The aim of this paper is to outline pharmacotherapy of the ‘third-line management of CML’ . Current management of CML with multi-TKI failure is reviewed. TKI  and non-TKI  drugs are available. The literature search was made in PubMed with particular focus on the clinical trials, recommendations, guidelines and expert opinions, as well as international recommendations. Progressing CML disease with multi-TKI failure should be treated with alloSCT based on the availability of the donor and EBMT transplant risk scores. The TKI and non-TKI drugs shall be used to get best promising  response. During the CP-CML phase of multi-TKI failure, 2nd generation TKIs (nilotinib or dasatinib) should be tried if not previously utilized. Bosutinib and ponatinib (3rd-generation TKIs) should be administered in double- or triple-TKI (imatinib and nilotinib and dasatinib) resistant patients. The presence of T315I mutation at any phase requires ponatinib or omacetaxine mepussecinate therapy before allografting. During the AP/BC-CML phase of multi-TKI failure, the most powerful TKI available (ponatinib or dasatinib if not previously used) together with chemotherapy should be given before alloSCT. Monitoring of CML disease and drug off-target risks (particularly vascular thrombotic events) are vital. Chronic myeloid leukemia (CML) is a slowly progressive clonal malignant disease characterized by myeloid neoplastic expansion with heterogeneous clinical manifestations.de novo or TKI-responsive patient with CML since TKIs could successfully modulate the disease course.1Tyrosine kinase inhibitors (TKIs) therapy induces high rate of response in the majority of patients. However, while a large proportion of patients attains a prolonged molecular response, and some of them could be considered cured, a not negligible number of patients show a resistance to TKIs therapy.Disease progression under TKIs is a ‘difficult-to-treat’ situation with the available drugs in CML.5The aim of this paper is to outline the perspectives for the drug therapy choices in the CML patient with progressive disease course after sequential multi-TKIs regimen. This clinical approach is known as ‘third-line management of CML’ in the current TKIs era.nd generation TKIs  had not been studied in well-designed proper prospective randomized clinical trials.The decision for the choice of TKIs drug depends upon the best available evidence obtained from randomized clinical trials (RCT), physician experience, and characteristics of the patient and his/her disease. In the “real world”, the management of resistant CML should have an integrative approach including: drug , the patient , and the status of local medical assistance .Disease duration is important in the pathobiology of CML. 5Terminal stage of this malignant neoplastic development is the accelerated phase (AP)/blastic crisis (BC) of CML. Late progressing chronic phase (CP), uncontrolled under TKIs, is also a precarious situation prone to AP/BC CML. The terrible end of CML makes ‘prevention of disease progression’ is the ultimate aim of TKI treatment. Thus, early and rapid reduction of BCR-ABL with acceptable TKIs control is a primary goal of CML therapy. Of course, this goal had already failed in the CML patients with progressive disease after the intake of sequential multi-TKI regimensde novo or TKI-responsive CML patient, based on the data obtained from numerous RCTs.Anyone of the remaining TKIs; allogeneic hematopoietic stem cell transplantation  recommended in all eligible patients ” quite similar to the suggestions in the AP/BC phase-CML “.. Anyone of the TKIs that were not used before progression (ponatinib in case of T315I mutation), then alloSCT in all patients. Chemotherapy is frequently required to make patients eligible for alloSCT”.2European LeukemiaNet (ELN) recommendations indicated the way of management in the Several clinical scenarios (and drug suggestions accordingly) can be generated to describe the ‘third-line CML’ from the ELN recommendations;The CML patient with failure of imatinib and dasatinib The CML patient with failure of imatinib and nilotinib The CML patient with failure of nilotinib and dasatinib The CML patient with failure of nilotinib and bosutinib The CML patient with failure of dasatinib and bosutinib The timing of alloSCT has changed to third- or fourth-line CML after failure of the second-generation TKIs.5Mutational analyzes shall be performed in all of the CML cases with multi-TKI failure during the drug treatment decision. BCR-ABL1 kinase domain point mutations are detectable in about 50% of patients with treatment failure and progression. The mutations detected during the TKI therapy may be resulted in drug switches based on the nature of the mutation. Dasatinib and nilotinib retain activity against most of the mutations that confer resistance to imatinib. Likewise, distinct mutations exhibit decreased sensitivity to dasatinib versus nilotinib.11The most challenging situations are failure to all available TKIs in the patients CML which cannot be transplanted or relapsing after allografting. These patients need effective and safer treatment options. Therefore, patient-centered clinical decision is necessary in this difficult situation.12Bosutinib is an oral dual Src/Abl TKI drug. It is recommended in 2nd, 3rd and later lines of CML treatment in both ELN and NCCN guidelines, where appropriate.14rd line  was 67% comparable to that achieved with dasatinib and nilotinib in earlier treatment lines (second line). In both ELN and NCCN guidelines, Ponatinib is recommended for use in 3rd line; with the ELN guidelines also indicating ponatinib for use also in 2nd line. Ponatinib is the only single TKI, which has been extensively studied in a large cohort of patients where imatinib and a 2nd generation TKI have failed. On the contrary, sequential use of nilotinib and dasatinib in third line has not been studied in appropriately designed clinical trials and the scarce available published evidence suggests a scarce efficacy. Garg and co-workers indicated that the use of second-generation TKI after failure to two TKIs may induce clinical responses, but those are usually not durable except in some CP-CML patients.rd-line CMLnd generation TKI has failed, the risk of disease progression is high, and ponatinib, which has demonstrated an unprecedented efficacy in difficult-to-treat patient population, may be considered as the therapeutic option, even if it presents an increased risk of thrombotic vessel occlusions. In fact, in October 2013, PACE trial was placed on partial clinical hold, due to observation of arterial thrombotic events in the ponatinib clinical program; following these events, a dose reduction was recommended. Serious arterial thrombotic episodes were observed in 19% of the ponatinib-treated patients and included cardiovascular 10%, cerebrovascular 7%, peripheral vascular 7% districts. Venous thromboembolic events too were signaled in 5% of the ponatinib-treated patients. Higher dose-intensity, older age, and cardiovascular risk factors were associated with higher likelihood of thrombotic events. However, Ponatinib could exhibit deep and durable responses in heavily pre-treated patients (58% received ≥3 prior TKIs) with relatively longer follow-up (median follow-up: 27.9 (0.1–39.5) months), particularly CP-CML. Initial data suggested that the response may be maintained after the dose reduction; however, longer follow-up is needed to understand impact on safety.22Ponatinib is an approved potent oral TKI active drug against native and the mutant BCR-ABL, including the resistant T315I mutant. The PACE trial evaluated the efficacy and safety of ponatinib (45mg qd) in CML and Ph+ ALL patients (n=449) resistant/intolerant to dasatinib or nilotinib or with the T315I mutation.Clinical trials in patients who have failed 2 previous lines of TKI suggest ponatinib may be more efficacious than bosutinib, but with a less favorable side-effect profile.12nd generation TKIs (nilotinib or dasatinib) are used if remaining. Bosutinib and ponatinib (3rd-generation TKIs) can be administered in double- or triple-TKI failed (Imatinib and nilotinib and dasatinib) patients. The presence of T315I mutation at any phase requires ponatinib or omacetaxine mepussecinate therapy before allografting. Combinations of TKI and interferon (IFN) or PEG-IFN are used in the everyday clinical practice for the unresponsive cases to TKI alone, but limited data is available for the combination approach.33Current standard practice is allografting for all of the CML cases with multi-TKI failure based on the availability of the donor and EBMT transplant risk scores. Before the alloSCT, all patients should be treated with the best available ‘remaining’ TKI in order to reach best promising response/remission land , complete cytogenetic response (CCyR), stable molecular response (MR)). For this aim all the drugs “Remaining”, bosutinib, ponatinib, dasatinib, nilotinib, and omacetaxine mepussecinate should be used. During the CP-CML phase of multi-TKI failure, 2


Mortality among leukemia patients admitted to the intensive care units remain high  and bleeThe basis for this study was a clinical observation of a high frequency of bleeding complications among ICU patients with leukaemia. We wanted to answer the following questions:1. How big is the problem?2. Can we predict bleeding or thrombosis using standard laboratory coagulation tests?3. Is bleeding and/or thrombosis associated with a higher ICU-mortality?Retrospective observational study of patients with AML, ALL or MDS admitted to the ICU at Copenhagen University Hospital 2008-12. Data was collected from the ICU electronic patient files, the National Blood Bank as well as the hospital's clinical chemistry laboratory database. Bleeding complications were graded according to the WHO classification.There was a high frequency of bleeding among these patients:- 66 patients (57%) had one or several bleeding episodes during their ICU stay- More than half of the bleedings were severe or debilitating (WHO grade 3-4)- One third of all the admitted patients had at least one major bleeding episode during their ICU stay- 10 deaths were directly related to bleeding episodes- 11 patients (9%) had thrombi clinically or on ultrasound/CT-scan9/L) there was no difference among bleeders and non-bleeders with regard to platelet numbers, INR levels, activated pro-thrombin time, fibrinogen concentrations and anti-thrombin levels.Among severely thrombocytopenic patients Bleeding was very common and associated with mortality in leukaemia patients in the ICU, but it was difficult to predict in the individual patient using traditional coagulation analyses.


Purpose: Although papillary thyroid carcinoma metastases to the parapharyngeal space are rare, the high amount of fat tissue allows tumors to grow clinically undetectable until they invade most of the parapharyngeal space. We describe for the first time a combined endonasal and transcervical approach for a parapharyngeal metastasis from a papillary thyroid carcinoma.Materials and Methods: A 51-year-old male with a previous history of papillary thyroid carcinoma presented with left ear fullness and left-sided facial numbness. Imaging revealed a 4x3 cm pre-styloid parapharyngeal space mass invading the foramen ovale and extending below the palate. Needle biopsy confirmed metastatic papillary thyroid carcinoma.Results: The lesion was resected with a combined endoscopic endonasal and transcervical approach. Postoperative MRI revealed gross total resection, and the patient recovered from his symptoms.Conclusion: This novel approach provides access to pre-styloid parapharyngeal tumors with superior extension to the skull base, avoiding more extensive traditional open approaches. The parapharyngeal space (PS) is the medial compartment of the infratemporal fossa. It extends from the skull base to the hyoid bone and is limited by the pterygomandibular raphe anteriorly, by the inner fascia of the masticatory space and the deep lobe of the parotid gland laterally, and by the buccopharyngeal and alar fasciae medially. The posterior limit of the PS is generally regarded to extend to the posterior fascia of the carotid sheath. The PS is divided into pre- and retro-styloid compartments by the tensor-vascular-styloid (TVS) fascia. The pre-styloid compartment contains fatty tissue, the salivary gland, the mandibular division of nerve V, and the internal maxillary and ascending pharyngeal arteries. The retro-styloid compartment contains the carotid artery, jugular vein, nerves IX-XII, sympathetic chain, and lymphatic nodes. Rouviere 1938) identified a consistent lymphatic trunk connecting the superior limit of the lateral thyroid lobe to the retropharyngeal lymphatic nodes [938 identLesions in the PS affecting the skull base with risk of intracranial invasion and spread to vital neurovascular structures require definitive intervention . The PS We describe a case of papillary thyroid carcinoma metastasis to the pre-styloid PS involving the skull base treated surgically using a combined endonasal transpterygoid approach and transcervical approach simultaneously. To our knowledge, this is the first reported case of a combined endoscopic endonasal approach (EEA) and a transcervical (TC) approach to the pre-styloid compartment of the PS.Informed patient consent was obtained prior to all treatments.A 51-year-old male with a previous history of metastatic papillary thyroid carcinoma presented with lower facial numbness and a fullness sensation on the left ear for four months. The patient’s medical history includes a total thyroidectomy and radiotherapy with Iodine-131 six years prior. Three years later, he developed metastatic disease to the left neck and internal jugular vein that was treated with a left neck dissection, including jugular vein resection followed by postoperative radiotherapy to 6600 cGy. Three more years later, he subsequently developed a left serous effusion and marked numbness in the left V3 territory. A myringotomy with fluid evacuation as well as magnetic resonance imaging (MRI) with gadolinium of the head and neck were performed. The MRI revealed a 4 x 3.3 cm highly vascularized tumor in the left pre-styloid PS with foramen ovale invasion , the combination of TC and EEA approaches described in the present case may allow achieving a larger resection safely with control of neurovascular structures in the management of the PS tumors with cranial base involvement.A combined EEA and TC approach is a good strategy for the surgical removal of large tumors of the PS with marked medial invasion. This strategy provides a 360-degree dissection of the pre-styloid PS tumors, allowing safe dissection of delicate structures of the pterygopalatine fossa, skull base, and the inferior aspect of the PS under direct visualization.


Alcohol consumption may be influenced by the local alcohol retailing environment. This study is the first to examine neighbourhood alcohol outlet availability  and alcohol-related health outcomes in Scotland. Alcohol-related hospitalisations and deaths were significantly higher in neighbourhoods with higher outlet densities, and off-sales outlets were more important than on-sales outlets. The relationships held for most age groups, including those under the legal minimum drinking age, although were not significant for the youngest legal drinkers (18–25 years). Alcohol-related deaths and hospitalisations were higher in more income-deprived neighbourhoods, and the gradient in deaths  was marginally larger in neighbourhoods with higher off-sales outlet densities. Efforts to reduce alcohol-related harm should consider the potentially important role of the alcohol retail environment. •Whether alcohol outlet availability influences health in Scotland was unknown•We assessed relationships with alcohol-related hospitalisations and mortality•More hospitalisations and deaths occurred in areas with greater outlet availability•Off-sales outlets were more important for health than on-sales outlets•Efforts to reduce alcohol harms should consider the alcohol retail environment GeograpOne geographical factor that may influence alcohol consumption is the availability of alcohol retail outlets. Neighbourhood availability of alcohol retailing may influence local consumption patterns and health outcomes through a number of pathways. Greater local availability of alcohol retailers, and increased visibility of their advertising and promotions, can increase the physical availability of alcohol, reduce the prices of alcohol products due to retailer competition, and shape and reinforce local attitudes and norms around drinking behaviours and drunkenness . IncreasDifferent types of alcohol outlet are likely to encourage distinct types of drinking behaviours and hence may influence health in varying ways. A key distinction is whether the outlet is licensed to sell alcohol for consumption on the premises  or off the premises . While political and media attention has often been concerned with binge drinking in public spaces dominated by on-sales premises, such as city-centre ‘entertainment districts’, less attention has been paid to the less visible drinking conducted at home, with alcohol purchased from off-sales premises . Indeed,Scotland has one of the highest levels of alcohol-related harm in Western Europe . In the Scotland also has a marked social gradient in both hospitalisations and deaths due to alcohol , contribGiven the magnitude of the health, social and economic costs of alcohol-related harm in Scotland, and that the health burden is falling disproportionately upon the most socially disadvantaged communities, there is an urgent need for a better understanding of potential policy levers for reducing high levels of alcohol consumption . ScotlanThe current Scottish study aimed to address the absence of neighbourhood-level work on alcohol outlet density and chronic health outcomes from countries other than the US or Australia. To date, little is known about the role of the alcohol retail environment in alcohol consumption and health in Scotland. In Australia and the US different urbanisation patterns have tended to result in lower population densities and higher levels of social and ethnic segregation. It might be anticipated that in these national contexts this may result in a lower density of outlets but with a greater social discrepancy due to the concentration of targeted consumers and alternative land use planning strategies. The only Scottish research in this area is limited to research on adolescents in Glasgow . Our stuThe aims of this study were to: (a) examine the association between neighbourhood densities of on-sales and off-sales outlets and alcohol-related morbidity and mortality in Scottish cities; (b) determine whether the relationship differed by age group and sex; and (c) assess whether socioeconomic gradients in alcohol-related health outcomes differed by level of outlet density. In addition to investigating relationships with all alcohol-related health outcomes combined, we also focused on cirrhosis cases specifically as an indicator of chronic alcohol-related harm. We hypothesised that cirrhosis would be more strongly related to off-sales than on-sales outlet densities, because prolonged excessive drinking is likely to be facilitated by cheaper alcohol, such as that sold for off-premise consumption . We expe22.1Alcohol outlet density measures were calculated for the Scottish ‘datazone’ administrative geography. Datazones are the Scottish Government׳s smallest geographical unit for the dissemination of administrative data, and were designed to contain populations between 500 and 1000. We then assessed the relationship of outlet density with alcohol-related hospitalisation and mortality, after accounting for relevant covariates.2.2Postcode-referenced records of licensed premises (correct as of 2008) were obtained from the Liquor Licensing Boards of the four largest cities in Scotland: Glasgow, Edinburgh, Aberdeen and Dundee. Resource constraints precluded collecting outlet data for the whole of Scotland , hence we selected the largest concentrations of outlets and population. The records distinguished premises licensed to sell alcohol for consumption either off or on the premises . A separate on and off-sales licence was also granted in some cases, but we were advised that sales from these premises would be largely on-premise , hence they were treated as on-sales outlets.Outlet density measures are often calculated for administrative geographies such as zip codes, census tracts or postcodes . Implici2 ‘kernels’. Second, for each kernel the number and proximity of outlets within a 1 km search radius were used to calculate a density measure, with outlets nearer the kernel being given greater weight. Density surfaces were produced for on-sales and off-sales premises, providing a proximity-weighted estimate of the density of each outlet type per km2 . The outlet data were analysed using ArcMap 9.3 Geographic Information System (GIS) software .We created a measure of alcohol outlet density per unit area rather than population, on the basis that an individual׳s alcohol consumption is more likely to be influenced by the absolute physical availability of an outlet rather than how many people the outlet is shared between. An alcohol outlet density surface was then produced for each city using KDE. First, the cities were divided into 50×50 mWe extracted outlet density values at the population-weighted centroid of each datazone in the four licensing board areas. Datazones  were the smallest spatial units for which sufficiently large health outcome counts could be obtained, to enable reliable statistical modelling. The four areas were not adjacent and we did not have outlet data for surrounding licensing board areas, hence potential density underestimation was avoided by restricting our sample to the 1360 datazones with population-weighted centroids at least 1 km from the landward edge of each licensing board area.2.3Anonymised individual occurrences of alcohol-related hospitalisation and death between 2000 and 2009 were obtained from NHS Scotland׳s Information Services Division (ISD). Each record included age group  and sex. The ISD definition of alcohol-related health outcomes  captures2.4For each datazone we extracted population counts (2001) by age group and sex, and a measure of area-level deprivation. The Scottish Index of Multiple Deprivation (SIMD) quantifies multiple deprivation on seven domains – employment, income, crime, housing, health, education and access – at the datazone level. The overall score includes an aggregate measure of alcohol-related hospitalisations, hence we selected the SIMD׳s Income Deprivation domain from 2006. This domain quantifies the percentage of the population in receipt of income support benefits.2.5Alcohol outlet densities and the covariates were appended to each record by ISD, based on the individual׳s datazone of residence at the time. ISD then removed the datazone identifiers before providing the linked dataset. The release of individual health data in this form was given ethical approval by the NHS Privacy Advisory Committee.2.6The health data covered 2000–2009, to ensure sufficient counts, although the alcohol outlet data were collected in 2008. We assessed whether 2008 data would adequately capture the alcohol environment over a wider period by comparing with outlet data collected for the same cities in 2012 . We foun2.72 for on-sales and 10.6 for off-sales. On-sales and off-sales outlet density measures were highly correlated , hence could not be included in the same models because of multicollinearity issues. Separate models were run for on- and off-sales densities, and Akaike׳s Information Criterion (AIC) values were compared to assess which of the models produced the best fit to the data.The relationships between alcohol outlet density and health outcomes  were modelled using Poisson regression in Stata/IC 11.1 . We modelled counts rather than rates because the datazones had small populations, which meant that rates would have been highly sensitive to small differences in the numerator. Baseline models were adjusted for age group, sex, datazone-level income deprivation rate, and city. City was included in the models to check whether the relationships varied between the cities. Age- and sex-specific population counts (from 2001 census) were used as the denominator. Subsequent models were stratified by sex and age group and by outlet density quartiles. Incidence rate ratios were calculated to represent the relative increase in the incidence rate of the health outcome associated with an interquartile range (IQR) increase in alcohol outlet density. The IQRs – the difference between the 25th and 75th percentiles – were 11.9 proximity-weighted outlets per kmSuch analyses may be biased due to non-independence of the geographical units, or ‘spatial autocorrelation’, hence it is typical to run spatial error models to adjust for this. However, due to the sensitive nature of these small-area data the geographic identifiers had been removed by the data provider, precluding any assessment of spatial autocorrelation. We note, however, that even where alcohol outlet and harm data have been found to be significantly autocorrelated the corrected results are often not substantively different to those without a spatial error term .33.1Descriptive statistics for the 1360 datazones included in the study are given in The included datazones had a combined population of 1.1 million in 2001, or 22% of the Scottish population. From this population a total of 45,444 individuals were hospitalised for alcohol-related conditions between 2000 and 2009, of whom 3970 (9%) were hospitalised for cirrhosis. Alcohol-related deaths numbered 7064, of which 3952 (56%) were attributable to cirrhosis. Nil or low counts restricted the age groups that could be included in the models for each health outcome: all ages for alcohol-related hospitalisations, 18+ for cirrhosis hospitalisations, and 25+ for all alcohol-related or cirrhosis deaths.3.2Higher densities of on- and off-sales outlets were related to significantly higher incidence of all alcohol-related health outcomes . Effect 3.3Stratified models revealed clear variation in the relationships between outlet densities and health outcomes by age and sex . On- andUnderage (<18 years) hospitalisations for all alcohol-related outcomes were significantly related to outlet densities, and the associations were strongest for females. An IQR increase in off-sales outlet density was associated with a 19% increase in alcohol-related hospitalisations for females . For females the relationship with alcohol-related hospitalisation was strongest in this age group.p<0.001) such that an IQR increase in densities of off-sales outlets was associated with 7% fewer male alcohol-related hospitalisations. No significant associations with hospitalisation were found for 25–29 year old males or females, but rates of all alcohol-related mortality for 25–29 year old males were significantly higher in neighbourhoods with higher outlet densities.All alcohol-related hospitalisations among 18–24 year olds were lower in areas with higher outlet densities. The relationship was significant for males  or cirrhosis (p=0.048), but neither for hospitalisations, nor for on-sales densities. Hence the socioeconomic gradients in alcohol-related and cirrhosis deaths were slightly steeper in datazones with higher densities of off-sales premises. In datazones with below-average off-sales outlet densities, for example, a 1% increase in income deprivation rate was associated with a 4.2% increase in all alcohol-related deaths, whereas the equivalent figure was 4.6% for datazones with higher than average densities.We used interaction models to investigate whether the socioeconomic gradients in alcohol-related health outcomes were affected by outlet densities. Continuous-by-continuous interaction models showed small but significant positive interactions for off-sales outlet densities in the relationship between income deprivation and mortality from all alcohol-related conditions combined . The relative importance of on- and off-sales densities to the relationship could not be assessed, although model diagnostics suggested that on-sales densities were acting as a proxy for off-sales densities. This supports the claim of Contrasting our findings for deaths and hospitalisations may shed light on the relative importance of the alcohol environment for chronic versus acute alcohol-related harms. Outlet densities were more strongly related to alcohol-related deaths than hospitalisations. Most alcohol-related deaths were due to chronic causes  hence the finding suggests that the local alcohol environment – and most likely off-sales rather than on-sales outlets – is particularly important for the long-term excessive drinking that initiates the development of chronic alcohol-related illness. High densities of alcohol outlets may simultaneously increase the availability of alcohol and normalise alcohol consumption behaviours for local communities.There is growing concern about increasing levels of alcohol consumption by young people which have led to increases in chronic alcohol-related illness at younger ages . OutletsWe expected the local alcohol environment to have least importance for alcohol-related health consequences of young legal drinkers because the riskiest drinking behaviours among this group may occur in out-of-neighbourhood entertainment districts (clusters of pubs and clubs) . AccordiWe also investigated whether the associations between neighbourhood outlet densities and the health outcomes varied by social group and hence might be a potential factor in explaining health inequalities. An Australian study attributed widening socioeconomic inequalities in cirrhosis mortality to increasing availability of alcohol over time . In our Our study had limitations. First, whilst our outlet density measures indicate the neighbourhood availability of alcohol retailing, we were not able to include other attributes of retailing that may influence alcohol consumption. Future work could usefully include information on prices, trading hours, venue type and on-sales capacity. Second, our kernel density measures were necessarily based on ‘as-the-crow-flies’ radii, which could not account for barriers to movement. While this meant that not all outlets captured by the measure could be accessed on a 1 km journey on roadways or paths the proximity-weighting applied meant that the measure reflected the density of closer outlets more than those further away. Third, to ensure accurate representation of outlet density we excluded areas on the periphery of each licensing board area – largely suburban areas with low outlet densities – which will have introduced some bias towards inner city locations. Fourth, exposure misclassification, a common issue for ecological studies, was inevitable because we considered only the outlet densities within the neighbourhood of residence at the time of hospitalisation or death. In reality individuals move between areas with different alcohol outlet densities (over daily and longer time periods). Future work could usefully incorporate a variety of geographical settings to which individuals are exposed over the course of their daily lives . Work in the field of physical activity that tracks individual ‘activity spaces’, often using GPS technology, offers significant potential to research on alcohol environments. Longer term – and often longer distance – residential movements could also be studied using longitudinal datasets. Nonetheless, the fact that outlet densities in the place of residence at the time of hospitalisation or death were positively related to cirrhosis rates is noteworthy, given the long lag period of the disease. The alcohol retail environment in the individual׳s place of residence at the time of their hospitalisation or death might therefore be indicative of the alcohol environment they had experienced during the development of the disease. Fifth, causal inference is not possible because of the cross-sectional study design. Longitudinal analysis is required to ascertain the mechanisms underlying the relationships we found, recognising that supply and demand dynamics interact to shape alcohol outlet availability and alcohol consumption patterns .Our findings have direct implications for alcohol policy in Scotland. In particular, the association between off-sales alcohol outlet density and alcohol-related hospitalisations and deaths highlights the potential for MUP  to reduce alcohol-related harm at a population level. This potential is particularly relevant for efforts to reduce harmful alcohol consumption in more deprived neighbourhoods, where the impact of MUP on off-sales is likely to be most pronounced . The strReducing alcohol outlet densities – particularly of off-sales outlets – could potentially help to reduce population-wide alcohol-related harm. Alcohol licensing regulations offer one possible mechanism for reducing outlet density, as recognised in WHO Europe׳s action plan on alcohol . Scotlan5Our study helps to further understanding of how social and spatial factors influence alcohol issues. Health problems attributable to alcohol were more prevalent in neighbourhoods with more alcohol outlets, which may indicate that outlet availability influences alcohol consumption. Availability of off-sales outlets appeared to drive these relationships, in line with the increasing dominance of off-premise drinking in UK. Alcohol-related hospitalisations of those under the legal minimum drinking age were also related to outlet densities, suggesting a need for greater enforcement of alcohol retail legislation. However, among the youngest legal drinkers alcohol-related health outcomes were somewhat decoupled from the residential alcohol environment. Tackling alcohol-related problems in this subgroup of the population is likely to require a wider focus, as alcohol outlets outside the local neighbourhood may be more important. Efforts to reduce alcohol-related harm should consider the role of the alcohol retail environment.


The ubiquitin-proteasome pathway (UPP) of protein degradation has many roles in synaptic plasticity that underlies memory. Work on both invertebrate and vertebrate model systems has shown that the UPP regulates numerous substrates critical for synaptic plasticity. Initial research took a global view of ubiquitin-protein degradation in neurons. Subsequently, the idea of local protein degradation was proposed a decade ago. In this review, we focus on the functions of the UPP in long-term synaptic plasticity and discuss the accumulated evidence in support of the idea that the components of the UPP often have disparate local roles in different neuronal compartments rather than a single cell-wide function. Many years of research on synaptic plasticity using various model systems, from slugs and flies to mammals, has yielded a wealth of information on mechanisms that underlie both short- and long-term synaptic plasticity . It is nPreviously one of us proposed a role for local proteolysis by the UPP in synaptic plasticity . The gisHow might local protein degradation be achieved in neurons? A simple way would be to restrict the protein substrate or the enzymes of the UPP to a subcellular location. For example, proteins whose expression is largely restricted to the synapses could be locally degraded because all the requisite UPP components are present at the synapse. In addition, substrates can be made susceptible (or resistant) to ubiquitination by locally controlled phosphorylation in neurons. Likewise ubiquitin ligases can be activated or inactivated locally by phosphorylation or other posttranslational modifications  neurons which survive to adulthood (Drosophila inhibitor of apoptosis 1 (DIAP1) an E3 ubiquitin ligase. DIAP1 is required for degradation of a caspase called Dronc. Therefore, degradation of DIAP1 enables activation of the Dronc caspase locally in dendrites. Because the Dronc caspase is critical for severing dendrites of C4da neurons, restricted dendritic activation of this caspase allows preservation of C4da neurons while removing their dendrites  causes a substantial increase in number of synapses. The protein product of the hiw gene contains a RING finger domain which is a key part of some ubiquitin ligases  called delta-like homolog 1 (DLK-1) which is also localized to the periactive zone like RPM-1. Inactivation of the DLK-1 cascade suppresses RPM-1 loss-of- function phenotypes whereas DLK-1 overexpression causes synaptic aberrations similar to the ones seen with RPM-1 mutations , which lacks the C-terminal ligase domain, revealed that the Phr1 protein is localized to axon shaft and is largely excluded from growth cones and distal processes. The substrate of Phr1 is most likely DLK in mice as well. Distribution of DLK is non-overlapping with that of Phr1; DLK is present in growth cones with only low levels in axon shaft  gene functions in synapse formation. During development of the Drosophila nervous system, faf overexpression leads to overgrowth of synapses and disruption of synaptic transmission. A similar phenotype is observed when a yeast DUB is expressed in the fruit fly CNS  mutation, a recessive mutation characterized by severe tremors, hind limb paralysis, and postnatal lethality , was applied in the culture medium to sensory-motor neuron synapses, this resulted in enhanced LTF and an increase in neurite outgrowth in isolated sensory neuron  in n of LTF . Later sy neuron . The resy neuron . The disy neuron . In a giy neuron  supportsy neuron . Blockiny neuron .Aplysia nervous system as well as the mouse brain. Furthermore, the synaptic and nuclear proteasome activities are differentially regulated by protein kinases with a key role in synaptic plasticity such as PKA, PKC, and MAP kinase  in the murine hippocampus , which modulates translation of a subset of mRNAs in dendrites. Moreover, proteasomal regulation of FMRP is required for metabotropic glutamate receptor-dependent LTD .Figure 1A).Investigations on hippocampal L-LTP showed that the proteasome inhibitor application to hippocampal slices prior to induction of L-LTP caused an increase in the magnitude of the early, induction phase but an inhibition of the late, maintenance phase . What isbrain-derived neurotrophic factor (BDNF) mRNA by chemically induced LTP (cLTP) or L-LTP induced by a theta-burst protocol .How might proteasome inhibition block transcription? One possibility is that normally the UPP aids the degradation of transcription repressors. Hence proteasome inhibition would result in accumulation of these repressors thus blocking transcription. Consistent with this concept, it was found that a CREB repressor ATF4 is degraded by the UPP during cLTP and β-lactone application to hippocampal slices prevents degradation of ATF4. Furthermore, ATF4-ubiquitin conjugates accumulate during cLTP when the proteasome is inhibited (CaMKIIα, is degraded by the proteasome in an NMDA- and activity-dependent manner (These studies have also revealed the changing role of the proteasome even in dendrites through progression of L-LTP. Application of β-lactone to isolated dendrites also blocks maintenance of the dendritic L-LTP . Under tt manner .Other studies have investigated the effect of proteasome inhibition on LTP. These studies failed to discern differential roles of the proteasome in LTP because one investigation used MG-132  which isThe evidence accumulated over the past decade has further supported the importance of local protein degradation in synaptic plasticity during brain development as well as in the adult brain. What has been lacking is research on the possible mechanisms by which local proteolysis is regulated in neurons. Looking ahead, we can expect to see exciting new discoveries on the local roles of protein degradation in the normal nervous system as well as in many neurodegenerative diseases.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.


While large-scale cancer genomic projects are comprehensively characterizing the mutational spectrum of various cancers, so far little attention has been devoted to either define the antigenicity of these mutations or to characterize the immune responses they elicit. Here we present a strategy to characterize the immunophenotypes and the antigen-ome of human colorectal cancer.We apply our strategy to a large colorectal cancer cohort (n = 598) and show that subpopulations of tumor-infiltrating lymphocytes are associated with distinct molecular phenotypes. The characterization of the antigenome shows that a large number of cancer-germline antigens are expressed in all patients. In contrast, neo-antigens are rarely shared between patients, indicating that cancer vaccination requires individualized strategy. Analysis of the genetic basis of the tumors reveals distinct tumor escape mechanisms for the patient subgroups. Hypermutated tumors are depleted of immunosuppressive cells and show upregulation of immunoinhibitory molecules. Non-hypermutated tumors are enriched with immunosuppressive cells, and the expression of immunoinhibitors and MHC molecules is downregulated. Reconstruction of the interaction network of tumor-infiltrating lymphocytes and immunomodulatory molecules followed by a validation with 11 independent cohorts  identifies BCMA as a novel druggable target. Finally, linear regression modeling identifies major determinants of tumor immunogenicity, which include well-characterized modulators as well as a novel candidate, CCR8, which is then tested in an orthologous immunodeficient mouse model.The immunophenotypes of the tumors and the cancer antigenome remain widely unexplored, and our findings represent a step toward the development of personalized cancer immunotherapies.The online version of this article (doi:10.1186/s13059-015-0620-6) contains supplementary material, which is available to authorized users. Recent studies using large cohorts and next-generation sequencing (NGS) technologies are providing a wealth of information and have revealed the genomic landscapes of common human cancers . But so Several types of immunotherapies have been shown to have great clinical impact, including adoptive T-cell transfer therapy, cellular vaccines, and checkpoint blockade inhibitors, such as the FDA-approved anti-CTLA4 monoclonal antibody (ipilimumab) and antibodies that block signaling through PD-1 and PD-L1 ,3. HowevWithout doubt, given the exciting development of these immunotherapeutic strategies, the importance and clinical relevance of intratumoral immune landscapes and cancer antigenomes is becoming increasingly appreciated. In a seminal paper, epitope prediction algorithms were used to identify candidate tumor antigens . Only reIn order to comprehensively characterize the antigenicity and immunogenicity of human CRC, we developed an analytical strategy and examined genomic data sets from The Cancer Genome Atlas  . We firsAn overview of the analytical strategy and the methods used is shown in Figure S1 in Additional file The enrichment analysis of TIL subpopulations using metagene expression profiles indicated that TILs are present in 96.6% of the tumors and that 70% of the tumors show a T-cell phenotype Figure A, previoThe quantification of TIL subpopulations showed that TILs were associated with distinct molecular phenotypes related to three features  or combinations thereof Figure B. It shoWe have previously shown that individual TIL subpopulations are associated with overall survival (OS) and disease-free survival (DFS), including CD8+ T cells  and B ceP = 0.002, r = -0.16). The progression of the tumor was characterized by distinct immune patterns. For example, activated CD8+ cells were significantly enriched from stage I to stage III tumors and depleted in stage IV tumors  since there were only two MSI-H and MSS^ patients with stage IV tumors. In the MSS patients the number of mutations increased during progression from stage I to stage IV  were detectable in all patients Figure A. About P = 0.05; Figure In strong contrast, the neo-antigens were infrequently shared between patients Figure B. From tOur analysis of the antigenome landscape of CRC demonstrated that the majority of the patients, including patients with lower mutational load (MSS), express neo-antigens and, hence, cancer vaccination may be effective also in these patients. However, tumor mutational heterogeneity adds another layer of complexity and may hinder personalized strategies . Within We then characterized the immunophenotypes for the genetically different tumors and visualized the immune subpopulations, which were enriched and depleted compared with normal tissue, using volcano plots Figure D. Major The genetic and immunophenotypic variability of the CRC tumors shown above impose the question of whether different tumors use different mechanisms of tumor escape. This knowledge is of utmost importance for making decisions about the most appropriate immunotherapy regimen. We carried out systematic analysis of the escape mechanisms and examined the enrichment of immunosuppressive cell types (MDSCs and Tregs), and the expression of five classes of molecules: key immunoinhibitory genes which may be upregulated to produce tumor escape , key immunostimulatory genes , which may be downregulated to avoid immune destruction, as well as major histocompatibility (MHC) class I, MHC class II, and non-classical MHC molecules. We included all previously reported immunomodulators ,24 and tIn the hypermutated tumors (MSI-H and MSS^) Tregs and MDSCs were depleted, whereas in the non-hypermutated tumors (MSS clusters) either Tregs cluster 1) or MDSCs (clusters 2 to 4) were enriched Figure D. The ex or MDSCsA number of immunoinhibitory and immunostimulatory molecules are currently under investigation for immunotherapy in various cancers . In an ain vivo. The microenvironment of the tumor can be immunosuppressive and prevent anti-tumor immunity. Our previous study [P < 0.001) were included in the final model. The model was evaluated by a 10-fold cross-validation procedure. The identified features disclosed the major determinants, which were ranked as follows: MDSCs, Tregs, CD86/B7-2, CCR8, CXCR5, and PD-L2) , and HLus study . Tumor eus study , immunosus study , and gerus study . We examus study  and only) Figure C.All but one of these determinants have been previously implicated as modulators of the tumor microenvironment. MDCSs and Tregs are well-known immunosuppressors . CD86/B7P = 0.069; Figure We then analyzed the available data and provide here several lines of evidence for the involvement of CCR8 in tumor progression in human CRC: 1) CCR8 is downregulated in metastatic tumors compared with non-metastatic tumors in the TCGA cohort Figure D; 2) patThere are conflicting results in the literature on the expression of CCR8 in various cell types. Originally it was shown that CCR8 is expressed in Th2 cells and is involved in eosinophil recruitment . In a reWe have developed an integrated strategy and for the first time characterized the immunophenotypes and the antigenome of human CRC. Our approach to deeply mine large datasets enabled us not only to disentangle tumor-immune cell interactions, but also to devise strategies for cancer immunotherapy in CRC. The dissection of the complex tumor microenvironment suggests several important biological conclusions.First, the results support the notion that the subpopulations of TILs mirror the underlying molecular phenotypes. Moreover, based on the enrichment of TIL subpopulations, specific characterization of all three studied molecular phenotypes  or the combinations thereof is possible. Hence, a single assay (RNA sequencing) enables refined molecular taxonomy of the tumors. One possible explanation for this observation is that the number of features  with cell type-related expression patterns is two orders of magnitude larger than the features used for traditional molecular phenotyping. Notably, the use of well-characterized expression of gene signatures in pure cell types reduces the noise inherent in high-throughput expression studies. With the advent of single cell sequencing techniques  and the Second, the antigenicity of the tumors and the intratumoral immune landscape evolve in parallel during tumor progression, with decreasing antigen frequencies, alleviated adaptive immunity response, and intensified innate immunity. The data also indicate that the mutational load has a major impact on the number of TILs as observed in the MSI-H and MSS^ groups. In the patient group with lower mutational load (MSS), it appears that tumor antigenicity decreases to a larger extent, as shown by an increasing number of mutations and decreasing neo-antigen frequencies during tumor progression. It should be noted that although longitudinal genomic data are currently not available, the large number of patients enabled the use of the concept of a statistical ensemble, that is, averaging large numbers of patients per stage represents a snap shot of the underlying process.Third, the results of the quantification of the antigenome in CRC showed that the number and expression levels of cancer-germline antigens were similar across tumors with different genomic instabilities, mutational loads, epigenetic instabilities, or TIL enrichments, whereas the neo-antigen landscape was highly diverse. It is intriguing to speculate that the T-cell responses might be primarily directed against neo-antigens rather than against cancer-germline antigens. It is noteworthy that the quality of the T-cell responses directed against neo-antigens and cancer-germline antigens has not been compared so far and future experimental studies are needed in order to validate our observation. Such studies would require isolation of neo-antigen and cancer-germline antigen-specific T cells from patient material and experiments with autologous tumors.Finally, one striking observation was how the genetic basis of the tumors determines the tumor escape mechanisms. As expected, hypermutated tumors (MSI-H and MSS^ tumors) showed high intratumor heterogeneity, suggesting that the greater mutational load in these tumors results in a higher load of neo-antigens, and promotes T-cell activation and infiltration . These tBeyond these biological insights, the results from this study also have important implications for clinical translations.The results of our systematic analyses of genomic data have several critical implications for cancer immunotherapies in CRC. Most importantly, we propose the use of two genetic features, that is, mutational load and tumor heterogeneity, to stratify the patients for targeted immunotherapy. In our analyses we used whole-exome NGS and SNP array data to estimate the mutational load and the tumor heterogeneity. A new algorithm  allows tThe set of major determinants of tumor immunogenicity in CRC we identified included not only immunosuppressive cells and chemokine receptors but also molecules which are currently tested in clinical trials: B7-2 (ligand of CTLA4) and PD-L2 (ligand of PD-1). It will be important to identify predictive markers for these therapies in order to select the patients most likely to respond to therapy. For example, preliminary evidence for a correlation between PD-L1 expression on tumor cells and the likelihood of response to anti-PD-1 therapy was reported in a patient with CRC . Thus, tIn addition, our integrative analyses led to the identification of two novel targets for therapy: BCMA and CCR8. BCMA was identified using a network reconstruction approach and validated in 11 independent cohorts. The function of this receptor is well characterized and both agonists and antagonists are available to study the effects on tumor progression. CCR8 was identified using a feature reduction method and the prognostic power was validated using independent cohorts. The function of the CCR8-CCL1 axis is less clear. In melanoma CCR8-CCL1 controls the egress of tumor cells from the afferent lymphatics into the lymph node . Here weThe quantification of the antigenome and both antigen classes, cancer-germline antigens and neo-antigens, of CRC has major clinical consequences. The sparsity of the neo-antigen space suggests that neo-antigens are rarely shared among patients and advocates against the development of off-the-shelf vaccines. Based on the results from our analyses, the size of the peptide library which could be required for vaccination of 50% of the MSS patients would be around 70. Paradoxically, it seems that a patient group with a high mutation rate (MSI-H) is amenable for off-the shelf vaccination: a vaccine with five peptides would cover 50% of these patients. Thus, personalized cancer vaccination strategy is required in which whole-exome NGS is performed to identify somatic mutations, followed by bioinformatics analyses to identify neo-antigens, and synthesis of peptide- or DNA/RNA-based vaccines. Viability of such personalized cancer vaccination strategy was recently demonstrated in clinical studies in melanoma patients . These pHowever, patients should be stratified for cancer vaccination based on the evaluation of the intratumoral immune landscape rather than on the molecular phenotype . As shown in this study, comprehensive characterization of both the genomic and intratumoral immune landscapes is possible using whole-exome NGS and RNA sequencing data, and can pave the way towards personalized cancer immunotherapy. Moreover, in order to increase the likelihood that a neo-antigen vaccine will induce T-cell responses and prevent other clones from outgrowing, multi-epitope vaccines seem obligatory. It was recently shown that using this type of multi-epitope vaccine regimen up to 12 peptides can be used . HoweverThe approach we show here to characterize the immunophenotypes and the antigenomes of tumors using NGS data can help to identify mechanisms of tumor progression and disentangle the complex tumor-immune cell interactions not only in CRC but also in other solid cancers. Over and above, it can help to improve therapeutic efficacy, even in the absence of immunotherapy. Understanding the molecular basis of the interactions between cytotoxic chemotherapeutics or targeted anticancer agents and the immune system is essential for the development of optimal therapeutic schemes  and in tP < 0.05). For the remaining genes, we ranked median mRNA expression levels within each immune cell type from highest to lowest. A two-fold change cutoff from the highest expression group to the next highest expression cell type was considered as a criterion for cell type-specific expression. For each gene in each cell type, we fixed the median expression value of the highest group and permuted group data and then calculated differential expression in the real data set compared with the permutation sets . The specificity of gene expression relied on the Jensen-Shannon divergence, which is an entropy-based measure that quantifies the similarity between a gene expression pattern and another predefined pattern that represented an extreme case in which a gene is expressed in only one tissue [P < 0.01) between all specific immune genes in the same cell type [In order to build a compendium of immune genes related to specific immune cell subpopulations, we collected expression profiles for different immune cell types from 36 studies from the Gene Expression Omnibus  and Arrae tissue . Due to e tissue  and in ae tissue : we seleell type . This thell type  , somatic mutations (n = 522), germline mutations (n = 468), methylation data (n = 582), SNP arrays (n = 573), and RNA-seq expression profiles (n = 598) of colon and rectum adenocarcinoma patients (last update July 2014). Raw sequencing data and alignments were downloaded from the Cancer Genomics Hub (cgHub) repository (September 2013). Details on data generation are available in the original study [, somaticIc, which is proportional to the absolute number of cells of a specific immune cell type c in a heterogeneous tumor sample, was estimated by calculating in each cell type:We used GSEA  to identnc is the number of immune metagenes per cell type c, i (TPM), and i in cell type c from microarray expression data, which was used to identify immune related genes.where The CTDatabase  was querMolecular phenotypes were defined using three features: MSI status, mutation rates, and methylation subtypes. For all three molecular phenotypes we used the most recent update of the raw sequencing TCGA data set. The MSI status was experimentally determined and provided with the clinical dataset (n = 590). The tumors assigned with MSI-low status (n = 93) were subsumed to the MSS group (n = 427). In the original publication, mutation rates were provided for 224 tumors . AdoptinFrom the 222,169 somatic mutations, we selected mutations that reside within exons and do not span exon borders. The DNA changes were mapped on the protein level. Start codon changes and splicing mutations were excluded. Along the missense, nonstop, frameshift, and non-frameshift mutations, we slid windows with lengths 8 of 11 nucleotides. To estimate the HLA haplotype of each patient, the raw RNA-seq data were analyzed using HLAminer’s method for prediction of HLA class I alleles . The HLAEpitope databases were screened in order to evaluate the predicted neo-antigens (last update Dec 2014). Deposited peptidic epitopes, T-cell assays and MHC ligand assays were downloaded from the Immune Epitope Database , Dana FaThe ABSOLUTE algorithm  was usedP < 0.05). Patients were dichotomized based on the median relative number of immune cells and the gene expression of immunomodulator molecules based on optimal cutoff method, respectively. The nodes of the network were colored according to the hazard ratio for OS and marked with a blue border if genes or cell types were significantly (log-rank) associated with OS. The size of the immune cell nodes was encoded by -log10. The network was visualized using Cytoscape [Based on the gene expression data we reconstructed an immune cell-gene (immunostimulatory/immunoinhibitory molecules) network. The edge weights of the network are based on the Pearson correlation coefficient between the relative number of immune cells and the expression of the immunomodulatory molecules  were included in the final model. The model performance was then evaluated by a 10-fold cross-validation procedure.A linear regression model with 221 parameters including the neo-antigen frequencies, immunomodulatory factors, classical and non-classical HLA class I molecule, chemokines, cytokines, immunosuppressive TILs, and cancer-germline antigens was applied to predict the presence of cytotoxic cells . The number of features included in the linear model was reduced by lasso regularization . Only vapost hoc tests were used. Normality of the distributions was tested with Shapiro-Wilk test. The information on all follow-ups for each patient was extracted from the clinical XML files in the complete clinical set. The overall survival time was defined using the latest information. For survival analysis, the patients were dichotomized based on gene expression levels. The optimal cutpoints were searched within the inner 80% selection interval and chosen based on a minimal corrected P-value as proposed by Altman et al. [survival).To test for differential expression across two  or more groups (adaptive clusters), we used the EBSeq  and DESen et al.  and basen et al. . Kaplan-4 to 105) were injected into the submucosa. During subsequent weeks, tumor growth was analyzed via colon endoscopy. After 2 to 4 weeks, the mice were sacrificed and the tumors excised.All animal experiments were performed according to national guidelines and approved by the government of Middle Franconia, Germany. C57Bl/6 wild-type and Rag1-/- mice were kept on a 12-h light/12-h dark cycle and housed according to institutional guidelines. Endoscopic injection of murine MC38 colon tumor cells in the submucosa of the colon of the wild-type and RAG1-/- mice: the needle was positioned inside the working channel of the endoscope so that there would be no damage to the colonic mucosa. After insertion of the endoscope, the tip of the needle was carefully inserted through the mucosa into the submucosa, and a low number of cells  using Trizol™  and reverse transcribed using SuperScript III and Random Priming (Life Technologies). SYBR green-based quantitative PCR was performed on a Viia 7 Lightcycler (Life Technologies) with the following primers:


A new sensing approach utilizing Mie resonances in metamaterial arrays composed of dielectric resonators is proposed. These arrays were found to exhibit specific, extremely high-Q factor  resonances at frequencies corresponding to the lower edge of the array second transmission band. The observed resonances possessed with features typical for Fano resonances (FRs), which were initially revealed in atomic processes and recently detected in macro-structures, where they resulted from interference between local resonances and a continuum of background waves. Our studies demonstrate that frequencies and strength of Fano-type resonances in all-dielectric arrays are defined by interaction between local Mie resonances and Fabry-Perot oscillations of Bloch eigenmodes that makes possible controlling the resonance responses by changing array arrangements. The opportunity for obtaining high-Q responses in compact arrays is investigated and promising designs for sensing the dielectric properties of analytes in the ambient are proposed. Until recently, the phenomenon of Fano resonance (FR) has been attributed to a special type of electron scattering in atomic systems. In the last decade FRs have been also revealed in planar periodic structures, such as slabs of photonic crystals (PhCs), 2D metamaterials (MMs) and plasmonic structures systems, however, there have been only a few reports about FR observations at microwave and mm-wave frequencies and only in structures of specifically combined resonators with broken symmetry.i.e., the phenomenon comparable to Electromagnetically Induced Transparency (EIT) known in atomic physics [Although FRs demonstrate a peculiar type of line shape, they can originate from conventional resonances of Lorentz-Mie type under special conditions provided by interference between locally initiated scattered waves and a continuum of background modes formed in multi-resonator systems under wave incidence ,2,3. In  physics ,4. The possibility to control the line-shape of FRs and to provide very high Q-factors of these resonances opens up exciting opportunities for advancing the science of resonance sensing. Since the efficiency of resonance sensing depends on the Q-factor of the employed resonance, low-loss dielectric resonators (DRs) represent desirable components of resonance sensors. However, typical Q factors for isolated DRs exhibiting Mie/Lorentz-type resonance rarely exceed 150. Therefore, even though DR-based microwave sensors are widely used for detecting the presence of various liquids and gases in the ambient media and in customized solutions, the sensitivity of these sensors is far not sufficient for advanced applications, especially in biochemistry and biomedicine ,7,8. TheIn this work we propose a new MM-based sensing approach for microwave testing. The proposed sensors utilize high-Q resonances  that proI is the normalized magnitude of resonance oscillations, ω is driving frequency, and resω and γ are the spectral position and the half-width of the resonance response (at the 3 dB level).Resonances are known to be a universal characteristic of many types of classical and quantum systems. The spectral dependence (line shape) of elementary resonances including the Mie resonance  is usualq is the Fano parameter characterizing the degree of asymmetry. The formula suggests the presence of one minimum (to one side of resω) and one maximum (to the other side of resω) in the Fano profile due to, respectively, destructive or constructive interference between components of responses. The switch in the character of interference can be related to changing the phase of resonance scattering by π at the resonance frequency.In the 60 s a new type of resonance called later FR was revealed in the studies of inelastic electron scattering from helium atoms ,21. Scatq| → ∞ asymmetry disappears and the Lorentz profile is observed, while at q = 0 the anti-resonance defines the line shape. Typical asymmetrical Fano profile is seen at q = 1, when maximal and zero scattering are shifted in opposite directions with respect to the resonance frequency by 1/q and q, respectively.As seen from 2 particles, which were neither perfectly spherical, nor uniform in size or homogeneous in dielectric permittivity. Following the discovery of FRs in ,21, theyet al. [Fano resonances in 2D MMs were first detected in structures composed of split rings representing two wire arcs of different lengths . At normet al. , interfeet al. .i.e., at slow waves. It is worth mentioning here that sharp FR-related transmission peaks were first observed in perforated dielectric slabs with simple symmetric unit cells [Following ,26, variit cells , while iit cells . The autit cells  confirmeit cells  and showf = fres by 180°, that, in turn, should reverse the sign of the medium effective parameter (permeability at the magnetic resonance) to a negative one [f = fmp thus causing the stop-band at frequencies fres < f < fmp. In frames of this approach no other resonances above fmp, i.e., near the upper edge of the stop-band were expected in MMs.Unusual resonance responses in DR arrays have been first detected by our group at the investigation of 3D MM structures a composetive one . This nei.e., characteristic for EIT.k-vector. A piece of rod was placed in the center of such unit cell along the wave H-field direction. The stacks of planar layers were modeled by linear arrays of such unit cells.The results of our recent studies of high-Q resonances in all-dielectric MMs have shown that these resonances apparently represent one of two states, in which Lorentz-Mie resonances can emerge in periodic resonance structures. This conclusion can be illustrated with the simulation results for dielectric rod arrangements formed by stacking planar rod arrays (PRAs) along the wave propagation direction. Each planar array of infinite rods was modeled by a unit cell with periodic boundary conditions (PBC) in two directions normal to As seen from Resonances observed near two opposite edges of the stop band formed different modes along the direction of wave propagation . For thek-vector and to ∂ω/∂k → 0 at the edge of the second transmission band. Such independence on k-vector is usually characteristic for local resonances weakly coupled to incident waves, i.e., for dark and slow transmission modes. These properties of even modes are in agreement with coherence of resonances and EIT-like high transmission, which should be free from scattering loss, if resonances are weakly coupled to incident waves.It should be noted that photonic energy band diagrams simulated for infinite rod arrays have shown that even modes are always observed at frequencies corresponding to close-to-zero values of The described results are in favor of Fano-type specifics of unusual high-Q resonances observed in DR arrays above the stop-band. It should be noted here that our earlier studies of Mie scattering by high-index dielectric resonators  have coni.e., by the same phenomenon as the EIT-like peaks in planar MMs [The fact that the FR-like resonances observed in DR arrays above the transmission gap are accompanied by full resonance transmission and, thus, can be considered as related to transmission resonances, which are responsible for fringes in transmission spectra of PhCs , let us anar MMs ,45.k-direction. This means that the array application in sensors a relatively long extension of array lattices along the wave propagation direction should be provided. The signal spectra obtained at H-field probe monitoring of local resonances in the center cells of linear arrays used to model 3D DR arrays extended in k-direction (From the presented above data demonstrating the formation of high-Q resonances above the stop-bands of 3D DR arrays  it folloirection a illustrirection b. This rAnother challenge for practical sensing is seen in realizing proper excitation of DR arrays comparable with plane wave excitation used in simulations. A conventional practice at employment of microwave resonators involves using standard microwave waveguides as feeding system. Waveguides, however, are relatively bulky; therefore, feeding microwave sensors by miniaturized microstrip lines or coplanar waveguides becomes more popular . To a pi21 spectra (A possibility of FR-based sensing at waveguide feeding is exemplified by  spectra . To testThe design of array based sensor employing microstrip line feeding is exemplified by 11 spectra (Simulation results presented in  spectra b demonst11 spectrum demonstrates similar behavior as it does in simulations. When polymer phantoms of analyte were applied, the dip in the spectrum shifted to the lower frequency. Because of the fabrication tolerance, the prototype device worked at lower frequencies compared to those in the simulation model and the dips were not sufficiently sharp. Prototyping based on a variety of models and experiments are currently in progress.The results of preliminary experiments with the prototype are shown in Conducted studies have demonstrated that MM arrays of DRs are capable of responding to microwave irradiation by the formation of resonances with extremely high-Q factor up to 15,000. These resonances are apparently related to the even or anti-bonding resonance mode, which represents one of two energy states for magnetic Mie resonances in the arrays. Thus, the characteristic frequency of the above mode corresponds to the bottom of the second transmission band in the energy diagram of the DR array. We infer that the high-Q factor, specific Fano-type shape, and remarkable strength of the observed resonances are defined by the interaction of local DRs with Fabry-Perot oscillations of slow background waves. The latter oscillations reveal themselves in accompanying transmission resonances, at which the distribution of local resonance magnitudes along the array corresponds to the shape of Fabry-Perot standing waves. Such mechanism opens up an opportunity for controlling the Q factor and the strength response at high-Q resonances by changing DR arrangement in arrays. Performed modeling and prototyping confirmed the possibility to design microwave sensors operating due to Fano-type resonances excited in compact DR arrays. The conducted studies create a conceptual basis for future designing of a new class of efficient sensors for a wide range of applications in microwave and mm-wave ranges. It is also worth noting that prototyping and characterization of DR-based MMs for incorporating in microwave range sensors is more feasible and cheaper than similar procedures with MMs for optical range.


Approximately 83 % of patients with bladder cancer have achieved a complete response after undergoing a novel bladder preservation therapy involving balloon-occluded intra-arterial infusion chemotherapy (BOAI) using a four-lumen double-balloon catheter, known as the Osaka Medical College regimen. This study aimed to show the quantitative difference in hemodynamics of the bladder arteries using syngo iFlow , which provides an automatic tool for quantitative blood flow analysis between double BOAI (D-BOAI) and conventional single BOAI (S-BOAI).Fifty patients were included. The catheters were introduced into both posterior trunks of the internal iliac arteries via contralateral femoral artery access. A side hole between the distal and proximal balloons was placed at the origin of each bladder artery to allow clear visualization of angiographic flow of the injected agent into the urinary bladder. Digital subtraction angiography was used during analysis with the syngo iFlow to evaluate the hemodynamics of the contrast medium in the pelvic arteries during BOAI. The comparative change in the amount of contrast medium in the bladder arteries between D-BOAI and S-BOAI was assessed using syngo iFlow.One-hundred pelvic sides were analyzed. The amount of contrast medium in the bladder arteries using D-BOAI was more than twice that using S-BOAI .The amount of contrast medium in the bladder arteries using D-BOAI was higher than that using conventional S-BOAI. This may increase the anticancer drug concentration in the affected bladder, leading to a good clinical response. The gold-standard therapy for locally invasive bladder cancer has long been radical cystectomy with pelvic lymph node dissection. However, approximately 50 % of all patients with invasive bladder cancer die, and the outcome of this disease is closely related to its pathologic stage . A highlA novel bladder preservation therapy known as the Osaka Medical College (OMC) regimen has resulted in good clinical responses –9. This Previous studies have angiographically confirmed more accumulation of contrast medium in the tumor site with D-BOAI than with S-BOAI, which might be related to the good clinical response. However, no clinical studies have objectively evaluated the hemodynamics associated with BOAI for patients with bladder cancer. The syngo iFlow  is a software application that uses all of the scenes in a given digital subtraction angiography (DSA) sequence to generate a color map that displays the time of maximum opacification. The further function of iFlow allows retrieval of time–intensity curves for a given point or region of interest (ROI) on the color map. Strother et al. previously described the underlying mathematic model for iFlow . The synWe performed D-BOAI in patients with bladder cancer who presented for radical cystectomy from November 2013 to September 2014 but who wished to preserve bladder function. Eligible patients had histologically confirmed carcinoma in situ or stage T1, T2, T3, or T4 muscle-invasive bladder cancer without distant metastasis. Imaging studies, including chest and abdominal computed tomography, abdominal/pelvic magnetic resonance imaging, and bone scintigraphy, were performed before the start of therapy. Based on the DSA series collected for the 89 patients, 50 patients  were successfully postprocessed using quantitative DSA with syngo iFlow. In all, 39 patients were excluded because severely tortuous arteries prevented placement of the catheter in the correct position. The clinical characteristics of the patients are presented in Table For the intra-arterial infusion procedure, we used an intra-arterial catheter equipped with two 6-Fr occlusion balloons  Fig. . This caThe catheter was introduced into the posterior trunk of the internal iliac artery via contralateral femoral artery access. After the distal balloon had passed through the furcation of the anterior trunk of the internal iliac artery, the distal and proximal balloons were inflated and immobilized to isolate the anterior trunk of the internal iliac artery, which lies upstream of the target vessels (BAs) between the balloons Fig. . Using DAll angiograms were obtained using the same angiography equipment , and the same injection parameters after bilateral catheters were connected to one high-pressure-resistant extension tube from a contrast medium injector . DSA was performed using the following parameters: 10 mL of iopamidol (370 mgI/mL) injected at a rate of 1.5 mL/s in an anteroposterior position at 4 frames per second. The DSA series were then postprocessed for analysis using the syngo iFlow.Both D-BOAI and S-BOAI DSA data Fig.  were traThe ROIs were placed on both side holes of the catheters, superior gluteal arteries (SGAs), inferior gluteal arteries (IGAs), and BAs on the color-coded single S-BOAI image Fig. . Each ROThe results of this study are shown in Table The syngo iFlow system provides a method for extracting more objective and quantitative information when comparing subjective and qualitative evaluations involving the accumulation of contrast medium during standard DSA. The software tool displays dynamic information in a colorful static image in which different-colored scales mark the history of the contrast medium through vessels. The quantitative measurements of the hemodynamic condition are recorded and depicted using syngo iFlow. Previous reports have also described hemodynamic analysis in the head and neck or liver using syngo iFlow –14.The OMC regimen—a novel therapy for bladder preservation in patients with invasive bladder cancer that involves D-BOAI of an anticancer agent using a 4L-DB catheter—has been used in our hospital for two decades. We have observed improved treatment outcomes using this regimen relative to conventional treatment entailing radical cystectomy with pelvic lymph node dissection –9. In paThe use of syngo iFlow provides several advantages . First, In the present study, it was quantitatively proved that more contrast medium accumulates in the BAs and less accumulates in the SGAs with D-BOAI than with S-BOAI. We assume that the alteration in the accumulation of contrast medium in the BAs with D-BOAI leads to a higher concentration of the anticancer drug in the bladder tissue using the OMC regimen, resulting in a better clinical response.The present study had several limitations. First, in patients with prominent stenosis of the internal iliac artery or tortuosity of the superior gluteal artery, it may not be possible to introduce a double-balloon catheter into the superior gluteal artery. D-BOAI would not be possible in these cases. Thus, these patients were inevitably excluded from the study. Second, the difference in the clinical response to the treatment with D-BOAI versus S-BOAI could not be evaluated because all patients in this study were treated using D-BOAI. Third, all parameters assessed using syngo iFlow were not absolute values. They were comparative values. Therefore, setting the ROI on the Ref-ROI should be required to calculate the AUC of one particular ROI. In this study, the AUC of the Ref-ROI with D-BOAI was regarded as identical to that with S-BOAI because the Ref-ROI was placed on the side hole of the same catheter. However, as can be seen in Fig. In conclusion, compared with S-BOAI, D-BOAI quantitatively increased the amount of contrast medium in the BAs by more than a factor of two. This may result in a higher concentration of anticancer drug in the affected bladder, leading to a good clinical response. However, the long-term clinical outcomes of the patients in this study have not yet been assessed. Further studies are needed to clarify the relation between drug concentration and clinical response.


Primate visual systems process natural images in a hierarchical manner: at the early stage, neurons are tuned to local image features, while neurons in high-level areas are tuned to abstract object categories. Standard models of visual processing assume that the transition of tuning from image features to object categories emerges gradually along the visual hierarchy. Direct tests of such models remain difficult due to confounding alteration in low-level image properties when contrasting distinct object categories. When such contrast is performed in a classic functional localizer method, the desired activation in high-level visual areas is typically accompanied with activation in early visual areas. Here we used a novel image-modulation method called SWIFT (semantic wavelet-induced frequency-tagging), a variant of frequency-tagging techniques. Natural images modulated by SWIFT reveal object semantics periodically while keeping low-level properties constant. Using functional magnetic resonance imaging (fMRI), we indeed found that faces and scenes modulated with SWIFT periodically activated the prototypical category-selective areas while they elicited sustained and constant responses in early visual areas. SWIFT and the localizer were selective and specific to a similar extent in activating category-selective areas. Only SWIFT progressively activated the visual pathway from low- to high-level areas, consistent with predictions from standard hierarchical models. We confirmed these results with criterion-free methods, generalizing the validity of our approach and show that it is possible to dissociate neural activation in early and category-selective areas. Our results provide direct evidence for the hierarchical nature of the representation of visual objects along the visual stream and open up future applications of frequency-tagging methods in fMRI. Neural processing in the ventral visual pathway is fundamental for object recognition in primates. Standard models of visual processing have proposed that different hierarchical processing steps are needed to extract information from simple features in early visual areas to invariant category representations in higher-level areas within the visual pathway ,2. WhileTo distil the invariant neural responses to object categories, we previously designed a novel method called SWIFT (semantic wavelet-induced frequency-tagging), which allows manipulating the high-level image semantics without changing the principal low-level image features . SpecifiHere, we measured blood-oxygen-level dependent (BOLD) signals with fMRI to extend the previous study  by assesWe validated the assumptions of the SWIFT method and provided a proof of concept, confirming these predictions for faces and scenes using both a fixed statistical threshold as well as criterion-free analyses. Furthermore, we found that the higher in the hierarchy, the more voxels SWIFT activated in a frequency-tagged fashion: steady and flat responses in V1 and V2, strong and periodic responses in category-selective areas, and intermediate responses in V3 and V4. The localizer, on the other hand, activated low- and higher-level areas more similarly compared to SWIFT. SWIFT category-related activations are consistent with models that propose gradually refined representations of visual objects along the visual hierarchy.Experimental procedures were approved by the Monash University Human Research Ethics Committee (CF12/2542–2012001375). Written consent was taken from nine paid participants , one of them was excluded due to failure in maintaining adequate levels of arousal during the experiment. Seven out of the remaining 8 participants were tested in Session 1 and all 8 participants were tested in Session 2. Sessions 1 and 2 were conducted on different days, several weeks apart. Session 1 and 2 contained different versions of the SWIFT experiments (see below). In addition to the SWIFT experiments, Session 2 contained the functional localizer as well as a retinotopic mapping experiment. All participants had normal or corrected to normal vision.Scanning was performed at the Monash Biomedical Imaging facility, Melbourne, Australia, in a 3 Tesla MRI scanner (Siemens Magnetom Skyra) using a 32-channel Head Coil. T2*-weighted functional images were acquired using a gradient-echo echoplanar imaging (EPI) sequence . Forty-four contiguous sagittal slices were acquired covering the whole brain. For the structural MRI, 256 T1-weighted sagital slices covering the whole brain were acquired using magnetization prepared rapid acquisition gradient echo (MP RAGE) sequence . Two structural MRIs were averaged to obtain the final structural image. For one participant who was tested only in Session 2, two structural MRIs acquired in Session 2 were averaged. For the rest of participants, one structural MRI obtained in Session 1 and another in Session 2 were averaged. Additional magnetic field (B0) mapping was acquired for off-line distortion correction of EPI sequences due to B0 inhomogeneity.https://www.google.com/imghp) to find images with “free to use, share or modify, even commercially” usage rights. Semantic wavelet-induced frequency-tagging (SWIFT) was used to modulate pictures' semantic information at a fixed temporal frequency while conserving the principal physical features of the image )Our successful BOLD category selective frequency-tagging opens up a new venue for fMRI research. In particular, a phenomenon called intermodulation between the two tagging frequencies has been successfully employed as a marker of integrative neuronal processing with EEG/MEG –51. CombAcross stimulus categories, the activation patterns by SWIFT were more similar to the patterns obtained by the scramble- than the category-contrast from the localizer data . This isA unique and non-trivial advantage of SWIFT is that it achieves the steady baseline activation by a large set of scrambled images that uniformly sample different combinations of the identical set of locally-conserved low level-features. This can be appreciated on the time course of the relative image similarity in Of the two main aims of this study, our primary interest was to test if SWIFT can periodically activate high-level areas while keeping the low-level areas constant. Accordingly, the other aim, whether SWIFT can activate the high-level area as specifically and selectively, was of less importance. We thus used a limited number of image exemplars in our experiment (n = 9), which was sufficient to address the primary question. The smaller set of instances per stimulus category makes it difficult to compare our results with those obtained with more variable sets of stimuli. Nonetheless, we believe it merits some discussion as to the difference we observed among categories in SWIFT 2 and 3 experiments, keeping in mind that some of the effects reported here might be due to our specific stimuli.While general results with faces were remarkably similar between SWIFT 2 and 3 Figs  and 6, tRelatedly, while the results with SWIFT were compelling for faces, and to a lesser extent, for scenes, those for objects were not Figs –6. WhileThe first explanation is due to heterogeneity of our pictures in the object category . This could yield a poor category response as each image would activate different subpopulations within the LOC. A more homogeneous set of objects, such as tools, might have produced more compelling responses.Another possible explanation might be that cortical modules that represent the object categories in an invariant manner may be less robust compared to those for faces. For the face category, converging evidence from clinical, intracranial recordings and neuroimaging data suggests the existence of invariant face representations in the FFA e.g., ,54,55). ,55. 54,5While our sub-optimal results with scenes and especially objects are consistent with the above mentioned studies, the response properties of these high-level category-selective areas are highly debated and under active investigation. Invariant neural responses in face-selective areas have been re-evaluated by studies showing strong influence of low-level properties in the face responsive areas –60. On tSWIFT engaged the ventral visual pathway gradually, with the weakest responses in the early visual areas and the strongest in the category-selective areas. This confirmed our design principle of SWIFT and it is in agreement with models of object representations which predict a gradual emergence of selectivity and invariance of category representations along the ventral visual pathway ,63–65, wThe most likely neural mechanism underpinning our results is gradual refinement of downstream readout in tuning. It is plausible that neurons in V3 and V4 are more preferentially activated only when a particular subset of neurons in V1 and V2 are activated, especially at the time when the original image is revealed. Given that fMRI can be more sensitive to synaptic inputs to a given area than output spikes from the area , our resThese results are important for the validation and refinement of current models of visual perception. In addition, SWIFT opens new avenues to resolve longstanding questions on the functional architecture of the visual cortex.One question concerns the role of horizontal connections in the coding of natural images. Horizontal connections, especially in V1, are implicated in contour processing  and theyIn the same vein, feedback activation has been shown to modulate the early visual areas via top-down attention –73 and, Future fMRI studies may be able to isolate subtle horizontal and feedback activations generated by SWIFT and thus better understand the role of these connections in the coding of natural images. This can be done, for example, by utilizing directed functional connectivity measures, such as Granger causality  and dynaS1 Movie(GIF)Click here for additional data file.S1 Supporting Information(PDF)Click here for additional data file.