Autism spectrum disorders (ASDs) affect ~1% of the population and are characterized by impairments in social interaction and communication, as well as by repetitive and restricted behaviours. ASDs include mild to severe levels of impairment, with cognitive function ranging from above average to intellectual disability (ID), and are often accompanied by seizures and other medical problems. There is an ~4:1 male to female gender ratio in ASD.ASDs are highly heritable and genomic studies have revealed that a substantial proportion of ASD risk resides in rare variation of high effect, ranging from chromosome abnormalities and copy number variation (CNV) to single nucleotide variation (SNV). These studies have highlighted a striking degree of genetic heterogeneity, implicating both de novo germline mutation and rare inherited variation in ASD distributed across numerous genes. De novo CNVs are observed in 5%–10% of screened ASD cases, some of which have proven to alter high-risk genes now tested for in the medical diagnostic setting. Exome-sequencing studies estimated another ~6% contribution to ASD, and an additional 5% conferred by rare inherited recessive or X-linked loss-of-function SNV. A genetic overlap between ASD and other neuropsychiatric conditions has also been increasingly recognized.Interestingly, CNV testing and exome-sequencing have so far yielded mostly non-overlapping genes, which may reflect different mutational mechanisms, though they may still perturb connected biological pathways. While numerous ASD genes have been recognized to date, they only account for a small fraction of the overall estimated heritability, consistent with predictions that there are ~1000 loci underlying ASD and that many causal genes and risk variants remain to be identified.Here, I will discuss our latest genomic experiments to identify and characterize additional ASD risk genes, and to identify the biological relationships and common pathways they share. Our combined results suggest that rare variants affecting ASD risk in the population collectively encompass hundreds of genes some being influenced by the gender of the mutation carrier. Despite significant heterogeneity, genes group in a relatively small number of interconnected functional networks, particularly related to neuronal signalling/development, synapse function and chromatin regulation. All of these findings, ultimately, contribute to our understanding of the cellular pathways involved in ASD, providing new diagnostic and therapeutic targets.


Growing evidence from task-based functional magnetic resonance imaging (fMRI) studies consistently indicates network abnormalities in Alzheimer’s disease (AD) patients ,2. Whilein vivo diffusion tensor imaging studies, while functional connectivity changes have been observed using other methods, such as rs- and task-based fMRI and positron emission tomography (PET).Resting state functional connectivity (RS FC) changes in the AD brain involve both decreased and increased connectivity within specific networks. Interpretation of such alterations is far from trivial, and it remains largely unknown how they relate to continuing cognitive decline as the disease advances. Decreased RS FC presumably indicates lesion-driven disconnection processes, whereas increased connectivity indicates compensatory and adaptive connectivity mechanisms ,12. WhilRs-fMRI is an excellent tool for assessing functional connectivity of distant brain areas in AD, because it does not require task performance in the scanner, which may be overwhelming to patients. In addition, the length of scanning in collecting rs-fMRI data are relatively short compared to task-based scanning sessions. The standard scanning duration in collecting rs-fMRI data are 5-7 min, although recent evidence suggests that scanning sessions that take 9-13 min result in improved reliability . DespiteIt appears then that intrinsic brain activity assessed by rs-fMRI contains important information on the changes in AD brain that can be traced from preclinical and prodromal stages to dementia. Since aberrant patterns of RS FC in prodromal and dementia stages of AD have been associated with specific cognitive symptoms, as discussed above, these associations can be used in clinical settings to support diagnosis, monitoring of disease progression, and assessment of efficiency of disease-modifying treatments. The rest of the article discusses the relevance of thalamic RS FC with frontal areas for general verbal fluency in AD.AD patients regularly perform considerably worse on verbal fluency tests than cognitively healthy elderly subjects . In thesVerbal fluency tests are often considered the tests of executive function, but as simple lexical tasks – cued word generation – they also implicate language. Even though some evidence suggests that category fluency better reflects verbal abilities whereas letter fluency better reflects executive function, not all findings support such a distinction . It has Attempts to determine the neural basis of verbal fluency indicate a special role of Broca’s area in these tasks, while also implicating a number of other areas. An early task-based fMRI study of covert cued word generation in healthy subjects that used both letter and category fluency tests  reportedA seminal study on RS FC of language networks that involved 970 healthy participants scanned at 22 institutions across the world suggests that the brain at rest reveals an extended language network, implicating the classical language areas  as well as a range of prefrontal, temporal, and parietal regions along with subcortical structures . Other sIn addition, the thalamus appears to be implicated in a range of language-related tasks, eg, word generation, speech production, picture naming, reading, and syntactic and semantic processing ,32-35. TEarly evidence for the role of the left pulvinar and some sections of the left ventrolateral nucleus in word retrieval comes from electrostimulation studies with patients undergoing awake brain surgery for the treatment of dyskynesias ,37. An eSince considerable atrophic changes in frontal areas do not appear in early stages of AD , whereasIn conclusion, it is important to better integrate cognitive neuropsychological and rs-fMRI data in clinical settings. This may provide a better insight into transition toward advanced stages of AD and help in the evaluation of treatments intended to slow down progression of the disease. Such integration of data are expected, given the increasing role of neuroimaging assessment in diagnosis and monitoring of AD, and the relative ease of incorporating rs-fMRI scanning into general scanning protocols.


Cylindromatosis gene, CYLD is a tumor suppressor gene that was originally identified as a gene mutated in familial cylindromatosis and related diseases . CYLD beRecently, we have demonstrated a significant role of CYLD in differentiation of neuroblastoma . NeuroblRecent work from our laboratory, demonstrated that all-trans retinoic acid (ATRA)-induced differentiation of neuroblastoma cells. It, operates by increasing in the expression of the small ubiquitin-like modifier (SUMO) protein that can affect downstream signaling inside the cells by quickly switch on or off different cellular processes, including survival, proliferation, and differentiation. The SUMOylation is reversible through the action of SUMO specific proteases (SENPs) Figure . We obsein-vitro approaches [In this context, maintaining high expression levels of CYLD and SUMO proteins together can be used promising tools for future therapeutic strategies for neuroblastoma patients. Indeed, cellular death induced by oxidative stress via overexpression of SUMO-1 was previously shown to be blocked by the activation of deSUMOylated enzyme SENP1 . Beside proaches . Future


Induced pluripotent stem cells (iPSCs) allow researchers to make customized patient-derived cell lines by reprogramming noninvasively retrieved somatic cells. These cell lines have the potential to faithfully represent an individual’s genetic background; therefore, in the absence of available human brain tissue from a living patient, these models have a significant advantage relative to other models of neurodevelopmental disease. When using human induced pluripotent stem cells (hiPSCs) to model X-linked developmental disorders or inherited conditions that undergo sex-specific modulation of penetrance , there are significant complexities in the course and status of X chromosome inactivation (XCI) that are crucial to consider in establishing the validity of cellular models. There are major gaps and inconsistencies in the existing literature regarding XCI status during the derivation and maintenance of hiPSCs and their differentiation into neurons. Here, we briefly describe the importance of the problem, review the findings and inconsistencies of the existing literature, delineate options for specifying XCI status in clonal populations, and develop recommendations for future studies. Approximately one third of autism spectrum disorder (ASD) patients have an identifiable pathogenic genetic variant, and as sequencing becomes more widespread, this percentage increases . AlthougAn important aspect of any cellular model of disease involves the issue of whether the cells faithfully reflect the normative process of X chromosome inactivation. Although only a minority of ASD susceptibility loci reside on the X chromosome, X-linked mutations are well-known causes of related developmental disorders. Perhaps even more importantly, there is growing evidence that supports the hypothesis that there is a “female protective effect” in which the pronounced sex ratio universally observed in ASD is resolvable to the interaction between female sex and inherited autism liabilities, which are predominantly polygenic and autosomal in nature –9. A proRandom X chromosome inactivation in female humans occurs early during the developmental process, resulting in the equalization of X chromosome-linked gene dosages across sexes. There are four steps associated with X chromosome inactivation (XCI), referred to as counting, choosing, initiation, and maintenance. All but maintenance are orchestrated by the X-inactivation center (Xic), which contains the noncoding RNA-encoding X-inactive specific transcript (Xist) locus. Xist RNA surrounds the X chromosome selected to be inactivated and is necessary for the initiation of its silencing. Although the exact mechanism involved in counting remains elusive, there is evidence to suggest that the cell utilizes the ratio between X chromosome number and haploid autosomal chromosome number in order to ensure that only one X chromosome is active . This prEarly in development, when Xist plays the major role in inactivation, XCI is reversible. Once the initiation phase transitions to the maintenance phase, however, the inactivation status is sustained . This prGenerally, X-inactivation is a random process that results in equal expression of both X chromosomes in tissue . HoweverThe extent to which these processes are recapitulated in the development of human-induced pluripotent stem cell-derived neuronal cell models is currently poorly understood and, while many papers discuss the modeling of neurodevelopmental disorders in hiPSCs, the literature is scarce regarding the status of XCI in hiPSCs and the potential impact on neurodevelopmental disease modeling –18. In tGeneration of hiPSCs has been performed by reprogramming of somatic cells through induced expression of four transcription factors—Oct4, Sox2, Klf4, and c-Myc. These four pluripotency transcription factors induce the pluripotent state of the reprogrammed cells by resetting transcriptional patterns as well as altering DNA methylation and histone modifications . The traThe current literature regarding X chromosome reactivation during reprogramming is contradictory and complex. This lack of consistency in results is apparent in research modeling Rett syndrome in human hiPSCs. For example, while results from Marchetto et al. indicate that hiPSCs undergo X chromosome reactivation (XCR), Ananiev et al. yielded results bolstering reports that the inactivation status of the somatic cell is maintained during reprogramming –26. It iAs depicted in Fig. It is important to note that there is a fundamental difference between mouse and human iPSCs: mouse iPSCs (miPSCs) and mouse ESCs (mESCs) have routinely been observed to contain two active X chromosomes, while human iPSCs, as discussed above, have a much more variable X-inactivation/reactivation status . This haThe differences in XCR state among hiPSCs appear to reflect differences in the pluripotent state of the reprogrammed cells, with at least two different forms of hiPSCs resulting from reprogramming. The first human iPSC type more closely resembles murine ESCs, which are derived from the inner cell mass of the early embryo, while the second type instead has greater similarity to epiblast-derived stem cells (EpiSC). In rodent models, EpiSC are derived from the epiblast at the post-implantation stage and therefore represent a later developmental cell state than ESCs in vivo. While both types of hiPSCs are considered pluripotent, based upon several experimental criteria, ESC-like hiPSCs exhibit more naïve or “ground state” characteristics: having undergone XCR, they biallelically express the X chromosome. By contrast, EpiSC-like hiPSCs have undergone XCI and exhibit a primed rather than naïve pluripotent status . These nWhile the status of the X chromosome appears to differ between hiPSCs with a more naïve versus primed pluripotent character, it remains unclear to what extent hiPSCs that retain XCI versus undergoing XCR differ in their capacity for or quality of subsequent differentiation. Teratoma assays are often performed on human hiPSCs to confirm that the cells have the capacity to differentiate into multiple germ layers. Both hiPSCs that have retained XCI and those that have undergone XCR can differentiate into all germ layer derivatives in such teratomas, but further research is required to determine whether differences in differentiation capacity exist that are not appreciable by teratoma assays , 23.Furthermore, the stability of XCR status in ESC-like hiPSCs is contested in the literature. In some work, XCR status is reported to be stable, enduring hiPSC line freezing and thawing as well as many passages . HoweverAnother possible fate for the X chromosomes of hiPSCs is erosion, which is similar to the third potential outcome of reprogramming presented above, in which the initially inactivated X chromosome loses Xist and repressive H3K27me3 modification but is not fully reactivated . During When studying X-linked genetic mutations, a straightforward method for determining which X chromosome is active is to assess expression of the allelic variant of the gene. This can be assayed by reverse transcription, polymerase chain reaction (PCR), and sequencing. Alternative assays may also be employed to determine the status of XCI. For example, an assay that takes advantage of androgen receptor (AR) polymorphisms utilizes methylation sensitive restriction enzymes that specifically digest unmethylated DNA; in this method, only the AR variant on the inactivated X chromosome is amplified via PCR . There aHuman iPSCs represent an ethically acceptable, increasingly widely available resource for developing cellular models of disease. These models are particularly important in autism research, given the relative lack of availability of the affected primary tissue (brain) for biological studies, marked heterogeneity in the genetic influences on ASD across affected individuals , the prevalence of the condition, and the availability of effective technologies for reprogramming noninvasively derived cells from individual patients . By recaTo date, there have been few published results of hiPSC models of nonsyndromic ASD , 36. GriWe conclude that variabilities in the status of X-inactivation and X-reactivation in hiPSCs are significant factors to be considered when implementing cellular models of autism and related neurodevelopmental disorders. The multiple variations in XCI status of clonal hiPSC populations include complete reactivation, complete inactivation, mixed inactivation and reactivation, and erosion. These complexities indicate that variation in reprogramming procedures and/or failure to precisely characterize XCI status can have significant consequences for the validity of a cellular model . This isThe discrepancies in XCR status in human hiPSCs reviewed here underscore the need for a greater understanding of how the parameters involved in reprogramming and culturing of hiPSCs affect their pluripotent cell state, differentiation potential, and other phenotypic properties relevant to their use for modeling human disease. While it may yet take years to fully define the molecular basis of reprogramming and factors that influence acquisition and maintenance of particular pluripotent states, it is important to identify specific conditions under which hiPSCs with stable XCI status can be obtained and maintained. This will provide a reliable and standardized method for researchers to generate hiPSC models that can more faithfully recapitulate the state of the cells in vivo and that therefore have higher utility for cellular modeling of human disease states.AR, androgen receptor; ASD, autism spectrum disorder; ESCs, embryonic stem cells; FISH, fluorescence in situ hybridization; H3K27me3, histone H3 trimethyl lysine 27; hiPSCs, human induced pluripotent stem cells; iPSCs, induced pluripotent stem cells; LIF, leukemia inhibitory factor; MEF, mouse embryonic fibroblast; mESCs, mouse embryonic stem cells; miPSCs, mouse induced pluripotent stem cells; PcG, polycomb group complex; PCR, polymerase chain reaction; XCI, X chromosome inactivation; XCR, X chromosome reactivation; Xic, X-inactivation center.


Deserts are generally regarded as lifeless and inhospitable ecosystems despite the general awareness of extremophilic microorganisms. An amazing microbial diversity and a huge biotechnological potential were unraveled during the last decade using molecular approaches. Hot and cold deserts were shown to host peculiar microbial assemblages able to cope with hostile environment and/or to rapidly adapt to changing conditions. This adaptation is inferred to particular community structure behavior and specific metabolic capacities allowing cells to overcome water stress, fluctuating temperature, and high salinity. Therefore, such microbes could constitute a source of novel metabolites, biomolecules, and enzymes potentially useful for environmental biotechnologies. With the global climate change, the aridification and creeping desertification that constitute a worldwide serious threat directly affecting agriculture and crop production, and the growing food demands, desert microorganisms could hold the key for green biotechnology and future applications into soil bioreclamation and plant growth promotion for vulnerable regions across the world.This special issue was focused on the desert microbial resource management (MRM) and how to explore and exploit these resources from hot and cold deserts as well as from arid areas. Aspects of this MRM concept are included in this special issue and they are highlighting (a) the microbial diversity and community structure behavior in desert environments and the identification of novel extremophiles, (b) the influence of the biotic and abiotic factors on microbial communities shape and dynamic and the functional networking including mechanism of adaptation and plant-microbes interaction under extreme or changing conditions, and (c) potential and case applications of desert microbes and/or mixed cultures, such as in soil bioreclamation, reverse-desertification, agriculture, and biomining. Micromycetes from Sebkha El Melah, a Saharan Salt Flat in Southern Tunisia,” reported the isolation of 21 alkali-halotolerant Ascomycetes assigned to the 6 genera Cladosporium, Alternaria, Aspergillus, Penicillium, Ulocladium, and Engyodontium, basing on morphological and molecular markers. Beside their salt and pH tolerance, these saline-system fungi were shown to resist to oxidative stress and low temperature and to produce extremozymes, namely, cellulase, amylase, protease, lipase, and laccase, active in high salt concentrations, which highlight their biotechnological potential. The paper authored by M. del C. Montero-Calasanz et al., “Geodermatophilus poikilotrophi sp. nov.: A Multitolerant Actinomycete Isolated from Dolomitic Marble,” described a new species within the genus Geodermatophilus. Strain G18T, isolated from site near the Namib Desert, is characterized by its resistance to heavy metals, metalloids, hydrogen peroxide, desiccation and ionizing, and UV-radiations. Even though 16S rRNA sequence of strain G18T showed 99% similarity with other Geodermatophilus species, its taxonomic position and species definition was inferred basing on polyphasic approach and its multitolerance towards environmental stresses, justifying the original given epithet “poikilotrophi.”This special issue contains one review and eight research articles that address the three main aspects indicated above. The paper of A. Jaouani et al., entitled “Diversity and Enzymatic Profiling of Halotolerant Frankia Microsymbionts and the Disjunct Actinorhizal Coriaria Species.” The investigation was achieved on five Coriaria host species sampled from sites covering the full geographical range of the genus . The reported findings argue that Frankia, the nitrogen-fixing actinobacteria microsymbionts, have not evolved jointly with their host plants and had probably dispersed globally as a proto-Frankia, a free living nonsymbiotic ancestor. The authors hypothesized also that cospeciation may have occurred but subsequently lost after bacteria mixing and fitness selection in the presence of indigenous symbionts. Frankia sp. was further investigated in terms of nitrogen fixation under different oxygen tensions. This work authored by F. Ghodhbane-Gtari et al. was conducted on the actinorhizal plant Casuarina and its compatible Frankia sp. strain CcI3. By studying the growth of the strain, vesicle production, and several genes expression, the authors confirmed the correlation between the biomass and the vesicle production with elevated oxygen tension. It was also shown that oxygen levels influenced nitrogenase induction and that Frankia protects nitrogenase by the use of multiple mechanisms including the vesicle-hopanoid barrier and increased respiratory protection. Clearly, the microbial assemblages selected by the plant roots in desert and arid soils are shaped by the ecological biotic and abiotic drivers but with the prerequisite of providing rhizosphere services and specific functionalities.Four papers were dedicated to the ecological drivers that shape microbial communities and functionalities. A nice example of “cold desert” is presented by S. Ciccazzo et al., “Safe-site Effects on Rhizosphere Bacterial Communities in a High-Altitude Alpine Environment.” In this work, the authors investigated rhizobacterial communities associated with floristic consortia in different safe-sites located in deglaciated terrain. Using DGGE and ARISA, they demonstrated a clear correlation between soil maturation and bacterial diversity and a plant-specific effect leading to the selection of specific rhizobacterial communities by the pioneer plants. Another model ecosystem was investigated by R. Ferjani et al., in the paper “The Date Palm Tree Rhizosphere Is a Niche for Plant Growth Promoting Bacteria in the Oasis Ecosystem.” The work focused on the characterization of the bacterial communities in the soil fractions associated with the root system of date palms cultivated in seven oases in Tunisia using culture-independent and dependent approaches. It was shown that the date palm rhizosphere bacterial communities were rather complex and correlate with geoclimatic and macroecological factors along a north-south aridity transect. However, with the wide diversity of cultivable strains detected, interesting common features of plant growth promoting (PGP) activity and abiotic stress resistance were detected. The authors concluded that palm root system and rhizosphere soil represent a reservoir of PGP bacteria involved in the regulation of plant homeostasis. The third example of plant-microbe interaction is the paper by I. Nouioui et al., in which they demonstrated, as written in the title, the “Absence of Cospeciation between the Uncultured Janibacter sp. FAS23. The strain was isolated from Sebkha El Naoual, a saline ecosystem in southern Tunisia. Using HPLC analysis, FAS23 was shown to be able to degrade high concentration of PCP (up to 300 mg/l) and to tolerate salt fluctuation. PCP degradation was further enhanced in the presence of glucose and nonionic surfactant tween 80. The strain is considered as a candidate for PCP bioremediation in polluted soils in arid areas. In another paper authored by O. Selama et al., the isolation of haloalkalitolerant and haloalkaliphilic bacteria from Algerian Sahara Desert soil was reported. Thirteen selected isolates, mainly filamentous Actinobacteria, were screened phenotypically for antibacterial, antifungal, and enzymatic activities and by PCR for putative antitumor compounds genes. The isolates were assigned to the genera Streptomyces, Nocardiopsis, Pseudonocardia, Actinopolyspora, and Nocardia, with this latter constituting possibly a new branch in the Actinomycetales order. Beside secreted extremozymes and bioactives, several isolates showed antitumorigenic potential. Another paper presented in this special issue is a review article nicely written by A. Azua-Bustos and C. González-Silva, who focused on the Atacama Desert microbes and their current biotechnological applications. A large-scale application in Chile is the copper bioleaching or biomining mediated by indigenous halotolerant and acidophilic chemolithotrophic bacteria like Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans. Other potential applications in arsenic bioremediation and in biomedicine, including the discovery of new antibiotics, antioxidant, antifungal, and immunosuppressive compounds, were cited. The authors reported also application from eukaryotic microorganism as in the case of the halophilic biflagellate unicellular green alga Dunaliella that produce beta-carotene.Biotechnological potential and applications of desert microbes have been reported in three papers. In one, A. Khessairi et al. described a novel efficient pentachlorophenol- (PCP-) degrading halotolerant actinobacterium,Definitely, desert environments represent a tremendous reservoir where more efforts, relying not only on metagenomics but also on culturomics, should be dedicated to unravel the hidden potential. The second decade for desert biotechnology has just begun.


Worldwide, attending physicians train residents to become competent providers of patient care. To assess adequate training, attending physicians are increasingly evaluated on their teaching performance. Research suggests that personality traits affect teaching performance, consistent with studied effects of personality traits on job performance and academic performance in medicine. However, up till date, research in clinical teaching practice did not use quantitative methods and did not account for specialty differences. We empirically studied the relationship of attending physicians' personality traits with their teaching performance across surgical and non-surgical specialties.We conducted a survey across surgical and non-surgical specialties in eighteen medical centers in the Netherlands. Residents evaluated attending physicians' overall teaching performance, as well as the specific domains learning climate, professional attitude, communication, evaluation, and feedback, using the validated 21-item System for Evaluation of Teaching Qualities (SETQ). Attending physicians self-evaluated their personality traits on a 5-point scale using the validated 10-item Big Five Inventory (BFI), yielding the Five Factor model: extraversion, conscientiousness, neuroticism, agreeableness and openness.P = 0.02). Openness was negatively associated with scores on feedback for surgical specialties only  and conscientiousness was positively related to evaluation of residents for non-surgical specialties only .Overall, 622 (77%) attending physicians and 549 (68%) residents participated. Extraversion positively related to overall teaching performance (regression coefficient, B: 0.05, 95% CI: 0.01 to 0.10, Extraverted attending physicians were consistently evaluated as better supervisors. Surgical attending physicians who display high levels of openness were evaluated as less adequate feedback-givers. Non-surgical attending physicians who were conscientious seem to be good at evaluating residents. These insights could contribute to future work on development paths of attending physicians in medical education. Residents carry out much of daily patient care, while being learners at the same time. Therefore, patient care quality benefits from adequate supervision of residents Research suggests that attending physicians who are younger, female and spend more time on teaching and conducting research, are more favorably evaluated on their teaching performance What are personality traits and in what way could they affect teaching of attending physicians? Personality traits can be categorized in five comprehensive domains, called the Five Factor Model: conscientiousness, extraversion, emotional stability, agreeableness and openness Extraversion means being sociable, talkative, outgoing and active Emotional stability involves high levels of self-esteem, positive affect and low levels of stress and anxiety. Indeed, a lack of emotional stability is associated with high levels of stress, anxiety and neuroticism Another personality trait that remains unexplored in the context of clinical teaching is agreeableness. Agreeableness refers to friendliness and includes being kind, cooperative, flexible and tolerant. Research suggests that agreeableness has positive relations with work performance where social interaction is part of the job, especially when it involves helping and cooperating with others Finally, openness is a personality trait that refers to being open and receptive to experience. Openness is associated with being imaginative, cultured, curious, and broad-minded. Findings suggesting that curiosity benefits teaching effectiveness Although personality traits characterize individuals, attending physicians do not function as individuals only — they work in teams within departments, delivering specialized patient care and medical training. The clinical specialty establishes a specific professional context, not only for the nature of patient care that varies across specialties, but also for interpersonal behaviors towards and interactions with patients Overall, since previous research suggests that personality traits could affect teaching performance in (mostly) non-clinical settings, there is a critical need for examining these in residency training. Moreover, the little existing research done in clinical teaching settings used qualitative methods only, making it nearly impossible to make inferences based on quantitative evidence. Moreover, nothing is known about differences between specialties in terms of plausible links between personality traits and teaching performance. Therefore, the objective of this study is to examine the relationship of personality traits with teaching performance of attending physicians within and across surgical and non-surgical specialties. We hypothesize that conscientiousness, extraversion, emotional stability, agreeableness, and openness all positively affect teaching performance. Since the differences between surgical and non-surgical specialties on this matter had not been documented in the literature, we had no specific expectations, electing for an explorative approach to this issue.This multicenter study was conducted at 61 different residency programs, covering 7 surgical and 18 non-surgical specialties, in 2 academic and 16 non-academic medical centers in the Netherlands, from May to December 2012. We invited 815 residents and 819 attending physicians by email and mentioned the formative purpose and use of the evaluations and stressed the confidential and voluntary character of participation.To measure teaching performance, we conducted a survey using the well-published System for Evaluation of Teaching Qualities (SETQ), a system for continuous evaluation of attending physicians, which is used by approximately 6000 residents and attending physicians representing 45 medical centers in the Netherlands. The details of instrument development are described elsewhere, showing that the instruments provide reliable and valid evaluations of the teaching qualities of attending physicians Attending physicians self-reported their personality traits using the shortened version of the Big Five Inventory (BFI-10),Taking into account BFI authors' recommendations, we added an extra item for the subscale agreeableness in order to safeguard internal consistency for this subscale, as it showed less internal consistency than the other personality subscales Attending physicians' gender was considered as a confounding variable, as research showed gender differences in personality as well as in teaching performance First, we aggregated teaching performance evaluations of different residents on the level of individual attending physicians, which resulted in average scores on teaching performance items for each attending physician. We calculated means and medians for the overall as well as domain sum scores when at least two third of the items were completed.  Next, we described the study sample using applicable analytical techniques. Using a random half of the sample, we then explored whether the five-domain structure of the original BFI-10 also applied to our sample data by conducting a principal components factor analysis, with promax rotation and retaining only factors with eigenvalue >1. With the second half of the sample data, we conducted confirmatory factor analysis of the factor structure that emerged from the previous step. We assessed goodness of fit using Comparative fit index (CFI), Tucker-Lewis Index (TLI), root mean square error of approximation (RMSEA), and standardized root mean square residual (SRMR). CFI >0.95, TLI >0.95, RMSEA <0.06 and SRMR <0.08 were taken to indicate good fit. We also accounted for clustering within hospitals and assessed equivalence across groups in the sample .The six scores for overall teaching performance and its five domains were used as outcomes in subsequent multivariable analysis. To quantify the impact of attending physicians' personality traits on their teaching performance as evaluated by residents, we performed multivariable adjusted regression analyses using generalized estimating equations (GEEs). Using GEE was appropriate because of their capacity to account for data nesting or clustering at different levels, such as attending physicians being part of specific departments in different hospitals. We, therefore, regressed (i) overall teaching performance on BFI-subscales, and (ii) each of the five domains of teaching performance on BFI-subscales, for all specialties combined. These two sets of models were repeated for surgical and non-surgical specialty groupings separately. To test for differences between the samples, we performed fit models with interaction terms. For this purpose, we used product terms involving each personality trait and surgical specialty . We controlled for gender and age by conditioning on them in the regression models. To deal with the varying number of resident evaluations of teaching performance per attending physician, we checked whether the results were sensitive to this variable, by adjusting the analyses for this. Because not all participating attending physicians filled out the questionnaires (participation was voluntary), we controlled for a possible selection bias by reweighting the analyses for selection probability. All analyses were conducted in IBM SPSS Statistics version 20 , Stata version 13.1 for Mac OS , and R version 3.0.2 .The institutional ethical review board of the Academic Medical Centre of the University of Amsterdam (AMC) waived ethical approval for this study.In total, 560 (68%) residents filled out 4305 evaluations of 805 attending physicians: 622 (77%) attending physicians participated, of whom 515 (83%) self-reported their personality traits see . The mear = 0.22. Results of the CFA supported the foregoing structure with acceptable goodness of fit indices: CFI  = 0.977; TLI  = 0.959; RMSEA  = 0.032; SRMR  = 0.034. There were no differences in factor structure across groups such as specialties.According to the factor analysis, the original five-factor structure of the BFI-11 was confirmed for our specific study sample see . The posoverall teaching performance , it could be concluded that openness was negatively associated with scores on feedback for surgical specialties and not for surgical specialties  Another point of self-reported personality traits is that they might have provided other information about personality traits than observer-reports would have. Indeed, it has been shown that self-reported and observer-reported personality traits each have unique variance Up till date, self-reported personality traits in relation to teaching performance are less common in research than other-reported personality traits.The reported positive effects of extraversion are consistent with previous research In line with expectations, conscientiousness turned out to be a positive trait for some specific teaching skills, i.e. the creation of a motivating learning climate and communication of learning goals. In general, conscientious people tend to be active learners In addition, we found that attending physicians who reported higher levels of conscientiousness, were perceived as more adequate in evaluating the knowledge and skills of residents, however, this only applied to non-surgical attending physicians. An explanation for this finding could be found in the fact that non-surgical residents find evaluation a more important teaching skill than surgical residents Research on good teaching in medical education highlights that good teachers are personable, approachable and respectful We found that surgeons with high levels of openness are less good feedback providers. Corresponding to Netlle's cost-benefit trade-off model Contrasting existing evidence In providing adequate training to residents, attending physicians could reflect upon the role of their personality and specialty in relation to their teaching. Teaching teams could ensure quality of teaching by creating heterogeneous teams in terms of personality of attending physicians, as different personality traits have been shown to facilitate different teaching skills. This way, residents can, for example, enjoy the superior professional attitude of their agreeable teachers and discuss learning goals with their conscientious supervisors. Future research could examine the interplay between personality of the resident and personality of the attending physician, as personality of the resident could determine their preference for supervision of specific attending physicians.As personality traits are broad domains, future research could use extensive personality questionnaires that give more detailed information about the specific personality traits having effect. Then it could become clear, for example, that it is the specific aspect assertiveness of extraverted attending physicians that serves their communication of learning goals. Insights resulting from future research could give direction to development or expansion of training or coaching of (teaching) competences (e.g. assertiveness) of attending physicians.This study found that extraverted attending physicians were favorably evaluated on overall and domain-specific teaching performance, signifying the importance of interpersonal and communicative capacities for teaching of attending physicians in residency training. Teaching qualities represent a variety of skills and appear to be served by a variety of personality traits as well. Lastly, what works in one specialty does not seem to work for another specialty. Future research could focus on specific specialties beyond broad categorizations into surgical versus non-surgical specialties, in order to provide more nuanced and detailed inferences.Figure S1The associations between mean scores on openness and mean scores on feedback, separately for surgical and non-surgical specialties.(TIF)Click here for additional data file.Figure S2The associations between mean scores on conscientiousness and mean scores on evaluation of residents, separately for surgical and non-surgical specialties.(TIF)Click here for additional data file.Figure S3The associations between mean scores on openness and mean scores on evaluation of residents, separately for surgical and non-surgical specialties.(TIF)Click here for additional data file.


Studies have linked cognitive functioning to everyday social functioning in psychotic disorders, but the nature of the relationships between cognition, social cognition, symptoms, and social functioning remains unestablished. Modelling the contributions of non-social and social cognitive ability in the prediction of social functioning may help in more clearly defining therapeutic targets to improve functioning.In a sample of 745 patients with a non-affective psychotic disorder, the associations between cognition and social cognition at baseline on the one hand, and self-reported social functioning three years later on the other, were analysed. First, case-control comparisons were conducted; associations were subsequently further explored in patients, investigating the potential mediating role of symptoms. Analyses were repeated in a subsample of 233 patients with recent-onset psychosis.Information processing speed and immediate verbal memory were stronger associated with social functioning in patients than in healthy controls. Most cognition variables significantly predicted social functioning at follow-up, whereas social cognition was not associated with social functioning. Symptoms were robustly associated with follow-up social functioning, with negative symptoms fully mediating most associations between cognition and follow-up social functioning. Illness duration did not moderate the strength of the association between cognitive functioning and follow-up social functioning. No associations were found between  cognition and follow-up social functioning in patients with recent-onset psychosis.Although cognitive functioning is associated with later social functioning in psychotic disorder, its role in explaining social functioning outcome above negative symptoms appears only modest. In recent-onset psychosis, cognition may have a negligible role in predicting later social functioning. Moreover, social cognition tasks may not predict self-reported social functioning. Although there is some variability in the literature, most studies suggest psychotic disorder is characterized by a stable, diminished performance on the majority of cognitive domains ,2, whichThe cognitive deficits associated with psychotic disorders are linked to everyday social functioning and may limit the rate of functional improvement . CognitiDespite the large body of evidence linking cognitive abilities to social functioning, literature shows mixed findings for the relevance of cognitive functioning as a prognostic factor for social functioning, that is, independent of clinical symptoms and negative symptoms in particular –17. WherAdditionally, the predictive value of cognitive functioning in the early phase of psychosis is unclear ,19. AlthFurthermore, processes may not be generalizable across different stages of the illness. Illness duration and treatment effects may influence associations between cognitive functioning and functional outcome and prognostic factors may have different predictive values in early psychosis ,19. ThusThe aim of the present study was to investigate the contribution of cognition and social cognition in the prediction of social functioning, by first comparing the longitudinal association between baseline cognitive and social cognitive performance and three-year follow-up social functioning in patients with psychotic disorder versus healthy controls; and subsequently by analysing the associations in patients, integrating symptoms in a longitudinal model to account for the mediating role clinical symptoms may play in the cognition-social cognition-functional outcome relationships. To further clarify the associations, cross-sectional analyses were presented to explore concurrent associations with social functioning. We also investigated whether associations between cognitive functioning and social functioning varied in strength depending on illness duration.0) and first follow-up (T1) measurements of the longitudinal ‘Genetic Risk and Outcome in Psychosis’ (GROUP) study (see )Illness duration did not significantly moderate the association between cognitive functioning and social functioning, suggesting that the associations are similar over the course of psychotic disorder. However, in contrast to the outcome in the patient group as a whole, sensitivity analyses in recent onset psychosis patients yielded inconclusive findings with respect to cognitive as longitudinal predictors of social functioning. In line with the latter findings, it has been suggested that cognition assessed within the first episode of the illness may not be a reliable predictor of functional outcome, explaining considerably less variance in outcome compared with previous cross-sectional studies with chronic patients . FurtherThe difference between the present results and previous findings may be related to differences in sample due to the recruitment procedures. Even though the majority of patients 84%) were diagnosed with schizophrenia-related disorders, the decision to include patients with other non-affective psychotic disorders may have attenuated effect sizes . Further% were diThe present study is unique in its naturalistic cohort design, with a broad neuropsychological assessment in a large sample of patients with a psychotic disorder and healthy controls. Nevertheless, some limitations need to be taken into account. First, baseline social functioning was not assessed; therefore, crossed-lagged analyses could not be performed. Because we did not have the possibility to evaluate this, we could not establish whether cognitive functioning may predict decline in or recovery of social functioning nor can we exclude that poor social functioning negatively impacts on cognitive performance rather than cognition impacting negatively on social functioning.Second, follow-up data were not available for a substantial number of patients. Drop-outs had significantly poorer social and non-social cognitive skills than patients who remained in the study. Moreover, not all subjects had complete baseline cognitive test scores. Finally, a self-report questionnaire was used to assess functional outcome. Self-report is inevitably subjective and may be driven in part by within-person factors such as psychopathology. However, self-report may arguably still be better than observer-rated social behaviour because the latter assessment method also comes with a degree of interpretation and may result in a substantial extent of missing data.The association between negative symptoms and social functional outcome might be partially explained by measurement overlap, e.g., the PANSS item Passive/Apathetic Social withdrawal has conceptual overlap with the SFS subscale social engagement/withdrawal. Measurement overlap may have resulted in an inflated correlation between negative symptoms and social functioning outcome  and may Thus, although neuropsychological assessment gives an impression of current strengths and weaknesses, the results of the present study suggest that the prognostic value appears limited for most cognitive domains. Neuropsychological assessment may predict longer-term social functioning, but the present findings suggest that for most cognitive domains, the predictive value is modest and only information processing speed and attention/vigilance may provide additional prognostic information above assessing current symptom severity.S1 Table(DOCX)Click here for additional data file.


Accurate measurement of vessel flows in the Total Cavopulmonary Connection (TCPC) can lead to better estimation of hemodynamics, which has been related to clinical outcomes in single ventricle patients. CMR is usually acquired in breath held conditions; however, respiratory effects can be considerable since the blood is passively routed to the lungs in Fontan patients. Here, we assessed the effect of respiration on caval and aortic flows using real time CMR images.Eight single ventricle patients with TCPC palliation underwent resting cardiac magnetic resonance imaging at both free breathing (FB) and breath held (BH) conditions. Real time, through-plane phase encoded velocity mapping (PC-MRI) was performed in superior (SVC), and inferior (IVC) vena cava, and ascending (AAO) and descending (DAO) aorta. FB and BH resting flow (Q) waveforms were then obtained at each vessel Figure . RespiraMean flows in all the vessels trended toward an increase with respiration compared to the BH conditions; however, only the increase in SVC flow was significant . IVC and SVC flows were markedly phasic with the respiratory cycle Figure , and theA novel technique of measuring flow and tracking respiration with the same slice plane was presented, obviating the need for respiratory bellows. Using this technique, we demonstrated that respiratory effects dominate the venous flow but have little effect on systemic arterial flow in Fontan patients. However, the effect of breath holding on mean flows is fairly small, supporting the routine use of breath holding in assessing flows in single ventricle patients.This study was supported by the National Heart, Lung, and Blood Institute Grants HL67622, HL098252, and HL089647.


Background and Aims: Attentional bias for drug-related stimuli is a key characteristic for drug addiction. Characterizing the relationship between attentional bias and brain reactivity to Internet gaming-related stimuli may help in identifying the neural substrates that critical to Internet gaming disorder (IGD).Methods: 19 IGD and 21 healthy control (HC) subjects were scanned with functional magnetic resonance imaging while they were performing an addiction Stroop task.Results: Compared with HC group, IGD subjects showed higher activations when facing Internet gaming-related stimuli in regions including the inferior parietal lobule, the middle occipital gyrus and the dorsolateral prefrontal cortex. These brain areas were thought to be involved in selective attention, visual processing, working memory and cognitive control.Discussion and Conclusions: The results demonstrated that compared with HC group, IGD subjects show impairment in both visual and cognitive control ability while dealing with gaming-related words. This finding might be helpful in understanding the underlying neural basis of IGD. Internet gaming disorder (IGD) is rapidly becoming a prevalent mental health concern around the world over the last few decades  in users of alcohol and a variety of other substances, including nicotine, cannabis, opiates, and cocaine  studies have identified that a number of brain regions are associated with addictive substance-related stimuli  = 0.694, p > 0.05]. Only males were included due to higher IGD prevalence in men than that of women ). The IGD subjects and HC group did not significantly differ in age  with the mean RT of gaming-related words being shorter, compared with neutral ones for all participants together. The interaction effect was not significant . Although IGD subjects showed longer RT than the HC group, it did not reach statistical significance . For accuracy rate, no main effects or interaction effect  were found. The IGD and HC group did not significantly differ on accuracy rate . And the accuracy rate for gaming-relate words and the neutral one showed no significant difference . In addition, to compare the attention bias between these two groups, an independent sample t-test was performed. The results revealed that the IGD subjects showed more obvious attentional bias than the HC group, although it did not reach statistical significant .Firstly, RT and accuracy rate were conducted with two factors repeated measures ANOVAs separately  2/3) and left temporal gyrus (BA 37) were significantly decreased for IGD subjects compared with HC group. We next determined the main effect of word type in brain regions in inferior frontal gyrus (BA 45) and the right pre-central gyrus (BA 6), with significant increased activation in gaming-related words than neutral words.The blood oxygen level-dependent (BOLD) data for attention bias demonstrated significant between-group differences. Greater BOLD signals were found in the inferior parietal lobule, middle occipital gyrus and DLPFC in the gaming-related words relative to the neutral words, when comparing the IGD subjects to the HC group during the addiction Stroop process  - ) Figure ; Table 2p=0.192)    in IGD subjects. The correlation between attentional bias and brain activity in the inferior parietal lobule among IGD subjects is 0.313  Figure . The cor) Figure . The cor) Figure .In behavioral performance, consistent with previous studies which have demonstrated that attentional bias toward substance-related words across different types of addictions, such as alcohol use, smoking and gambling (Janes et al., Although we observed the un-significant behavioral results, however, in neuroimaging results, this study revealed several important findings that could deepen our understanding about the neural activities in IGD. We observed greater brain activation in IGD subjects relative to the HC group in the inferior parietal lobule, the middle occipital gyrus and the DLPFC during the addiction Stroop task. Our findings are in accordance with researchers using other addicted groups such as substance addiction and pathological gambling (Dannon et al., The DLPFC was demonstrated to be mainly related to cognitive control processing (MacDonald et al., A significant positive correlation was found between attentional bias and activation of middle occipital gyrus in IGD subjects, which suggested that the more endeavors were engaged toward gaming-related words, the higher the brain activities in middle occipital gyrus. The occipital activity may be associated with word reading and visual attention processes (Tang et al., In summary, the current study shows that the gaming-related words are more interference to IGD subjects and they need to engage more endeavors to perform better for their attentions paid to the semantic rather than the color of the gaming-related words during the addiction Stroop task. More importantly, present study reveals that several abnormalities of brain regions might be implicated in underlying the pathophysiology of IGD in terms of neural results.YZ programmed the experiment, analyzed the data, and wrote the first draft of the manuscript; XL and HZ contributed to data collection, and XL, JX revised and improved the manuscript. GD designed this research, revised and improved the manuscript. XD contributed to fMRI data collection. All authors contributed to and have approved the final manuscript.The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.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 elderly are more likely to suffer from fracture because of age-related trabecular bone loss. Different bone loss locations and patterns have different effects on bone mechanical properties. Extended finite element method (XFEM) can simulate fracture process and was suited to investigate the effects of bone loss on trabecular bone. Age-related bone loss is indicated by trabecular thinning and loss and may occur at low-strain locations or other random sites. Accordingly, several ideal normal and aged trabecular bone models were created based on different bone loss locations and patterns; then, fracture processes from crack initiation to complete failure of these models were observed by XFEM; finally, the effects of different locations and patterns on trabecular bone were compared. Results indicated that bone loss occurring at low-strain locations was more detrimental to trabecular bone than that occurring at other random sites; meanwhile, the decrease in bone strength caused by trabecular loss was higher than that caused by trabecular thinning, and the effects of vertical trabecular loss on mechanical properties were more severe than horizontal trabecular loss. This study provided a numerical method to simulate trabecular bone fracture and distinguished different effects of the possible occurrence of bone loss locations and patterns on trabecular bone. Given rapid increase in the elderly population, age-related fracture has become an important public health issue , 2. ManyAge-related changes in the microstructure of trabecular bone, such as decrease in trabecular thickness and loss of trabecular connectivity, may decrease bone volume fraction (BV/TV) and weaken bone microstructure , 5. GiveSince significant relationship between age-related changes in trabecular microstructure and its fracture risk has been found, it is therefore important to understand the effects of such changes on the mechanical properties of trabecular bone , 12. To In addition, finite element (FE) method has become a highly efficient technique to estimate bone strength and fracture risk , 19. SevAccordingly, this study aimed to simulate the fracture processes of ideal trabecular bone models based on XFEM analysis. Several ideal normal and aged trabecular bone models were first created based on different bone loss locations and patterns, and then the effects of these different locations and patterns of age-related bone loss on the mechanical properties of trabecular bone were compared. These simulations may assist in explaining the age-related fracture mechanism by analyzing the variations in trabecular microstructure and provide a theoretical basis for prevention of age-related fracture.Trabecular bone is composed of trabeculae in the form of rods and plates, and trabecular bone microstructure may vary across anatomical locations , 24. AccAging may result in trabecular bone loss at two types of regions: random sites and low-strain locations , 16, 29.Regardless of the locations where trabeculae were lost, the process of age-related bone loss includes two steps: thinning of the trabecula and eventual loss , 16, 32.The fracture process of trabecular bone structure is generally controlled by strain-based criterion, and both of the tissue yield and crack initiation strains are asymmetric in tension and compression –36. ThusAlthough compressive strain may occasionally introduce breakage , for XFEμm and 126 μm [μm was selected, and eight-node hexahedron C3D8 element was used.Once crack initiation condition was met, crack began to grow obeying bone damage propagation law. The energy-based criterion was selected in damage evolution, and the energy release rates of trabecular bone tissue were all set to 0.33 N/mm , 41. Alld 126 μm , 30. Thed 126 μm , the ave2 [To validate the method in modeling ideal trabecular bone and XFEM analysis used in this study, four rapid prototype (RP) models of Model-rod A and four RP models of Model-plate A were manufactured, respectively, by RP laser sinterstation . Due to the limitation of sinterstation resolution, each edge of the ideal normal trabecular bone model was scaled up by 10 times so that trabecular features in the RP models can be observed . Polyami2 , 43. The2 –46.Comparison of the deformations and fracture patterns between the trabecular FE models and the corresponding RP models was shown in Figures The apparent stress-strain curves of the three rod-like models were shown in The apparent stress-strain curves of the three plate-like models were shown in Different trabecular bone degeneration patterns caused by trabecular thinning or loss both led to decrease in the mechanical properties of normal trabecular bone models, whereas the mechanical properties were more sensitive to trabecular loss than to trabecular thinning; furthermore, the mechanical properties of trabecular bone were less affected by loss of horizontal trabeculae than by loss of vertical trabeculae . For theThis study utilized a numerical simulation method to predict the fracture processes in normal and aged trabecular bone models. The fractures of these models were simulated using XFEM analysis embedded subroutine UDMGINI, in which the principal strains in tension and compression were used to control the crack initiation and propagation. In the simulation process, when the principal strain in the aged trabecular bone model exceeded the crack initiation strain of trabecular bone tissue, crack in the aged model would be initiated, and then the crack began to grow obeying the bone damage propagation law until complete fracture occurred in the aged model. According to the XFEM analyses in this study, different fracture processes from crack initiation to complete failure for the aged trabecular bone models were primarily observed; then, apparent ultimate stress and fracture strain of the aged trabecular bone models were obtained, and the mechanical properties of different aged trabecular bone models were compared; finally, the effects of bone degeneration locations and patterns on the mechanical properties of aged trabecular bone models were analyzed quantitatively.At present, many numerical methods can simulate crack initiation and propagation of bone structure , 47, 48.In this study, the apparent fracture strain of Model-rod A was 1.94%, and that of Model-plate A was 2.79%. These were consistent with the previous investigation, which shows that the apparent fracture strains are 2% and 2.7% in the normal rod-like and plate-like trabecular bone models . SeveralWith respect to the roles of rod-like and plate-like trabeculae in fracture process, rod-like models may participate in initiation and progress of fracture at the sites with low bone density, which are more susceptible to large deformation or buckling failure; by contrast, plate-like models may be located at sites with high bone density, which are most likely to bear bending loads , 52, 53.As shown in Figures Regardless of the locations where trabeculae are lost, age-related bone loss is derived from trabecular thinning or loss. Thus, it is necessary to determine the crucial degeneration factor by identifying the relative effects of trabecular thinning and loss on mechanical properties. As shown in For the plate-like structures, trabecular loss was generated by resorption cavities, and the connectivity within the structure was not totally lost, so that the load can still be carried and transmitted, which had better mechanical properties compared with rod-like structures . HoweverSeveral limitations were associated with the ideal trabecular bone models and simulation process in this study. First, there are some differences in microstructure between ideal and actual trabecular bone models. Understanding the effects of different degeneration locations and patterns on mechanical properties of trabecular bone was of great significance. However, actual trabecular microstructure is very complex, which may lead to difficulties in identifying different degeneration locations or patterns in actual trabecular bone models. To overcome this problem, ideal trabecular bone models were used in this study. These ideal models allow artificially inducing bone losses which were manufactured from normal models and direct comparison between the normal and aged models without confounding variations in tissue properties that are inherent in actual trabecular bones , 43. AltAge-related trabecular bone loss occurring at low-strain locations was more detrimental to trabecular bone structure than that occurring at other random sites; meanwhile, the decrease in trabecular bone strength caused by trabecular loss was higher than that caused by trabecular thinning, and the effects of vertical trabecular loss on the mechanical properties of trabecular bone were severer than horizontal trabecular loss.


Cancer is now viewed as a stem cell disease. There is still no consensus on the metabolic characteristics of cancer stem cells, with several studies indicating that they are mainly glycolytic and others pointing instead to mitochondrial metabolism as their principal source of energy. Cancer stem cells also seem to adapt their metabolism to microenvironmental changes by conveniently shifting energy production from one pathway to another, or by acquiring intermediate metabolic phenotypes. Determining the role of cancer stem cell metabolism in carcinogenesis has become a major focus in cancer research, and substantial efforts are conducted towards discovering clinical targets. Cancer, above all other diseases, has countless secondary causes.But even for cancer, there is only one prime cause …: metabolism.Otto WarburgAdult stem cells, in contrast to most cells in our body, which are differentiated and have a specific role, are rare cells that harbour unique biological properties such as a lack of differentiation and indefinite self-renewal. Stem cell asymmetrical division into one new stem cell and a committed progenitor, which can give rise to a functionally mature progeny, helps maintain tissue homeostasis .Cancer is characterized by an unrestrained proliferation of malignant cells that are morphologically and functionally different. Two models have been proposed in order to explain this cellular diversity within tumours. The traditional, stochastic way of explaining cancer initiation and development is through sequential accumulation of mutations, each of which promotes the loss of specific tissue traits until dedifferentiation and regression into a more primitive phenotype occurs. According to this clonal evolution model, each cancer cell has a similar potential to grow a tumour. A second model, the cancer stem cell (CSC) hypothesis, postulates that a reduced group of stem-like cells is responsible for the development of the disease. Accordingly, tumours are hierarchically organized and sustained by a distinct self-renewal subpopulation of cancer cells. These tumour-initiating cells (TICs) with stemness properties are located at the apex of a pyramid and are responsible for the generation of a varied progeny of highly proliferative cells forming the bulk of the tumour , 2. BothCSCs share numerous properties with normal stem cells besides their ability to renew themselves by remaining in an undifferentiated state: the expression of surface markers, such as CD44, CD133 or the enzyme aldehyde dehydrogenase (ALDH), the activation of particular cell signalling pathways, such as Wnt, Notch or Hedgehog, a relative quiescence or an active DNA repair capacity . Given tMetabolic adaptation is believed to be one of the hallmarks of cancer cells . The rolGlycolysis is the enzymatic conversion of glucose into lactate, which concomitantly produces 2 molecules of ATP per molecule of glucose. In the presence of oxygen, cells generally adopt oxidative phosphorylation (OXPHOS) as the main pathway to produce energy, which is more efficient than glycolysis because it theoretically generates 36 molecules of ATP per molecule of glucose. Cancer cells can generate ATP via glycolysis even under normoxic concentrations in what is known as the Warburg effect. In fact, gycolysis can more rapidly produce ATP compared with OXPHOS in the presence of abundant levels of glucose , a switcStudies of mitochondrial morphology and mitochondrial DNA levels indicate that stem cells have fewer mitochondria, which are less mature and relatively inactive compared with those of differentiated cells, resulting in reduced reactive oxygen species (ROS) levels . Low amoGlucose seems to be an essential nutrient for CSCs, as its presence in the microenvironment significantly increases the amount of stem-like cells in the overall cancer cell population. On the other hand, glucose deprivation induces the depletion of CSCs in vitro . However1 regulates stemness of cancer cells and mitochondrial DNA copy number [OCT4, TERT and MYC decreases [Several reports suggest that CSCs are more glycolytic than other differentiated cancer cells in vitro and in vivo. These studies were performed in many tumour types including osteosarcoma, glioblastoma, breast cancer, lung cancer, ovarian cancer and colon cancer –18. Glucy number , 22. Alsecreases . InsteadIn clear contrast with these publications, growing evidence shows that CSCs have a preference for mitochondrial oxidative metabolism Fig. . AccordiN-acetyl-cysteine (NAC) reduces ROS, suppressing the differentiation of CSCs and increasing metastatic burden [During differentiation under hypoxic conditions, CSCs from several tumour types are able to switch from an oxidative to a glycolytic metabolism in order to compensate for deficient mitochondrial machinery . Likewisc burden , 39. Furc burden . Indeed,c burden , 41. HenFinally, CSCs seem to be vulnerable to mitochondria-targeted drugs and the inhibition of OXPHOS seems to inhibit tumour formation , 42–44. One possible explanation for the discrepancies in CSC metabolism reported in the scientific literature may be found in the metabolic adaptability that CSCs show following microenvironmental fluctuations. For example, most in-vitro studies are carried out in non-physiological high glucose and oxygen concentrations, which favour a glycolytic phenotype. Ideally, the optimal experimental conditions to keep the metabolic traits of CSCs intact would be to isolate them directly from patients and analyse them immediately or within the first steps of in-vitro culture. In fact, when patient-derived, low-passage CSCs are used, OXPHOS seems to be the preferred metabolic pathway for the energy production of CSCs .Breast CSCs in mouse and human tumours have a more glycolytic phenotype compared with their differentiated progeny . CSCs frThis observed adaptive metabolic plasticity might allow CSCs to survive in changeable, sometimes hostile, environments or unfavourable circumstances encountered during tumour progression, such as at metastatic sites. In support of this metabolic malleability scenario, some publications show that CSCs are able to switch to a glycolytic metabolism when OXPHOS is blocked , 59.CSCs have been shown to adapt to starvation and hypoxia by upregulating glucose transporters and switching to a more glycolytic phenotype to outcompete their differentiated progeny , 60. HypK-Ras ablation-resistant pancreatic cells with stem-like characteristics are unable to increase compensatory fluxes such as glycolysis following OXPHOS inhibition, despite being more resistant to nutrient deprivation and other environmental stresses. This lack of plasticity may be attributed to the shutdown of the K-Ras-driven metabolic programme . Ont theNormal stem cells and iPS cells utilize glycolysis, while CSCs have the capacity to shift to OXPHOS and mitochondrial metabolism. One can speculate that differences in signalling pathways mediated by OCT3/4, SOX2, KLF4, MYC in iPS cells and by SHH, NOTCH, WNT, PTEN, MAPK, KRAS, HIF and TP53 in CSCs drives these divergent metabolic phenotypes. Understanding the metabolic differences between normal stem cells and CSCs and their regulators will be important for the process of developing novel therapeutics that metabolically target, but preserve, the key functions of normal stem cells.hi/CS24– surface markers and a slow-cycling, quiescent state [hi/CS24– CSCs [The CSC phenotype may not necessarily be uniform between cancer subtypes or even between tumours of the same subtype. For instance, the preferred metabolic pathway to produce energy may depend on the metastatic site, indicating extensive metabolic variability . Moreovent state . Mesenchnt state , 67. Indnt state . Convers24– CSCs . Finally24– CSCs . AnotherThese results suggest that the dual blockade of glycolysis and mitochondrial respiration may represent a better way to eradicate CSC heterogeneity than focusing exclusively on glycolysis inhibition or suppression of mitochondrial respiration. Indeed, combined inhibition of glycolysis and mitochondrial respiration has been shown to be effective in suppressing tumour growth and metastasis .TP53 and also frequently display deletions or epigenetic silencing of the PTEN tumour suppressor gene. In addition to HER2 gene amplification, HER2-positive breast cancers frequently display deletions in PTEN, and indeed this is a likely cause of resistance to HER2-targeted therapies [BRCA1 germline mutations or epigenetic silencing of the BRCA1 locus are most frequently associated with TNBCs [Genetic analysis of breast cancers has demonstrated different mutational profiles across the subtypes of breast cancer. For example, the most frequent genetic alteration found in luminal breast cancers is mutational activation of PI3K signalling . In contherapies –75. BRCAth TNBCs . All of The effects of the niche on CSC metabolism are also starting to be recognized. High catabolism in the microenvironment with NF-κB, HIF-1α and TGF-β activation coincides with glycolysis and ketogenesis, and promotes CSC features –80. A moMetabolic stresses also induce significant changes in non-malignant cells within tumours. T cells can preferentially develop into the immunosuppressive regulatory subtype (Tregs) following glucose restriction, which promotes tumour growth , 87. HypConversely, inflammatory cytokines generated by the tumour microenvironment (such as IL-6 and IL-8) with activation of NF-κB induce glycolysis with activation of PI3K and AKT and stimulate CSC self-renewal, which then may promote tumour growth and metastasis , 90–92. The role of cell metabolism has evolved into an active area of research during the last decade, with a strong focus on glucose metabolism. Unfortunately, scarce attention has been directed to amino acid and lipid metabolism. A recent report shows that pancreatic CSCs are glutamine dependent. Inhibiting glutamine availability by targeting glutaminase or glutamine oxaloacetic transaminase (GOT), accountable for the conversion of glutamine into oxaloacetate, reduces the expression of stemness genes, inhibits self-renewal and sensitizes CSCs to radiation therapy via accumulation of ROS in vitro and in vivo . Colon aOver the years, substantial evidence for the existence of CSCs has strengthened the view that these cells are accountable for cancer development. CSCs renew themselves and at the same time generate progenitors that lose their stemness, ultimately giving rise to the bulk of the tumour. In addition, the last decade of research highlights that metabolism is not a mere player subordinated to CSC physiology, but actually may orchestrate it. Given that changes in metabolism precede changes in stemness, a perturbation in the metabolic phenotype of CSCs could be essential for the acquisition of the CSC state. Conventional therapy targets rapidly proliferating cancer cells that make up the bulk of the tumour without necessarily having an effect on the CSC population. CSCs possess special metabolic traits that distinguish them from the bulk of the tumour and that may constitute the basis for the development of new therapeutic strategies to eradicate them. From a clinical point of view, targeting the particularities of CSC metabolism is more likely to translate into permanently curing cancer or at least providing long-term disease-free survival, because these cells are responsible for resistance to therapy and metastasis, the main cause of cancer-related deaths. The interest in exploiting CSC metabolism for drug targeting is therefore gaining ground. However, a well-defined portrait of the singularities of CSC metabolism still needs to be depicted and CSC metabolism remains a controversial issue, with studies supporting a glycolytic phenotype of CSC and others stating that CSC metabolism is mainly oxidative.Many other unresolved issues need to be addressed. Elucidating the differences in metabolism between CSCs and non-stem cancer cells, and between CSCs and normal progenitor stem cells, will be crucial to develop new therapies and may reveal new ways to distinctively target these TICs. Whereas normal stem cells rely more on glycolysis, CSCs might depend more on mitochondrial oxidative metabolism. If this is the case, why would the stemness state of cancer cells require a different metabolic state than normal stem cells? In contrast with normal physiological development, tumourigenesis tends to be highly disorganized and cancer metabolic malleability could provide a niche more prone to CSC survival. Nevertheless, the stability or plasticity of the CSCs phenotype needs to be verified. Are CSCs really able to metabolically adapt depending on microenvironmental fluctuations? Is the CSC population metabolically heterogeneous or does it exhibit different degrees of stemness-related phenotypes? During tumourigenesis, characteristics of the CSCs might mutate, and distinct CSC populations could eventually emerge in what it would be a metabolically changeable or versatile target. In such a scenario, future therapies designed to eradicate CSCs via targeting their metabolism might need to simultaneously block glycolysis and mitochondrial respiration.We therefore need to overcome multiple obstacles before we can effectively eliminate CSCs. First of all, to properly recognize CSCs and differentiate them from other cell types, greater efforts should be made towards the identification of specific CSC markers, because none of the markers so far defined is unique for CSCs. Likewise, a combination of markers could greatly improve the purity of CSCs for research purposes. Finally, it is important to note that CSCs are settled in a niche formed by multiple other cell types and cancer heterogeneity is clearly more complex than originally thought. Hence, studying the metabolism of CSCs in experimental settings that do not reflect the heterogenic architecture of tumours such as the absence of a pertinent microenvironment is not ideal. Better models that preserve the CSC physiological state and structure should be developed.Despite the limited information we currently have on the role of metabolism in the ability of CSCs to self-renew, initiate tumours, metastasize and survive therapy, targeting CSCs by blocking their metabolic singularities holds great potential in improving current cancer treatments. In practice, combinational treatments involving both a standard cytotoxic therapy and a CSC-targeted therapy will probably be required to ablate all cancer cells Fig. .


The hallmark of embryonic development is regulation – the tendency for cells to find their way into organized and ‘well behaved’ structures – whereas cancer is characterized by dysregulation and disorder. At face value, cancer biology and developmental biology would thus seem to have little to do with each other. But if one looks beneath the surface, embryos and cancers share a number of cellular and molecular features. Embryos arise from a single cell and undergo rapid growth involving cell migration and cell-cell interactions: features that are also seen in the context of cancer. Consequently, many of the experimental tools that have been used to study embryogenesis for over a century are well-suited to studying cancer. This article will review the similarities between embryogenesis and cancer progression and discuss how some of the concepts and techniques used to understand embryos are now being adapted to provide insight into tumorigenesis, from the origins of cancer cells to metastasis. Summary: This article reviews the similarities between embryogenesis and cancer progression and discusses how the concepts and techniques of developmental biology are being applied to provide insight into all aspects of tumorigenesis. The cancer research community has relied on some of the same tumor-modeling systems for decades, namely cell lines, xenografts and, more recently, genetically engineered mouse models (GEMMs). Mouse models are widely considered to more accurately represent the human tumor microenvironment compared to cell line injections and xenografts; however, even GEMMs have limitations when it comes to recapitulating the more dynamic aspects of tumor biology. For example, clonal relationships, invasive properties and cell-cell interactions are not readily apparent upon histological examination of a murine tumor, when one is staring at a field of seemingly identical cancer cells. Furthermore, GEMMs can only be used to model cancers characterized by one or a few specific genetic mutations, but many cancers, especially hematological malignancies, are caused by large-scale chromosomal rearrangements that cannot be easily replicated in a murine system. Cancer researchers have recently turned to some classic developmental biology techniques to overcome these limitations. This Review will highlight some of the common cellular and molecular mechanisms shared by developing organisms and tumors, and then provide a detailed description of how developmental biology tools, specifically lineage tracing, live imaging and cellular reprogramming, have been repurposed to study various aspects of tumor progression.The process of embryogenesis requires precise spatial and temporal activation of developmental signaling pathways . Re-actiDuring development, Wnt signaling is involved in cell fate specification, proliferation and migration, and in the adult this pathway is involved in maintaining homeostasis in tissues such as the intestine, where Wnt signaling is critical for stem cell function . There aEctoderm: the outer layer of the three germ layers.Endoderm: the inner layer of the three germ layers; forms the lining of the gastrointestinal tract, pancreas, liver, lungs and other glands.Epiblast: upper layer of the mammalian blastoderm; eventually forms the embryo proper as well as amniotic ectoderm and extraembryonic mesoderm.Epithelium: a sheet of adherent, polarized cells lying on a basement membrane.Gastrulation: phase during early embryogenesis characterized by dynamic cell movements when the blastula differentiates into three germ layers .Mesenchyme: motile, mesoderm-derived cells scattered throughout extracellular matrix.Mesoderm: the middle layer of the three germ layers.Morphogen: a secreted factor that depends on local threshold concentrations to determine cellular signaling output.Neural crest: a transient, motile embryonic cell population that develops into numerous cell lineages, including melanocytes, smooth muscle, neurons and craniofacial mesenchyme.Parenchyma: the functional tissue of an organ.Patterning: spatial and temporal organization of cell fates.Pluripotent stem cell: a cell with the ability to self-renew and produce all embryonic cell types but not extraembryonic cell types.Primitive streak: thickened layer of epiblast cells that becomes mesoderm during gastrulation.Specification: when a cell or tissue is committed to a particular cell fate but the decision can still be reversed.Stroma: the supportive tissue of an organ, including connective tissue and blood vessels.Essential Developmental Biology, Adapted from Canonical Wnt signaling is active in nearly all developing tissues and plays a crucial role in body axis patterning , stem ceThe Hedgehog (HH) pathway, like the Wnt pathway, plays a critical role in the development of many organs, including but not limited to the patterning of the central nervous system (CNS), tooth development and limb formation . The thrThe Notch pathway is similarly involved in embryonic cell fate decisions; specifically, it is crucial for the development of organs including but not limited to the CNS, pancreas, bone and heart . This paA recurring motif in development is reciprocal signaling between neighboring cell populations: crosstalk that facilitates morphogenesis of the emerging tissue. Reciprocal signaling between developing epithelium and mesenchyme see  occurs rEmbryogenesis entails dramatic morphological changes and cellular movements that are recapitulated within tumors. One of the most notable of these is epithelial-to-mesenchymal transition (EMT), a process in which epithelial cells lose their epithelial characteristics, including apical-basal polarity and cell-cell adhesion, and take on the motile features of fibroblasts. The primary role of EMT is during embryonic development, where it is crucial for gastrulation and other developmental events; however, cancer cells exploit this property of increased motility to facilitate spread. During gastrulation, WNT, TGFβ and FGF orchestrate primitive-streak formation by promoting EMT via the activation of the transcription factors Snail and Twist . These dIn conclusion, the molecular cues used to pattern an embryo are harnessed by tumor cells to enhance growth, recruit stromal cells and coordinate spread from the primary tumor. Because of the many commonalities between development and cancer at the molecular and cellular levels, it has been possible to use the tools of developmental biology to address fundamental questions about tumor biology. Below, we highlight some classical developmental biology techniques that have been repurposed in new and innovative ways to expand our understanding of cancer.Fate mapping, also known as lineage labeling or lineage tracing, was originally developed to visualize the fate of individual cells and their progeny, referred to as clones, during embryogenesis, but it has also proven useful for the study of tumor-initiating populations and dynamic cellular movements in the context of cancer. Early fate-mapping experiments employed vital dyes, fluorescent dyes or radio-labeling to mark a specific region, lineage or even a single cell of an embryo and follow it throughout development as cells divide and migrate away from their original positions. These studies allowed embryologists to generate detailed fate maps for a number of model organisms, including frog, zebrafish and chick .loxP and flippase/flippase recognition target (FLP/FRT) systems, it became possible to genetically label and track a cell lineage, facilitating fate mapping in mammals, which, unlike lower model organisms, are not transparent and contain many more cells and lineages. Cre and FLP are both recombinases and, although they recognize different genetic sequences, they function similarly by inducing DNA recombination to facilitate DNA manipulation at target sites  and targets these for recombination, which, depending on the orientation of the sites, can result in deletion, inversion or translocation of intervening sequences  Importantly, because cancer cells can, in principle, acquire stem cell properties as a consequence of mutation or epigenetic remodeling, they need not arise from stem cells. The ability of tumors to emerge in tissues in which it is questionable whether stem cells exist (e.g. the kidney) is further evidence that cancers can arise from fully differentiated cells.+ cells labeled with Cre-based technology durably gave rise to all the differentiated cell types of the intestinal villi (Apc in mice in either the stem cell compartment (using Lgr5-Cre) or the non-stem-cell ‘transient amplifying’ compartment – capable of short-term self-renewal only –   that exhibited unimpeded growth, Apc deletion in the transient amplifying cells resulted in microadenomas (tumors less than 10 mm in diameter) whose growth rapidly stalled  . Whereas stalled . This rein vivo. By using tamoxifen-induced Cre systems such as CreER and titrating the dose of tamoxifen, one can limit recombination to rare, sparse cells within a tissue, providing the resolution to identify clonal populations. Utilizing this approach, Driessens and colleagues were able to identify cancer stem cells and their progeny in a chemical-induced carcinogenic model of squamous cell carcinoma (SCC)  . Using ama (SCC) .Such lineage-tracing experiments support the general notion that cancer is the result of having the ‘right’ mutations in the ‘right’ cell at the ‘right’ time. In other words, certain cell populations (stem cells or non-stem cells) might be susceptible to the oncogenic effects of certain gene mutations that have no effect in other cell populations. Additional factors, such as the local environment (e.g. inflammation), could contribute to the susceptibility or resistance of various cell types to the cancer-causing effects of a given mutation. Similar lineage-tracing experiments have identified cells-of-origin in a number of contexts . From thGenomic analyses have confirmed that tumors are composed of numerous ‘sub-clones’, or clones with distinct mutations in addition to the original tumor-driving mutation(s) . Such tuloxP sites (loxP sites and oriented in such a way that excision or inversion results in the (somewhat) random expression of one of the four lineage labels. Using this system, it has been possible to identify ‘bottlenecks’ during the clonal evolution of a tumor in vivo. For example, premalignant progression in pancreatic cancer from acinar-to-ductal metaplasia (ADM) to more advanced pancreatic intraepithelial neoplasia (PanIN) is accompanied by a shift from polyclonal to monoclonal lesions. This model has also revealed differences in metastatic potential between different tumor sub-clones, providing evidence for inter-clonal cooperation during tumor dissemination  made up only a small fraction of a naïve tumor, but, upon treatment with temozolomide, an alkylating agent that is used to treat some brain cancers, these cells and their progeny expanded to become the most abundant tumor clone  lineage label into a well-established GEMM of pancreatic cancer, it became possible to unambiguously identify cells that had undergone EMT because such cells still bore the lineage marker confirming their epithelial origins, despite acquiring a mesenchymal phenotype .+ cells could be observed in the lung; at 2 months, YFP+ micrometastases were evident; and, by 3 months, gross metastases were found in 100% of animals  has revolutionized stem cell biology and opened up new avenues for regenerative medicine. Since the initial discovery that four transcription factors can reprogram terminally differentiated cells to a pluripotent state , investiTo date, most studies involving cancer-derived iPSCs provide proof-of-principle, demonstrating that patient-derived tumor specimens  and humaTP53 tumor suppressor gene. The disorder is characterized by early onset of a diverse range of malignancies, including osteosarcoma (OS), breast cancer and leukemia; however, the precise molecular mechanisms driving tumorigenesis in these varying tissues – bone, mammary gland and blood – are unclear. in vitro and, unlike iPSCs derived from unaffected relatives, LFS-iPSCs formed tumors in nude mice (a commonly used model that is unable to mount an immune response). Using this tool, the authors were able to identify the imprinted H19 locus, a gene that is crucial for osteoblast differentiation, as a target of altered p53 activity in individuals with LFS and an important repressor of OS development (Li-Fraumeni syndrome (LFS) is an autosomal-dominant inherited syndrome driven by mutations in the elopment .EZH2, which is frequently mutated in MDS. Cellular reprogramming thus allowed the authors to accurately model the human disease from its early stages and zero in on the functionally important genes within the 7q locus.iPSCs also fill a void in the study of human malignancies that arises because of a lack of synteny between the mouse and human genomes. An example is provided by the myelodysplastic syndromes (MDSs): disorders of the hematopoietic system that predispose affected individuals to acute myeloid leukemia (AML) and are characterized by large chromosomal deletions. Loss of the long arm of chromosome 7 (7q) is a frequent occurrence in MDS. Genes within human 7q map to four different murine chromosomes, precluding the generation of GEMMs of MDS. To address this, Although many cancers, such as colon and breast cancer, have well-defined and readily detectable pre-malignant stages, the precursor lesions associated with other cancers, such as pancreatic adenocarcinoma (PDA), are hard to detect, resulting in a lack of early screening methods. As noted above, PDA is thought to arise via ADM and the generation of PanINs, histological lesions that are not readily seen by imaging. Hence, it has been nearly impossible to identify biomarkers associated with early pancreatic disease in humans, and thereby to diagnose PDA early enough to slow its aggressive progression.Recently, Kim et al. sought to address this unmet need by generating iPSCs from primary PDA surgical specimens and matched normal pancreatic tissue . Althoug. (2014) took advantage of a well-defined neural differentiation protocol for human embryonic stem cells (hESCs). After generating neural progenitor cells (NPCs), the authors transformed them with lentiviruses containing the most frequently implicated genetic alterations in DIPG: constitutively active platelet-derived growth factor receptor α (PDGFRα), histone H3.3 with a lysine-to-methionine alteration at residue 27 (K27M) and a p53 short-hairpin RNA (shRNA) knockdown construct. They found that the triple combination of lentiviruses (termed P5K) significantly increased the proliferation of NPCs, but not undifferentiated hESCs or differentiated astrocytes. The P5K condition also prevented NPCs from differentiating into glial cells, maintaining them in a progenitor state with high self-renewal capacity. When injected orthotopically into immunocompromised mice, P5K-NPCs were capable of forming tumors that had a similar histopathology to low-grade DIPG. Importantly, the authors were able to use these hESC-derived DIPG-like cell lines to perform a small-molecule screen and found that these cells were particularly sensitive to inhibitors of a tumor suppressor named Menin both in vitro as well as in vivo , an aggressive pediatric brain-stem cancer . Surgery in vivo . The cur in vivo , so the Collectively, the iPSC-based studies discussed above demonstrate the many ways in which our understanding of pluripotency and directed differentiation can be used to study cancer in a different light. Although the use of iPSC technology in cancer research is in its infancy, we foresee its utility in uncovering molecular mechanisms of tumorigenesis in other difficult to model cancers and pre-malignant stages.in vivo. Recently, live imaging has become an important tool for studying development because it reduces the need for inference and allows researchers to directly follow cellular behavior in real time. Live imaging has elucidated the dynamics of neural crest cell migration in chick embryos  – have permitted real-time observations of tumors, including cell-cell interactions within primary tumors, migration of cells to metastatic sites, and chemotherapeutic responses. Two-photon excitation microscopy, the most commonly used type of MM, uses two long-wavelength, low-energy photons to excite a fluorophore, resulting in less autofluorescence, significantly reduced phototoxicity and deeper tissue penetration (up to 1 mm) compared to confocal microscopy  see Box.Box 3. Another obstacle to long-term intravital tumor imaging is anatomic location. In particular, imaging tumors within the abdominal cavity or beneath the skull is difficult not only because of relative inaccessibility but because of artifacts introduced by animal respiration. These issues have been addressed by constructing durable imaging windows in anesthetized animals  and by cThese advances have provided a more dynamic picture of the tumor microenvironment than had been surmised from fixed tissue sections. For example, IVM has revealed that leukocytes traffic in and out of primary tumors and interact with each other and cancer cells, facilitating invasion and metastasis . In recein vitro, ruling out cell-intrinsic mechanisms for resistance. Returning to the intravital model, the authors found that, upon doxorubicin administration, myeloid cells were recruited to sites of necrosis, where they facilitated remodeling of nearby tumor blood vessels, causing them to become less leaky and therefore less efficient at drug delivery, sparing nearby tumor cells , where it must survive and proliferate in a foreign, potentially hostile, microenvironment .Many questions remain about the cellular and molecular mechanisms governing the establishment of micrometastatic lesions, and intravital imaging has provided some insight. For example, loxP variants and two fluorescent proteins, green (GFP) and red (RFP), to sparsely label cells in a mosaic fashion, resulting in four potential recombination outcomes: GFP+ mutant, RFP+ wild type, double-positive heterozygote and colorless heterozygote  . The mosgote see .iPSC technology has only just begun to be applied to questions of cancer biology and tumor progression. It is becoming apparent from numerous cancer-iPSC studies that primary tumor cells are significantly less efficient at generating iPSCs compared to untransformed cells , a problKFP allele into a cancer GEMM might permit investigators to track metastatic cells in real time. This type of technology would vastly improve our ability to vet anti-metastatic therapies by helping to identify metastasis-bearing animals and monitor their progress during treatment.Live imaging and IVM are now being used to illuminate the cellular dynamics of tumor progression and metastasis. However, there is an unmet need for autochthonous GEMMs that allow for real-time metastasis monitoring. One recently developed imaging tool is a new reporter mouse that permits noninvasive whole-animal imaging by employing the far-red Katushka fluorescent protein (KFP) . This stAs tools such as lineage labeling, cellular reprogramming and live imaging are improved and further harnessed to provide new insight into a variety of human diseases, cancer – with its close relationship to developmental biology – will likely remain at the top of the list.