Cell and animal studies have demonstrated that circadian rhythm is governed by autonomous rhythmicity of clock genes. Although disturbances in circadian rhythm have been implicated in metabolic disease development, it remains unknown whether muscle circadian rhythm is altered in human models of type 2 diabetes. Here we used human primary myotubes (HPM) to investigate if rhythmicity of clock- and metabolic gene expression is altered in donors with obesity or type 2 diabetes compared to metabolically healthy donors. HPM were obtained from skeletal muscle biopsies of four groups: type 2 diabetic patients and their BMI- and age-matched obese controls and from lean, healthy and young endurance trained athletes and their age-matched sedentary controls. HPM were differentiated for 7 days before synchronization by serum shock followed by gene expression profiling over the next 72 hours. HPM display robust circadian rhythms in clock genes, but REVERBA displayed dampened rhythmicity in type 2 diabetes. Furthermore, rhythmicity in NAMPT and SIRT1 expression was only observed in HPM from trained athletes. Rhythmicity in expression of key-regulators of carbohydrate and lipid metabolism was modest. We demonstrate that in human skeletal muscle REVERBA/B, NAMPT and SIRT1 circadian rhythms are affected in donors of sedentary life style and poor health status. Skeletal muscle plays an important role in maintaining whole body energy and substrate metabolism and is responsible for ~80% of postprandial glucose uptake in humans2+), can activate sirtuin 1 (SIRT1), which in turn represses the active limb by deacetylation of both activating and repressing limb components 78To anticipate such metabolic adjustments at the cellular levels, peripheral tissues generate circadian oscillations by an intricate transcriptional-translational feedback loop, known as the molecular clock. This clockwork involves an activating limb, comprising the heterodimer of Brain and muscle ARNT-Like 1 (BMAL1) and circadian locomotor output cycles kaput (CLOCK). This complex promotes gene expression at E-boxes, and controls thereby the expression of period (PER), cryptochrome (CRY) and nuclear receptor subfamily 1, group D, member 1 (REVERBA). The repressing limb comprises REVERBA, REVERBB and the heterodimer of PER with CRY. The timely shifted translocation of the PER/CRY heterodimer into the nucleus inhibits the activating limb. Independently, REVERBA inhibits the expression of BMAL15Apart from keeping time, this clockwork governs the transcription of clock-controlled genes, such as tissue-specific transcription factors of carbohydrate- and lipid metabolism1011121310151617In recent years, evidence is emerging that disturbances in the circadian clock may lead to metabolic diseases. For example glucose homeostasis was severely disturbed in rodent knock out and deficiency models of BMAL1192021Also in human studies, clock gene expression is associated with metabolic health, as adipose tissue clock gene expression was shown to correlate with waist circumference2930This cell study examines circadian rhythmicity in human primary myotubes cultured from 4 phenotypically different donor groups: young endurance trained athletes , their age-matched young, lean sedentary controls , healthy, obese subjects (OB) and patients with type 2 diabetes (T2D) that were matched for BMI and age with OB. Characteristics of the donors are provided in We first determined gene expression profiles of the core molecular clock components to examine circadian rhythmicity. BMAL1 expression showed 24-hour rhythmicity in all four donor groups as detected by JTK_CYCLE .Next, we measured the transcripts of genes that have been reported to be under the transcriptional control of CLOCK and BMAL1, and to be involved in the fine-tuning of the circadian clock. Both for REVERBA and REVERBB, 24-hour circadian rhythmicity was observed in athletes, lean sedentary and obese subjects  in all gA circadian rhythm was observed for SIRT1 only in athletes (We next measured transcript abundance of genes that are key regulators in cellular glucose metabolism. Circadian rhythmicity determined by JTK_CYCLE was found in gene expression of glycogen synthase 1 (GYS1) for type 2 diabetes patients only, but not for its inhibitory regulator – the glycogen synthase kinase 3 beta (GSK3b). For hexokinase II (HKII) JTK_CYCLE indicated rhythmicity in sedentary lean and tendencies in obese and type 2 diabetic donors  that exchanges ATP and ADP in the mitochondrial matrix. Further, we quantified the peroxisome proliferator-activated receptor alpha (PPARα) and delta (PPARδ), which both are involved in regulating lipid metabolism and mitochondrial biogenesis. Circadian rhythmicity was found in gene expression of ANT in sedentary lean and for PPARα in athletes (JTK_CYCLE p < 0.05), but one-way ANOVA did not reveal significant differences in time for any of the groups .The donors of the biopsies that were cultured for this study were grouped phenotypically to represent a group of young endurance trained athletes , aged-matched, lean sedentary controls , normoglycemic middle-aged obese subjects  and BMI and age-matched (with OB) patients with type 2 diabetes . In these groups, insulin sensitivity  progressively deteriorated from TL > UL > OB > T2D . To linkRodent studies have revealed the importance of the CLOCK/BMAL1 component in the regulation of autonomous circadian rhythm in skeletal muscle14et al.34The aim of the current study was to examine if circadian rhythmicity in skeletal muscle is affected by the characteristics of the donors, whom were ranging in metabolic health from endurance trained athletes to metabolically-compromised T2D patients. We find that serum shock is capable of inducing synchronization in human primary myotubes from a skeletal muscle biopsy. In line with the study of Balsalobre in vivo muscle phenotype in terms of insulin sensitivity29in vitro models than in muscle biopsies, we previously did not observe this in our myotubesin vivo phenotype in culture, indicates that cultured myotubes from phenotypically characterised donors appears a valid and valuable model in our hands. In the current study, we confirm that also after the procedure of serum shock, mitochondrial function was reduced in type 2 diabetic patients compared to obese controls , a couple of limitations deserve to be mentioned. First we did not quantify sleep quality, nor did we assess the circadian phase of the donor at the time of the biopsy. Hence, we cannot exclude a putative carry over effect of any abnormalities in sleep or rhythmicity to the cells in culture. We did, however, take all muscle biopsies between 8.00 and 9.00 PM from patients without recognized sleep abnormalities. Second, we assessed metabolic data just prior to taking the biopsy and did not follow-up the putative rhythmic profile of metabolic data of the donors for 72 hours, which would have permitted a direct comparison between In conclusion, we demonstrate, that circadian components of the molecular clock, namely REVERBA/B, SIRT1 and NAMPT, that are associated with mitochondrial function and metabolic health, showed lack of circadian rhythmicity in human primary myotubes from type 2 diabetic patients. Modest circadian rhythmicity was observed in few downstream genes of the molecular clock system, such as metabolic genes involved in glucose and lipid metabolism. Future studies are needed to examine if circadian rhythmicity can also be observed in physiological measures such as insulin sensitivity or mitochondrial function in cultured human muscle cells of insulin resistant subjects.In the present study, we included human primary myotubes from subjects that had previously participated in our studies37et al.2/min) and was corrected for fat-free mass (FFM).Peripheral insulin sensitivity was measured by a two-step hyperinsulinemic-euglycemic clamp, according to Sparks 2 max) uptake and carbon oxide production (VCO2) was performed under standardized laboratory conditions. Exercise was stopped when the subjects were unable to maintain cadence >60 RPM, heart rate exceeded 220 minus age or at a respiratory exchange ratio >1.10.A stepwise progressive exercise test until exhaustion was performed on a stationary bike to measure whole body oxygen uptake as a reflection of physical fitness. Simultaneous breath gas analysis to measure maximal oxygen ; Minimum Essential Medium α  supplemented with 2% FBS  and fetuin . Every other day differentiation media was changed.As described beforein vivo observations we observed compromised mitochondrial function in T2D, measured as lower basal as well as maximal mitochondrial oxygen consumption rate in T2D than in OB . RNA was further purified by the RNeasy kit from Qiagen . The quality and yield of RNA was assessed using a NanoDrop spectrophotometer . The high-capacity RNA-to-cDNA kit from Applied Biosystems  was used for transcribing 0.5 μg RNA to cDNA. Transcript abundance was determined using a 7900HT Fast Real-Time PCR System . Three housekeeping genes (HKG), namely RPL26, GUSB and CYPB, were identified to be stably expressed in synchronized multi-nucleated myotubes throughout time  by both To determine whether cultures of the four donor groups kept the donor phenotype, human primary myotube were plated and differentiated on a Seahorse Bioscience 96-well plate. One hour prior to measurement, differentiation medium was changed to Seahorse Biosciences XF media according to the XF Cell Mito Stress Test Kit . Oligomycin, FCCP and rotenone with antimycin A was added to the cells to determine the oxygen consumption rates for basal mitochondrial respiration, maximal respiration and mitochondrial proton leak . OCR wasResults are presented as means ± SEM derived from three independent cultures of each donor group. Gene expression was tested for rhythmicity using the JTK_CYCLE package for R in Windows 8How to cite this article: Hansen, J. et al. Synchronized human skeletal myotubes of lean, obese and type 2 diabetic patients maintain circadian oscillation of clock genes. Sci. Rep.6, 35047; doi: 10.1038/srep35047 (2016).


Sarcopenia is the loss of muscle mass accompanied by a decrease in muscle strength and resistance and is the main cause of disability among the elderly. Muscle loss begins long before there is any clear physical impact in the senior adult. Despite all this, the molecular mechanisms underlying muscle aging are far from being understood. Recent studies have identified that not only mitochondrial metabolic dysfunction but also mitochondrial dynamics and mitochondrial calcium uptake could be involved in the degeneration of skeletal muscle mass. Mitochondrial homeostasis influences muscle quality which, in turn, could play a triggering role in signaling of systemic aging. Thus, it has become apparent that mitochondrial status in muscle cells could be a driver of whole body physiology and organismal aging. In the present review, we discuss the existing evidence for the mitochondria related mechanisms underlying the appearance of muscle aging and sarcopenia in flies and mice. Loss of muscle mass and muscle wasting are clinical symptoms associated with many chronic diseases as well as with the aging process. The loss of muscle mass accompanied by a decrease in muscle strength and resistance which occurs in the elderly is termed sarcopenia. In the population over 65 years of age, this decay in muscle function is particularly associated with increased dependence, frailty, and mortality. In fact, sarcopenia is the main cause of disability among the elderly , 2. As tThe molecular mechanisms underlying muscle aging are of multifactorial etiology –7. AmongTo better understand and untangle the complexity of the molecular mechanisms driving sarcopenia and the contribution of muscle decay to the integral aging process, more studies using model organisms are required in the future. In the present review, we discuss the existing evidence for the mitochondria related mechanisms underlying the appearance of muscle aging and sarcopenia in flies and mice. Drosophila, it has been observed that naturally occurring variations in mtDNA can have a significant influence on mitochondrial bioenergetics and longevity. Variations in mitochondrial respiration on permeabilized muscle fibers were directly linked to naturally occurring mtDNA haplotypes, even in young flies. As flies aged, an increase in ROS production and copy number of mtDNA were observed in all strains and the percentage increase in each strain could be associated with the mtDNA haplotype and with longevity [ Drosophila, using naturally occurring mtDNA haplotypes in an isogenic nuclear background, have recently shown that mtDNA affects both copy number of mitochondrial genomes and patterns of expression of mitochondrial protein coding genes. Strikingly, these experiments showed that a high expression of ND5  inversely correlates with longevity in males but not in females [Reactive oxygen species (ROS) are produced in the mitochondria as a byproduct of an inefficient transfer of electrons through the Electron Transport Chain (ETC) . During ongevity . These e females . Conside females . An incr females . One pos females . Contras females . New tec females –28 or mu females –31 along females , 28. Suc Superoxide dismutase 1 (Sod1) age prematurely and develop severe sarcopenia [ Sod1 indicating a high apoptotic potential. Furthermore, the levels of the proapoptotic factors Bax and Bak are significantly elevated in mitochondria without Sod1, whereas Bcl-XL and Bcl-2 were lowly regulated [ mitochondrial catalase (mCat) overexpression, they improved tetanic Ca2+ in skeletal muscle, reduced sarcoplasmic reticulum Ca2+ leak, and increased sarcoplasmic reticulum Ca2+ load in muscle from aged mCat mice, thus providing significant evidence for a direct role of mitochondrial ROS in the appearance of pathological signs of sarcopenia. Other hypotheses have emerged as more molecular participants have been described. For example, Diolez et al. [Increased ROS species in the cell have also been associated with diminished ROS scavengers activities during aging –34 Tabl. Seminalrcopenia . Susceptegulated . These segulated  describez et al.  proposedSeveral questions remain open regarding the behavior of ROS during organism and muscle aging. For example, when in lifespan do ROS first appear in the muscle? or which concentrations of ROS are required to alter the gene and protein networks that ensure mitochondria and muscle quality functions? These are still matters to be addressed.fuzzy onions” and is expressed specifically in male testes [ dMFN (Drosophila Mitofusin) or Marf  [ Marf in flies causes second-instar larval lethality, while somatic or cardiac muscle specific downregulation causes a mitochondrial fragmentation phenotype, like in vertebrates [ Opa1 homolog is also present in flies. Opa1 mutations cause embryonic lethality in flies. Interestingly, like in the case of Marf downregulation, Opa1 specific downregulation in somatic or cardiac muscle leads to a mitochondrial fragmentation phenotype [ Opa1, assessed using eye specific mutant somatic clone experiments, also induces a mitochondrial fragmentation phenotype, confirming in vivo the role of fly OPA1 in the fusion process [ Drosophila RNA interference library search for mitochondrial morphology alterations in S2 cells [ Drp1 mutants, mapped by complementation analyses, harbor fewer mitochondria at the neuromuscular junction which affects ATP production at this location leading ultimately to impairment of synaptic vesicle trafficking during prolonged stimulation [ Drp1 using Drp1 RNA interference or assessing muscle cells of Drp11/2 mutants show elongated mitochondrial morphology and increased average mitochondrial area in the tissue [ in vivo genetic screens are still missing in order to understand whether there are tissue specific components in the fusion/fission machinery. For instance, underlying mechanisms of the fission/fusion balance in different cell types could be given by tissue specific interactors and regulators of this machinery. Supporting this idea, it has been found that Mfn2 interacts with other Mfn2 molecules or with Drp1 through different regions in the Mfn2 protein to regulate fusion/fission balance [ PGC-1α, Mfn1/2, Fis1, Opa1, and Drp1, in 12 months old rats and older age groups, suggesting that mitochondrial dynamics are central in order to maintain muscle quality [On the past decades, mitochondria, once thought as a single double membrane organelle, have been redefined as a continuous and dynamic interconnected membrane-bound network . Such coe testes . However factor) . Ubiquittebrates , 56–58. henotype , 59. Fur process , 60. On  process . RecentlS2 cells . In fliemulation . Moreovee tissue , 42. Alt balance . Altoget quality .The complexity of the mitochondrial network architecture in skeletal muscle fibers has been approached by defining two subgroups of mitochondria depending on their localization, intermyofibrillar and subsarcolemmal, each with different capabilities and functions according to cell ATP requirements , 65. Dif Mfn1/Mfn2 double knockout mice die of heart failure due to ventricular dilatation and decreased ejection performance after nine weeks of phenotype induction [ Drosophila mutant models for Pink1 or parkin show that the thoracic muscle degeneration and locomotor deficit can be rescued by promoting fission of mitochondria via expression of Drp1 or ablation of Opa1 in these cells [ Mfn1/2, Fis1, Opa1, Drp1, and PGC-1α. Nonetheless, little is known about the upstream stimuli and regulators which trigger mitochondrial dynamics. Observations from flies and mice suggest that it is the balance of fusion and fission rather than one event or the other that it is important to maintain a healthy aging. Further, from the data available at the moment from model organisms, it is tempting to speculate that movement or exercise and diet could be upstream regulators governing the mitochondria dynamics balance and even constitute a positive feedback mechanism for healthy aging.The relevance of mitochondrial dynamics to cell homeostasis and to overall organisms' physiology is also illustrated in age-associated diseases . Severalnduction . In thisnduction . Geneticse cells , 75. These cells . Consistse cells , 78. In 2+ ion channels located at the ER or at the outer mitochondrial membranes (OMM) like the Inositol 1,4,5 trisphosphate Receptor (IP3R) [ dry is the sole ryanodine receptor of the fly [ dip codes for the only Drosophila IP3R [Muscle fibers are well-organized and compact cells, the Endoplasmic Reticulum (ER) and mitochondria networks are strategically localized to supply energy and quality control in muscle cells. Moreover, both ER and mitochondria undergo constant remodeling in response to cellular demands , changinr (IP3R)  and voltr (IP3R) , enzymesr (IP3R) , as wellr (IP3R) . Notewor the fly , dip codila IP3R , and PORila IP3R . VDAC1/Pila IP3R . Hsc70-52+ bridges between the ER and the mitochondria and thus may be directly involved in regulating mitochondria oxidative metabolism, cell death pathways [2+ regulator proteins settled in the MAMs like IP3R, and VDAC and the uniporter mitochondrial Ca2+ channel (MCU) are well described as the interchanger axis between ER and mitochondria. The entry of Ca2+ into the mitochondria occurs through MCU, which is strategically located in the inner mitochondrial membrane [2+, thereby preventing its entry into the mitochondria under normal cytoplasmic concentrations. It is at this point that the location of the organelle becomes important. Once Ca2+ enters into the mitochondria, it is used by various enzyme systems, namely, Pyruvate DeHydrogenase (PDH), Glycerol-3-Phosphate DeHydrogenase (G3PDH), isocitrate dehydrogenase, and oxoglutarate dehydrogenase, as a cofactor for Krebs cycle reactions, thus contributing to the maintenance of mitochondrial metabolism [2+ into mitochondria significantly decreases cell metabolism [2+ impairs mitochondrial function due to dissipation of the mitochondrial membrane potential and increased ROS production [2+ transfer between the two organelles. While a role for mitochondrial dysfunction and decreased metabolism in aging muscle has been extensively described, evidence about the participation of mitochondrial calcium uptake from the ER and the IP3R-VDAC-MCU axis is still lacking. Recent data from Mammucari et al. [2+ handling by the MCU regulates skeletal muscle mass through signaling pathways involving protein kinase B (Akt) and PGC-1α4. Moreover, overexpression of MCU in skeletal muscle fibers protects from denervation-induced atrophy [2+ regulation is highly involved in anabolic pathways and could effectively control muscle loss. Furthermore, defective sarcoplasmic reticulum- (SR-) mitochondria crosstalk has been causally linked to the abnormal mitochondrial Ca2+ uptake in failing hearts and may underlie their increased oxidative stress [2+ release with caffeine was followed by a rapid increase in mitochondrial Ca2+ uptake and this increase was severely decreased in cardiomyocytes from old hearts. Likewise, in fruit flies, downregulation of MARF in the heart leads to a phenotype with increased contraction-associated and caffeine-sensitive Ca2+ release, showing that there is a physical association of SR and mitochondria which occurs through MARF/Mfn2 and is essential for normal Ca2+ signaling in cardiomyocytes [Among other functions, MAMs have been described to be important Capathways , and muspathways   [2+ release in SR vesicles [2+ release units. They described an increase in damaged mitochondria together with reorganization of the SR-mitochondria interaction, with a significant decrease in tethers and misplaced mitochondria from the normal triad position, possibly resulting in reduced metabolic efficiency and a consequent decline of skeletal muscle performance. Enhancement of antioxidant activity of mitochondria results in an improvement of aged mice muscle function due to decreased intracellular Ca2+ leak and increased SR Ca2+ load. In these same experiments, there were differences in the RyR oxidation levels between young and old mice when submitted to genetically enhanced antioxidant activity [2+ leakage and accumulation occurs in muscle type I fibers of old subjects as a consequence of reversible oxidative modifications of RyRs [2+ homeostasis towards the EC-coupling with the consequent perturbation of mitochondrial metabolic function precede the appearance of sarcopenia.In skeletal muscle, the tight link between Caresearch . Impaireic force . These fs, DHPR)  and reduvesicles , 46, posvesicles  proposedactivity . Compati of RyRs . On the Mitochondrial dynamics not only is governed by fission and fusion processes but also implies processes of mitochondrial biogenesis and degradation. The balance between these last two processes gives the cell new pools of mitochondria and confers mitochondrial quality control. Deficiency in mitochondrial fission processes during aging could promote mitochondrial dysfunction and a subsequent accumulation of damaged mitochondria. In fact, confocal microscopy experiments following mitochondria into autophagolysosomes and mitochondrial potential registration point to mitochondrial fission processes preceding mitophagy to maintain homeostasis of these organelles . To unve Drosophila, Spargel (srl) the homolog of PGC-1α coordinates mitochondrial biogenesis in fat bodies in response to insulin [ srl role in the aging muscle. More about the role of PGC-1α is known from mouse skeletal muscle. Several studies have reported that an increase in PGC-1α, the major regulator of mitochondrial biogenesis, may suppress atrophy symptoms [α in skeletal muscle induces a fast (type II) to slow (type I) fiber type conversion and increases mitochondrial content and oxidative capacity through the modulation of genes involved in metabolism like Citocrome c, COXII, and COXIV [α effectively prevented muscle disuse-induced atrophy. Further studies have unveiled a strong relationship between PGC-1α and mitophagy, suggesting a dual role for this protein in mitochondrial turnover [α exhibited less mitochondrial population together with autophagy deficient mechanisms and altered muscle phenotype [α KO animals, resulting in reduced autophagy and mitophagy flux.In insulin ; howeversymptoms , 101. Ovnd COXIV . Oppositnd COXIV . On the nd COXIV  reportedturnover . AnimalsConsidering that aging develops an inflammatory scenario, one line of evidence suggests that inflammation could directly affect mitochondrial clearance and further enhance aging effects on skeletal muscle through the macrophage migration inhibitory factor and the subsequent inhibition of mitochondria dependent death pathways . Drosophila, will be crucial for in vivo validations of these high throughput networks and for the study of the evolution of these networks throughout lifespan. Further insights into the mechanisms that govern the aging process and how muscle quality contributes to the overall organism homeostasis will come from the understanding of whether these regulatory networks are tissue specific and when in lifespan are they perturbed.To date, only a few genetic interactors have been found to drive mitochondrial function and dynamics. With the refinement of transcriptomic and proteomic techniques, it has become a pressing matter to elucidate the gene and protein networks controlling function and dynamics of mitochondria in order to find new players of these organelles homeostasis. The role of model organisms, in particularCurrently, exercise is the only known effective method to stop muscle degeneration. Thus, future research should address the question on how genes and proteins from the mitochondrial network are expressed during exercise and how do these changes correlate with the functional capacity of skeletal muscle fibers. Further, in vertebrates, it is becoming apparent that mitochondrial dynamics are tailored to the fiber type metabolic status, indicating that there might be differential regulators of mitochondrial homeostasis in different fibers. Perturbations in these gene/protein interaction networks could lead to the finding of key fiber specific regulators which may be used as therapeutic targets for sarcopenia and muscle degeneration.


Gaze direction is a common social cue implying potential interpersonal interaction. However, little is known about the neural processing of social decision making influenced by perceived gaze direction. Here, we employed functional magnetic resonance imaging (fMRI) method to investigate 27 females when they were engaging in an economic exchange game task during which photos of direct or averted eye gaze were shown. We found that, when averted but not direct gaze was presented, prosocial vs. selfish choices were associated with stronger activations in the right superior temporal gyrus (STG) as well as larger functional couplings between right STG and the posterior cingulate cortex (PCC). Moreover, stronger activations in right STG was associated with quicker actions for making prosocial choice accompanied with averted gaze. The findings suggest that, when the cue implying social contact is absent, the processing of understanding others’ intention and the relationship between self and others is more involved for making prosocial than selfish decisions. These findings could advance our understanding of the roles of subtle cues in influencing prosocial decision making, as well as shedding lights on deficient social cue processing and functioning among individuals with autism spectrum disorder (ASD). Prosocial behaviors are the cornerstone of a harmonic society , posterior cingulate cortex (PCC), medial prefrontal cortex (mPFC), temporal-parietal junction (TPJ) and amygdala  method to investigate brain responses when participants were making either prosocial or selfish choices against anonymous counterparts in a novel economic exchanging game task  participated in this study. Only females were recruited to avoid confounding gender influence in social decision making , the proposed portion of repayment to the counterpart , and the location (left or right) of the bars representing two options were balanced across the different types of photos. Before getting into the scanner, each participant was given detailed instructions and completed a minimum of eight practice trials to ensure task comprehension. The photos used in the practice trials were different from those used in the formal task. All participants reported after the task that they believed they were playing with real human-being when the human eye stimuli were shown. The participants were debriefed after the experiment. Each participant was awarded 200 Chinese Yuan as compensation and also 0–100 Chinese Yuan  as a task bonus. The visual stimuli presentations and response collections were performed through the integrated functional imaging system (IFIS).2, voxel size = 3 × 3 × 4 mm3). A high-resolution anatomical 3D T1-weighted MPRAGE image  was also acquired.All images were acquired using a 3-Tesla Siemens Trio Tim MR scanner with a 12-channel head coil. T2*-weighted functional images were obtained using an EPI pulse sequence without inter-slice gap  = 192 × 192 mmImages were preprocessed by using the CONN toolboxps > 0.287). The four contrasts of interest were prosocial choice  accompanied with direct gaze, selfish choice  accompanied with direct gaze, prosocial choice accompanied with averted gaze, and selfish choice accompanied with averted gaze. The frequency of choice and RTs were then respectively analyzed by a 2 (gaze direction: direct and averted) × 2  repeated-measures ANOVA model.Participants made actions (either accept or reject) in more than 94% trials. For our research aims, conditions involving interactions with robot served as a control conditions representing non-social-related processes such as general value-based decision making. We thus subtracted frequency of choice and mean reaction time (RT) related with robot counterpart from the corresponding data associated with human counterparts. No difference in choice frequencies was observed for human and robot counterpart trials  was used to examine the experimental effects across task events within each participant. The onset of the decision phase was modeled by six regressors with 3-s duration which were combinations of eye stimuli  and action (accept and reject offers). In addition, one regressor modeled the choice response, one modeled the onset of the outcome phase (3-s duration), and six extra regressors modeling residual head motions were included as nuisances. These regressors were convolved with the SPM canonical hemodynamic response function. High-pass temporal filtering with a cut-off of 128 s was employed to remove low-frequency drifts.p < 0.001 and survived family-wise error (FWE) cluster-level correction (p < 0.05) within the whole brain. To specifically test our a priori hypotheses, we also reported findings with voxel-level height threshold at p < 0.001 and survived FWE correction (p < 0.05) within regions of interests (ROIs) including the PCC  and posterior TPJ  in spheres with a radius of 8 mm  × 2  flexible factorial model. Results were voxel-level height thresholded at p < 0.001, FWE cluster-level corrected at p < 0.05, and contained more than 5 voxels. Mean beta values of gPPI contrasts were extracted for further analyses from the significant cluster through the MarsBaR toolbox. Bonferroni method was employed to correct for multiple comparisons during post hoct tests and behavior-fMRI correlation analyses.We further investigated the functional coupling between the seed region and the rest of the brain, especially in the ROIs of ToM areas. The seed area was the region showing significant interaction between gaze direction and choice in the fMRI analyses. We performed a generalized psychophysiological interaction (gPPI) analysis through the gPPI toolboxFs < 2.292, ps > 0.142) or RT . Comparisons between human (including both direct and averted gaze conditions) and computer counterparts were organized in the supplementary document.Mean and standard deviations of behavioral measures were organized in Table p value = 0.018) than direct gaze, while there was no significantly stronger activation for direct than averted gaze. No main effect of choice was detected significant. Importantly, a significant interaction between gaze direction and choice was detected in the right STG  = 4.583, p < 0.001 corrected) but not direct (t(26) = −0.459, p > 0.6) gaze was presented , see Supplementary Table S2.Averted gaze elicited stronger activations in right STG  = 3.703, p = 0.006 corrected) but not direct (t(26) = −0.672, p > 0.5) gaze was presented  choices when perceiving averted gaze. The PCC has been widely reported in studies on self-referential processing  who exhibit impairments in reciprocal social interactions  and the center of faces (Blais et al., In conclusion, our study provides the first evidence that the neural processing of social decision making is influenced by perceived gaze direction. Our findings suggest that, when the perceived gaze direction signals lack of social contact, making prosocial vs. selfish decisions is associated with greater neural processing of inferring others’ intention and understanding the relationship between self and others. These findings also shed light on the deficiencies in processing subtle social cues and social functioning in ASD individuals.DS, RS, ZW and TMCL made substantial contributions to the conception or design of the work or the acquisition, analysis, or interpretation of data for the work; drafted the work or revised it critically for important intellectual content; approved the final version to be published; agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Substitution urethroplasty for the treatment of male stricture disease is often accompanied by subsequent tissue fibrosis and secondary stricture formation. Patients with pre-existing morbidities are often at increased risk of urethral stricture recurrence brought upon in-part by delayed vascularization accompanied by overactive inflammatory responses following surgery. Within the context of this study, we demonstrate the functional utility of a cell/scaffold composite graft comprised of human bone marrow-derived mesenchymal stem cells (MSC) combined with CD34+ hematopoietic stem/progenitor cells (HSPC) to modulate inflammation and wound healing in a rodent model of substitution urethroplasty. Composite grafts demonstrated potent anti-inflammatory effects with regards to tissue macrophage and neutrophil density following urethral tissue analyses. This was accompanied by a significant reduction in pro-inflammatory cytokines TNFα and IL-1β and further resulted in an earlier transition to tissue remodeling and maturation with a shift in collagen type III to I. Grafted animals demonstrated a progressive maturation and increase in vessel size compared to control animals. Overall, MSC/CD34+ HSPC composite grafts reduce inflammation, enhance an earlier transition to wound remodeling and maturation concurrently increasing neovascularization in the periurethral tissue. We demonstrate the feasibility and efficacy of a stem cell-seeded synthetic graft in a rodent substitution urethroplasty model. Male urethral stricture disease is prevalent with an incidence estimated between 200 and 1200 cases per 100,000 individuals and has a substantial impact on quality of life and healthcare costs245With advances in the field of tissue engineering, multiple strategies have been proposed for urethral regeneration and reconstruction. These include the use of synthetic and biologic scaffolds in the presence or absence of exogenous cell seeding. These models have been utilized with variable success rates in several animal models but have been hampered by inherent variability in composition, compliance and structural reliability of available scaffolds. This has been coupled with the inability to modulate inflammation in response to graft material while concurrently promoting tissue regeneration79We hypothesize that by using this scaffold seeded with specific stem cell populations, we may be able to enhance the healing and reconstitution of the urethra postoperatively when the graft is incorporated. An associated decreased risk of stricture and scarring would allow us to improve the rates of long-term success of urethroplasty and reduce the need for repeated surgeries.2 followed by 25,000 bone marrow derived CD34+ HSPC/graft . If cell-seeded scaffolds demonstrated less than ideal cell growth as determined by light microscopy, re-seeding of either MSCs or CD34+ HSPCs was performed as needed to achieve cellular confluence prior to urethroplasty. Nude athymic male rats  were anesthetized with inhaled isoflurane (2%) and underwent urethroplasty procedure. Cotransplantation of MSCs and CD34+ HSPCs populations was performed based on previously published data establishing clear synergistic effects102 overdose followed by cervical dislocation. In total, 21 rats were used for this study . Animal studies were approved by the Institutional Animal Care and Use Committee at the Stanley Manne Research Center, Chicago, with all methods performed in accordance with the relevant guidelines and regulations.The elastomeric scaffold POC, was synthesized and cell-seeded as previously described10Urethral patency was assessed using four parameters: 1) daily assessment for clinical voiding patterns ; 2) void spot on filter paper at the time of euthanization; 3) urethral catheterization using a 22Fr catheter after euthanization; and 4) abdominal dissection and confirmation of bladder decompression post-mortem.Formalin-fixed tissue was gradually dehydrated and embedded in paraffin before generating 5 μm sections using a microtome as previously described11°C for 30 minutes before boiling in antigen retrieval solution  for 15 minutes, cooling to room temperature for 50 minutes, followed by blocking with bovine serum albumin (BSA)  for 15 minutes. Primary antibodies for myeloperoxidase (MPO) at 2 μg/mL (rabbit anti-rat), CD68 at 10 μg/mL (mouse anti-rat), TNFα at 9 μg/mL (rabbit anti-rat), γ-tubulin at 20 μg/mL (mouse anti-human), IL-1β at 20 μg/mL (rabbit anti-rat) and IL-10 at 5 μg/mL (rabbit anti-rat) in 1% BSA were incubated for 1 hour. After washing with phosphate-buffered saline (PBS), slides were incubated with secondary antibodies  for 30 minutes at a dilution of 1:400. Following this, slides were rinsed with PBS and mounted with Vectashield . All primary antibodies were obtained from Abcam .Preparation of slides for immunofluorescence staining was performed as previously describedBrightfield and polarized microscopy images were analyzed using Adobe Photoshop CC . Vascular quantification was achieved by analyzing the Masson’s Trichrome images, counting number of vessels, pixel area and diameter of vessels per high power field (HPF) on circumferential images . Picrosirius slides were evaluated under polarized light, with images divided into red, green and blue channels, analyzing the area of red-yellow (collagen I) or green (collagen III) birefringence per HPF on circumferential images .http://rsbweb.nih.gov/ij/), once again on circumferential images . The integrated cell counting function was used after setting appropriate color threshold, determining percent positivity for immunofluorescence.For neutrophil, macrophage, and cytokine detection, image analysis was performed using ImageJ, version 1.46r .Means and standard error are presented, and significances calculated with Student’s A total of 21 rats underwent substitution urethroplasty  with all subjects surviving to their pre-determined endpoint. All subjects but one  met both functional and anatomical measures for urethral patency at their endpoint. One subject in the seeded group was found to be in urinary retention at the 28-day endpoint, and had undergone urethroplasty by a different surgeon from other subjects, less familiar with the procedure. This rat was excluded from analysis given both surgical and functional differences from all other subjects.Human bone marrow-derived MSC and CD34+ HSPCs in the area of substitution urethroplasty were identified using human-specific γ-tubulin antibody immunohistochemistry postoperatively as previously described2) was seen in the seeded (MSC/CD34+ HSPC) group as evidenced by disparity by day 5 postoperatively . With longer follow-up, histologically, a change in size of vessels between groups is seen, with seeded animals demonstrating a shift towards fewer and larger-sized vessels compared to unseeded animals, in whom vessels remained relatively small (An increase in mean vascularity is seen in the immediate postoperative period (day 2 to 5) in all groups, although this initial change was not statistically significant . At these early time points, more robust neovascularization  and IL-1β , neutrophil marker MPO  and macrophage marker CD68  and the anti-inflammatory cytokine IL-10 . All components of the inflammatory phase demonstrated a statistically significant decrease in the healing urethra in seeded animals over time, while anti-inflammatory and pro-regenerative cytokine IL-10 was upregulated. In comparison, while neutrophils showed a slight decrease by day 5 in the unseeded subjects, no further significant drop is seen and TNFα and macrophage levels remained stably elevated throughout the entire healing process in the unseeded group. Comparative trends in marker levels on are shown in Tissue maturation was determined by the ratio of collagen III to collagen I in the periurethral region using picrosirius staining. We demonstrate a significantly elevated and increasing ratio of collagen III:I during urethral healing in the unseeded group in contrast to the stabilized and lower ratio seen in the seeded animals . Early iThe balance between essential versus prolonged inflammation seen with post-surgical healing is required for an environment promoting proper tissue regeneration and limiting sequelae of pathologic inflammation and scar formation. While the tissue injury with surgery stimulates an innate immune response including the recruitment of granulocytes and macrophages, abnormally persistent elevation of a pro-inflammatory response in healing tissues may result in poor tissue reformation and immature architectural remodeling13Despite advances in tissue engineering over the last several decades, use of graft tissues remain limited to date by immunogenic host response, as well as limitations of vascularity and availability and variability of scaffolding tissues, all factors which increase graft failure. Although MSC are known to exhibit potent anti-inflammatory properties and CD34+ HSPCs have demonstrated the promotion of angiogenesis in ischemic myocardium, it is not until recently that the combination of these stem cell populations has been investigated9101516910To our knowledge, this work demonstrates the establishment of a novel substitution urethroplasty model in rats corroborated by data that supports both the feasibility of the surgical procedure and urethral patency at a minimum of four weeks. Urethral wound healing has been previously characterized in rats, and provides a framework for comparison with the addition of POC scaffold and cell-seedingThe anti-inflammatory effect of MSC/CD34+ HSPC cells was seen consistently across multiple inflammatory markers at both early and late urethral healing . SpecifiProtracted or inappropriate inflammation in the setting of healing tissues can result in pathologic wound healing and scar formation, best illustrated in lichen sclerosis, which is a condition characterized by chronic inflammation resulting in progressive and recurrent urethral strictures19212223Sufficient vascularization of grafted tissue is imperative for adequate healing and exchange of nutrients and gases, preventing graft necrosis10This study provides an unprecedented examination of the histological changes after substitution urethroplasty in a rat model, characterizing the striking and beneficial effects on urethral healing in the presence of MSC/CD34+ HSPC. Only specific aspects of the innate immune system were studied, providing important, albeit limited, information; therefore, other parts of the immune response must be examined to fully characterize the relationship between cell-seeding and tissue regeneration. While examination of collagen ratio as a proxy for maturation and remodeling of scar may limit this study, long-term outcomes assessing clinical obstruction will be a more telling outcome in future studies. Furthermore, while this study provides conceptual groundwork for strategies to optimize tissue engineering in urethroplasty, as with any animal model, translation of these results using a rat model will require further animal and eventual human studies.Cell-seeding of the POC scaffold with the combination of MSC and CD34+ HSPC demonstrates a striking modulation of the inflammatory milieu, increased vascularization and acceleration of scar maturation and remodeling. These effects are demonstrable as early as 2 and 5 days postoperatively compared to controls, with continued favorable effects with increasing divergence from controls at 4 weeks. Favorably altering the urethral process to prevent pathologic inflammation and hypertrophic scar, while enhancing blood supply to graft tissue could have wide-reaching implications for the future of substitution urethroplasty and reduction of stricture recurrence. We furthermore demonstrate both feasibility and efficacy of substitution urethroplasty in a novel rat model.How to cite this article: Liu, J. S. et al. Bone Marrow Stem/Progenitor Cells Attenuate the Inflammatory Milieu Following Substitution Urethroplasty. Sci. Rep.6, 35638; doi: 10.1038/srep35638 (2016).


To review the advances in our understanding of the genetics of inclusion body myositis (IBM) in the past year.HLA-DRB1 alleles, and identified certain amino acid positions in HLA-DRB1 that may explain this risk. A suggestive association with CCR5 may indicate genetic overlap with other autoimmune diseases. Sequencing studies of candidate genes involved in related neuromuscular or neurodegenerative diseases have identified rare variants in VCP and SQSTM1. Proteomic studies of rimmed vacuoles in IBM and subsequent genetic analyses of candidate genes identified rare missense variants in FYCO1. Complex, large-scale mitochondrial deletions in cytochrome c oxidase-deficient muscle fibres expand our understanding of mitochondrial abnormalities in IBM.One large genetic association study focusing on immune-related genes in IBM has refined the association within the human leukocyte antigen (HLA) region to The pathogenesis of IBM is likely multifactorial, including inflammatory and degenerative changes, and mitochondrial abnormalities. There has been considerable progress in our understanding of the genetic architecture of IBM, using complementary genetic approaches to investigate these different pathways. Sporadic inclusion body myositis (IBM) is the most common acquired muscle disease presenting in people over 50 years of age. Clinically, it is characterized by slowly progressive weakness and muscle wasting predominantly of the quadriceps and long finger flexor muscles.In IBM, inflammatory features in muscle biopsy specimens suggest an immune-mediated component to disease pathogenesis. In addition, circulating anti-Ro autoantibodies may be found in around 20% of patients and recent work has identified cytosolic 5′-nucleotidase 1A (anticN-1A) autoantibodies in around one-third of patients. However, unlike other idiopathic inflammatory myopathies (IIMs), such as polymyositis and dermatomyositis, IBM is unresponsive to conventional immunosuppressive treatments. This lack of response may be explained by evidence of impaired autophagic processes, including rimmed vacuole formation and the accumulation of misfolded proteins. Whether these degenerative processes represent a primary or secondary involvement is unclear.Some hereditary diseases may mimic clinical features of IBM. These diseases may also exhibit similar pathological features, such as rimmed vacuoles and protein accumulations ,2. TheseAlthough the primary cause of IBM remains unknown, genetic factors likely influence disease susceptibility. This article reviews advances that have been made in the past year in our understanding of the genetic component of IBM and potential future approaches for research in this rare disease. There has been considerable progress in our understanding of the genetic basis of IBM. Two different approaches have been used recently in IBM; large genetic association studies and smaller targeted sequencing studies.The study design of genome-wide association studies (GWAS) and contemporary genetic association studies frequently test hundreds of thousands, if not millions, of single nucleotide polymorphisms (SNPs) across the genome. Results from GWAS in autoimmune diseases suggest that most associated variants reside in regulatory regions, exerting their low effect sizes (odds ratio <1.1) on the expression of immune-mediated genes ,6. The cOther approaches to investigate the genetic component of IBM have relied on detecting rarer variants through sequencing studies that commonly involve fewer individuals than GWAS. To date, these have focused on candidate genes taken from related neuromuscular or neurodegenerative diseases. However, novel approaches are also being used, including targeting of genes identified in proteomic studies. Variants discovered through these studies likely will be rarer, with a larger effect size than those discovered by GWAS. Therefore, GWAS and sequencing approaches test complementary hypotheses to investigate the genetic architecture of IBM.+ cytotoxic T cells surrounding major histocompatibility complex (MHC) class I-expressing fibres [▪▪].There are multiple lines of evidence for inflammation as a key pathway in the pathogenesis of IBM. These include the presence of CD8g fibres , the preg fibres ,10 and ag fibres –13. Receg fibres ,15. In lg fibres ▪▪.∗03 : 01, HLA-DRB1∗01 : 01 and HLA-DRB1∗13 : 01. Although HLA-DRB1∗03 : 01 is known to be associated with polymyositis and dermatomyositis, the association with HLA-DRB1∗01 : 01 and HLA-DRB1∗13 : 01 is unique to IBM within the IIMs. Unlike many other diseases . This suggests that there is no strong HLA association with the antibody over and above the general association with HLA-DRB1∗03 : 01 in IBM. Similarly, a study in 24 anticN-1A positive IBM patients did not detect a unique HLA class II association independent of HLA-DR3 . However, three other loci reached a suggestive level of significance, one of which was assigned to the CCR5 gene. The authors hypothesize that the protective effect of this association may be due to a frameshift mutation in CCR5 which inhibits its function as a chemokine receptor involved in T-cell migration. CCR5 previously has been associated with other autoimmune diseases lending support to the hypothesis of an immune-mediated component to IBM . They report four rare missense variants in SQSTM1 and three variants in VCP. This represented 4.0% of the cohort, and is the first time potential pathogenic variants in SQSTM1 have been observed in IBM patients. As these variants may cause diseases that mimic IBM, it was confirmed that none of these patients had developed symptoms of PDB, FTD or ALS and all fulfilled diagnostic criteria for IBM. As SQSTM1 is involved in the autophagy pathway, and VCP is involved in proteasomal degradation of misfolded proteins, this further supports the role of autophagic alterations and aggregation of proteins in the pathogenesis of IBM. These studies suggest that there is merit in the targeted sequencing of genes previously associated with hIBM and other inherited muscle disorders.A recent study used whole exome sequencing (WES) in 181 IBM patients focusing on p62, also named sequestosome 1 (SQSTM1), and  and FTD ▪▪. They ▪▪]. Two hundred and thirteen proteins showed a statistically significant overrepresentation in rimmed vacuole samples compared to controls. Many proteins already known to be involved in IBM or other protein aggregate myopathies overlap with the proteins identified, validating this approach. The 173 novel proteins not described before in IBM warrant further investigation. Proteins that were present in at least 50% of rimmed vacuole samples (131 genes) were taken forward for genetic analysis using WES data from 62 patients with IBM. Hundred missense or loss of function variants were identified in 52 genes. Genetic data from ALS patients were then used to identify variants statistically enriched that are specific to IBM. Rare missense or loss of function variants in FYCO1 were enriched in IBM patients (11.3%) compared to ALS patients  or healthy controls . FYCO1 is involved in autophagosome/endosome trafficking. Along with the VCP and SQSTM1 associations described above, this provides further evidence for autophagosome processing as a basis for future mechanistic studies. Novel treatments targeting protein dyshomeostasis are currently in development [Rather than using related diseases, a novel way of identifying candidate genes is using proteomic analysis. We know that p62/SQSTM1 accumulates in inclusions of IBM muscle fibres . A recen33elopment .SQSTM1 and VCP were found, or in genes that would explain the observed clinical phenotype. A subsequent case−control association analysis identified seven SNPs enriched in the Finnish IBM population with P < 0.005. Reassuringly, two of these were within the HLA region; however, the other associations were in novel genes; STARD3, SGPL1 and SETD4. Results from a small association study are to be treated with caution, and will need to be replicated.In contrast to the studies outlined above, a smaller sequencing study from Finland did not find any rare missense genetic variants . This stVCP, SQSTM1 and FYCO1 validate the use of a sequencing approach, and have given us greater mechanistic insight in to IBM cause. It is worth noting, however, that in all of these cases, patients lacked a family history of IBM or weakness, indicating that these inherited variants alone are not sufficient for disease pathogenesis. There are likely many more variants that predispose to IBM. Identification of novel genes will come from WES and whole genome sequencing (WGS) studies that detect rarer coding variants with potentially larger effect size. Additional WES studies in IBM are currently being undertaken [As discussed above, associations with dertaken . Future Cytochrome c oxidase (COX)-deficient muscle fibres are a common histopathological feature of IBM, and there is increasing evidence that mitochondrial abnormalities may play a role in the cause of this disease. Recent research has shown increased mitochondrial DNA (mtDNA) deletions in COX-deficient muscle fibres, and that the proportion of these deficient fibres correlates with severity of T-lymphocyte infiltration and muscle fibre atrophy suggesting a mechanistic link between these disease processes ,34. In aMyofibres may harbour clonally expanded large-scale mtDNA deletions responsible for respiratory chain deficiency that is thought to be central to IBM pathogenesis ,41. PrevFurther evidence for mitochondrial dysfunction in IBM is provided by research that sought to characterize mitochondrial phenotype at a genetic, molecular and functional level in 30 IBM patients ▪. The auet al. in Fig. There has been considerable progress in our understanding of the genetic architecture of IBM. Evidence suggests a complex interplay between inflammatory and degenerative processes and mitochondrial abnormalities. A model starting with inflammation within muscle and subsequent deposition of amyloid and other proteins because of overloaded protein degradation systems has been proposed by Benveniste None.This work was funded by an MRC Partnership Grant (MR/N003322/1).There are no conflicts of interest.▪ of special interest▪▪ of outstanding interestPapers of particular interest, published within the annual period of review, have been highlighted as:


The effects of spatial attention and part-whole configuration on recognition of repeated objects were investigated with behavioral and event-related potential (ERP) measures. Short-term repetition effects were measured for probe objects as a function of whether a preceding prime object was shown as an intact image or coarsely scrambled  and whether or not it had been attended during the prime display. In line with previous behavioral experiments, priming effects were observed from both intact and split primes for attended objects, but only from intact (repeated same-view) objects when they were unattended. These behavioral results were reflected in ERP waveforms at occipital–temporal locations as more negative-going deflections for repeated items in the time window between 220 and 300 ms after probe onset (N250r). Attended intact images showed generally more enhanced repetition effects than split ones. Unattended images showed repetition effects only when presented in an intact configuration, and this finding was limited to the right-hemisphere electrodes. Repetition effects in earlier (before 200 ms) time windows were limited to attended conditions at occipito-temporal sites during the N1, a component linked to the encoding of object structure, while repetition effects at central locations during the same time window (P150) were found for attended and unattended probes but only when repeated in the same intact configuration. The data indicate that view-generalization is mediated by a combination of analytic (part-based) representations and automatic view-dependent representations. A central question in the study of visual cognition concerns the nature of the mental representations mediating the recognition of familiar objects. To investigate this issue, many experiments measure priming: Objects are typically recognized faster and more accurately when they are seen a second time . PrimingStudies using scalp recorded event-related potentials  have been observed following immediate or short lag repetition as sensitive to variation in shape or view between an initial and repeated exposure with an object 1.More consistent and long-lasting ERP repetition effects for visually presented objects are reported for time windows after 200 ms post stimulus onset . Henson The domain-sensitivity reflected in the functional characteristics and neural distribution of the N250r component is consistent with the activation of cortical generators that operate as perceptual representation sub-systems, for example, a structural description system (SDS), word form descriptions, and face recognition units . Martin-Taken together, the time course of reported ERP repetition effects provide compelling evidence for both early visuo-perceptual neural operations that underlie the rapid categorization of objects in image sensitive views (P150) and later neural representations that code abstract structural descriptions that can account for the constancy of object recognition across numerous views and viewing conditions (N250r). In general, observations indicate that the magnitude of ERP repetition effects depends on the amount of view similarity between initial and subsequent presentation of an object  and add Figure 1). In addition, unlike structural description theories the hybrid model predicts priming for unattended objects if they are shown in the same view across repetitions  in which the halves swap locations. This manipulation, which affects the holistic configuration while leaving the part-based description largely intact, has been shown to distinguish between part-based and view-based representations .The second important factor in testing the hybrid model concerns visual attention. The hybrid model predicts that repetition effects should occur for probes following attended intact and split objects, whereas probe objects following unattended prime images should show repetition effects only in intact views. This prediction has not yet been tested directly using ERP repetition effects. The present study examined ERP repetition effects for an intact object image (the probe object) following immediately preceding images of either the same or a different object (the prime object). The variable of prime view (intact vs. split) was factorially crossed with its cued location (cued vs. uncued), a manipulation that has been shown to successfully direct spatial attention to the cued object  the termTo our knowledge this study is the first to investigate the predictions of the hybrid model  using ERFigure 1 depicts the priming predictions derived from common object recognition models. Figure 2 presents a schematic overview of the prime–probe trial procedure.The literature reviewed previously broadly suggests that ERP repetition effects elicited prior to 200 ms post stimulus onset (P150) are sensitive to changes in image input parameters and present only when objects are repeated in the same view, whilst later ERPs following 200 ms (N250r) reflect neural representations involved in object constancy and the representation of invariant structural descriptions. An implicit property of the hybrid model is that holistic representations are generated fast, whereas an analytic representation takes more time to be established, because it relies on synchronized firing of separate units coding parts belonging to the same object. Therefore, following the hybrid model, we predicted that if the current paradigm detects very early repetition effects for objects , these would be view-specific and independent of attention  were 20–39 years old (mean age 25.7 years), were all right-handed, had either normal or corrected to normal vision and were native English speakers. The Ethics Committee of the Department of Psychology at Goldsmiths approved the experimental procedures.Figure 2). Stimuli were presented on a CRT monitor with the timing set to synchronize with screen refreshes. E-Prime 1.1  was used for stimulus presentation and behavioral response collection. Stimuli were presented at a viewing distance of 100cm against a light gray background and were standardized in size such that they subtended 4° of visual angle .The stimulus set consisted of 616 asymmetrical black and white line drawings of familiar objects derived from different sources . For eacProbe objects were always shown in an intact configuration. The relationship between the prime and the probe display was manipulated to produce six conditions: probe objects could be preceded either by a prime display containing the probe object , or by a prime display containing only different objects to the probe object . The resulting six experimental conditions were therefore: Attended-Intact, Unattended-Intact, Unprimed-Intact, and Attended-Split, Unattended-Split, Unprimed-Split.Figure 2 for a schematic of the prime/probe sequence), followed by a mask (495 ms). The participant’s task was to covertly name the cued prime object and then the probe object, by making a button press to coincide with their silent naming. Trials in which participants did not recognize either the prime or the probe object (and no button press was recorded) were excluded from the analyses.The experiment was divided into 11 blocks, comprising one practice block and 10 experimental blocks. Each block consisted of 24 prime/probe trial presentations . The allocation of stimuli to conditions was fully counterbalanced across participants, and the order of priming conditions was randomized within blocks, as was the presentation of attended (cued) objects to the left or right of fixation in each prime display. Each trial consisted of a prime and a probe display. The prime display began with a fixation cross (500 ms) followed by a cueing square which subtended 4.45° × 4.45° visual angle. The cue was presented to the left or the right of fixation for 50 ms. The center of the cue was 4° from the mid-point of the screen. Next, two objects were presented either side of fixation, and one of these appeared inside the cue square (attended object) while the other appeared on the uncued side (unattended object). Neither of the object images exceeded 4° degrees of visual angle. Both the attended and unattended prime stimuli remained on the screen for 150 ms followed by a random line mask subtending the whole screen (495 ms). Following an inter-stimulus interval (1995 ms blank screen) and fixation cross (495 ms), a single probe object was presented for 165 ms , relative to a 100 ms prestimulus baseline. Epochs with activity exceeding ±30 μv in the HEOG channel reflecting horizontal eye-movements or ±60 μv at FPz  were excluded from analysis, as were epochs with voltages exceeding ±80 μv at any other electrode. Following artifact rejection, participants’ average ERPs were computed for intact probe objects quantified separately on the basis of the preceding prime condition . Resulting grand average ERPs were derived for each of the six experimental conditions. Importantly, to reiterate, all probe objects were presented in an intact configuration and only trials on which a response was collected were retained for analyses.Electroencephalogram was recorded from 64 (Ag/AgCl) electrodes mounted in an elastic cap distributed over the head surface according to the extended 10-20 EEG system  with a BEvent-related potential analyses were carried out on the probe trials only. We first assessed the impact of object repetition, followed by an analysis investigating effects of attention and configuration. ERP analyses were focused on those electrodes and time intervals that have been associated with ERP repetition priming in previous studies at occipital–temporal posterior electrodes and at fronto-central sites . These eInitial repeated-measures analyses of variance (ANOVAs) included the factors repetition , hemisphere , and electrode site . There were two separate analyses to establish the presence of reliable ERP repetition priming effects for probes primed either by their intact (same) or split (different) counterparts in the prime display. A second set of analyses focused on attention and configuration effects using difference ERPs computed by subtracting mean amplitudes to unprimed trials from those to the respective (intact and split) primed trials. For analyses at posterior electrodes, resulting difference ERPs were subjected to 2 × 2 × 2 × 2 ANOVAs with factors attention , configuration , hemisphere  and electrode site . For fronto-central analyses, the factor of hemisphere was not included and the site factor was replaced with electrodes FCz/Cz. Greenhouse–Geisser adjustments to the degrees of freedom were applied to analyses of ERPs where appropriate to correct for violations of sphericity.Table 1). There was no significant difference in the number of trials that involved a valid response between the six experimental conditions ; see Table 1 for the percentage of correct responses. An analysis of the behavioral reaction time (RT) data was carried out to assess priming across conditions. RTs to each of the attended primed conditions for intact and split, as well as the unattended intact and split were compared with RTs to unprimed-intact probes and unprimed-split conditions, respectively. Significant priming effects were found for attended intact , attended split , and unattended-intact  conditions. RTs to unattended split objects were not significantly different to those for unprimed objects (p > 0.33).Priming was operationalized as the difference in naming latencies for repeated compared with unrepeated probe objects . Trials in which the behavioral response was greater than 2000 ms or shorter than 250 ms were removed from both behavioral and ERP analyses, along with any trials on which a response was not recorded  = 20.53; p < 0.001] and configuration  showing that probe RTs to previously attended objects were faster than to previously unattended objects, and probe responses were faster following intact primes than following split prime objects. The interaction between attention and configuration was also significant  which reflected the reliable RT difference in priming between intact and split objects for attended conditions  compared with a non-significant difference for unattended conditions (p > 0.32). The behavioral results confirm previous findings  using a within-subjects ANOVA with the factors attention (attended/unattended) and configuration (intact/split), analog to previous behavioral work . Resultsfindings  that theFigures 3 and 4 show grand average ERPs derived from voltages recorded at posterior electrodes P7/P8 and PO7/PO8, and from fronto-central electrodes FCz/Cz, respectively. ERPs were triggered to intact probe objects as a function of the preceding prime trial type: attended, unattended or unprimed in either a split or intact configuration. Statistical evaluation of early ERP repetition effects (<200 ms post stimulus onset) was focused on mean amplitudes recorded at posterior and fronto-central electrodes during the time window 140–180 ms post stimulus onset and corresponding to the N1 and P150 components, respectively.F = 3.98; p = 0.025], that did not interact with hemisphere or site . Follow-up analyses revealed significantly enhanced N1 amplitudes (ERP priming) triggered to attended-intact probes in the contrast with unprimed-intact , see Figures 3 and 5A, whilst ERPs to unattended-intact did not differ to unprimed . The second 3 × 2 × 2 ANOVA on N1 mean amplitudes triggered to probes preceded by split primes revealed a non-significant effect of prime-condition  , but a significant interaction between prime-condition and electrode site . In neither the intact nor the split factorial ANOVA did prime-condition interact with recording hemisphere . Follow-up analyses confirmed a reliable ERP repetition effect, identified as an enhancement of N1 amplitudes triggered to attended-split trials in the contrast with unprimed-split trials, however, this priming effect was focal to left lateral electrode site P7 . N1 amplitudes at PO7, P8, and PO8 did not differ between attended split and unprimed split trials. Furthermore N1 amplitudes did not differ between unattended split and unprimed split4 at P7, P8, PO7, and PO8 To establish the magnitude and onset of ERP repetition effects at posterior electrode sites during the N1 component (140–180 ms) two repeated measures 3 × 2 × 2 ANOVAs were each focused on N1 mean amplitudes derived from intact and split trials separately. The first of these was a 3  × 2  × 2  ANOVA which revealed a significant main effect of prime-condition  or right lateral posterior electrodes , see Figure 5A. Importantly, no posterior N1 ERP priming effects were found for unattended trials following either intact or split primes.Taken together the results suggest that attention is a prerequisite for posterior N1 (140–180 ms) ERP priming effects. Furthermore, priming for attended intact (same configuration) repeated probes was found at bilateral posterior electrode sites, whilst attended split (different configuration) trials produced ERP priming at left lateral posterior sites only. Interestingly, despite the lateralized pattern of N1 ERP priming effects for attended trials that depend on configuration of the preceding primes, follow-up contrasts showed N1 amplitudes did not differ between attended intact and attended split trials at either left  that did not interact with electrode site . Follow-up analyses conducted separately on attended-intact and unattended-intact trials against unprimed-intact conditions revealed an enhanced P150 that reflected ERP effects of repetition for both attended-intact  and unattended-intact trials  ; in neither contrast did prime-condition interact with electrode site (both p’s > 0.15).Analyses of ERP repetition effects were focused on the P150 (140–180 ms) measured during the same time window as the posterior N1 but recorded at fronto-central sites (FCz and Cz). Similar to the previous analyses, two within-subjects ANOVAs were conducted on ERPs to probes preceded by either intact or split primes. The first 3  × 2  ANOVA revealed a significant effect of prime-condition  or interaction between prime-condition and site . Follow up contrasts confirmed this result with the finding that P150 amplitudes did not discriminate significantly between attended-split and unprimed trials  nor unattended split and unprimed  furthermore, prime-condition did not interact with electrode site in either contrast . Taken together the results demonstrate that P150 amplitudes index ERP repetition effects for intact objects regardless of whether they had been at the focus of attention during the preceding prime trial . Topographic scalp maps of ERP differences between significantly primed and unprimed trials in the 140–180 ms time interval are shown in Figure 5C.A very different pattern of ERP repetition effects was found in the analyses for split trials at fronto-central electrodes during the P150. Results revealed no significant effect of prime-condition   or configuration , or interactions involving attention, configuration, hemisphere or site . Next, P150  difference ERPs were computed for each of the four repeated conditions. Repeated measures ANOVA 2  × 2  × 2 Site  showed no main effect of attention  or configuration . There was no two-way interaction between attention and configuration and no three-way interaction with site . (See Table 2.)To assess the combined impact of attention and configuration on mean amplitudes triggered to probe objects, difference ERP waveforms were computed for each of the priming conditions during the N1/P150 (140–180) time interval. For example, ERPs on unprimed trials were subtracted from ERPs to each of the four repeated conditions . The four resulting difference ERPs (priming conditions) were subjected to factorial within-subjects ANOVA. First, a 2  × 2  × 2  × 2  ANOVA on N1 amplitudes revealed no significant main effects of attention  that marginally interacted with hemisphere  but not with electrode site . Follow-up analyses confirmed the presence of reliable N250r (ERP priming) effects for attended-intact compared with unprimed at both right  and left  posterior sites. For unattended-intact trials the N250r differed to unprimed at right lateral posterior electrodes  but not in the left sites . In neither analysis did prime-condition interact with electrode site (p’s > 0.1), see Figure 6A. The same analyses for split trials revealed a main effect of prime-condition  that did not interact with hemisphere  or electrode site . Follow-up analyses confirmed ERP priming on attended-split trials in the contrast with unprimed-split  but demonstrated an absence of ERP priming during the N250r for unattended-split . To summarize, ERP repetition effects during the N250r were present at bilateral posterior electrode sites for attended-intact and attended-split probes, and at right lateral posterior sites for unattended-intact probes .Event related potential repetition effects following latencies greater than 200 ms after probe onset  = 43.27; p < 0.001] and configuration  moderated by a significant interaction between attention and configuration . There were no interactions involving attention or configuration with the factors recording hemisphere or site . Follow-up analyses for attended trials revealed significantly enhanced N250r amplitudes for attended-intact compared with attended-split at both left and right posterior sites . Whereas N250r amplitudes to unattended-intact trials did not differ from unattended-split at either left or right posterior sites  In summary, N250r amplitudes at lateral posterior electrodes were significantly different on attended-intact compared with attended-split trials, and unattended-intact did not differ to unattended-split trials. (See Table 2.)Event related potential effects of attention and configuration were assessed by computing difference waveforms for repeated conditions . The resulting N250r difference ERPs were then subjected to a 2  × 2  × 2 , × 2 (electrode: PO7/P7 vs. PO8/P8) repeated measures ANOVA. Results revealed significant main effects of attention [Table 2). For occipital–temporal sites enhanced amplitudes during the N1 time-window revealed very early (less than 200 ms after onset of the probe object) repetition effects. The magnitude of N1 ERP repetition effects for attended trials found at left posterior sites did not differ for attended-intact and attended-split objects, a result that likely reflects the activation of attention sensitive neurons that code for view-independent features of objects. However, ERPs in this early time window at right posterior sites revealed priming for attended-intact probes only. Importantly, during the same time window (140–180 ms) an enhanced P150 recorded over central and fronto-central scalp sites demonstrated reliable ERP repetition effects for attended and unattended objects repeated in the same intact configuration. Crucially, P150 ERP priming effects were not revealed for split conditions, which suggest that neural generators that contribute to the P150 reflect the activation of ‘view sensitive’ cells that operate independent of attention. The functional pattern of early repetition-sensitive ERPs provides evidence for temporally overlapping neural mechanisms differentially sensitive to the allocation of spatial attention and the constancy of image features across initial and repeated presentations. Important to note is that early ERP priming effects for repeated same-image objects do not reflect image priming afforded by simple pixel-by-pixel overlap, as objects were repeated at different locations in the visual field between first and second presentation on all trials.The present study revealed behavioral short-term repetition priming of the naming of intact objects from previously attended objects in both intact and split views. By contrast, repetition priming from unattended objects was strictly view-dependent. Similar repetition effects were found using ERP measures, during time windows 140–180 ms (associated with the N1/P150) and in particular in the time window 220–300 ms (N250r) (see During the time window between 220 and 300 ms (N250r) following the onset of probe objects pronounced ERP object repetition effects were observed as more negative-going amplitudes at occipital–temporal scalp sites in all but the unattended-split condition, with attended-intact images showing more enhanced repetition effects than split ones, although for unattended-intact conditions this ERP priming was only observed in the right hemisphere.5. Importantly, as predicted, repetition effects were obtained even for probes that had been unattended in the preceding prime trial, but only when the related prime objects were shown in an intact configuration . Across the two time-windows this pattern of priming and repetition effects is generally consistent with the concept of two qualitatively different parallel processing routes mediating object recognition: an analytic route, part-based and dependent on attentional resources, involving representations for the recognition of unfamiliar views of familiar objects , and a holistic route, view-dependent and automatic, in which view-based priming works without visual attention  and ERP repetition effects (during the N1/P150 and N250r time windows) mirror previous findings in fMRI studies , and alsttention .Is it possible that the repetition effects for unattended objects may have resulted from residual attentional processing? Arguably, the manipulation of spatial cueing may have not completely prevented a spill-over  or leakaFigure 1). We will discuss the implications of this outcome after reviewing how our data compare to previous EEG studies on object recognition.Importantly, the repetition effects reported here—both behavioral as well as their ERP correlates—indicate a pattern of priming that cannot be predicted by either strictly part-based  accounts of object recognition nor strictly view-based models alone  being in line with this model . HoweverSimilar to our results previous research has found that the most pronounced ERP repetition effects are observed during latencies after 200 ms post stimulus onset  and can Anterior positive-going deflections after repetitions have been attributed to semantic or conceptual priming of objects . SchendaIn general, our results are broadly consistent with previous findings on object repetition for attended images. The N250r , an observation that is generally consistent with theories that explain object constancy via a mechanism of view transformations or interpolations across 2D views  or by asTo account for structural properties of object recognition some view-based models have proposed templates for object “fragments” rather than view-based templates for whole objects e.g., . These fThe present evidence for repetition effects from split objects also provides only limited support for pure part-based accounts of object representations e.g.,  because Figure 1) predicted repetition effects indicating shape processing that can compensate for configurational changes, while, at the same time, repetition effects indicating strong automatic view-dependent processing in both attended and unattended conditions. The current behavioral and ERP priming effects are broadly consistent with the hybrid model’s assumption that a previously attended probe object in an intact view contributes input from both analytic as well as holistic (view-dependent) processing to priming, whereas a previously attended probe object in a split view only contributes the input of the analytic shape processing route to priming. The behavioral priming results and the repetition effects observed in the right occipital–temporal electrodes in the N250r not only mirror closely previous behavioral and imaging data . Indeed we found early repetition effects for probe objects around the N1/P150, which seem to fit with the hybrid models’ notion of a fast, holistic route of object recognition, because ERP repetition effects were not only found for attended-intact objects, but also for unattended objects in intact views . Thus, overall we found strong neural evidence for a view-based component that is established rapidly, as proposed by the hybrid model.The current findings support and extend previous observations of ERP studies showing (N250r) occipital–temporal repetition effects for objects in identical views even when they had been previously spatially unattended in a prime display. At the same time we observed repetition effects for novel views of familiar objects in early time windows (<200 ms post stimulus onset) indicating that view-independent object model selection begins earlier than had been previously suggested by ERPs e.g., . These rAll authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer JH declared a shared affiliation, though no other collaboration, with one of the authors AR-K to the handling Editor, who ensured that the process nevertheless met the standards of a fair and objective review.


Most cancer-associated deaths result from metastasis. However, it remains unknown whether the size, microenvironment or other features of a metastatic lesion dictate its behaviour or determine the efficacy of chemotherapy in the adjuvant (micrometastatic) setting. Here we delineate the natural history of metastasis in an autochthonous model of pancreatic ductal adenocarcinoma (PDAC), using lineage tracing to examine the evolution of disseminated cancer cells and their associated microenvironment. With increasing size, lesions shift from mesenchymal to epithelial histology, become hypovascular and accumulate a desmoplastic stroma, ultimately recapitulating the primary tumours from which they arose. Moreover, treatment with gemcitabine and nab-paclitaxel significantly reduces the overall number of metastases by inducing cell death in lesions of all sizes, challenging the paradigm that PDAC stroma imposes a critical barrier to drug delivery. These results illuminate the cellular dynamics of metastatic progression and suggest that adjuvant chemotherapy affords a survival benefit by directly targeting micrometastases. Metastatic colonization—the growth of single metastatic cells or small lesions into gross metastases—is poorly understood. Here, the authors characterize the natural history of metastatic growth and assess the impact of chemotherapy in an autochthonous model of pancreatic cancer. Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival rate of less than 6%, a dismal outcome related to late detection and a high rate of spread at the time of diagnosis13One process thought to facilitate metastasis is epithelial-mesenchymal transition (EMT), whereby epithelial cells lose their adhesive contacts with neighbours and take on the migratory phenotype of mesenchymal cells781112Like primary tumours, metastases are a conglomerate of cancer cells, stromal cells and extracellular matrix (ECM). In primary pancreatic cancer, a particularly dense (desmoplastic) stroma containing leukocytes, fibroblasts and ECM makes up a large portion of the tumour mass, while the density of blood vessels tends to be lowHere, we have taken advantage of a lineage-labelled autochthonous model of pancreatic cancer to carefully catalogue the changes that occur within metastatic cells and their immediate microenvironment during metastatic colonization and growth. We report that the process is highly dynamic, as both tumour cells and the stroma undergo marked changes during progression from singly seeded cells to micro- and macro-metastases. Furthermore, we show that treatment with gemcitabine and nab-paclitaxel results in killing of metastatic tumour cells despite the presence of an ostensibly protective microenvironment. These results provide insight into the process of metastatic colonization and challenge the hypothesis that stroma acts as a physical barrier to drug delivery.Cre-mediated recombination triggers mis-expression of mutant KrasG12D, deletion of one copy of p53, and activation of a YFP lineage label in pancreatic epithelial cells—allowing us to track tumour cells at the single cell level16n=40). We confined our subsequent analysis to the liver as it is the most frequent site of metastasis. Lesions were binned into five size categories according to the number of YFP+ cells: single, nano (2–10 cells), micro (11–100 cells), milli (101–1000 cells) and macro (greater than 1000 cells) (n=23). Most metastatic lesions contained 100 or fewer cells, with nano- and micro-metastases being the most abundant (We began by characterizing metastatic PDAC lesions in KPCY mice—in which 0 cells) . When exabundant . Detaile+ cells within each lesion) were not significantly different across lesions of all five metastatic categories (11–19%) and were comparable to the primary tumour (+). Primary tumours and larger lesions also exhibited low (∼1%), albeit significantly higher levels of apoptosis compared to single cells  exhibited rates of epithelial staining in the range of 60–80%, resembling primary tumours (21n=6 cases). Consistent with the murine data, CLDN7 staining was greater in primary tumours than micro-metastases while FSP1 staining was greater in micro-metastases compared to gross metastases and primary tumours  and Claudin-7 (CLDN7)1819 tumours . A complumours 21. Compariumours 21. These r tumours . Thus, i+ area at larger lesions , and contribute to the desmoplastic reaction in PDAC1425YFP) livers  livers , only MD) livers . In cont) livers . These r+ lumen-containing blood vessels per × 40 field). As expected given the high prevalence of vascular sinusoids in the normal liver, vessel density in the vicinity of small lesions (single cells and nano-metastases) was high, ranging between 25 and 30 vessels per field (+ vessel produced a similar trend: single cells (n=34) and nano-metastases (n=130 cells) were in close proximity to blood vessels  while cells within milli- (n=115 cells) and macro-metastases (n=60 cells) were far removed from the nearest blood vessel  similar to primary tumours   .+ vessels per micron. Consistent with our findings in the KPCY model, small but not large metastases had a higher vessel density compared to primary tumours  every four days for 2–4 weeks, for a total of 4–8 doses of chemotherapy, and were sacrificed when moribund. Compared to untreated historical controls  produced by the cancer cells either attract pre-existing myofibroblasts to the lesion or activate local stellate cells to differentiate into myofibroblasts. ECM deposition became more evident in advanced lesions, with macro-metastases exhibiting levels of collagen I, fibronectin, hyaluronic acid and SPARC comparable to levels present in primary tumours, consistent with a recent study reporting similar levels of myofibroblasts, collagen and hyaluronic acid in human primary pancreatic cancers and large metastasesThe vasculature also underwent dynamic changes during metastatic progression, with small lesions surrounded by blood vessels and larger lesions exhibiting hypovascularity, mirroring the hypovascular nature of primary PDACs in this model and in the majority of patients. Importantly, we found that human pancreatic tumours exhibit the same phenomenon, with small metastatic lesions having a higher vessel density than large lesions and primary tumours. Although the mechanism leading to vessel paucity in primary tumours and large metastases remains unknown, we recently showed that depletion of stromal fibroblasts  results in increased vessel density, suggesting that fibroblasts and/or ECM components exert an anti-angiogenic effectFor patients with metastatic PDAC, treatment with chemotherapy provides minimal improvement of survival362930et al. that human and murine mesenchymal PDAC cell lines are more susceptible to gemcitabine compared to epithelial lines414243There are several possible explanations for the apparent resistance of single cells to both long-term and short-term chemotherapy. The simplest possibility is that single disseminated cells are replenished by the primary tumour faster than they are being removed by chemotherapy. However, the observation that treated mice have significantly fewer CTCs would argue against this hypothesis. Mesenchymal tumour cells were acutely sensitive to chemotherapy, consistent with the observation by Collisson Our data also have implications for the treatment of patients with resectable PDAC. KPCY animals treated for only 2–4 weeks with gemcitabine/nab-paclitaxel exhibited a marked improvement in metastatic burden, yet no animals had complete absence of metastatic disease. Indeed, this failure of combination chemotherapy to demonstrate ‘curative potential' in KPCY mice mirrors a critical failing of adjuvant chemotherapy for patients with PDAC; namely, improvement in median survival compared to observation, yet only rare long-term remissions or cures. Thus, even as gemcitabine/nab-paclitaxel and other combinations are beginning to be tested in the adjuvant clinical setting, our findings provide additional motivation to evaluate novel, non-chemotherapeutic approaches that target residual micrometastatic disease.LSL−G12D, p53L/+, RosaYFP/YFP mice have been described previouslyYFP/YFP mice were used as controls. All vertebrate animal experiments were conducted in compliance with the National Institutes of Health guidelines for animal research and approved by the University of Pennsylvania Institutional Animal Care and Use Committee.Pdx1-cre, KrasTissues were fixed in Zn-formalin, paraffin embedded and stained as previously described+ lesions were quantified for five liver sections spaced 100 μm apart. Large metastatic lesions that were captured on multiple sections were only counted once.Gross metastases were confirmed by fluorescent microscopy using a Leica MZ16FA multi-colour fluorescent stereomicroscope. To determine metastatic burden, YFPPercent area was determined for a subset of stains  by first cropping the image within one cell diameter of each metastatic lesion. Using ImageJ, fluorescent channels were split and the channel of interest was thresholded to highlight positive staining while excluding background. The ‘analyse particles' tool was then used to calculate percent area.−1 Texas red-conjugated dextran  30 min prior to euthanasia. Tissues were embedded in OCT , cut into 5 μm sections and imaged on an Olympus IX71 inverted multicolour fluorescent microscope.Female tumour-bearing mice between 12–14 weeks of age were injected with 25 μg kgTissue samples were obtained from patients who consented to a research autopsy in association with the IRB approved Johns Hopkins Rapid Medical Donation Program (PMID: 19273710). Clinicopathological characteristics are listed in 2O2. Endogenous avidin and biotin were also blocked using an Avidin/Biotin Blocking Kit  according to the manufacturer's instructions. Sections were then blocked with 5% donkey serum in 0.3% Triton-X100  in PBS for 1 h at RT, then incubated with primary antibodies at 4 °C overnight. The next day, slides were washed in 0.1% Tween-20  in PBS (PBST) and incubated with a biotin-conjugated secondary antibody for 1 h at RT. Slides were washed in PBST and staining was revealed using ABC-HRP and DAB kits (Vector Labs) according to the manufacturer's instructions.Sections were deparaffinized in xylene, rehydrated and subjected to antigen retrieval. Endogenous peroxidases were blocked with 1.5% H2 (1500 × 1500 pixel) fields as TIFF images for further analysis. The fields were randomly selected in the area of interest using a low magnification view of the slide. To compare vessel density in primary and metastatic tumour, up to five fields were extracted from each slide. The extracted fields were then analysed with image analysis software written using ImageJ . Total tissue area was measured by manually thresholding a grayscale version of the original field. The CD31-positive area was then measured by first masking out any areas with background staining using manual drawing tools and then performing colour deconvolution to separate the DAB and hematoxylin staining2) in size were excluded to reduce the degree of large vessel fragmentation and the presence of single immunoreactive cells. CD31+ vessel density was calculated as the number of CD31 positive particles/total tissue area.Immunolabelled slides were scanned at × 200 total magnification at a resolution of 0.49 μm per pixel using an Aperio Scanscope CS digital slide scanner . Aperio ImageScope was used to extract 735 μm−1. The drugs were administered simultaneously once every four days until the mouse became moribund, between 2 and 4 weeks from enrolment. Each mouse received at least 4 (and a maximum of 8) doses.Tumour-bearing animals in healthy condition were enrolled once the tumour had reached an estimated × 1 cm in diameter based on palpation. With the knowledge that the average number of metastases per mouse in our historical, untreated controls is 50.7 (±64.9), we concluded that a large effect size of at least a 75% decrease in metastasis with a relatively small (≤10) standard deviation would be necessary to detect significant changes after treatment. Using these values, with an alpha error level of ≤5%, we determined that a sample size of eight mice would be sufficient. Gemcitabine-HCl  and nab-paclitaxel  were both dissolved in PBS and administered via intraperitoneal injection at 120 mg kg−/CD45−/YFP+ events were counted as CTCs.Blood was collected by cardiac puncture as previously describedpost hoc test to assess differences between ‘single cells' and all other groups. For experiments in which ‘single cell' means were binary (0 or 100%), differences between ‘single cells' and all other groups were analysed by one-sample t-test. All statistical analyses were performed using GraphPad Prism 6 . P≤0.05 denotes differences that are statistically significant.Differences between two groups were analysed by two-tailed Student's t-test with Welch's correction to account for unequal SDs, or by Mann-Whitney test for non-normally distributed data. Differences between three or more groups were analysed by one-way ANOVA with Tukey's multiple comparisons test used as a The data that support the findings of this study are available within the Article and How to cite this article: Aiello N. M. et al. Metastatic progression is associated with dynamic changes in the local microenvironment. Nat. Commun. 7:12819 doi: 10.1038/ncomms12819 (2016).Supplementary Figures 1-9, Supplementary Tables 1-3