Hum Brain Mapp 35:5083–5092, 2014. © 2014 The Authors. Human Brain Mapping Published by Wiley Periodicals, Inc.Despite advances in understanding basic organizational principles of the human basal ganglia, accurate in vivo assessment of their anatomical properties is essential to improve early diagnosis in disorders with corticosubcortical pathology and optimize target planning in deep brain stimulation. Main goal of this study was the detailed topological characterization of limbic, associative, and motor subdivisions of the subthalamic nucleus (STN) in relation to corresponding corticosubcortical circuits. To this aim, we used magnetic resonance imaging and investigated independently anatomical connectivity via white matter tracts next to brain tissue properties. On the basis of probabilistic diffusion tractography we identified STN subregions with predominantly motor, associative, and limbic connectivity. We then computed for each of the nonoverlapping STN subregions the covariance between local brain tissue properties and the rest of the brain using high‐resolution maps of magnetization transfer (MT) saturation and longitudinal (R1) and transverse relaxation rate (R2*). The demonstrated spatial distribution pattern of covariance between brain tissue properties linked to myelin (R1 and MT) and iron (R2*) content clearly segregates between motor and limbic basal ganglia circuits. We interpret the demonstrated covariance pattern as evidence for shared tissue properties within a functional circuit, which is closely linked to its function. Our findings open new possibilities for investigation of changes in the established covariance pattern aiming at accurate diagnosis of basal ganglia disorders and prediction of treatment outcome.  The steadily growing number of patients treated with deep brain stimulation of basal ganglia structures motivates further in‐depth investigation of the anatomy and function of the corticosubcortical circuits. Exemplified on the well‐established stimulation of the subthalamic nucleus (STN) for treatment of motor symptoms in idiopathic Parkinson's disease, we acknowledge the emergence of behavioral and cognitive side effects related to the specific site of stimulation [Mallet et al., Experimental and clinical findings confirm the existence of a certain degree of topological specialization within the basal ganglia corresponding to their implication in motor, associative, and limbic functions. As part of the corticosubcortical circuitry the STN is thought to follow this organizational principle in such a way that motivational and emotional content are attributed to anterior portions and motor control to its posterior part [Hamani et al., In humans, the functional anatomy of basal ganglia can be studied in vivo using magnetic resonance imaging (MRI) under the assumption of topological correspondence between anatomical connectivity patterns and functional subdivisions. Topology estimates of STN anatomical projections based on MRI sensitive to water diffusion suggested a tripartite organization of the STN; however, this notion was confirmed only in 14 of 24 STNs studied [Lambert et al., Alternatively, anatomical connectivity patterns in the brain can be studied on the basis of MRI‐derived regional volume covariance between putatively connected areas with common maturation pathways [Mechelli et al., Considering previous reports about gradients of iron content in the STN spatially overlapping with functional subregions [Dormont et al., Here, we present a detailed analysis of the regional covariance of myelin and iron content within limbic and motor basal ganglia circuits that combines different anatomical MRI modalities—probabilistic diffusion tractography and quantitative multiparameter mapping within the well‐established framework of voxel‐based quantification, VBQ [Dick et al., Thirteen subjects  underwent diffusion‐weighted imaging and quantitative multiparametric brain imaging on a 3‐T whole‐body MRI system  using a 32‐channel radiofrequency (RF) head receive coil and RF body transmit coil. Multiparametric brain imaging data were pooled together with data from a separate cohort to a total of 101 healthy subjects , previously acquired with the same protocol to study the effects of healthy aging [Chowdhury et al., α = 6°), T1 weighting , and MT weighting  contrast according to the previously published protocol [Weiskopf et al., The whole‐brain quantitative MR imaging (101 subjects) consisted of 3D multiecho FLASH datasets with predominantly proton density weighting , running under Matlab 7 . Probabilistic diffusion tractography was performed with the FDT diffusion toolbox in the framework of FSL [Behrens et al., Image processing was performed with the freely available Statistical Parametric Mapping software , MT saturation, and effective transverse relaxation rate (R2* = 1/T2*) parameter maps was performed as described previously in the context of VBQ [Draganski et al., To allow bias‐free definition of seed and target areas we performed supervised detection and labeling of the STN in subject‐specific native space. We used an approach based on affine transformation of the surface‐based digitalized Morel histological atlas [Krauth et al., Probabilistic tractography was performed in subject‐specific native space after affine registration and correcting for eddy currents using the default settings in FSL bedpostx with the following parameters: 5,000 originating tracts per voxel, curvature 0.2, and step length 0.5. Distributions of diffusion parameters were estimated at each voxel to model the directions of up to two tensors per voxel [Behrens et al., z‐score). We then built exclusive motor, associative, and limbic STN connectivity maps by assigning at the voxel level the highest probabilistic value to a particular area on the “winner‐takes‐all” principle after subtraction of probabilistic connectivity values from the two other subregions.Three STN connectivity maps were built for each side: (i) “motor” map—STN probabilistic connectivity distribution to primary motor cortex and the supplementary motor cortex; (ii) “prefrontal associative”—connectivity to prefrontal cortex including superior, middle, and inferior frontal gyri; and (iii) “limbic” map— connectivity distribution to ACC, hippocampus, amygdala, and orbitofrontal cortex including medial, anterior, posterior, and lateral orbitofrontal cortex, but not the medial surface of gyrus rectus. All maps were spatially registered to MNI space for further analysis by applying the spatial registration estimates from the previous step. To obtain a Gaussian distribution and to minimize variability across subjects, connectivity values were first normalized (The exclusive masks obtained from diffusion tractography (13 subjects) were used to delineate motor, associative, and limbic STN in multiparameter maps (101 subjects). This allowed for extraction of mean MT, R1, and R2* values from each STN subarea in each subject to be used for further analysis.t‐tests .Aiming at investigating differential tissue properties underlying STN functional specialization, we performed separate analyses of variance (ANOVAs) for each quantitative map values  extracted from STN subareas, followed by T‐statistics were computed to detect regional correlation for each parameter map regarding motor, associative, and limbic STN regional maps separately. Statistical thresholds were applied at P < 0.05 after family‐wise error (FWE) correction for multiple comparisons over the whole volume of the GM/WM mask. Trends were assessed by using an auxiliary uncorrected voxel threshold of P < 0.001 [Friston et al., To test the hypothesis of brain tissue property covariance within functional basal ganglia circuits, we computed voxel‐wise correlations between parameter values extracted from motor, associative, or limbic STNs and the rest of the brain. The usage of different datasets improves the generalizability of results, as radically different methods are used to delineate functional areas in STN (diffusion‐weighted imaging) and in the rest of the basal ganglia (covariance of relaxometry‐based multiparameter maps). We used a multiple linear regression model as implemented in the general linear model framework of SPM. Age and gender were included as additional variables in the design matrix. All parameter data  were included in the same model after concatenation using a mass‐univariate approach with single block‐diagonal design matrix structure. This ensures the same beta‐parameter estimates as in the separate component analyses. Different variance components were estimated for each block of data separately using REML nonsphericity estimations [Friston et al., 3.The semiautomated STN identification in native space was visually inspected for gross misalignments. The average seed volume of the STN was 173.84 ± 19.99 mmThe probabilistic diffusion tractography conducted on 13 subjects demonstrated STN connectivity patterns with distinct gradients in the posterolateral to anteromedial direction . t‐Tests confirmed the significance of differences between associative and limbic next to differences between motor and limbic subregions (P < 0.05). The summary of statistical results is presented in Table Mean values of multiparameter maps in the limbic, associative, and motor parts of the STN differed significantly across the 101 subjects' scans  and pars interna (GPi). R2* maps in the limbic STN covaried with the anteroventral internal and external pallidum  we aimed at investigating the robustness of our inferences and repeated the analysis seeding from a bipartite STN. We defined subsequently a “limbic–associative” (=limbic + associative) and a “motor” STN.R2* maps in the motor STN showed the highest covariance with posterior putamen, pallidum, and in a wide anterior thalamic area encompassing the anterior, mediodorsal, laterodorsal, ventral anterior, and ventral lateral nuclei . R1 maps in the limbic STN covaried with R1 values in the ventral–anterior caudate. The R2* maps of the limbic STN showed significant covariance with the ventral GPi and GPe.Here, we demonstrate in vivo that STN subregions can be characterized not only by specific anatomical projections but also by differential brain tissue properties measured with MRI. More importantly, the covariance of iron and myelin content between STN subregions and corresponding areas within basal ganglia circuits is interpreted as evidence for shared tissue properties within specific functional subcortical networks. Our findings open new possibilities to study the anatomical organization of the STN as part of the corticosubcortical circuitry and have potential impact on attempts to define intraindividually the optimal target for deep brain stimulation.Taking advantage of high‐resolution diffusion‐weighted sequences, we demonstrate gradients of connectivity to motor, associative, and limbic cortical regions, spanning along the entire length of the STN. Here, we did not observe clear‐cut boundaries among functional subregions. Corroborating the results from a recent tracing study in non‐human primates investigating the patterns of STN connectivity, we interpret our findings as evidence for spatial overlap of convergent projections [Haynes and Haber, Main finding of our study is the in vivo evidence for differential tissue properties within the STN consistent with lower myelin and higher iron content in limbic areas compared with motor and associative functional regions, which is in line with previous imaging and postmortem studies [Dormont et al., Under the assumption that regional covariance in local brain tissue properties reflects anatomical connectivity patterns we focused on investigation of corticosubcortical circuits integrating within the STN. Considering the absence of strong evidence for clear‐cut segregation of STN subregions [Keuken et al., Local parameter values linked to iron and myelin content within the motor STN subregion showed a positive correlation with the corresponding tissue property maps in the caudal–dorsal putamen and caudal pallidum Fig. . The spaParameter values in the anterior STN correlated positively with tissue properties in the dorsal and anterior–ventral caudate, including the nucleus accumbens, the anterior putamen, and ventral pallidum Figs.  and 3. SThe analysis based on bipartite and tripartite STN shows almost identical covariance patterns. An important distinction, however, is the detected R1 covariance between the associative STN and the dorsal pallidum considered as associative area [Shink et al., Even considering the high level of anatomical plausibility of our findings there are methodological limitations of this study to be considered. Led by our intention to provide a framework for optimal delineation of the STN subregions and their corresponding connectivity patterns in other basal ganglia structures our choice of limbic, associative, and motor cortical targets is mainly based on systematic, however potentially subjective selection of evidence from the literature. Together with the current relatively low spatial resolution of the MR images in relation to the STN size, this could explain why the dorsal caudate, receiving important projections from dorsal prefrontal regions, was not found to covary with the associative STN. Considering the known potential pitfalls of the probabilistic diffusion tractography method, we acknowledge the absence of strong evidence for direct connections from STN to hippocampus and amygdala in humans [Groenewegen and Berendse, In summary, our findings bring strong in vivo evidence for differential tissue properties in limbic and motor STN subregions. The demonstration of regional covariance of myelin and iron content within limbic and motor loops across all basal ganglia structures is a novel finding, which has the potential for practical implementation not only in the target planning phase of STN deep brain stimulation but also as a biomarker for early diagnosis in movement disorders caused by neurodegeneration.The authors are very grateful to Dr. Thomas Schönecker for his valuable help with defining the anatomical STN borders.


Mediation analysis revealed that the effects of ageing on RSFA were significantly mediated by vascular factors, but importantly not by the variability in neuronal activity. Furthermore, the converse effects of ageing on the rsMEG variability were not mediated by vascular factors. We then examined the effect of RSFA scaling of task‐based BOLD in the sensorimotor task. The scaling analysis revealed that much of the effects of age on task‐based activation studies with fMRI do not survive correction for changes in vascular reactivity, and are likely to have been overestimated in previous fMRI studies of ageing. The results from the mediation analysis demonstrate that RSFA is modulated by measures of vascular function and is not driven solely by changes in the variance of neural activity. Based on these findings we propose that the RSFA scaling method is articularly useful in large scale and longitudinal neuroimaging studies of ageing, or with frail participants, where alternative measures of vascular reactivity are impractical. Hum Brain Mapp 36:2248–2269, 2015. © 2015 The Authors Human Brain Mapping Published by Wiley Periodicals, Inc.In functional magnetic resonance imaging (fMRI) research one is typically interested in neural activity. However, the blood‐oxygenation level‐dependent (BOLD) signal is a composite of both neural and vascular activity. As factors such as age or medication may alter vascular function, it is essential to account for changes in neurovascular coupling when investigating neurocognitive functioning with fMRI. The resting‐state fluctuation amplitude (RSFA) in the fMRI signal (rsfMRI) has been proposed as an index of vascular reactivity. The RSFA compares favourably with other techniques such as breath‐hold and hypercapnia, but the latter are more difficult to perform in some populations, such as older adults. The RSFA is therefore a candidate for use in adjusting for age‐related changes in vascular reactivity in fMRI studies. The use of RSFA is predicated on its sensitivity to vascular rather than neural factors; however, the extent to which each of these factors contributes to RSFA remains to be characterized. The present work addressed these issues by comparing RSFA  to proxy measures of (i) cardiovascular function in terms of heart rate (HR) and heart rate variability (HRV) and (ii) neural activity in terms of resting state magnetoencephalography (rsMEG). We derived summary scores of RSFA, a sensorimotor task BOLD activation, cardiovascular function and rsMEG variability for 335 healthy older adults in the population‐based Cambridge Centre for Ageing and Neuroscience cohort (Cam‐CAN;  With the global demographic shift towards an older population, and the impact that age can have on cognition and dementia, there is a pressing need to understand the neurobiology of cognitive ageing. Functional brain imaging with functional magnetic resonance imaging (fMRI) has many advantages as a tool to study ageing and much of the current theoretical understanding of neurocognitive ageing has drawn on blood‐oxygenation level‐dependent (BOLD) based fMRI, linked to the cognitive ontologies from cognitive neuroscience. However, the BOLD signal is not a direct measure of neural activity, but is a complex convolution of neural and vascular signals  to correct for the vascular effects of age when studying task‐induced BOLD fMRI responses. First, we characterized age differences in RSFA and determined the effects of RSFA scaling on age‐dependent fMRI‐BOLD signal change in a sensorimotor task. We predicted (i) that spatial patterns of age differences in RSFA would be similar to those reported by other calibrating techniques; and (ii) that the use of RSFA scaling would reduce the apparent effects of ageing on BOLD‐fMRI responses, by revealing the underlying effects of age on neural response.Importantly, given the unique combination of fMRI, MEG and physiological data from the large number of participants afforded by the Cam‐CAN dataset, we were able to validate the use of RSFA as a scaling factor. We did this by using mediation models to determine the extent to which age differences in RSFA were dependent on vascular and neural sources of signal fluctuations. In the first two mediation models, we examined the dependency of interindividual differences in RSFA with measures of vascular performance  and neural activity . We predicted there would be significant mediating effects of HRV, but not of neural variability, on the age differences in BOLD response variability. In a final confirmatory mediation analysis we determined whether or not HRV mediates the effect of ageing on neural variability.A healthy, population‐based sample was collected as part of the Cambridge Centre Ageing and Neuroscience (Cam‐CAN). Ethical approval for the study was obtained from the Cambridgeshire 2  Research Ethics Committee. Participants gave written informed consent. Exclusion criteria included poor vision  = 256 × 240 × 192 mm3; resolution = 1 mm isotropic; accelerated factor = 2) with acquisition time of 4 min and 32 s.Imaging data were acquired using a 3T Siemens TIM Trio System at the MRC Cognition Brain Sciences Unit. A 3D‐structural MRI was acquired on each participant using T1‐weighted sequence ; repetition time (TR) = 2,250 ms; echo time (TE) = 2.99 ms; inversion time (TI) = 900 ms; flip angle α = 78°; FOV = 192 × 192 mm2; resolution = 3 × 3 × 4.44 mm3) during 8 min and 40 s. Participants were instructed to lie still with their eyes closed. The initial six volumes were discarded to allow for T1 equilibration. The preprocessing and statistical analyses were carried out in SPM 12 (http://www.fil.ion.ucl.ac.uk/spm). The preprocessing steps included (i) spatial realignment to correct for head movement and movement by distortion interactions, (ii) temporal realignment of all slices to the middle slice, (iii) coregistration of the EPI to the participant's T1 anatomical scan, (iv) unified‐segmentation‐normalization to normalize the T1 image to the MNI template, the parameters of which were then applied to the functional data and (v) spatial smoothing with an FWHM of 8 mm to meet the lattice assumption of random field theory and account for residual interparticipant structural variability. Further processing procedures of the resting state time series for estimation of RSFA were performed using a combination of the following steps : The first step was to apply a data‐driven approach for removing motion artefacts from fMRI data  data of 261 volumes were acquired with 32 slices , slice thickness of 3.7 mm with a slice gap of 20%  of 1.97 s (resulting in SOAs ranging from ∼2–26 s). Scanning parameters and preprocessing steps were the same as for resting state data. Event‐related fMRI analysis for the sensorimotor‐task used a canonical haemodynamic response function (HRF) and its temporal and dispersion derivatives  with a duration of 34 and 300 ms, respectively. In addition, eight unimodal  stimuli were randomly distributed throughout the whole run. For each trial, participants responded by pressing a button with their right index finger if they hear or see any stimuli. In addition, null events, comprised of a fixation cross in the centre of the screen, were interdispersed with events of interest. To maximize efficiency, real and null events were ordered using a 255‐length m‐sequence  and scaled  activation maps . To formally address the differences between analyses with scaled‐ and unscaled‐BOLD signal, we constructed an additional second‐level GLM analysis with activation maps estimated from the difference between scaled‐ and unscaled‐BOLD maps.MEG data were acquired approximately 8 weeks before or after fMRI acquisition, in a magenetically shielded room using a Vectorview system  with 102 magnetometers and 204 orthogonal planar gradiometers. Vertical and horizontal eye movement was recorded using paired electrooculography (EOG) electrodes. Cardiac rhythm was monitored using paired electrocardiogram (ECG) electrodes – one on the right scapula and one on the left lower rib. Head movements were continuously monitored with respect to the MEG sensors using five head‐position indicator (HPI) coils. Position of the HPI coils, of approximately 100 “head points” across the scalp, and of three anatomical fiducials (the nasion and left and right preauricular points) were recorded using a 3D digitizer . Data were sampled at 1 kHz with a band‐pass filter of 0.03–330 Hz. Participants were in seated position with eyes closed for approximately 9 min. The initial 30‐s recording interval was removed from further analysis to allow a steady state of the recorded signal, resulting in 504321 samples for each individual, and provide a resting state recording with a duration similar to the duration of the fMRI resting state recording (∼8.5 min).Preprocessing of the MEG continuous data was performed with MaxFilter 2.2 . This uses spherical basis functions to remove environmental noise based on magnetic fields outside a sphere enclosing the sensor array, by rejecting temporally correlated sources inside this sphere but outside a sphere enclosing the brain ; Taulu et al., To remove residual non‐brain physiological artifacts of ocular or cardiac origin, the data were subjected to independent component analysis (ICA). Each independent component (IC) was then correlated with the recorded vertical EOG, horizontal EEG, and ECG. ICs that showed a high temporal correlation with one of the recorded EOG or ECG channels, and  a high spatial correlation with pre‐defined template topographies for vertical and horizontal eye‐movement were projected out of the data. The topographies were created by ICA of independent data, where the template topographies corresponded to the spatial part of those ICs that were clearly blinks, horizontal eye‐movements, or cardiac . We then averaged those spatial components over ∼18 subjects. A high temporal correlation was defined as at least three times the standard deviation of all IC correlations with each EOG/ECG channel; a high spatial correlation was defined as at least two times the standard deviation of all IC correlations with a template topography.Finally, we wanted to derive a measure of neural variability to relate to the rsfMRI variability. Previous work suggests that Hilbert Envelope signal variability shows linear relationship with raw signal variability of MEG time series  in milliseconds. To further screen for outlier IBIs which may result from ectopic beats  and with the ECG (in the case of the MEG session). The sampling frequency for PulseOx data was 50 Hz, while the ECG data was sampled at 1 kHz. Preprocessing of the cardiac data was performed in Matlab . This included R‐waveEstimation of mean heart rate (HR) was based on the mean number of IBIs within each 60‐s interval during the recording. To estimate the HRV, we used the frequency‐domain information of IBI, which provides a measure of low‐ and high‐ frequency components of the HRV, relative to time‐domain alternatives, for example, the root mean squared difference of successive intervals (RMSSD) which pertains mainly to high‐frequency dynamics of HRV  were from different modalities, we computed summary measures for each of these metrics to allow statistical analysis without excessive risk of multiple statistical comparisons. We started by calculating the SD across time for fMRI/MEG resting state time series data . We then used ICA across participants to derive spatial patterns of RSFA across voxels and rsMEG variability across sensors expressed by the group in a small number of ICs. The number of ICs was estimated using PCA with a minimum description length (MDL) criterion . In brief, the fastICA algorithm was applied after the optimal number of sources explaining the variance in the data was identified using PCA with MDL criterion  a source matrix that maps independent components (ICs) to voxels (here referred to as “IC maps”), and (2) a mixing matrix that maps ICs to participants. The mixing matrix indicates the degree to which a participant expresses a defined IC, see Figure Z‐scores) and used in the subsequent between‐participants analysis with standardized summary measures from other modalities.RSFA maps were decomposed in a set of spatially independent sources using Source Based Morphometry toolbox , and  a mixingAnalogously to the RSFA, the number of components of MEG variability was estimated with an MDL criterion and PCA, but for each frequency band separately, followed by ICA using a fastICA algorithm , which was acquired simultaneously with the resting state BOLD data. As a summary measure of each individual's RSFA, we used the IC loading value for each of a number of RSFA‐ICs. Only RSFA‐ICs showing ageing differences in the loading values were included in Model 1.– Model 1 tested whether HRV mediates the effect of age on RSFA, where age was the independent variable, HRV was the mediator and RSFA was the dependent variable  mediated the effects of age on RSFA, where age was the independent variable, rsMEG was the mediator and RSFA was the dependent variable  RSFA and vascular markers, and (ii) RSFA and neural variability , as part of the assessment of the use of RSFA to correct for the effects of age on BOLD fMRI. Since several measures of interest were likely to be correlated due to shared age‐related variance, we used mediation pathway analysis Hayes, , to testWhole group analysis revealed relatively high numerical values of RSFA (relative to the average across the brain) in the frontal orbital, inferior frontal gyrus (IFG), dorsolateral prefrontal cortex (dlPFC), superior frontal cortex, anterior and posterior cingulate, and lateral parietal cortex see Fig. a,b.With respect to ageing, we observed significant decreases in RSFA as a function of age in the bilateral IFG, bilateral dlPFC, bilateral superior frontal gyrus (SFG), primary visual cortex, cuneus, precuneus, posterior and anterior cingulate, superior temporal gyrus, medial parietal cortex, and lateral parietal cortex Fig. c. RegionGLM analysis revealed that the group BOLD response to audiovisual stimulation versus interstimulus baseline included the primary visual cortex, bilateral auditory cortex, the left motor/sensorimotor cortex, and the supplementary motor area . After RSFA scaling, however, the observed age‐related decreases in the visual areas and extended portion of the auditory areas  were abolished  vascular health, that is, HR and HRV and (ii) neural activity, that is, rsMEG signal variability of the time course of standard frequency bands.From ICA of the RSFA data, there were 12 components according to the MDL criterion , cerebellum (IC 2), posterior association areas , and anterior association areas (IC 10). These components, except for IC2, were more strongly expressed by young adults , and subarachnoid space (IC11).The topographic distribution of mean rsMEG variability across all participants revealed spatial distributions with largest signal variability in fronto‐temporal regions for the subdelta and delta bands, in superior fronto‐parietal regions for the theta band, in occipito‐parietal regions for the alpha band, in central areas  for the beta band and in fronto‐temporal areas for the gamma band see Fig. .In terms of ageing, beta and gamma signal variability in frontal and temporal regions increased with age, whereas alpha band variability in visual areas decreased with age. Lower frequency bands  showed age‐related increases in signal variability in peripheral sensors Fig. .From ICA of the rsMEG power in each frequency band, the optimal number of components according to the MDL criterion was 3, 3, 2, 4, 3, and 2 for subdelta, delta, theta, alpha, beta, and gamma bands, respectively. The topographies of the ICs for each band, and their relationships with age are summarized in Figure PulseOx) was highly correlated with PC1 from the ECG data (PC1ECG), r = 0.741, P < 0.001.PCA analysis estimated the first principal component (PC1) to explain approximately 65% of the variance across the three summary measures of HR, whether those measures were derived from the PulseOx recordings in the MRI scanner, or the ECG recordings in the MEG scanner. The absolute contribution to PC1 was similar across measures, varying from 0.39 to 0.67 Table . As expeOne of the main goals of the present study was to investigate the differential contribution of vascular and neural signals to RSFA. For the purpose, we conducted three mediation models using a product‐of‐coefficients approach Hayes,  to deterPulseOx loading values) and RSFA  and the direct effect of HRV on RSFA, while additionally controlling for age (path b1). The model also estimated the indirect effect of age on RSFA by way of HRV (path ab1) and the direct effect of age on RSFA (path c'1) when accounting for path ab1.The mediation analysis provided estimates of the direct effect of age on HRV , and participants with low HRV (irrespective of their age) showed differential expression of the spatial patterns for all ICs of RSFA (significant path b), except for IC2 and IC10. The indirect effect of age on RSFA through HRV was reliably negative according to bootstrap 99% CIs (path ab). There was evidence that the direct effect of age on RSFA, independent of HRV, was significantly reduced, that is, the absolute value of path c' was smaller than path c, indicating that HRV significantly mediated the ageing effects on RSFA IC loading values.As can be seen from Table In a second set of analyses, we were interested in whether the effects of ageing on rsfMRI variability in neuronally meaningful areas  were mediated by measures of neural variability. In this analysis age, rsMEG variability and rsfMRI variability were treated as the independent variable, the mediator and the dependent variable, respectively. As a summary measure of rsMEG variability, we used the grand mean of time‐series variability across all sensors for six frequency bands , see Figure path a), that participants with large rsMEG variability showed greater expression of the RSFA ICs (positive path b), that the indirect effect of age on RSFA through rsMEG variability was significantly positive (positive path ab), and that the negative effect of age on RSFA significantly increased after controlling for the indirect effects of rsMEG variability , that is, rsMEG variability was a significant suppressor, not mediator, of the ageing effects on RSFA.Out of 36 mediation tests (six frequency bands x six ICs of RSFA maps over GM areas), only two exceeded the 99% CIs around zero. These were the models with rsMEG variability in the sub‐delta frequency band as a mediator and RSFA loading values in IC1 and IC5 as the dependent variable (Table Alpha band‐IC3 and IC4) or weak ageing effects in few sensors might be washed out by averaging with the remaining sensors . This resulted in 90 mediation tests (15 rsMEG ICs × 6 ICs of RSFA), of which five tests satisfied all conditions for mediation/suppression according to 99% CIs  as a scaling parameter in the analysis of the effects of age on task‐related BOLD‐fMRI activations. Our results demonstrated the importance of such scaling to identify the effects of age on brain function: without accounting for influence of RSFA, there appeared to be a significant age‐related decline in activation of the primary visual and auditory regions during a sensorimotor task, accompanied by an increase in ipsilateral primary motor cortex. After RSFA scaling, the effect of age in the visual areas was significantly attenuated, to the extent that it was no longer significant. The effect of age in the motor cortex remained significant. We confirmed the hypothesis that individual differences in RSFA were related to cardiovascular factors. In particular, mediation analyses revealed that age‐related associations of widespread RSFA decreases across the brain were mediated by an age‐related decline in vascular functions. Moreover, by comparing with neurophysiological MEG data from the same participants, we demonstrated that age‐related changes of neuronal variability did not mediate the age‐related changes in RSFA. There was one possible exception to the latter statement: a small proportion of variance attributed to age differences of RSFA in sensory areas was influenced by neuronal differences as measured by MEG variance in the beta band. However, this was a suppression rather than mediation of the Age‐RSFA relationship (as discussed below).The BOLD responses to the sensorimotor task indicated an age‐related decrease of BOLD in visual and auditory cortices, in accord with previous studies  sensory areas , (ii) the cerebellum, and (iii) association areas . These patterns of spatially distinct cortical areas might reflect segregation of cortical tissue composition, for example, delineation on the basis of cyto‐ or myelo‐ architectonic differences  showed an age‐related decrease in tempo‐central regions . We also observed an age‐related increase in the alpha band and a decrease in the beta band over superior temporo‐parietal sensors, which might suggest age‐related slowing of resting‐state oscillations not be restricted to alpha band  are in agreement with previous reports using spectral power and signal complexity measures  contributed positively to the RSFA signal in sensory areas, that is, individuals with large neural variability showed large RSFA, an effect that remained even after accounting for the direct effect of ageing. This suggests that there is an effect of ageing on RSFA with a neural origin, which should increase with age, despite the observed overall age‐dependent decrease in RSFA. In other words, there might be two effects  of ageing on RSFA in opposite directions, where an age‐dependent increase in RSFA of neural origin is concealed by an age‐dependent decrease in RSFA with vascular origin.This dual effect of ageing on RSFA was confirmed in the mediation analysis, where we found no evidence that age‐related variance in neural activity (indexed by rsMEG variability) mediated positive age differences in RSFA across the whole brain. Instead, the effects of ageing on RSFA in sensory areas was suppressed, with a small portion of variance attributed to rsMEG variability in the alpha and beta band. In other words, accounting for the effects of rsMEG variability, the ageing effects on RSFA increased; or that, the suppression effect of rsMEG variability masked a small portion of the ageing effects on RSFA dominated by vascular modulatory origin in the sensory areas. As mentioned above, the contribution of neural activity to the RSFA might be as a result of functionally unspecific unregulated enhanced neuronal excitability within regions with increased rsMEG variability at rest as a result of less efficient GABAergic inhibition in ageing , we propose that scaling task‐induced BOLD signal by RSFA can still account for differences in vascular function, without removing the principle signals of neuronal origin.Although MEG offers a direct measure of neural activity, there are practical issues when trying to relate MEG to fMRI signals on intra‐ and interindividual basis. For instance, one reason why we did not observe rsMEG variability mediating the effects of age on RSFA might be due to MEG's relative insensitivity to signals from radially oriented sources, and from deep cortical and subcortical regions  to late age (78–88 years). Although the population sampling method for this study sought to minimize age‐based cohort effects, future studies would be strengthened by longitudinal analysis.We propose that RSFA can be used as a robust scaling factor to control for vascular differences in task‐induced BOLD signal, particularly in the context of fMRI studies of ageing. RSFA is, therefore, a suitable alternative to other calibrating techniques (such as breath‐holding and hyper/hypocapnia), where hypercapnic challenge would be inappropriate . Without such scaling methods, fMRI studies of the effects of age on cognition may misinterpret effects of age as neurocognitive, rather than neurovascular, phenomena.


MSCs) are being assessed for ameliorating the severity of graft‐versus‐host disease, autoimmune conditions, musculoskeletal injuries and cardiovascular diseases. While most of these clinical therapeutic applications require substantial cell quantities, the number of MSCs that can be obtained initially from a single donor remains limited. The utility of MSCs derived from human‐induced pluripotent stem cells (hiPSCs) has been shown in recent pre‐clinical studies. Since adult MSCs have limited capability regarding proliferation, the quantum of bioactive factor secretion and immunomodulation ability may be constrained. Hence, the alternate source of MSCs is being considered to replace the commonly used adult tissue‐derived MSCs. The MSCs have been obtained from various adult and foetal tissues. The hiPSC‐derived MSCs (iMSCs) are transpiring as an attractive source of MSCs because during reprogramming process, cells undergo rejuvination, exhibiting better cellular vitality such as survival, proliferation and differentiations potentials. The autologous iMSCs could be considered as an inexhaustible source of MSCs that could be used to meet the unmet clinical needs. Human‐induced PSC‐derived MSCs are reported to be superior when compared to the adult MSCs regarding cell proliferation, immunomodulation, cytokines profiles, microenvironment modulating exosomes and bioactive paracrine factors secretion. Strategies such as derivation and propagation of iMSCs in chemically defined culture conditions and use of footprint‐free safer reprogramming strategies have contributed towards the development of clinically relevant cell types. In this review, the role of iPSC‐derived mesenchymal stromal cells (iMSCs) as an alternate source of therapeutically active MSCs has been described. Additionally, we also describe the role of iMSCs in regenerative medical applications, the necessary strategies, and the regulatory policies that have to be enforced to render iMSC's effectiveness in translational medicine.Mesenchymal stem cells ( Bone marrow‐derived MSCs (BMMSCs) have been used as a predominant source of MSCs. Bone marrow‐derived MSCs have been successfully used in a significant number of clinical and pre‐clinical applications In this regard, human‐induced pluripotent stem cells (hiPSCs) reprogrammed from human adult somatic cells, converge to a better‐defined ground state of pluripotency. Human‐induced pluripotent stem cells can be differentiated into all three germ layer cell types  of the organism and – while in the pluripotent state – can be cultured virtually indefinitely without significant signs of replicative senescence. A recent breakthrough in the generation of hiPSCs from human somatic cells by using defined factors, in vitro differentiation potential and teratoma formation The PSCs could serve as an alternate source for the generation of MSCs. Embryonic stem cells can be used as an efficient source to generate the MSCs almost indefinitely, attributed to tremendous proliferation potential of ESCs The discovery of hiPSCs has accelerated the regenerative medical research The establishment of a reliable source of autologous, transgene‐free progenitor cells have enormous potential in the field of cell‐based regenerative medicine Recent studies using RNA‐based technology in vitro and in vivo+, CD90+, CD105+, CD166+, CD31−, CD34− and CD45−) et al. has shown that iMSCs could be orderly derived in a single step from iPSCs on fibrillar collagen coating et al. has shown that treatment of iPSCs with SB431542, a transforming growth factor β pathway inhibitor to generate epithelial monolayer‐like cells in two‐dimensional (2D) culture system followed by induction of epithelial–mesenchymal transition lead to rapid and reliable differentiation into iMSCs + peripheral blood or cord blood into functional mesenchymal stromal cells The adult MSCs does not exhibit long‐lasting immunoregulatory functions et al. have demonstrated that iMSCs from mice exhibited characteristic mice MSC surface marker such as CD105, CD140a, Sca‐1 and CD44 as previously described The specific cell surface marker on the human MSCs remains to be properly elucidated. Currently, a panel of markers is used to characterize the MSCs isolated from different tissue sources, since there is no specific marker for identifying the bonafide MSCs. The iMSCs satisfies the essential criteria's such as plastic adherence, expression of key MSC surface markers and tri‐lineage differentiation capability properties as laid down by the International Society of Cellular Therapy via secretion of suitable bioactive molecules  profile. However, tissue‐specific and age‐related DNAm profiles of iMSCs were completely erased. Further, the iMSCs reacquired senescence‐associated DNAm. The study also contrastingly highlights that iMSCs reacquire incomplete immunomodulatory functions The iMSCs and ESC‐derived MSCs displayed similar strong immunosuppressive characteristics les Fig. . Giulianin vivo teratoma formation that is caused by the rapid proliferation and uncontrolled spontaneous differentiation of PSCs. Thus, controlled differentiation of hiPSCs into cells that resemble adult MSCs is an attractive approach to obtain a readily available source of progenitor cells for tissue engineering. Unlike previously reported methods that typically rely on the addition of soluble factors to affect PSC differentiation, a recent study by Liu et al. reports an alternative approach using a biomaterial coating on a cell culture plate made of fibrillar collagen Type I to promote the derivation of MSC‐like cells. This study has reported a collagen matrix that could potentially play a positive role in regulating the differentiation of hESCs and hiPSCs towards a multipotent mesenchymal progenitor cell et al. to generate MSCs from human iPSCs, and investigate the osteogenic differentiation of iMSCs seeded on biofunctionalized CPCs containing RGD (Arg‐Gly‐Asp) peptides, fibronectin (Fn), Fibronectin‐like engineered polymer protein (FEPP), Geltrex and platelet concentrate has also been reported The use of stem cells and biologically suitable scaffolds offer the full potential for tissue regeneration. Transplantation of lineage‐committed cells can obviate in vitro culture conditions Giuliani et al. has shown that human MSCs derived from the iPSCs considerably down‐regulated NK cell cytolytic capabilities et al. showed MSCs from iPSCs ameliorated diabetic polyneuropathy (DNP) in mice et al. indicated that iMSCs facilitated the periodontal regeneration coupled with newly formed mineralized tissue in periodontitis rat models et al. demonstrated that tumour necrosis factor alpha‐stimulated gene‐6 (TSG6) expressing iMSCs were capable of decreasing the inflammation in experimental periodontitis model and inhibiting alveolar bone resorption et al. revealed that human iMSCs attenuate limb ischaemia in mice et al. indicated human iMSCs could continuously proliferate for more than 32 passages without undergoing cellular senescence and displayed superior wound healing and pro‐angiogenic properties et al. have shown that iMSCs derived from Hutchinson–Gilford Progeria syndrome (HGPS) were helpful in studying the molecular pathology of HGPS et al. has successfully utilized iMSCs for modelling Fanconi anaemia. The Fanconi anaemia iPSC‐derived MSCs displayed premature senescence The iMSCs could be effectively used for diseases modelling, drug screening and therapeutic applications Fig. . The immet al., reported generated iPSCs devoid of C‐Myc enervated retinal ischaemia and reperfusion injury following transplantation in rat models in vivo functionality because of epigenetic memory. Until now, there are no data on the best starter cell type for a particular clinical application. Hence, more research needs to be conducted to determine suitable starter cell type based on the type of clinical application in vivo transplantation often leads to tumorigenicity and genomic instability might result in low clinical utility Safety and Efficacy of iMSCs are of paramount importance to succeed in the field of translational regenerative medicine. The viral vector‐based strategy for reprogramming might result in tumour formation as a result of insertional mutagenesis of the transgene. C‐Myc is a proto‐oncogene that has been shown to increase the efficiency of reprogramming by suppressing the tumour suppressor p‐53 gene. The overexpression of proto‐oncogene and moderating of tumour suppressor genes render hiPSCs beneficial results regarding higher proliferative advantage for downstream translational applications etc., are favourable as they have relatively longer life span and displays physiological similarities to humans albeit a limited number of disease models and inability to modify the genome with ease constitute major road block in the usage of large animal models The iPSCs present unique sets of technical and regulatory hurdles when compared to even ESCs for translational applications in vivo is not well documented. A few studies have demonstrated that transplantation of PSCs and differentiated cells resulted in poor survival of the cells in vivo following transplantation. Hence, suitable surgical/imaging techniques should be developed for in vivo fate mapping of the cells Efficacy of the transplanted cells Current good manufacturing protocol guidelines must be followed to generate and characterize iPSC‐based products Generation of iPSCs from somatic cells requires a significant amount of molecular manipulations Immune response against transplanted cells presents a critical challenge that can detrimentally affect the outcome of therapy. Some of the important questions as to why the donor cells pose a risk of immune response or genetic diseases, cell efficiency, cells exhibiting risk of contamination, effectiveness and safety of transplanted cells has to be answered, if they are perceived to develop into potential therapeutic agents One of the biggest advantages of iPSCs is the possibility of generation of patient‐specific autologous cell lines. Hence, the cumbersome procedure of screening against different cell lines for a proper match is excluded The invention of cellular reprogramming of adult cells from the terminally differentiated state of PSCs state with the help of transcription factors, biological factors and small molecules open up a large window of opportunity in the field of regenerative medicine. By incorporating the advantages of both iPSCs and MSCs, the resulting iMSCs are emerging as a novel stem cell population None.


We revisit Fauth et al.'s conclusion and discuss how the use of these terms has changed over time since their review. An updated analysis of term definition from a selection of popular ecological textbooks suggests that definitions have drifted away from those encountered pre‐1996, and slightly disagreed with results from a survey of 100 ecology professionals . Results suggest that confusion about these terms is still widespread in ecology. We conclude with clear suggestions for definitions of each term to be adopted hereafter to provide greater cohesion among research groups.Community ecology is an inherently complicated field, confounded by the conflicting use of fundamental terms. Nearly two decades ago, Fauth et al. (1996) demonstrated that imprecise language led to the virtual synonymy of important terms and so attempted to clearly define four keywords in community ecology; “ Ecology is a young but rapidly developing field of science. Unlike more established fields, such as mathematics and physics, ecologists are yet to create an established and unambiguous framework of terminology Hodges . Nearly community or assemblage are prone to subjective interpretations by ecologists. Variability in the use of these terms can impact the efficacy of comparisons across ecological datasets, which in a discipline that includes increasingly larger temporal and spatial scales may hinder the interpretation of more comprehensive ecosystem patterns  (see Online Appendix A1 for survey questionnaire). We advertised the survey on several popular ecology blogs, mailing lists  and social media outlets . During the sampling period of 7 days, approximately 400–500 people viewed the survey and provided 100 completed surveys (20–25% completion rate). We received responses from 11 countries and 31 states within the United States. Respondents were asked to report their profession  and field of study. Definitions were quantified using weighted rubrics of key words, which were designed to encompass the most important descriptive factors of each term (see Online Appendix A2). This survey was conducted in an attempt to observe if there existed differences in term understanding between students of ecology ranging from undergraduate level to professors. Additionally, we aimed to assess whether their interpretations correctly aligned with definition trends displayed in popular ecological textbooks , compared to 20% of undergraduates surveyed. This suggests an understanding of basic ecology terminology that is adjusted with career progression, but also provides the potential for a misunderstanding of basic definitions that may persevere over the span of a career.There was acceptance among all survey groups that community as a “group of species that occur together in space and time”  and biological  processes drive ecological processes and patterns .Survey results indicate that the most important defining factor of a guild into two subdefinitions, functional guild and taxonomic guild, so as not to repeat the patterns of generalized and ambiguous interpretations that have been observed in the use of the term community. We suggest taxonomic guild refer to “a group of taxonomically related species that exploit the same class of resources in a similar way”, while functional guild refers more broadly to “a group of functionally similar species that exploit the same class of resources in a similar way”  on Pacific and Caribbean reefs have very different functional roles depending on whether they bioerode reefs or not  should be used to inform environmental and physiological constraints.One pattern that has emerged repeatedly is that phylogenetically similar species can have very different functional roles Duffy . For exaensemble in ecology remains rare, although the term has experienced an increase in use over the past decade  at conceptualizing these terms; however, feel that the current usage of all terms has continued to deviate despite these efforts, and an updated review was needed. Remarkably, there was no complete agreement for any one definitive factor for any term in our survey of ecologists (range = 0–82.11%). This highlights that, although incredibly popular in current literature (Fig. Here, similar to Schoener , we haveNone declared.Table S1. Survey questionnaire.Table S2. Survey results quantification methods.Click here for additional data file.


This article highlights pre‐clinical and clinical studies into the field of wasting disorders that were presented at the 8th Cachexia Conference held in Paris, France December 2015. This year some interesting results of clinical trials and different new therapeutic targets were shown. This article presents the biological and clinical significance of different markers and new drugs for the treatment of skeletal muscle wasting. Effective treatments of cachexia and wasting disorders are urgently needed in order to improve the patients' quality of life and their survival. The development of preventive and therapeutic strategies against cachexia and wasting disorders including sarcopenia, are perceived as an urgent need by health professionals and have instigated intensive research on the pathophysiology of these syndromes.et al.  presented interesting data to the role of TWEAK‐Fn14 signalling in muscle wasting.in vivo. The cytokine TWEAK and its cognate receptor Fn14 are members of the TNF/TNFR superfamily and are upregulated in tumors. In recent literature, Johnston et al.et al.et al.Lactobacillus spp. levels were decreased in leukemic mice with cachexia, and the denaturing gradient gel electrophoresis analysis suggested that bacteria other than lactobacilli were affected by the disease.One interesting candidate in the pathway of muscle wasting, presented at the conference, is the tumor necrosis factor (TNF)‐like weak inducer of apoptosis (TWEAK). Kumar et al.n = 8) and weight‐stable (n = 8) gastro‐oesophageal cancer patients and healthy controls (n = 8). The number of protein species identified in cachectic samples was greater than that identified in weight‐stable cancer and control samples. Many of the proteins identified have not been reported previously in the urine of cancer patients. Proteins identified specifically in cachectic samples included muscle (myosin species), cytoskeletal  and microtubule‐associated proteins , whereas immunoglobulin κ‐light chain and zinc α‐2 glycoprotein appeared to represent markers of cancer. Using urinary proteomics they suggest myosin 2 as new potential marker. Thus they conclude that urinary proteomics can identify muscle‐specific and non‐muscle‐specific candidate biomarkers of cancer cachexia.A number of elegant models were presented in order to improve our understanding of pathways involved in the wasting process. Muscle wasting has received increasing research efforts in recent years. Thus, one of the hot topics of the meeting was the investigation of biomarkers using muscle proteome and urine proteome. Skipworth et al. et al.n = 13) or to the control group (n = 13). They showed the safety and suitability of such an intervention but whole body vibration training did not influence muscle strength in this intervention significantly. Maddocks et al.et al.P = 0.05). BIA may represent a useful non‐invasive tool for the clinical assessment of this population group–but more research is necessary. However the most amazing method was recently descripted by Stimpson et al. et al.  presented results of a clinical validation study demonstrating that the creatine dilution method is strongly associated with whole body MRI‐method. The dose of D3 creatine in adults is 30 mg and 2 mg in infants. The D3 creatine dilution method appears to be a valid non‐invasive measurement for muscle mass in infants2H2O, lean body mass and muscle mass can be measured non‐invasively. Importantly the measurement of muscle mass using the creatine dilution method is not affected by shifts in body water that occur with many cachectic diseases and that sometimes poses a confounder in other measures of skeletal muscle estimations.Most impressive data in the field of body composition and exercise were presented by Vanderbyl et al.  presented data on the efficacy of the specific antagonist of myostatin named REGN1033. REGN1033 was tested in 253 patients with sarcopenia. They were split into four groups: (i) placebo group (n = 55), (ii) 100 mg REGN1033 subcutaneously every 4 weeks (n = 63), (iii) 300 mg REGN1033 subcutaneously every 4 weeks (n = 65) and (iv) 300 mg REGN1033 subcutaneously every other week (n = 60). Compared with placebo, all doses of REGN1033 treatment were associated with a significant increase in total lean body mass from baseline to week 12. At the higher dose (300 mg) REGN1033 treatment resulted in significant decreases in total and regional fat mass. Mean values of strength and function tests generally tended to indicate a beneficial direction with REGN1033 treatment. REGN1033 was well tolerated at all 3 doses tested. The results showed that REGN1033 increases muscle mass, force production, and physical performance outcomes in patients with sarcopenia, and preventing the loss of muscle mass. Thus, the use of antagonists of myostatin may be worthwhile in clinical testing.Loss of skeletal muscle mass and strength plays a significant pathological role in the progression of a wide variety of disorders associated with aging and catabolic conditions. Neutralization of myostatin activity results in skeletal muscle hypertrophy and prevents atrophy in adult skeletal muscle. REGN1033 is a fully human monoclonal antibody administered subcutaneously that specifically binds myostatin and blocks its function. Donahue et al.  gave an update of recent enobosarm developments. They showed data from the International Pivotal Phase III Clinical Trials G300504 and G300505, which used enobosarm for prevention and treatment of muscle wasting in patients with non‐small cell lung cancer (NSCLC) stage III/IV at initiation of first line chemotherapy. Enobosarm (3 mg) was tested in patients with platinum and taxane‐based chemotherapy (G300504) and in patients with platinum and non‐taxane‐based chemotherapy (G300505), both against placebo. These trials confirmed that patients with advanced NSCLC have severe muscle loss and physical function impairment at diagnosis that decline further with platinum‐based chemotherapy treatment. Enobosarm treatment was associated with an increase in lean body mass compared with a decline in the placebo group in both studies. In G300504, enobosarm treatment was associated with better stair climb performance compared with placebo but not so in G300505. Lean body mass response was associated with an improvement of both physical function and survival, as well as maintenance of quality of life.Other therapeutic treatments include the selective androgen receptor modulators (SARMs). SARMs are a new class of non‐steroidal, tissue specific, anabolic agents that have the potential to increase muscle mass and improve physical function without the unwanted effects on the prostate, skin, or hair that are commonly associated with testosterone or other nonselective, synthetic anabolic steroids. Enobosarm is a nonsteroidal SARM that induces conformational changes in the androgen receptor upon binding, which selectively alters the interaction of the receptor with co‐activator and co‐repressor proteins that exist in different tissues and changes the receptor's ability to regulate gene expression.et al.  presented data from the Safety and Efficacy of Anamorelin HCl in Patients With Non‐Small Cell Lung Cancer‐Cachexia (ROMANA 1 and 2) trials with ghrelin receptor agonist anamorelin. Anamorelin is currently in phase III development, with two parallel trials in NSCLC cachexia recently completedet al.  identified a new potent ghrelin receptor agonist, RQ‐00433412. RQ‐00433412 increased growth hormone release in mice and rats, increased body weight in normal mice, attenuated decrease in body weight and increased food consumption in cisplatin treated rats. They showed attenuated decrease in body weight and muscle mass, without changes of food consumption in AH‐130 hepatoma rat cachexia model. Ghrelin and ghrelin receptor agonists are potential useful in cancer anorexia/cachexia.Ghrelin and ghrelin receptor agonists are being explored for their potential impact on clinical conditions such as anorexia and cancer cachexia. These compounds have been found to have clinical attributes, which make them well‐suited for the treatment of cachexia.α‐spectrin; nischarin) and microtubule‐associated proteins. For non‐invasive measurement of skeletal muscle mass the D3‐creatine dilution method can be applied repeatedly to measure total body creatine skeletal muscle mass change in longitudinal studies.From basic science new therapeutic targets were shown including the TWEAK–Fn14–NF‐κB–MuRF1– myosin heavy chain protein degradation cascade and the neutralization of tumour‐derived parathyroid‐hormone‐related protein, as well as the influence and the role of the gut microbiota in the therapeutic management of cancer and associated cachexia. Effective Treatments were REGN1033, Ghrelin and ghrelin receptor agonist anamorelin and enobosarm. Many new hopes have appeared on the cachexia treatment horizon recently, and several drugs have shown to increase muscle mass but failed to translate this effect into increased strength. Other substances remain in preclinical testing, but we are awaiting clinical testing to commence soon for RQ‐00433412. There is further need for attractive biomarkers as therapeutic target. Urine proteomics identified new potential biomarkers for example myosin species, proteins of the cytoskeletal (The authors N.E. and S.vH. declare that they have no conflict of interest.


Patients with a substance use disorder (SUD) and co-occurring attention deficit hyperactivity disorder (ADHD) or autism spectrum disorder (ASD) often start using substances in an attempt to cope with the stress related to their ADHD or ASD. To improve treatment for these patient groups, it is important to identify and compare the various coping styles between SUD patients with and without ADHD or ASD and with subjects from a general population sample.Cross-sectional study using the Utrecht Coping List (UCL) in 50 SUD patients, 41 SUD + ADHD patients, 31 SUD + ASD patients and 1,200 railway employees.Compared with the reference group, all three SUD groups showed a significant higher mean on the Palliative reaction, Avoidance, and Passive reaction subscales of the UCL. The scores for all UCL subscales of the SUD and the SUD + ADHD groups were very similar. However, the SUD + ASD group scored higher on Passive reaction and lower on Reassuring thoughts than the SUD and the SUD + ADHD groups and lower on Expression of emotions subscale in comparison with the SUD + ADHD group.Regardless of the presence of a co-occurring disorder, SUD patients reported more palliative, avoidant and passive coping when confronted than people in the general population. In addition, SUD patients with co-occurring ASD were different from other SUD patients in their coping and professionals should take this into account when working on more adaptive coping strategies with these patients. There are many reasons for why people start using substances, continue to use them and eventually become dependent on them. Speaking broadly, three core reasons for substance use can be distinguished: (a) positive reinforcement (i.e. instant pleasure and euphoria provided by substance use); (b) negative reinforcement ; and (c) habitual/compulsive substance use  –7.People with Attention Deficit/Hyperactivity Disorder (ADHD) are more likely to use substances and become addicted because they are either looking for thrills  or seeking relief from their ADHD symptoms  , 9. Canninitially started to use alcohol and/or drugs to cope with ADHD and ASD associated stress. Substance use may initially ameliorate the symptoms and the related stress but it may worsen the situation later [In a recent study, we have shown that patients with a substance use disorder (SUD) and co-occurring ADHD or ASD on later . An impoCoping refers to the cognitive and behavioural efforts of individuals to manage their internal and external demands which are appraised as taxing or exceeding the resources of the individual . People dysfunctional coping strategies was related to poor mental health while the use of what could be considered functional coping strategies was related to good mental health. Specific coping strategies and overall coping styles showed some stability but were nevertheless open to change over time and thus malleable.Unfortunately, there is no consensus on the classification of coping styles or the best model of human coping to be used . For exaalso used ‘positive reappraisal’ as an adaptive coping strategy intended to give some positive meaning to the stressful situation via a focus on personal growth and learning [Research has shown that ADHD patients use more confrontational and escape-avoidance behaviours for the management of stressful situations and less planned problem-solving strategies when compared to a control group of individuals without ADHD , 19. Howlearning . Finallylearning –23.To our knowledge, however, no studies have compared the coping styles of SUD patients with and without a co-occurring ADHD (SUD + ADHD) or co-occurring ASD (SUD + ASD). It can thus be asked (1) what coping styles are displayed by adult SUD patients with and without co-occurring ADHD or ASD and (2) whether significant differences emerge when the coping styles of these groups of SUD patients are compared to those of a reference group of individuals from the general population.Based on the literature summarized before, we hypothesize that SUD + ADHD patients will report more confronting behaviour and more positive reappraisal than SUD and SUD + ASD patients. In contrast, SUD + ASD patients will report more disengagement than SUD+ ADHD and SUD patients. We also hypothesize that SUD + ADHD patients will report more avoidance behaviour than SUD and SUD + ASD patients. Finally, we expect all of the patients to score similarly for socialization behaviours despite stronger limitations in the social interaction and communication capacities of ASD patients.To test these hypotheses, coping style data of SUD patients with and without a co-occurring ADHD or ASD were compared to each other and to existing coping style data for a general population reference group.A cross-sectional study was conducted using the Utrecht Coping List (UCL) in SUD patients with and without a comorbid ADHD or ASD and a group of healthy controls.The patient population consisted of SUD patients without and with a comorbid ADHD (SUD + ADHD) or comorbid ASD (SUD + ASD). Inclusion criteria: inpatients and outpatients seeking treatment for SUD; age 18–65; IQ >80; DSM-IV diagnosis of SUD with or without DSM-IV diagnosis of adult ADHD or ASD; and mastery of the Dutch language. Exclusion criteria: somatic complaints not directly related to SUD. All 122 patients also participated in the study previously reported by Kronenberg et al. . AlthougThe general population reference group consisted of 1,200 Dutch railway employees who were very similar to the patient groups in age (mean 43 years) and gender  .A certified medical ethics committee (Commissie Mensgebonden Onderzoek Regio Arnhem-Nijmegen) and the institutional review board of Dimence (Commissie Wetenschappelijk Onderzoek) approved the study. All patients received both oral and written information about the study and signed an informed consent form.In order to identify coping styles, we administered the Utrecht Coping List (UCL) to all patients. The UCL consists of 47 items constituting seven subscales: (1) Active problem solving , (2) Palliative reaction , (3) Avoidance , (4) Socialization , (5) Passive reaction , (6) Expression emotions , (7) Reassuring thoughts .All UCL items are rated on a four-point Likert scale ranging from 1 (=never) to 4(=very often). A higher score on a coping style thus indicates that this response style is used more often. Table In the SUD groups, the internal consistency of the subscales was found to be good for four scales  and acceptable for three scales .p < .05) was conducted on the UCL scores to test for significant differences between the three groups of patients. An additional series of ANCOVAs was performed to control for group differences in the living situations of the patients. Post-hoc independent T-tests (p < .05) were conducted to clarify the differences between the groups when significant differences were revealed by the ANOVAs or ANCOVAs. In addition, the standardized effect sizes (Cohen’s d) were calculated to facilitate the interpretation of significant outcomes: d < 0.20 no relevant difference; d =0.20–0.30 small difference; d = 0.30–0.80 moderate difference; and d > 0.80 large difference [The UCL scores were normally distributed for all groups. A series of one-way ANOVAs (fference . In ordep = 0.036 and cannabis F 4.684/p = 0.011). Based on these findings, the SUD group comparisons were adjusted for differences in living conditions (p = 0.025). We decided not to adjust for the substances that were used, because this could result in overcorrection and produce false negative findings.A total of 122 patients participated in the study: 50 patients with SUD only, 41 patients with SUD + ADHD and 31 patients with SUD + ASD. Most of these patients were in a substance abuse treatment programme at the time of assessment although a small percentage (13%) was not. There were substantial group differences in the living conditions of the patients compared to each other and compared to the healthy controls. There were also substantial differences between the three patient groups in the substances which were used . Similar findings were found when differences in the living situations of the patients were controlled for  (ANCOVA B-C-D). In particular, SUD + ASD patients reported more Passive reaction and used less Reassuring thoughts than both the SUD group (d = −0.534 and d = 0.560) and the SUD + ADHD group (d = 0.511 and d = −0.713). In addition, the SUD + ASD group reported less Expression of emotions than the SUD + ADHD group (d = 0.511). No significant differences were observed between the SUD and the SUD + ADHD group.Table Passive reaction , more Palliative reaction  and more Avoidance .From Table Palliative reaction, Avoidance and Passive reaction subscales. The scores for the SUD and SUD + ADHD groups are very similar on all UCL subscales. However, the SUD + ASD group scores are significantly higher on Passive reaction and lower on Reassuring thoughts compared to the SUD and SUD + ADHD groups and lower on Expression of emotions compared to the SUD + ADHD group.Compared to the reference group, all three SUD groups scores much higher on the Active problem solving behaviour than the other groups while their average score on Reassuring thoughts was higher than in the SUD + ASD group but not the SUD group.These findings only partly supported our first hypothesis, namely that patients with SUD + ADHD would show more confronting behaviour but also more positive reappraisal than patients with SUD and SUD + ASD. The patients with SUD + ADHD in our study did not show more Passive reaction and lower on Expression of emotions than the SUD + ADHD patients.Our second hypothesis was confirmed: patients with SUD + ASD in our study reported more disengagement behaviour compared to the patients with only SUD and those with SUD + ADHD. The SUD + ASD patients scored higher on Avoidance than the reference group with no significant differences detected between the three patient groups.Our third hypothesis that the SUD + ADHD group would report more avoidance behaviour than both the SUD and SUD + ASD groups was not supported. All three patient groups scored higher on Our fourth and final hypothesis that all patient groups would score similarly with respect to socialization behaviours (i.e.seeking comfort from other or asking for help) was supported: all patient groups scored similar with no significant between group differences.and ADHD . This would then explain why the SUD + ADHD group did not score higher than the other patients groups and reference group on confronting behaviour.Our initial hypotheses were derived from a very small body of studies involving mostly patients with ADHD or ASD and not from studies with SUD patients with a comorbid ADHD or ASD. Data from patients with a single diagnosis obviously cannot be applied automatically to patients with co-occurring diagnoses . It is cIn contrast, it was expected that the SUD + ADHD group would show avoidance as a particular coping style compared to the SUD and SUD + ASD groups since studies on both the single diagnosis of SUD –23 and tAs expected, the patient groups scored similarly on socialization behaviours despite the often strong limitations on the social interaction and skills of patients with SUD + ASD. Substance use, particularly for the patients with ASD, may have facilitated social interaction , 29.The results of the current study confirm previous results showing passive disengagement coping strategies to be associated with high levels mental health problems . HoweverThe present study has both strengths and limitations. The most important strengths are the relatively large subgroups of patients included in the study and the availability of a relatively well-matched reference group. A first limitation is the relatively low internal consistency of some of the UCL subscales in the patient group. This is probably due to some of the patients and especially those in the SUD + ASD group having difficulties distinguishing between the ‘sometimes’ versus ‘often’ response options. Research assistants were available to assist but may not have been approached. A second limitation is that comorbid conditions other than ADHD and ASD were not identified and utilized as an exclusion criterion. A third limitation is that we did not take into account the primary substance of abuse as a confounder for the differences in coping style between the three patient groups, because this may have lead to overcorrection and false negative findings. However, it can not be excluded that differences in coping style between the patient groups are partly explained by differences in the different patterns of alcohol and drug use. A fourth limitation is that patient groups differed considerably in their living conditions. However, statistical adjustment for these differences did not change the findings. In order to prevent false negative findings in this relatively small study, we decided not to correct for multiple comparisons. A fifth limitation is that males were highly overrepresented in all three patients groups and in the reference group. We therefore cannot extrapolate from the current findings to draw conclusions about female patients with SUD, SUD + ADHD or SUD + ASD. This is important, because it is also well known that males and females differ considerably in coping strategies . Finallyall SUD groups were Palliative reaction, Avoidance and Passive reaction. These coping styles may be effective in the short run because they decrease the jumble of emotions and mixture of agitation, distress and anxiety which can plague these patients [palliative, avoidance or passive coping style can interfere with the ability of patients to effectively deal with their impairments, anticipate possible problems and handle problems when they occur. Guiding patients towards a more active, adaptive and problem-focused manner of coping should be at least one goal of treatment.Overall, the preferred coping styles for patients . In the Active problem solving and Reassuring thoughts. For SUD + ASD patients, reducing their substance use might even be the primary treatment goal as substance use is known to increase passivity. However, in this patient group substance use may also enable socialization. The care professional might collaborate with the patient on the development of a relapse prevention plan, for example, and help the patient to anticipate problems and thus prevent them or be better prepared to deal with them in a functional as opposed to dysfunctional manner.In addition, total abstinence or reduced consumption should be a treatment goal for all patients with a combination of disorders. In SUD + ADHD patients, substance use may hinder Palliative reaction, Avoidance and Passive reaction coping styles when confronted with unpleasant events and problems than the general population. In addition, patients with SUD + ASD used more Passive reaction and less Reassuring thoughts than patients with SUD or SUD + ADHD. These findings are important for understanding SUD patients in general and those with a comorbid diagnosis in particular. Patients with SUD + ADHD frequently want to become totally abstinent because their SUD exacerbates their ADHD symptoms and lead to major problems in many domains of adult life. In contrast, patients with SUD + ASD often only want to reduce their drinking, because it can facilitate their social interaction.SUD patients in this study used more


This treatment enables the drugs to reach their intended subcellular target more efficiently and improves their efficacy. In this review we summarise the applications of this technique with the main emphasis placed on cancer chemotherapy.Photochemical internalisation (PCI) is a technique for improving cellular delivery of certain bioactive agents which are prone to sequestration within endolysosomes. There is a wide range of agents suitable for PCI‐based delivery including toxins, oligonucleotides, genes and immunoconjugates which demonstrates the versatility of this technique. The basic mechanism of PCI involves triggering release of the agent from endolysosomes within the target cells using a photosensitiser which is selectively retained with the endolysosomal membranes. Excitation of the photosensitiser by visible light leads to disruption of the membranes  DDSdrug delivery systemEGFRendothelial growth factor receptorEGP‐2endothelial glycoprotein‐2HER‐2human epidermal growth factor receptor 2HUVECshuman vascular endothelial cellsMDRmultidrug resistanceNPnanoparticlePCIphotochemical internalisationPDTphotodynamic therapyPSphotosensitiserRIPribosome inactivating proteinROSreactive oxygen specieset al. and is also an “active” DDS, since drug delivery is triggered by the application of light.Chemotherapy is routine in the treatment of a range of cancers, however limitations include systemic toxicity and multidrug resistance (MDR),via photooxidative damage thereby releasing the drug confined within the organelles . This treatment enables the drugs to reach their intended subcellular target more efficiently which enhances their efficacy. This release mechanism also serves to counteract enzyme‐induced degradation of some drugs within lysosomes. Because photosensitisers used for PCI are fluorescent, the photochemically induced disruption to the endolysosomes can be studied using fluorescence imaging.in vitro.et al. have also demonstrated in vivo light‐induced redistribution of a cytotoxic agent, gelonin, in treated rat liver using immunohistochemistry.The basic mechanism of PCI involves triggering drug release from endolysosomes within the target cells using a photosensitiser (PS) which is selectively retained with the endolysosomal membranes. Excitation of the PS using visible light in the presence of molecular oxygen leads to disruption of organelle membranes les Fig. a. This in vivo studies, the PS and bioactive agent are administered systemically but the enhanced drug delivery induced by PCI is confined to the zone of illuminated tissue, and PCI is therefore a site‐specific technique. PCI has been successfully used for delivery of anti‐cancer cytotoxins such as bleomycin, which is now being tested clinically as discussed below. With improved intracellular delivery of the bioactive agents, the administered dose of the agent can be reduced which is important in the chemotherapy induced toxicity.For b). PCI is a site‐specific drug delivery technique and thus, unlike PDT, the photosensitisers employed in PCI are not designed to induce a cytotoxic effect on their own. Besides, photooxidative damage to endolysosomes is not an efficient means of inducing cytotoxicity by itself.The development of PCI as a drug delivery technique stemmed in part from Photodynamic Therapy (PDT) which also involves the administration of a photosensitiser. Excitation of the PS results in the production of reactive oxygen species (ROS), particularly singlet oxygen which can oxidise important subcellular substrates including membrane components such as unsaturated lipids and cholesterol. Like PDT, PCI also relies on the presence of molecular oxygen for generation of ROS, although since oxygen solubility in organelle membranes is relatively high only strongly hypoxic cells should be unaffected by PCI.The photosensitiser should induce efficient generation of ROS and low aggregation propensity since monomeric PS is more photoactive, and exhibit strong absorption at red/near‐infrared wavelengths to enable deeper therapeutic efficacy. PCI is therefore distinct from other light‐triggered delivery systems which rely on UV excitation where tissue penetration is very limited. In practice, PDT is difficult to suppress in areas receiving the highest light dose, but PCI will dominate at the deeper regions of the tumour. Because the PS dose used for PCI is deliberately low, skin phototoxicity should be less likely.There are several different photosensitisers currently available for PCI based on phthalocyanine, porphyrin or chlorin aromatic dye molecules, typically amphiphilic sulfonated derivatives where the sulfonate groups are substituted adjacently on one side of the aromatic macrocycle. The opposite unsubstituted side of the macrocycle is therefore relatively hydrophobic and can reside within the lipid bilayer, allowing the ionic substituted side to localise at the interface of the lipid/aqueous phase.via adsorptive endocytosis with optimum localisation occurring after 18–24 hrs administration in vitro although longer times have been used in vivo. AlPcS2a  is a phthalocyanine‐based PS, was initially used for in vivo PCI owing to its strong absorption in the far‐red region (670 nm).2a (Amphinex®) has been developed, which contains far fewer regioisomers than AlPcS2a and is therefore easier to prepare reproducibly.2a has suitable photophysical and photobiological properties for PCI2a (meso‐tetraphenylporphin with two sulfonate groups on adjacent phenyl rings), has been extensively used in vitro with good results but is less suitable for clinical development since its red absorption is comparatively weak.et al. have shown that conjugation of a hydrophobic porphyrin to a cationic cell penetrating peptide rendered the porphyrin suitable for PCI.The resulting amphiphilic structure favours endolysosomal membrane localisation owing to uptake PCI is site selective owing to the light delivery, selective uptake of photosensitisers in tumours is generally insufficient to confer treatment selectivity. Enhanced selectivity may be conferred by passive targeting of the agents through the use of macromolecular drug carriers and the EPR effect (enhanced permeability and retention) or actively targeting the agents by conjugation with ligands such as antibodies. In the following section, we review cytotoxic cancer chemotherapy agents that have been used for PCI without active targeting followed by a review of results obtained using actively targeted drugs  which favours uptake et al. in 2005 performed PCI both in in vitro and in vivo human sarcoma, human colorectal adenocarcinoma and murine colon carcinoma models. TPPS2a (0.7 µg mL−1) and bleomycin (0.14 IU mL−1) significantly enhanced cytotoxicity by a factor of three compared to bleomycin alone in vitro2a, resulted in significant tumour growth delay compared to control groups without PCI. Furthermore, the initial weight loss seen in bleomycin treated animals, was reversed in the PCI group.in vivo study, Norum et al.2a. Using histology, a smaller area of viable peripheral tissue was seen post‐PCI for the treated animals than for PDT alone. The greater cell killing in this highly proliferating area was not due to higher levels of AlPcS2a as confirmed in fluorescence studies.Berg et al. demonstrated the advantage of bleomycin PCI using a range of chemotherapeutic agents in several human and non‐human bladder cancer cell lines in vitro.In vivo studies showed improved survival of animals bearing the F98 glioma model using a combination of bleomycin PCI and an epsilon prototoxin which is known to disrupt the blood‐brain barrier versus controls.PCI using bleomycin has also been investigated for bladder and brain cancer. Arentsen in vitro study on human head and neck cancer cell lines, bleomycin PCI has been shown to be effective when a lipophilic photosensitiser is administered using liposomal formulations which should favour uptake by endocytosis. However for this approach to work effectively in vivo the photosensitiser would have to remain confined to the liposome once in circulation.As discussed above, amphiphilic photosensitisers are generally used for PCI but in a recent et al. compared PCI in doxorubicin sensitive and doxorubicin resistant MCF‐7 breast cancer cell lines and showed that PCI overcame endosomal entrapment of the doxorubicin in the resistant cell line by promoting transport of doxorubicin to cell nuclei.et al.,via the ATP‐binding cassette transporter involved in MDR (ABCG2).PCI has also been used successfully with anthracycline drugs, doxorubicin (0.5 kDa) and mitoxantrone (0.4 kDa), which inhibit DNA and RNA synthesis. Although these are relatively small molecules, they are weak bases which may be retained within acidic lysosomes due to ion‐trapping of the protonated form. In 2006, Lou et al. used the NHIK 3025 cervical carcinoma cell line exposed to a combination of gelonin and photosensitisers (TPPS2a or AlPcS2a).et al.,2a to determine the difference in gelonin‐induced cytotoxicity using a light—“before” or —“after” approach. A light‐before strategy significantly enhanced gelonin's cytotoxicityet al. completely eradicated sarcoma induced in mice in 50% of gelonin PCI treated animals, and showed significantly delayed tumour regrowth in the remaining 50% compared to PDT or non‐treated groups.Type 1 ribosome inactivating protein (RIP) inhibitors such as gelonin and saporin (approximately 30 kDa MWt), which are plant‐derived are candidate chemotherapy agents but require cytosolic delivery. Gelonin and saporin are both highly toxic but subject to endolysosomal sequestration and degradation, and have therefore been widely investigated for PCI studies.et al. investigated cytotoxicity induced using liposomes containing toxins such as saporin by PCI.PCI is well suited for the delivery of macromolecular drug carriers, such as dendrimers and other nanocarriers, which owing to their size are taken up by endocytosis, thereby limiting the efficacy of the drug which then has to escape from the endolysosomes to reach its target site. Fretz et al. recently developed polymeric nanocarriers based on ketals which were co‐loaded with camptothecin (0.35 kDa) and the photosensitiser haematoporphyrin which were administered to HeLa cells.in vitro.Pasparakis et al. in 2006et al. in 2012EGFR targeted PCI has been the focus of several studies, where the RIP inhibitor saporin has been conjugated to anti‐EGFR molecules.2a and AlPcS2a), with a greater effect seen with the targeted MOC31‐gelonin in a small cell lung carcinoma cell line (NCI‐H146). Lower cytotoxicity was seen with MOC31‐gelonin alone, but were equally efficient on EGP‐2 antigen negative cells.Endothelial glycoprotein‐2 (EGP‐2), is overexpressed in most carcinomas.in vitro2a to treat mice bearing subcutaneous WiDr tumours, where 20 days after PCI treatment in 6/9 cases tumours were completely eliminated. Moreover, no weight variation was seen in the animals and skin damage was resolved 3 weeks after therapy.The same study was performed using WiDr human colorectal cells et al. in 2012versus PCI induced cell killing on either HER2+ or HER2− breast cancer cells.HER2 is overexpressed in ∼25% of all breast cancer cases and it is used to determine progression and prognosis.et al.2a and TPPS2a). This fusion toxin was used to demonstrate the potential of PCI for skin cancer treatment in vivo (A‐375 cells), as well as malignant glioma (U87MG) and lobular breast carcinoma (MA11) cells in vitro.et al.,The progenitor marker gp240 has been found to be relevant in lobular breast carcinomas, gliomas and melanomas.in vitro were exposed to saporin bound to antibodies against two different epitopes of CD133, combined with TPCS2a and light.in vitro, were able to delay in vivo tumour growth in 2/3 cases and no tumour regrowth occurred in 1/6 cases. PDT studies in vitro seemed to trigger cellular proliferative signals, which would correlate with the tumour regrowth observed in vivo.PCI has been used to successfully target cells expressing CD133, a surface molecule which has been described as a marker for stem‐like cancer cells exhibiting resistance to chemotherapeutic agents and resulting in a poor prognosis.high, 95% expression), a pancreatic cancer cell line with a small population of CD133+  and a prostate cancer cell line which is CD133low expressing, .pm of unconjugated saporin, were similar to PDT.in vitro and in vivo; thus, targeted PCI could be in this case be a highly effective treatment against aggressive, and rapidly metastasising tumours.A similar study was conducted on WiDr colorectal cells  have also been the target of PCI treatment, confirming the efficacy of relevant immunotoxins based on saporin.et al. who used encapsulation of PS in micellar structures, and in addition to overcoming doxorubicin resistance in MCF‐7 resistant cells both in vitro and in vivo, they found in the doxorubicin resistant tumours, the greatest antitumour effect was seen by treating with light‐before PCI. It was thought that P‐glycoprotein membrane transporter shutdown may facilitate intracellular and nuclear accumulation of doxorubicin.In previous sections we have reviewed several studies showing that PCI is effective in treating drug‐resistant cell lines.et al. demonstrated higher efficacy of bleomycin PCI compared to PDT towards the periphery of a murine fibrosarcoma.et al. demonstrated that PCI with surgery was able to induce a significant delay in tumour growth compared to surgery, and PDT in combination with surgery.Within a tumour non‐proliferating cells are usually found in the centre, whereas proliferating cells generally reside in the periphery of an actively growing tumour mass. Norum et al. also confirmed vascular shutdown post‐PCI occurring at a later stage than for PDT.2a or AlPcS2a with saporin was recently compared using human vascular endothelial cells (HUVECs) and fibrosarcoma cells.For cancer therapy PCI has generally been aimed at enhancing delivery within the tumour cells. However, recent work has shown that PCI is also capable of affecting vascular structuresClinicalTrials.gov identifier: NCT00993512). No adverse reactions were found so a multi‐centre phase II study  is now being carried out between several European centres , which will focus on both superficial and deeper head and neck tumours using interstitial illumination. In addition, a phase I/II dose escalation study of PCI of gemcitabine using Amphinex®, followed by gemcitabine/cisplatin chemotherapy has recently commenced for advanced inoperable cholangiocarcinomas .A single‐centre  dose‐escalation phase I clinical trial was successfully completed on 22 patients in 2013 with superficial skin and head and neck neoplasms combining bleomycin and Amphinex® (Many research groups worldwide are investigating PCI, and there is growing evidence supporting its potential as a drug delivery system in cancer treatment. PCI is a very versatile technique which has been shown to be effective for delivery of drugs exhibiting diverse physicochemical properties and sizes and may find application for cytosolic delivery in the emerging field of “nanomedicine.” Site‐specific light application to the target lesion results in the focal delivery of therapeutic compounds, thereby minimising damage to normal adjacent tissue