intermittent claudication. We did not image the brains of patients in this study making further assessment of this impossible.The findings from this study should be interpreted in light of methodological limitations particularly in relation to the size and representativeness of the sample. The predictive power of the findings must also be received with some caution. Statistical error must be considered with any research, however, studies with small samples are particularly vulnerable to sampling errors leaving uncertainty about the negative results on depression in this study.Another limitation which should be considered is the role of gender. Gender amongst age and education is generally acknowledged as a mediating factor in cognitive function Further study in this area would be to first replicate the current findings using a larger sample of participants with a greater emphasis on gender and socio-economic status. Whilst the current study has identified observable cognitive differences between genders, the sample remains too small to make any broad generalisations to the general population. Another aspect which was not examined in this study is the relationship between specific cytokines and cognitive function in patients with AAA. In particular, systemic inflammatory markers resistin and osteopontin have been implicated in the pathogenesis of AAA, however, their role in cognitive functioning is currently unknown. Further research needs to closely examine this potential relationship.In conclusion, this study suggests that AAA and aortic diameter are associated with cognitive performance. Systemic inflammatory and vascular changes related to AAA could account for this association, however further studies are required to examine this.


Many of these EMBRYO-DEFECTIVE (EMB) genes encode proteins with an essential function required throughout the life cycle. This raises a fundamental question. Why does elimination of an essential gene in Arabidopsis often result in embryo lethality rather than gametophyte lethality? In other words, how do mutant (emb) gametophytes survive and participate in fertilization when an essential cellular function is disrupted? Furthermore, why do some mutant embryos proceed further in development than others? To address these questions, we first established a curated dataset of genes required for gametophyte development in Arabidopsis based on information extracted from the literature. This provided a basis for comparison with EMB genes obtained from the SeedGenes dataset. We also identified genes that exhibited both embryo and gametophyte defects when disrupted by a loss-of-function mutation. We then evaluated the relationship between mutant phenotype, gene redundancy, mutant allele strength, gene expression pattern, protein function, and intracellular protein localization to determine what factors influence the phenotypes of lethal mutants in Arabidopsis. After removing cases where continued development potentially resulted from gene redundancy or residual function of a weak mutant allele, we identified numerous examples of viable mutant (emb) gametophytes that required further explanation. We propose that the presence of gene products derived from transcription in diploid (heterozygous) sporocytes often enables mutant gametophytes to survive the loss of an essential gene in Arabidopsis. Whether gene disruption results in embryo or gametophyte lethality therefore depends in part on the ability of residual, parental gene products to support gametophyte development. We also highlight here 70 preglobular embryo mutants with a zygotic pattern of inheritance, which provide valuable insights into the maternal-to-zygotic transition in Arabidopsis and the timing of paternal gene activation during embryo development.The SeedGenes database ( DrosophilaCaenorhabditisEssential genes have long played an important role in microbial and medical genetics. In recent years, several factors have contributed to the establishment of large datasets of essential genes in microorganisms: the failure of mutant cells to proliferate is readily detected in high-throughput gene disruption experiments; the products of essential genes define promising targets for novel, antimicrobial compounds relevant to human health; and lethal mutations in essential genes help to define the minimal gene set required to sustain a living cell emb) mutants of Arabidopsiswww.seedgenes.org), which presents detailed information on genes required for embryo development in ArabidopsisEMB genes with an essential cellular function required to produce a normal, mature embryo. Many EMB proteins are likely to be required throughout the life cycle. Mutants exhibit defects in embryogenesis because that is when the absence of a functional gene product first becomes critical.Our desire to establish a comparable dataset of essential genes in a model plant Arabidopsis through the isolation and characterization of mutants defective in gametophyte development ArabidopsisOther research groups have pursued a complementary approach to the analysis of essential genes in In order to characterize the cellular disruptions that lead to defects in embryo rather than gametophyte development, we first established a curated dataset of gametophyte essentials that could be compared with the embryo essentials found in the SeedGenes database. We then used this dataset in combination with an updated SeedGenes dataset to determine what factors most often distinguish mutants defective in embryo development from those defective in gametophyte development. Specifically, we wanted to understand how mutants defective in embryo development are able to survive gametophyte development when an essential function required throughout the life cycle is disrupted, and conversely, why some mutants exhibit gametophyte lethality instead. In addition, we sought to explain why some mutant embryos reach a later stage of development than others, and what general conclusions might be drawn about the relationship between gene function and mutant phenotype.Arabidopsis. Although past work has elegantly demonstrated the diversity of transcripts found in male and female gametophytes Arabidopsis, pollen tubes travel short distances in order to reach the ovule, which increases the likelihood that male gametophytes lacking an essential gene product can survive and participate in fertilization. By contrast, equivalent gene disruptions in plants with long pollen tubes, most notably maize, are more likely to result in male gametophyte lethality.This analysis led us to conclude that pre-meiotic gene expression in diploid microsporocytes and megasporocytes is likely to be an important feature of reproductive development in This raises another important question: how can mutant embryos disrupted in an essential gene survive beyond the globular stage of development, given that levels of gene products stored in mutant gametophytes are insufficient to support unlimited growth? Several additional factors, including gene redundancy, residual gene function, delayed activation of specialized pathways, and diffusion of products from maternal tissues likely influence the stage of development reached by mutant embryos before seed desiccation. In addition, some gene products may perform a more limited role in an essential cellular process than others, and this can allow further development than might otherwise be expected. We conclude that the datasets of genes required for embryo and gametophyte development presented here provide a useful resource for comparative studies with other multicellular eukaryotes and valuable insights into the molecular basis of reproductive lethality in a model plant.Arabidopsis appears to contain 750 to 1000 EMB genes located throughout the genome EMB loci in Arabidopsis. Many of the EMB gene identities revealed through work in our laboratory have not been published before. The current dataset, which excludes six atypical or problematic SeedGenes loci, likely represents about 40% saturation. This is sufficient to begin addressing the diversity of protein functions required for cell viability and embryo development. Regarding the level of certainty that each gene identified is responsible for the mutant phenotype described, we recognize two distinct categories of EMB genes: those labeled as confirmed, either through molecular complementation or the analysis of additional mutant alleles disrupted in the same gene, and those labeled as not confirmed, where robust genetic data alone indicate close linkage between the gene and mutant phenotype (www.seedgenes.org). Approximately 100 of the EMB genes in Based on the abundance of embryo-defective mutants and the frequency of duplicate alleles in mutagenesis experiments, EMB loci associated with additional defects in gametophyte development, we divided the remaining 352 “true” EMB loci into terminal phenotype classes, ranging from preglobular to cotyledon. The results are shown in EMB genes are required at the earliest, preglobular stages of embryo development. Although such broad descriptions fail to capture important phenotype details, they demonstrate on a global scale that EMB genes differ in how far embryo development can proceed when their functions are disrupted. Furthermore, as discussed later, the existence of a substantial number of embryo-defective mutants with a Mendelian pattern of inheritance, characterized by 25% mutant seeds in siliques of selfed heterozygotes, along with a preglobular stage of embryo arrest, has implications for the timing and extent of the maternal-to-zygotic transition in gene expression during plant embryo development.To assess the terminal stage of development reached by mutant embryos prior to seed desiccation, we evaluated more than 700 mutants classified in SeedGenes as having an embryo-defective mutant phenotype. When more than one mutant allele was available, we chose what appeared to be the strongest allele based on phenotype and known location of the mutation. After removing 44 Arabidopsis has been vague and subjective. Some embryo mutants exhibit defects in gametophyte development, and some gametophyte mutants, when maintained as heterozygotes, produce a low frequency of viable mutant gametes. Embryo mutants with defects in male gametophyte development often exhibit a reduced percentage and nonrandom distribution of mutant seeds in heterozygous siliques. This “certation” effect was originally noted by Müller Historically, the distinction between embryo and gametophyte mutants in EMB loci to distinguish them from “true” EMB genes that lacked knockout defects in gametophyte development. We also examined publications dealing with SeedGenes loci to identify additional examples of EMB genes associated with defects in gametophyte development. By definition, mutants assigned to the EMG subclass are known or predicted to produce at least 10% defective seeds following self-pollination of heterozygotes and have either a reduced frequency of mutant seeds overall, too few mutant seeds at the base of the silique, or an excessive number of aborted ovules. We then established another subclass of embryo essential genes with more severe defects in gametophyte function, labeled GEM (Gametophyte-Embryo), to recognize cases where heterozygotes are known or predicted, based on reduced gamete transmission of the mutant allele, to produce between 2% and 10% mutant seeds. Both the EMG and GEM subsets of EMB genes, therefore, exhibit a combination of embryo and gametophyte defects. The difference lies in the extent of these defects and their impact on fertilization and embryo development. We chose 2% mutant seeds as the baseline for the GEM subclass because this corresponds to a single mutant seed per silique, on average, which is marginally above the background rate of spontaneous seed abortion Arabidopsis, recorded male and female transmission ratios when documented, and noted information about frequencies of mutant embryos produced. This provided the foundation for the curated dataset presented here of genes required for gametophyte development.Because the SeedGenes database includes information, when available, on mutant seed ratios and distributions, we assembled a list of SeedGenes loci that appeared to have defects in both embryo and gametophyte development. These were assigned to an EMG (Embryo-Gametophyte) subclass of EMB dataset found at SeedGenes, we excluded unconfirmed, putative gametophyte loci where only a single mutant allele was characterized and where flanking sequence was not obtained from both sides of the insert. This eliminated from consideration a sizeable number of candidate genes. But it also increased the likelihood that genes identified as being responsible for the gametophyte defects were indeed correct. The resulting dataset, shown in EMB genes devoid of gametophyte defects  of the Arabidopsis proteome. EMB proteins lacking a match with at least an e-30 level of significance were viewed as likely to be unique. Those with more significant matches were classified as potentially redundant, although in many cases these genes may still be functionally distinct. We chose an e-30 cutoff value to be relatively stringent in our selection of unique genes because we wanted to identify those EMB genes where survival of mutant gametophytes was least likely to result from genetic redundancy. The resulting dataset of 152 “unique true” (UT) EMB loci is presented in Two potential explanations for how mutants defective in embryo development might survive gametophyte development involve genetic redundancy and mutant allele strength. The first model proposes that duplicated genes with redundant functions compensate for the loss of essential EMB loci were associated with one or more mutant alleles that contained an exon insertion in the first or middle third of the gene. Another 23 had an intron insertion in the same (A/B) region, and 19 contained an insertion in the final third of the gene. When we compared the terminal phenotypes of 89 mutants carrying A/B exon disruptions with those of 42 mutants containing A/B intron or C region disruptions, we found no significant differences  in the frequencies of early versus late embryo phenotypes (data not shown). We also found no significant differences  in the phenotype distributions obtained when the unique and potentially redundant datasets of “true” EMB genes were compared  and middle (B region) of a gene may be more likely to eliminate protein function than those at the end of a gene (C region). We also distinguished insertions found in introns from those located in exons, although when a single flanking sequence was available and localized to an intron, we were uncertain whether an adjacent exon might also be disrupted. Overall, 89 of the 152 UT-compared , althougEMB loci have been documented in the literature. We have uncovered several cases, including EMB1989/NRPB2, EMB2779/LCB1, RSW1/CESA1, EMB24/AtASP38, and EMB2776/PRP4/LISEMB genes that complete embryo development and exhibit other defects as seedlings or adult plants , late expression of a transcriptional regulator (wox2), delayed requirements for specialized pathways in metabolism , and peripheral roles of EMB proteins in essential cellular processes . Other notable features of this dataset include the abundance of genes encoding chloroplast-localized proteins (15 loci) and proteins with unknown cellular functions (12 loci). We conclude that in some cases, the late embryo phenotypes observed in these knockouts are consistent with known protein functions that become important late in embryo development. In other words, elimination of these specialized functions would not be expected to result in lethality immediately after fertilization. In other cases, the protein product likely performs a peripheral role in an essential process required throughout growth and development. The survival of mutant gametophytes in emb/EMB heterozygotes is best addressed, therefore, using EMB genes with knockout phenotypes at early stages of embryo development, because in those cases, survival of the mutant gametophyte contrasts with the documented, early lethality found in the mutant embryo.Disruptions of some UT-EMB genes with a preglobular or zygotic terminal phenotype, which is more consistent with an essential gene product required throughout development  and tRNA modification in the cytosol  to chromosome dynamics  and microtubule assembly . Surprisingly, chloroplast-localized proteins are missing from this dataset and genes encoding proteins with unknown functions are reduced in occurrence. We propose that in some of these knockouts, residual gene products from the wild-type allele transcribed in diploid sporocytes enable mutant gametophytes to participate in fertilization. Development of homozygous mutant seeds arrests a short time later, when these reserves become depleted. Diffusion of nutrients from surrounding maternal tissues cannot be the primary source of metabolites for all stages of male gametophyte development because pollen-tube growth in Arabidopsis can proceed in vitro on a simple, defined medium We then focused on a dataset of 25 UT-elopment . Once agArabidopsis. Mutant alleles unable to support this essential process are likely not transmitted through male or female gametes in vitro is blocked in the presence of translation inhibitors In principle, the gene products contributed by diploid sporocytes to mutant gametophytes could be either RNA or protein, or a combination of both. Male gametophytes have long been known to be active in transcription. Post-meiotic gene expression was first demonstrated in maize by analyzing isozyme patterns for dimeric enzymes in pollen from heterozygous plants SHORT SUSPENSOR (SSP) locus, where a transcript produced in male gametophytes is translated in the zygote and modulates an important signaling pathway early in embryo development Knockout mutants disrupted in essential subunits of RNA polymerase are unable to transmit the mutant allele through female gametes and exhibit reduced transmission of the mutant allele through male gametes EMB genes in haploid gametophytes by comparing the published transcript profiles Arabidopsis: (a) 59 GAM or GEM loci with severe defects in male transmission  48 smission ; and (c)25 loci;  or globu25 loci;  stage ofEMB loci with early defects in seed development are more likely to be absent from mature pollen  than transcripts from gametophyte loci with severe defects in male transmission. This suggests that survival of emb mutant pollen tubes often depends on proteins synthesized during pollen development and not on stored mRNAs translated after pollen maturation. This model predicts that EMB proteins should be present in mature pollen grains when their transcripts are absent. Unfortunately, the published proteome datasets for ArabidopsisEMB genes with early stages of embryo arrest are included in these datasets, and even the GAM, GEM, and EMG classes are infrequently represented  is not significantly higher for UT-EMB genes with early embryo defects than for male gametophyte essentials , or for all genes with transcripts detected in microsporocytes . We therefore propose that in most cases where transcription of an EMB gene in microsporocytes supports gametophyte development, survival of mutant (emb) gametophytes results from relatively modest levels of residual transcript contributed to young microspores and subsequently translated during pollen development. By contrast, male gametophyte defects in gametophyte lethals arise when post-meiotic expression of an essential gene is required to supplement whatever functional transcript remains from pre-meiotic expression in microsporocytes. Confirmation of this model could be achieved in the future by determining whether down-regulation of an EMB gene in microsporocytes changes an embryo lethal into a gametophyte lethal.This model is further supported by our analysis of the dataset of Yang et al. We focused our analysis on genes required for male gametophyte development because data for female meiocytes are more difficult to obtain and evaluate. One could argue that because the number of confirmed, female gametophyte mutants identified to date is fewer than the number of male gametophyte mutants, megasporocytes may be more effective than microsporocytes at contributing functional gene products to progeny haploid cells. This cannot, however, be true for all essential genes, because RNA polymerase knockouts are not transmitted through the female side, which indicates that some essential genes must be transcribed in female gametophytes. While it seems logical that knockouts of genes required for rapid tip growth should exhibit male rather than female gametophyte lethality, the mechanism responsible for severe female defects in knockouts with minimal disruption of male gametophytes remains obscure. In some cases, gene duplication may be involved, with a redundant gene supplying the missing function on the male side when the paralog required for female development is disrupted.Arabidopsis megasporocytes EMB genes with early embryo defects, 62% produced transcripts detected in at least three of four megasporocyte samples analyzed 2 = 0.24; p = 0.62) from that observed for the 75 UT-EMB genes with early phenotypes, whose knockouts survive gametophyte development. This result mirrors the trend noted above for microsporocyte transcripts involving male gametophyte essential genes. Furthermore, we found no significant difference  in the likelihood of transcripts being present in megasporocytes, and available in the egg cell for contribution to the zygote, when we compared our datasets of UT-EMB genes with preglobular, globular, and cotyledon mutant phenotypes. We have therefore been unable to establish a role for pre-meiotic transcripts in supporting the continued growth of mutant embryos to a globular stage and beyond. By contrast, observed differences in the severity of phenotypes between male and female gametophyte mutants disrupted in essential genes, and between embryo and gametophyte lethals overall, appear in some cases to reflect differences in the amount of residual, essential gene products donated from sporocytes to developing gametophytes. The practical applications of these different patterns of reproductive lethality in Arabidopsis remain to be explored.The recent publication of transcriptional profiles for Arabidopsis, we utilized the dataset of EMB genes presented here to address another central issue in plant development related to stored, essential gene products. Two fundamental questions have figured prominently in recent discussions of gene expression during plant embryo development. One involves the extent of paternal allele contributions to the developing embryo and endosperm tissue. The other concerns when during development the transition from maternal to zygotic gene expression takes place. Both of these questions have been widely studied in maize ArabidopsisIn addition to evaluating sporocyte transcription and gametophyte viability in embryo-defective mutants of emb173) with an unusual pattern of inheritance: heterozygous plants produced 50% mutant seeds regardless of pollen genotype MEA, were isolated and characterized elsewhere, the gene was shown to encode a polycomb group protein that regulates cell proliferation, and the paternal allele was found to be imprinted by DNA methylation, particularly in the endosperm www.seedgenes.org] but differing seed phenotypes and mechanisms of maternal influence have also been described EMB genes with a normal, zygotic pattern of inheritance.Our interest in this field began almost 20 years ago, when we isolated and characterized a mutant  in transcripts produced following reciprocal crosses between accessions seemed to support these observations. This model was soon challenged through further work on ArabidopsisArabidopsisWe were therefore surprised by a report 12 years ago that claimed widespread, delayed activation of the paternal genome during seed development in Arabidopsis embryos at the 2–4 cell stage indicated that genome-wide, transcripts from the maternal allele predominate over those from the paternal allele through at least the globular stage of development. Following a cross between Columbia and Landsberg erecta accessions, almost 90% of the 135,000 informative sequence reads derived from 4,000 loci genome-wide appeared to be of maternal origin at the 2–4 cell embryo stage. Overall, the paternal contribution increased somewhat by the globular stage, and some loci produced equal amounts of transcripts from both maternal and paternal alleles, but early in development, the vast majority of loci seemed to be under maternal control. The authors stated that their work reconciled observations made by different laboratories of both maternal and zygotic effects during embryogenesis in Arabidopsis. But their data still conflict with the existence of large numbers of embryo-defective mutants with a Mendelian pattern of inheritance and cellular defects observed well before the globular stage.Recently, a large-scale RNA sequencing effort Arabidopsis have cited at most a handful of EMB genes with known defects early in development, we decided to establish a more robust dataset of known EMB genes with arrested embryos that fail to reach the globular stage. The resulting collection of 70 genes, corresponding to about 18% of all 396 EMB genes identified to date, is presented in EMB30/GNOM, where the initial defects are observed long before embryo development becomes arrested, and other mutants with late terminal phenotypes where early defects remain to be uncovered. Nevertheless, with this dataset in hand, we were intrigued to learn of another study on the maternal-to-zygotic transition in Arabidopsis that followed an approach similar to Autran et al.EMB genes listed in Because all of the publications on the maternal-to-zygotic transition in Arabidopsis, and that unlike the situation observed in most animal systems, early expression of essential genes is required to support a wide range of cellular functions beginning shortly after fertilization. From a historical perspective, elucidating the role of paternal gene expression in plant embryo development provides a compelling illustration of the value of large datasets of genes with essential functions during growth and development. A more detailed picture of the molecular basis of reproductive lethality in Arabidopsis should emerge as additional genes with essential functions are identified in the future.When these results are evaluated in the context of several decades of research on embryo-defective mutants, we believe there is compelling evidence for early activation of the zygotic genome in EMB genes EMB genes that were missed in past screens EMB genes EMB genes that had previously been mapped but not cloned EMB genes identified in our laboratory and found in The last published, comprehensive dataset of Arabidopsis. We began with a list of all known genes with a mutant phenotype published 8 years ago www.arabidopsis.org) that were thought to be associated with phenotype information, and performed a variety of PubMed searches (www.ncbi.nlm.nih.gov) with relevant keywords . Publications describing the large-scale identification of genes required for male and female gametophyte development 1 plants) / (number of wild-type F1 plants). Subclasses of genes with different mixtures of embryo and gametophyte defects  and levels of transmission reduction  are defined in the second and third tabbed spreadsheets in Several approaches were taken to identify and evaluate genes reported to be required for gametophyte development in Table S1Updated SeedGenes dataset of 396 EMB genes of Arabidopsis.(XLS)Click here for additional data file.Table S2173 Arabidopsis genes with a gametophyte-defective phenotype.(XLS)Click here for additional data file.Table S3352 true EMB genes without evidence of gametophyte defects.(XLS)Click here for additional data file.Table S472 GEM and EMG genes with both embryo and gametophyte defects.(XLS)Click here for additional data file.Table S5152 unique true EMB genes of Arabidopsis.(XLS)Click here for additional data file.Table S6Examples of EMB genes with other phenotypes in weak alleles or RNAi lines.(XLS)Click here for additional data file.Table S736 unique true EMB genes with late phenotypes in putative knockout alleles.(XLS)Click here for additional data file.Table S859 GAM or GEM genes with severe defects in male gamete transmission.(XLS)Click here for additional data file.Table S948 GEM or EMG genes with moderate defects in male gamete transmission.(XLS)Click here for additional data file.Table S1050 unique true EMB genes with early globular to globular phenotypes.(XLS)Click here for additional data file.Table S11Detection of proteins required for male gametophyte and embryo development in pollen.(XLS)Click here for additional data file.Table S1270 EMB genes where mutant embryos arrest at the preglobular stage of development.(XLS)Click here for additional data file.


Spatial cueing has been used by many different groups under multiple forms to study spatial attention processes. We will present evidence obtained in brain-damaged patients and healthy volunteers using a variant of this paradigm, the hybrid spatial cueing paradigm, which, besides single-target trials with valid and invalid cues, also contains trials where a target is accompanied by a contralateral competing stimulus . This allows one to study invalidity-related processes and selection between competing stimuli within the same paradigm. In brain-damaged patients, lesions confined to the intraparietal sulcus result in contralesional attentional deficits, both during competition and invalid trials, according to a pattern that does not differ from that observed following inferior parietal lesions. In healthy volunteers, however, selection between competing stimuli and invalidity-related processes are partially dissociable, the former relying mainly on cytoarchitectonic areas hIP1-3 in the intraparietal sulcus, the latter on cytoarchitectonic area PF in the right inferior parietal lobule. The activity profile in more posterior inferior parietal areas PFm and PGa, does not distinguish between both types of trials. The functional account for right PF and adjacent areas is further constrained by the activity profile observed during other experimental paradigms. In a change detection task with variable target and distracter set size, for example, these inferior parietal areas show highest activity when the stimulus array consists of only one single target, while the intraparietal sulcus show increased activity as the array contains more targets and distracters. Together, these findings lead us to the hypothesis that right PF functions as a target singleton detector, which is activated when a target stands out from the background, referring both to the temporal background (expectancy) and the momentaneous background . Spatial attention encompasses a wide set of divergent processes that govern the distribution of attentional weights over locations that are, or may be, occupied by objects. A powerful concept in spatial attention research, stemming from neurophysiology and computational neurobiology, is the “attentional priority map”. The attentional priority map refers to a topographic representation of attentional weights  is consistently more active compared to single stimulus conditions , has been implicated by numerous functional imaging studies . This information is incorporated in the probabilistic maps  has a critical role in selection between competing stimuli (Vandenberghe et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This dogma has been seriously challenged by several independent laboratories in recent years [Both the premature and age-induced ovarian failure are associated with significant medical and quality of life complications. Moreover, millions of children and adults suffer from iatrogenically-induced premature gonadal failure as a result of chemotherapy treatments. In the case of chemotherapy, commonly used cancer drugs such as doxorubicin or cyclophosphamide result in apoptotic death of primordial follicle reserve -3, probant years -5, even While there has not been direct laboratory evidence in humans supporting or refuting the concept of germ cell renewal, our clinical and laboratory observations lend support to the existence of oocyte regeneration in human ovaries -7. Ovariet al [et al observed. Perhaps higher androgen levels in the circulation from younger males are responsible for increased follicular atresia that the authors observed, as this is a known effect of potent androgens. As the androgen levels drop in the senescing mice, regenerative effects of androgens on germ cells become predominant, resulting in overall increase in primordial follicle reserve. This hypothesis is also consistent with recent clinical observations that weak androgens may be associated with improved ovarian follicle reserve. DHEAS, a weak androgen, is associated with improved ovarian response to fertility drugs and enhanced ovarian reserve in aging women [Our clinical observations are in line with the parabiotic model reported by Niikuraet al , in thisng women , and Polng women .As such the recent work by the Tilly laboratory  and our


In a previous meta-analysis across almost 200 neuroimaging experiments, working memory for object location showed significantly stronger convergence on the posterior superior frontal gyrus, whereas working memory for identity showed stronger convergence on the posterior inferior frontal gyrus . As similar locations have been discussed as part of a dorsal frontal—superior parietal reach system and an inferior frontal grasp system, the aim of the present study was to test whether the regions of working-memory related “what” and “where” processing show a similar distinction in parietal connectivity. The regions that were found in the previous meta-analysis were used as seeds for functional connectivity analyses using task-based meta-analytic connectivity modelling and task-independent resting state correlations. While the ventral seed showed significantly stronger connectivity with the bilateral intraparietal sulcus (IPS), the dorsal seed showed stronger connectivity with the bilateral posterior inferior parietal and the medial superior parietal lobule. The observed connections of regions involved in memory for object location and identity thus clearly demonstrate a distinction into separate pathways that resemble the parietal connectivity patterns of the dorsal and ventral premotor cortex in non-human primates and humans. It may hence be speculated that memory for a particular location and reaching towards it as well as object memory and finger positioning for manipulation may rely on shared neural systems. Moreover, the ensuing regions, in turn, featured differential connectivity with the bilateral ventral and dorsal extrastriate cortex, suggesting largely segregated bilateral connectivity pathways from the dorsal visual cortex via the superior and inferior parietal lobules to the dorsal posterior frontal cortex and from the ventral visual cortex via the IPS to the ventral posterior frontal cortex that may underlie action and cognition. Concepts on the organisation of human memory are mainly based on the long-held dichotomy of short-term (STM), respectively, working memory (WM) and long-term memory (LTM)  tasks. In particular, there was a clear distinction between brain regions that are reliably activated in tasks requiring the subjects to remember object identity and object location, respectively . This approach to functional connectivity assesses which brain regions are co-activated above chance with a particular seed region in functional neuroimaging experiments. This method takes advantage of the fact that functional imaging studies are normally presented in a highly standardized format using ubiquitously employed standard coordinate systems, and the emergence of large-scale databases, which store these information. The first step in MACM is to identify all these experiments in a database that activate the seed region. After that, quantitative meta-analysis is employed to test for convergence across the foci reported in these experiments. As experiments are selected by activation in the seed, the highest convergence will be observed in the seed region. Significant convergence of reported foci in other brain regions, however, indicates consistent co-activation, i.e., functional connectivity with the seed , which contained at the time of analysis approximately 10,000 neuroimaging experiments. We only included studies that reported group analyses of functional mapping experiments of healthy subjects. Those studies that dealt with disease or drug effects were excluded. No further constraints  were enforced, yielding approximately 6,500 experiments for analysis. Note that we considered all eligible BrainMap experiments because any pre-selection of taxonomic categories would have constituted a fairly strong a priori hypothesis about how brain networks are organized. This was a conservative approach, given that an understanding of how psychological constructs, such as action and cognition, map on regional brain responses remains elusive  and transformed into Z scores for display.To delineate task-based functional connectivity, i.e., co-activations of the regions implied by the previous meta-analysis  Fig. a, we thuZ scores obtained from the inspected MACM maps. The experiments contributing to either analysis were then pooled and randomly divided into two groups of the same size as the sets of contrasted experiments  and dorsal (where) seeds were established by first calculating the voxelwise differences of the 2, 36 axial slices (3.1 mm thickness) covering the entire brain]. The first four scans were excluded from further processing analysis using SPM8 (http://www.fil.ion.ucl.ac.uk/spm). The EPI images were first corrected for movement artefacts by affine registration using a two pass procedure in which the images were first aligned to the initial volumes and subsequently to the mean after the first pass. The obtained mean EPI of each subject was then spatially normalized to the MNI single subject template . For each subject, 300 resting state EPI images were acquired using blood-oxygen-level-dependent (BOLD) contrast  and dorsal extrastriate visual areas (V3d and the cortex in the vicinity of V5) as well as with the posterior superior frontal gyrus.In contrast, the IPS, i.e., the parietal region more closely connected to the ventral seed, featured significantly stronger task-based and task-independent functional connectivity with bilateral ventral extrastriate visual cortex, the anterior insula, (pre-) SMA, and the posterior inferior frontal gyrus. Moreover, significant differences in connectivity with the right posterior lateral prefrontal cortex (LPFC) and the right cerebellum (Lobule VI) were also found Fig. a.Fig. 6Finally, we calculated the same MACM and resting state functional connectivity differences for the parietal regions showing differential connectivity with the two frontal seed regions (IPS vs. SPL/IPL) using the very same frontal seed regions as inclusive masks. The procedure revealed that, indeed, the significant distinction in connectivity patterns was reciprocal. That is, those regions that were defined by stronger connectivity with the ventral frontal seed also featured significantly stronger connectivity with this region when used as seeds themselves. In turn, regions that were defined by stronger connectivity with the dorsal seed also showed significantly stronger connectivity with this region Fig. b. While As described before, the frontal seed regions show stronger connectivity with distinct parietal regions, respectively. The functional characterization of the networks found by these seeds and their respective parietal connections is shown in Fig. In contrast, the ventral seeds and the IPS featured a significant overrepresentation of behavioural domains related to visual shape and configuration  processing as well as those related to general visual/attentive functions. The paradigm class analysis then revealed that the latter could be attributed to tasks involving passive and active (recognition) viewing of images as well as following film presentations Fig. a. When cIn a previous coordinate-based meta-analysis of functional neuroimaging studies  time-series, in MACM the unit of observation is thus represented by a particular neuroimaging experiment. Large-scale databases such as BrainMap have facilitated the assessment of such task-based functional connectivity analyses, which should present an optimal complement to resting-state functional connectivity analyses. Whereas the former delineates interactions during an externally driven, task-based state, the latter reveals interactions in an endogenously driven, task-free state (Eickhoff and Grefkes It is important to appreciate that both approaches not only differ in the (presumed) confounds [task-based (MACM) functional connectivity is intrinsically constrained to tasks that may be performed in a scanner environment and influenced by spatial uncertainty inherent to neuroimaging results whereas task-free (resting state) functional connectivity may be more susceptible to preprocessing and physiological confounds]. Rather, they also represent different conceptual models: While MACM networks are externally controlled and targeted at stimuli and reactions, resting state networks reflect ongoing, endogenously controlled cognition.Given this complementary nature, a combination of both approaches allows the delineation of functional connectivity independently of the current “mode” of brain function . Thus, a robust estimation of the functional connectivity patterns of a particular seed region may consist of assessing task-based (MACM) as well as task-free (resting-state) functional connectivity and constraining inference to those regions showing convergent evidence in both approaches. It may be argued that convergent findings across both approaches, as presented in the current study, should represent the core of the respective network-interactions because the ensuing regions show the respective coupling pattern across two fundamentally different “modes”  parietal lobule. What is remarkably, though, is that we based our seed regions on a contrast of different working-memory tasks rather than any motor-related behaviour and still observed patterns of parietal connectivity very much like those known for action-related circuits. The convergence between the connectivity patterns of regions supporting memory for object identity and object location and those for ventral and dorsal premotor areas as well as the performed functional characterization Fig.  hence prThe hypothesis voiced in the conclusion to the previous section is corroborated by a comparison of the two seed regions with the functional definition of the vPMC and dPMC provided in a large-scale meta-analysis of motor control tasks  visual cortex cf. Fig. a. When sHere it is important to note, that our analysis did not aim at identifying analogues to previously reported dorsal and ventral visual streams or even the classical dorsal/ventral stream proposed by Ungerleider and Haxby . Rather,Interestingly, we did not find a clear segregation of the LPFC in our analysis in spite of previous evidence towards such distinction  the human LPFC is responsible for more abstract global processes, or in other words, occupies a more integrative role rather than being part of a “what” and “where” distinction.It could be argued that the present study showing connectivity of the posterior inferior frontal gyrus with the IPS and ventral visual cortex as well as connectivity of the posterior superior frontal cortex with the IPL/SPL,and dorsal/medial visual cortex is primarily a replication of previous work on the organisation of premotor-parietal networks in humans and monkeys. While we were indeed able to show similar connections as previous studies on the premotor cortex, it is important to empathize that these findings were obtained when seeding from regions showing differential involvement in WM tasks. That is, premotor networks were replicated in spite of the fact that the seeds were defined by differential engagement in cognitive (WM) functions rather than motor behaviour. The current study thus showed that regions in the posterior frontal cortex preferentially engaged by memory for object location and identity, respectively, are part of similar networks as discussed for the ventral and dorsal premotor cortex. This strongly argues for a congruence between the seed regions defined by WM functions and the premotor cortex and thereby sheds an interesting light on the relation of cognitive and motor systems.Our seeds were defined by assessing differential neural correlates of working memory for object identity or object location, that is, they were specified exclusively by their cognitive functions. Nevertheless, their location and parietal connectivity suggests a close homology to premotor areas in monkeys and humans (vPMC/dPMC). Functionally, this distinction in the primate premotor system has been related to differential involvement in reaching movements in space as opposed to fine motor skills for manipulation  and location  centred visual-parietal-premotor systems are shared by both cognitive and action-related processes and hence the same neural system may support memorizing object location in space and reaching towards it, while another system may underlie both memory for object properties and identity  and finger positioning for manipulation. In such scenario, the visual nodes would provide the analysis of the sensory input as well as first conceptual representations. Potentially, parietal areas may code supra-modal representations arising not only from visual but also auditory and somatosensory “what” and “where” information and at the same time tie  motor actions towards these. Premotor regions would then most likely hold the most utilizable or output-ready representation, which may be used to perform an action—or judge whether the probe matches the previously memorized object in the relevant dimension. From an evolutionary perspective, it is well conceivable that these pathways were originally primarily devoted to action-related processing  and have assumed more abstract cognitive functions in primates and particularly humans. That is, the decisive step in the evolution of neural systems for action and cognition may be the decoupling of action execution from the preparatory aspects, using exactly the same brain systems to address abstract problems. Thus, many cognitive functions may be simulations of motor preparation after detaching the need for sensory input and movement output.


Most natural behaviors are arranged hierarchically, with complex actions composed of serial combinations of more basic motor gestures. The learning of courtship song in birds presents an ideal model system for understanding the neural mechanisms underlying such learning. In the most commonly studied species, the zebra finch, songs are learned by young males from their fathers or other male tutors. They are highly stereotyped by adulthood, and yet vary greatly between individual birds. The basic element of song is generally considered to be the syllable, a period of continuous expiratory vocalization separated by silence and a brief period of inspiration. These brief silences make it relatively easy to segment the acoustic signal into syllables, and previous studies have examined song development by extracting acoustic features from the sound spectrogram and then averaging these features over syllables . However, despite experimental evidence that syllables form behavioral units, the syllabic structure of song is not readily apparent in many electrophysiological recordings. Individual neurons in the song motor pathway generally produce 5-10 msec bursts of activity that are aligned to song production with millisecond precision and have no obvious relationship to syllable onsets or offsets.We have developed a suite of software that enables semi-automatic the tracking of sub-syllabic features in a large corpus of acoustic data recorded from developing zebra finches with live tutors. Our approach is based on three basic ideas. First, we segment song into syllables and annotate individual syllables based on averaged feature values in a manner similar to previous approaches. Second, we use a coarse temporal alignment to average across renditions and produce an averaged ‘template’ for each syllable type. Then we use dynamic time warping to align each syllable rendition to this averaged template. Finally, we measure how well each rendition matches the template, and use this measure to perform semi-automated screening of possible outliers and syllable misclassifications.We are currently applying this approach to track the development of song features during the later stages of song development. To quantify the degree to which the mechanisms of song development respect a division of song into syllable-based units, we will measure song-by-song correlations between different features in different sub-components of the same syllable and compare these to feature correlations across syllables.


Perceptions are changing concerning the functional role of rhythmic brain activity. There is increasing evidence that low-frequency (2–25 Hz) neural oscillations are not simply epiphenomena, but reflect an essential mechanism for coordinating brain function. A leading mechanistic hypothesis for how rhythms might directly influence neural circuit function is by synchronously modulating the membrane potentials of many neurons such that mutual dendritic input between members of this population is more likely to induce action potentials at specific times  are slower than many of the timescales of perception and action. Secondly, low frequency oscillations are coherent across centimeters of the cortical surface, and thus can exert a similar influence on local circuits that implement dramatically different computations. Thirdly, the modulation of population firing by low-frequency rhythms is usually greater during rest conditions than during perceptual or motor tasks, when rhythms are often suppressed (desynchronized)—such that rhythmic entrainment is most pronounced during disengaged cortical states Miller, . Thus, tFindings are now emerging concerning the higher order spatial and temporal properties of rhythmic entrainment as captured by PAC. In a recent article van der Meij et al. reported on the spatial distribution of PAC across human cerebral cortex  a more clearly elucidated physiological basis for rhythmic entrainment processes and (ii) systematic and targeted studies of how PAC changes in relation to specific perceptual and behavioral events.Interestingly, the rhythmic entrainment may itself have oscillatory nesting on much slower (infraslow) timescales. For example, in posterior-medial regions of the default-mode network, there is a slow 0.1 Hz fluctuation of the rhythmic entrainment, paralleling the hemodynamic fluctuations observed by neuroimaging from the same areas (Foster and Parvizi, The findings discussed here point to a large variety of interacting motifs within and between rhythmic phenomena in the brain, spanning many spatial and temporal scales. Constrained experiments and analyses are needed if we are to disentangle and definitively answer the questions that now present themselves. Specifically, we propose studies in which only one or two brain rhythms are empirically defined: the changes in rhythmic dynamics should then be characterized with respect to one associated behavior, and results should be segregated by anatomic boundaries for clear association between brain loci and physiology (Foster and Parvizi,


We discuss here a series of testable hypotheses concerning the role of chromosome folding into topologically associating domains (TADs). Several lines of evidence suggest that segmental packaging of chromosomal neighborhoods may underlie features of chromatin that span large domains, such as heterochromatin blocks, association with the nuclear lamina and replication timing. By defining which DNA elements preferentially contact each other, the segmentation of chromosomes into TADs may also underlie many properties of long-range transcriptional regulation. Several observations suggest that TADs can indeed provide a structural basis to regulatory landscapes, by controlling enhancer sharing and allocation. We also discuss how TADs may shape the evolution of chromosomes, by causing maintenance of synteny over large chromosomal segments. Finally we suggest a series of experiments to challenge these ideas and provide concrete examples illustrating how they could be practically applied. In recent years considerable progress has been made to further our understanding of chromosome organization. Blossoming applications of Chromosome Conformation Capture 3C) have played a central role in fostering these advances C have pltransHigh-throughput 3C approaches Box 1The chromosome territory represents the relative positioning of topologically associating domains (TADs) in the three-dimensional space of the nucleus. The relative arrangement of TADs may differ between cell-types, but is generally related to their transcriptional activity, so that groups of active TAD coalesce to form the A-Type genomic compartment while the inactive TADs are segregated away in the B-Type compartment DrosophilaIn spite of these achievements, chromosomal organization at the sub-Mb scale has remained elusive – partly because of the technical compromise between genomic resolution and coverage imposed by 3C technologies. These limitations are starting to be overcome thanks to the increase in sequencing depth of Hi-C data Drosophila and up to hundreds of kb in mammals, within which physical contacts, as measured by 3C, occur much more frequently than between domains . This mechanism is thought to involve physical contact between these regulatory elements and their target promoters, which is permitted by the folding pattern of the chromatin fiber. Examples illustrating the diversity by which enhancers mechanistically stimulate promoter activity have been reviewed elsewhere cis-regulating elements. In support of this notion, most if not all of the previously reported cases of very long-range (>500 kb) functional enhancer-promoter communications are found to occur within TADs Sonic Hedgehog and the Lmbr1 intron (1 Mb away) TADs are, by definition, chromosomal regions within which DNA sequences most often establish long-range physical contact. This means that any given promoter will be contacted most frequently by sequences belonging to its TAD. It is therefore expected that enhancers, and other regulatory elements that act through physical contact with target promoters, should mainly impact the promoters of the same TAD. For these reasons it is tempting to speculate that the TAD of a given promoter embodies its regulatory domain, meaning the chromosomal region containing its Applications of high-throughput 3C, such as Hi-C and 5C, have revealed that promoters can be contacted by a large set of sequences within their TAD. What can seem surprising is that promoters are not specifically contacted by enhancers, in general – as defined genetically or according to their epigenomic signature Sonic Hedgehog (Shh) locus, where various enhancers within the Shh TAD can be active in different tissues, depending on the trans-acting factors that bind and activate them Using state-of-the-art genetics and genomics approaches Duboule and colleagues Iroquois clusters or the proximal HoxD/Evx2/Lnp domain  and its targets lie in the same TAD . Such Given the relative pervasiveness of chromosomal contacts within TADs, how can we explain that a given enhancer is able to find its specific promoter target? Actually, we would argue that there is little evidence showing that, in their native genomic context, distal enhancers are generally able to distinguish promoters in order to exert their control over a specific target. Rather, several lines of evidence suggest that within TADs, enhancers can exert broad regulatory effects that are often loosely targeted. A consequence of this is that rapid activation of control elements – for example during exposure to a given stimulus – can lead to extensive pervasive regulatory spills. This situation is exemplified by the ripple effects on transcriptional activation that are observed upon cell stimulation with growth-factors What is the structural basis for the communication between regulatory elements and promoters within TADs? A common textbook representation of enhancers is that they come into contact with promoters thanks to extensive looping out the intervening sequences. As recently discussed in depth by Fudenberg and Mirny The apparent specificity that accompanies the preferential engagement of an enhancer with a given promoter may therefore rely on their inherent ability to maintain this contact, even temporarily, rather than to establish it. The sensitivity of 3C-based assays, which sample millions of cells, may reveal a tendency in the cell population but detection of an association does not necessarily reflect the existence of a stable chromosomal structure. This also implies that the structural mechanisms that control chromatin conformation, and the probability of enhancer-promoter encounters, act upstream of promoter-enhancer regulatory communication. It also means that genomic elements (and the factors bound to them) controlling chromatin architecture can be distinct from the factors involved in modulating the actual process of transcription E.Xist/Tsix locus has been reported to impair spatial insulation, leading to regulatory bleed-through from one domain to the other and transcriptional mis-expression Given these considerations, it might therefore be expected that disrupting folding in TAD, either by genomic alterations or changes in the epigenomic makeup, can lead to redirection of regulatory influences. Indeed, deleting a TAD boundary at the Fgf8 locus is sufficient to redirect underlying enhancers to different promoter targets HoxD locus prevents distal regulatory elements from accessing and properly regulating the HoxD cluster, without creating a new TAD boundary These considerations also predict that altering the internal organization of TADs that contain enhancers that do not have an intrinsic specificity for their promoter target should alter the orchestration of long-range regulation. A compelling example of this is that shuffling genomic organization at the HOTTIP produces an RNA that partners with chromatin-modifying enzymes and regulates transcription of the portion of the HOXA locus that lies in the same TAD Enhancers are not the only genomic elements that use chromatin architecture to convey regulatory information from afar. For example, some non-coding RNA loci also appear to rely on a similar process to modulate transcription. Human Altogether these findings suggest that rather than being highly specific of a target promoter, enhancers should be considered as elements that can come into contact with a wide set of sequences, including but not limited to their target promoters, thanks to the underlying chromosomal architecture. Their radius of action would therefore be determined by the mechanisms that control three-dimensional chromatin organization. It is expected that long-range regulatory contacts within TADs will depend on the kinetics of chromatin diffusion. Determining the actual frequency and duration of these contacts .It is well known that the presence of DrosophilaXist/Tsix locus Such considerations also have implications for the genomic (or epigenomic) features that may define given sequences to act as TAD boundaries. For example, the observation that promoters of house-keeping genes are often found close to TAD boundaries has been proposed to reflect a possible mechanistic explanation for boundary formation and topological insulation. An alternative – and non-exclusive – hypothesis is that housekeeping genes would tend to become positioned close to TAD boundaries during evolution because their presence within TADs would interfere with their constitutive expression and might also perturb the proper functioning of long-range regulatory landscapes. The same reasoning holds for SINE elements or even CTCF binding sites, as well as for the reported tendency of P-element transgenes to insert close to TAD boundaries in cis-regulatory landscapes it helps to create. Mapping of TADs in diverse species will open the possibility to further explore this exciting avenue.In the context of such considerations it seems highly likely that segmental folding of chromosomes has an impact on genome evolution, mainly because of the evolutionary pressure exerted by the cis-regulatory domains? Approaches involving genome engineering to alter chromosome conformation would be one way of addressing such questions.How might one test whether chromosome segmentation in TADs actually underlies domain-wide chromatin states? What experiments could address the role served by such modularity in the definition of First, disrupting folding in a TAD could provide an opportunity to address what happens to other domain-wide features of chromatin. For example fusing two neighboring TADs, which can be achieved by deleting the boundary between them , 13, couAnother approach would be to study situations where genomic landscapes are reshuffled without affecting TAD organization. This would enable the testing of how autonomous a given genomic region is in setting up its chromatin make-up, as opposed to being influenced by its chromosomal neighborhood. Engineering large chromosomal inversions, for example using CRE/Lox-mediated recombination techniques cis-regulatory landscapes, then it is to be anticipated that the primary candidates for the relevant gene targets are those lying within the same TAD as the variant identified by GWAS. Given the stability of TAD positioning along chromosomes during development Finally, we would like to consider the possible practical applications that chromatin folding into TADs can bring. Over the years genome-wide association studies (GWAS) have implicated many non-coding DNA sequence variants, such as single nucleotide polymorphisms or small copy number variants, as possible drivers of a wide range of traits, including a plethora of human diseases. Understanding how such variants lead to transcriptional mis-regulation and ultimately to a disease state is a great challenge. The first bottle-neck is to identify the gene target(s) that are transcriptionally mis-regulated when the driver sequence variants are present. If TADs can define cis-regulatory landscapes, it would be expected that transgenes covering most of the TAD harboring a locus of interest would be the most likely to recapitulate appropriate gene expression. An illustration of this can be found with the Xist/Tsix locus, where single copy transgenes harboring only part of the Xist TAD or of the Tsix TAD are unable to recapitulate proper expression during development Drosophila, where TADs are of a smaller size, ∼100 kb on average, and for which libraries of TAD-sized transgenes are readily available.Transgenesis approaches are frequently used to define gene function and expression patterns. This represents another practical application where knowledge of the segmental architecture of chromosome into TADs could be critical. In the context of our discussion of the implications of TADs for Another important consideration is that flanking transgenes by TAD boundaries could likely insulate their expression from the influence of neighboring control elements and minimize position effects. Along the same lines, the choice of transgene insertion site is critical. Insertions of insulator-containing transgenes might disrupt TAD folding, as these sequences might act as ectopic boundaries, and disrupt the underlying regulatory landscape. Targeting transgene insertions close to endogenous TAD boundaries may therefore represent the safest strategy to house such transgenes.In this paper, we have discussed a series of hypotheses concerning the manner in which chromosome segmentation into TADs might relate to, and maybe even underlie, several aspects of chromatin architecture and metabolism, as well as long-range transcriptional regulation and genome evolution. This opens up several exciting avenues that could be explored in order to advance our understanding of chromosome structure and function. What are the molecular mechanisms that drive such folding patterns? What is the temporal fluctuation of this organization, in terms of chromatin motion, and how does this relate to the transcriptional dynamics of the underlying loci? What are the consequences of disrupting this organization, and do such alterations underlie diseases? Tackling these questions, using combinations of genomic engineering, genome-wide approaches as well as single-cell and live imaging approaches, with the ultimate aim of understanding the functional impact of chromatin architecture, represents an exciting challenge.HoxD locus has provided evidence that genes lying close to a TAD boundary can actually fold with either one of the two neighboring domains, in a tissue and developmental stage specific fashion. This folding shift was shown to provide a modular mechanism for HoxD exposure to two distinct regulatory landscapes during limb development A recent study at the