Autism is a heterogeneous collection of disorders with a complex molecular underpinning. Evidence from postmortem brain studies have indicated that early prenatal development may be altered in autism. Induced pluripotent stem cells (iPSCs) generated from individuals with autism with macrocephaly also indicate prenatal development as a critical period for this condition. But little is known about early altered cellular events during prenatal stages in autism.iPSCs were generated from 9 unrelated individuals with autism without macrocephaly and with heterogeneous genetic backgrounds, and 6 typically developing control individuals. iPSCs were differentiated toward either cortical or midbrain fates. Gene expression and high throughput cellular phenotyping was used to characterize iPSCs at different stages of differentiation.A subset of autism-iPSC cortical neurons were RNA-sequenced to reveal autism-specific signatures similar to postmortem brain studies, indicating a potential common biological mechanism. Autism-iPSCs differentiated toward a cortical fate displayed impairments in the ability to self-form into neural rosettes. In addition, autism-iPSCs demonstrated significant differences in rate of cell type assignment of cortical precursors and dorsal and ventral forebrain precursors. These cellular phenotypes occurred in the absence of alterations in cell proliferation during cortical differentiation, differing from previous studies. Acquisition of cell fate during midbrain differentiation was not different between control- and autism-iPSCs.Taken together, our data indicate that autism-iPSCs diverge from control-iPSCs at a cellular level during early stage of neurodevelopment. This suggests that unique developmental differences associated with autism may be established at early prenatal stages. SEE COMMENTARY ON PAGE 419Autism spectrum conditions (henceforth referred to as autism) are a genetically heterogeneous spectrum of neurodevelopmental conditions , 2, 3. AThe use of induced pluripotent stem cells (iPSCs) has made it possible to study prenatal cellular behavior in autism , 10, 11.In this study, we generated iPSCs from autistic individuals without macrocephaly from 3 independent participant cohorts to capture a wider population of individuals with autism. Initial RNA-sequencing studies indicated that early neurodevelopment may be affected. To further investigate the source of atypical gene expression, we undertook extensive cellular phenotyping experiments. The goal of this study was to understand whether there was a fundamental difference between typical and autistic prenatal neurodevelopment, focusing primarily on early neuroectodermal structures and cell types that constitute the developing cerebral cortex.Further information can be found in Participants were recruited and methods carried out in accordance to the Patient iPSCs for Neurodevelopmental Disorders (PiNDs) study (NHS Research Ethics Committee No. 13/LO/1218). Informed consent was obtained before recruitment from all subjects for participation in the PiNDs study. Ethical approval for the PiNDs study was provided by the NHS Research Ethics Committee at the South London and Maudsley (SLaM) NHS Research and Development Office. Participants with autism were selected based on Autism Diagnostic Observation Schedule or Autism Diagnostic Interview–Revised scores, while typical control subjects were selected from the population if they had no diagnosis of any psychiatric condition.All participants provided consent to report and publish their data.https://www.pluritest.org); and genome integrity was assessed by an Illumina Human CytoSNP-12v2.1 beadchip array and analysed using KaryoStudio software   were generated from hair keratinocytes as previously described ,16. Detaego, CA) .iPSCs were differentiated to cortical neurons using a dual SMAD inhibition protocol that recapitulates key hallmarks of corticogenesis ,16. iPSChttps://isocpp.org/) and R-based  programs.RNA-sequencing was performed from a subset of our cohort, using 2 clones from each participant , with 2 technical replicates per clone. Poly(A) containing messenger RNA was purified and libraries were prepared using TruSeq Stranded mRNA kit (Illumina). Unstranded libraries with a mean fragment size of 150 base pairs were constructed and underwent 50–base pair single-ended sequencing on an Illumina HiSeq 2500 machine. Bioinformatics analysis was performed using C++-based –glutamatergic progenitor cells and neuronal function with a macrocephaly-associated cell proliferation phenotype ,17. ThusThe differences in cell fate acquisition observed between control- and autism-iPSCs could be due to genetic differences between control- and autism-iPSCs. Alternatively, this variation could be due to stochastic fluctuations in activation of key transcription factors during differentiation, as reported during iPSC differentiation toward a cortical fate . HoweverNRXN1 deletions (109NXM and 092NXF) were grouped on the same branch. Three participants with syndromic autism  did not group together with the nonsyndromic participants. Lastly, the 2 autism samples 004ASM and 010ASM seemed to group on the same branch based on not only the cellular data points but also gene expression patterns in The findings in this study indicate that there may be a link between autism and prenatal cortical development. Our HCS approach has allowed us to collect large data sets of multiple cellular readouts at a number of developmental time points for each iPSC line. Hierarchical clustering approaches allow for the identification of similar patterns between samples by placing them into cluster sets . Using tIn this study, we investigated whether iPSCs generated from autistic individuals display differences during prenatal cortical development. Previous studies have indicated that prenatal development is a critical period for the emergence of phenotypes associated with autism , 13, 14.First we found that autism-iPSCs generated atypical neural rosettes, indicating an alteration in neural differentiation. Consistent with this, autism-iPSCs showed significant differences in development of early neural progenitor cells. This effect persisted at a late precursor cell stage, although to a lesser degree. No differences in proliferative capacities were observed between control- and autism-iPSCs, indicating that this was not the cause of altered neurogenesis in autism-iPSCs. Examination of cortical neuron subtypes revealed a divergence in the development of dorsal forebrain or excitatory precursors and ventral forebrain or inhibitory precursors from an early stage of development. Conversely, control- and autism-iPSCs demonstrated the same ability to differentiate into mFFP cells. This indicates that atypical neurogenesis predominantly impacts the development of cortical lineages in autism-iPSCs. Finally, based on all the temporal cortical data points acquired in this study, the participants were grouped separately into control and autism groups, with further unbiased branching within the autism cohort. Together, these data suggest that unique developmental differences associated with autism may be established at early prenatal stages.We were particularly interested in modeling divergent patterns of development in the autistic cortex. We used a cortical differentiation protocol that recapitulates cortical precursor development from iPSCs and that yielded primarily excitatory cortical neurons . This enIt is of interest that in the current study the phenotypic changes occurred without the presence of proliferative differences between control- and autism-iPSCs. This suggested that cell type and structural anomalies previously reported using autism-iPSCs ,13 may bAnother consideration we faced during cellular phenotyping of iPSCs being differentiated toward a cortical fate was the high degree of variability between experimental replicates. This variability is due in part to stochastic fluctuations in transcription factor activation during cortical differentiation ,43. We oIn this study, we used iPSCs generated from independent cohorts and from individuals with autism but without macrocephaly. Using unbiased methods, we identified that differentiation of autism-iPSCs toward a cortical, but not a mesencephalic, fate results in atypical neurogenesis characterized by premature maturation and abnormal specification of neural progenitor cells. These effects occur in the absence of altered proliferative activity between control- and autism-iPSCs. Identification of these cellular/molecular phenotypes enabled us to find common cellular pathways in a cohort having heterogeneous genetic background. In future, similarly designed studies will help identify which cellular pathways underlie these phenotypes and may help to improve diagnosis and develop a greater understanding of the origins of autism.


Dascyllus aruanus, D. reticulatus, and Pomacentrus moluccensis), explicitly testing whether these fishes can successfully move and recolonize nearby coral hosts. Responses of fishes to localized coral loss was studied during population irruptions of coral feeding crown-of-thorns starfish, where starfish consumed 29 (34%) out of 85 coral colonies, of which 25 (86%) were occupied by coral-dwelling damselfishes. Damselfishes were not tagged or individually recognizable, but changes in the colonization of different coral hosts was assessed by carefully assessing the number and size of fishes on every available coral colony. Most damselfishes (> 90%) vacated dead coral hosts within 5 days, and either disappeared entirely (presumed dead) or relocated to nearby coral hosts. Displaced fishes only ever colonized corals already occupied by other coral-dwelling damselfishes (mostly conspecifics) and colonization success was strongly size-dependent. Despite movement of damselfishes to surviving corals, the local abundance of coral-dependent damselfishes declined in approximate accordance with the proportional loss of coral habitat. These results suggest that even if alternative coral hosts are locally abundant, there are significant biological constraints on movement of coral-dwelling damselfishes and recolonization of alternative coral habitats, such that localized persistence of habitat patches during moderate or patchy disturbances do not necessarily provide resilience against overall habitat loss.Increasing degradation of coral reef ecosystems and specifically, loss of corals is causing significant and widespread declines in the abundance of coral reef fishes, but the proximate cause(s) of these declines are largely unknown. Here, we examine specific responses to host coral mortality for three species of coral-dwelling damselfishes ( Declines in the quantity or quality of habitat patches, as well as increasing distances among habitat patches (habitat fragmentation), can all have significant effects on the local abundance and persistence of habitat-associated species2. Local persistence of such species is conditional upon recolonization of vacant habitat patches and/or movement between habitat patches in accordance with changes in habitat condition4. On coral reefs, many fishes occupy discrete coral colonies7, and the composition, size and abundance of specific coral hosts exert major constraints on local and geographic abundances of these fishes10. Declines in the abundance and composition of coral habitats following acute disturbances invariably lead to rapid and pronounced declines in the local abundance of coral-dwelling reef fishes, especially for species with very specific resource requirements15.Many species exist as metapopulations (or metagroups) occupying fragmented patches of suitable habitat19, causing sustained and ongoing declines in the abundance of habitat-forming corals. Causes of coral loss vary regionally, and are increasingly being compounded by anthropogenic climate change22. One of the major contributors to sustained declines in coral cover in the Indo west-Pacific are population irruptions of coral feeding crown-of-thorns starfish (CoTS), Acanthaster spp.25. Aside from causing extensive and widespread coral depletion26, Acanthaster spp. feed disproportionately on certain coral types and can have strong selective effects on the structure of coral assemblages28.Coral reefs are currently subject to unprecedented levels of disturbance29, but also avoid certain corals because they are defended by coral-associated organisms. Xanthid crabs, and mainly Trapezia spp., are the predominant organisms implicated in defending corals from CoTS32. However, the effectiveness of these crustacean guards may be enhanced by activities of coral-associated fishes34. Weber and Woodhead33 report seeing Dascyllus aruanus bite the leading tube feet of a CoTS in Fiji, which together with activities of crabs inhabiting the same coral, ultimately prevented CoTS from feeding on their host colony of Stylophora. Several species of territorial damselfishes  have also been observed to attack and effectively repel CoTS35. Coral-dwelling damselfishes are known to feed on larval CoTS potentially regulating the local abundance of these organisms37, but may also play a role in deterring adult CoTS from feeding on their host colonies. If not, responses of coral-dependent fishes to coral loss caused by Acanthaster spp. (or any other disturbances) will depend on the specific overlap in patterns of habitat use versus habitat vulnerability38.Crown-of-thorns starfish have inherent feeding preferences41, focusing mainly on changes in the abundance or diversity of fishes following large-scale disturbances that cause extensive coral loss. While increasing environmental changes that are emerging as predominant cause of coral loss22 also have direct effects on coral reef fishes44, there has been little research to date on the specific behavioral responses of fishes to acute coral loss46. It is implicitly assumed, for example, that declines in the abundance of reef fishes following extensive and widespread coral loss are due to elevated rates of individual mortality, linked to resource depletion and loss of individual condition and/or increased susceptibility to predation48, combined with reduced levels of population replenishment50. Coker et al.51 showed that coral-associated mesopredators (Pseudochromis fuscus) were almost twice as likely to strike at potential prey fishes associated with the stark white bleached or recently dead corals compared to equivalent prey on strongly pigmented, unbleached, corals. Even if predation does not cause increased in situ mortality, it is likely that increased exposure to predators will provide significant motivation for coral-dwelling fishes to rapidly vacate bleached and/or dead coral hosts51.Many studies have explored the effects of coral loss on coral reef fishes53. Damselfishes from the genera Dascyllus and Pomacentrus tend to occupy all-purpose home ranges or territories within the immediate vicinity of a single branching coral colony56. Whilst these damselfishes are critically dependent on their host corals15, the proximate causes of declines in their abundance following host coral mortality, remain largely unexplored. Of particular interest is timing of responses relative to changes in the physical and biological structure of host corals57. Feary et al.49 suggested that coral-dwelling fishes vacate host corals as soon as they bleach, let alone die. However, several other studies have recorded persistent associations between coral-dwelling damselfishes and their coral hosts even after coral bleach or die60. It is also unclear whether the disappearance of fishes from specific host corals necessarily represents mortality, or movement of individuals to new and alternative coral habitats. Knowledge of the capacity of site-attached fishes to relocate and colonize alternative habitats following habitat perturbations, and the identification of social, ecological and physical impediments to such recolonization45, is central to understanding patch dynamics and metapopulation resilience of fishes with strong microhabitat associations.Coral-dwelling damselfishes (family Pomacentridae) comprise a diverse and numerically dominant assemblage of fish species that are often found living in close association with live coral coloniesAcanthaster cf. solaris). Previous research has shown that coral infauna  may be effective in deterring A. cf. solaris from feeding on their host corals32, but it is unknown whether coral-dwelling damselfishes effectively defend host corals. Secondly, we examined the specific responses of damselfishes following host coral mortality, using intensive sampling to assess if and when these fishes vacate dead coral hosts. Specifically, we recorded the number and individual size of damselfishes that resided within all possible coral hosts to infer patterns of relocation, and test whether coral-dwelling damselfishes are generally resilient to host coral mortality given availability of alternative coral hosts within the immediate vicinity. This study is important in understanding the responses of site-attached fishes during moderate or patchy disturbances, whereby at least some corals persist62. However, it is unknown to what extent the responses of coral-dwelling fishes is moderated by the capacity of displaced fishes to colonize new and alternative coral habitats58. If so, we might expect an increase in the densities of damselfishes within surviving coral colonies or increasing use of previously unoccupied  coral hosts with no net change in the local densities of coral-dwelling damsels.The aims of this study were firstly, to test whether coral colonies occupied by coral-dwelling damselfishes are more or less likely to be consumed by the Pacific CoTS , hereafter referred to as P. cf. damicornis which accounted for 55.8% of total habitat area  were completely consumed by CoTS , but there was no difference in rates of consumption for corals that were or were not occupied by damselfish  of colonies consumed by ish Fig. A. MoreovDascyllus aruanus was found exclusively on Pocilloporidae corals, predominantly P. cf. damicornis , but also Stylophora pistillata and Seriatopora hystrix , S. pistillata and P. meandrina, but was also occasionally found on Acropora corals , there was no significant change in the habitat associations of D. aruanus during the course of this study  in the Lagoon up to 88.2% (15 out of 17 colonies) at Lizard Head. All damselfish species exhibited significant habitat selectivity at Day 0  size-classes compared to small (< 3 cm) or large (> 7 cm TL) individuals , for which smaller (< 3 cm TL) juveniles were under-represented at Lizard Head.Coral-dwelling damselfishes quickly disappeared from corals consumed by CoTS; The majority 165/182) of fishes persisted for less than 5 days on dead corals. Persistence on dead corals did not vary among the three species of damselfishes, but did vary consistently with body size  for P. moluccensis  for 26, which can have substantial flow on effects for coral reef fishes and especially highly specialized species that rely on corals for food, shelter or settlement38. However, effects of CoTS are (like many disturbances) often very patchy61, due partly to avoidance of corals that are actively defended by infaunal and associated organisms33. In this study, we did not observe any overt aggression towards CoTS by any coral-dwelling damselfishes, and there was no effect of damselfish occupation on whether or not corals were consumed. Rather, coral colonies used by coral-dwelling damselfishes were vulnerable to predation by A. cf. solaris.Population irruptions of CoTS represent the most significant biological disturbance on tropical coral reefs, killing up to 90% of coralsD. aruanus, D. reticulatus, and P. moluccensis) are considered to be dependent on live coral hosts, because (i) they are almost invariably found living in close association with live corals53 and (ii) decline in abundance following localized mortality of host corals64. However, the specific timing and proximate mechanisms leading to declines in the abundance of these species , following acute coral loss, are largely unknown. Notably, there are reports of coral-dwelling damselfishes living on dead coral hosts for protracted periods66. Sano et al.65, for example, recorded both D. aruanus and P. moluccensis living on dead coral colonies several months after extensive coral mortality caused by severe outbreaks of A. cf. solaris in Japan. Sano et al.65 concluded that the abundance of damselfishes living on coral colonies is generally unaffected by the loss of live coral tissue, and only declines when physical structure of host colonies is compromised. In contrast, we found that coral-dwelling damselfishes vacated coral hosts that had been consumed by CoTS within 12 days, disappearing (moving and/or dying) long before any changes in the physical integrity of the coral skeletons. Similarly, Coker et al.58 showed that D. aruanus rapidly vacate dead (but not bleached) coral habitats when alternative coral hosts are available. The extent to which coral-dwelling damselfishes persist on dead coral may therefore, depend on the availability of alternative coral hosts, which can be very limited following severe disturbances and in highly degraded reef environments65. Alternatively, differences in the persistence of coral-dwelling damselfishes on dead coral hosts may be attributable to changes in the abundance of predators, many of which are also vulnerable to extensive coral loss and habitat degradation67, such that fishes living in highly disturbed environments may actually experience much lower risk of predation.All three damselfish species  to nearby (1–2 m apart) healthy corals. In this study, we directly observed some damselfishes moving to very nearby corals (< 1 m apart) as soon as CoTS began feeding on their original host coral. Overall, 46.7%  of displaced fishes were recorded to re-colonize other nearby occupied coral hosts, adding to the number of fishes already established within these colonies. This capacity of coral dependent fishes to move between alternative coral hosts would be expected to confer increased resilience to disturbances, especially during moderate disturbances that cause limited coral loss45. However, displaced coral-dwelling damselfishes only ever took up residence in coral colonies that were already occupied by other coral-dwelling damselfishes, and mainly conspecifics. This is consistent with earlier findings that coral-dwelling damselfishes  preferentially colonize coral hosts that are already occupied by conspecifics69 and may actually cue in on the presence of conspecifics to identify suitable microhabitats. It is also rare to find solitary individuals of coral-dwelling damselfishes within any given coral colony. In this study for example, the mean number of damselfishes  found in occupied corals was 8.17 (± 1.1SE) and only 3 (out of 70) corals were occupied by a single damselfish, and always P. moluccensis. This suggests that there are significant benefits for group-living by coral-dwelling damselfishes70, which outweigh potential costs associated with joining established colonies or colonizing new and unoccupied coral hosts71. It is also possible that the few colonies (15 out of 85) of branching corals that were not already colonized by coral-dwelling damselfishes may be unsuitable as habitat for these fishes, as specialist coral-dwelling damselfish are not prepared to use alternative coral species even during a scarcity of coral hosts15. Even among the colonies of coral species that these species do normally use, unoccupied colonies may be unsuitable owing to their specific size or morphology73. We could not see any obvious differences between coral colonies that were and were not occupied by coral-dwelling damselfishes, though we did not explicitly quantify differences in habitat structure between these corals.There is increasing evidence that coral-dwelling fishes can and will move among alternative coral hosts75, reproductive opportunities76, and food resources71. Our observations show that movement and recolonization success is strongly size-dependent, being lowest for both the largest (> 7 cm TL) and smallest (< 3 cm TL) size classes. Notably, larger individuals of all three fish species tended to persist on recently dead corals much longer than smaller individuals, and larger individuals were only rarely detected on alternative host corals within the area of experimental quadrats, and never for D. aruanus  are more closely associated with specific coral growth forms than larger conspecifics80 and likely to be very vulnerable to predation while they remain on recently dead coral colonies51, potentially explaining why we did not record any such damselfishes living on dead colonies for > 4 days. However, these individual are also likely to be very vulnerable to predation when trying to move to alternative coral hosts78, thereby limiting their opportunity to try and integrate into established groups of coral-dwelling damselfish on alternative coral hosts.Explanations for the low probability of very small damselfishes successfully moving and recolonizing alternative colonies following the mortality (consumption) of their original coral hosts is likely to be very different from that of larger individuals. Most notably, smaller individuals are likely to be much more readily accepted into established coloniesD. aruanus, D. reticulatus, and P. moluccensis) through the course of this study. Moreover, net declines in the abundance of fishes were concordant with levels of host coral mortality, as recorded previously for many coral-dwelling damselfishes81. This suggests that the movement and redistribution of coral-dwelling species in responses to changes in the abundance and distribution of coral hosts does little to mediate longer-term effects of habitat loss. Observed declines in the abundance of these fishes are attributable to emigration  and/or mortality of fishes that persisted on dead coral hosts or attempted to move to alternative coral hosts. Given that no displaced fishes were relocated within suitable coral hosts in the buffer zone , we initially assumed that successful movement and recolonization of fishes to alternative coral hosts is likely to be spatially restricted. However, the effectiveness of the buffer zone for detecting fishes moving outside of experimental quadrats has to be questioned given that fishes never relocated to colonies that were not already occupied by conspecifics. Even so, we did record at least some displaced fishes (46.7%) recolonizing alternative coral hosts within the experimental plots. The fact these movements did not buffer against overall coral loss is attributable to limited persistence of elevated densities of coral-dwelling fishes on these surviving coral colonies, possibly reflecting inherent limits on the number of fishes that can be sustained within individual corals82. Given our inability to distinguish individual fishes of the same size we do not know whether fishes that moved ultimately succumbed, or whether these individuals effectively displaced other fishes of equivalent size45.Despite the movement of damselfishes and successful recolonization of alternative corals hosts, especially among medium sized individuals, there were net declines in the abundance of all damselfish species  as well as James Cook University Animal Ethics Committee.2) experimental plots were established at each of four different locations . All plots were established at the base of the reef slope (7–10 m depth) in areas with a rubble substrate where isolated colonies provided relatively independent units of habitat. To explore the responses of these fishes to host coral mortality, this study took advantage of high local densities of crown-of-thorns starfish, which naturally consumed corals within the immediate area of experimental plots. The four 100 m2 experimental plots together encompassed a total of 85 coral colonies, most of which (71/85 colonies or 83%) were occupied by one or more species of the coral-dwelling damselfishes, Dascyllus aruanus, D. reticulatus, and/or Pomacentrus moluccensis. Sites were selected where there were abundant CoTS (4–12 starfish per plot), but no apparent recent coral mortality. However, A. cf. solaris are only very occasionally observed within the lagoon86 and did not consume any corals within the experimental plot established at this location, effectively serving as a ‘control’. Because CoTS do not directly affect the physical structure of coral colonies39, and natural erosion and decomposition of dead coral colonies takes years rather than days40, changes in the condition of coral habitats were solely affected through the loss of live tissue with little or no change in physical integrity of coral hosts.This study was conducted at Lizard Island , on the northern Great Barrier Reef, Australia; 10 × 10 m (100 m2) around each of the plots, removing all damselfishes from within live coral colonies (using clove oil and hand nets) but leaving the now vacant coral colonies in place. Without having tagged all individual damselfishes, there was limited capacity to assess long-distance movement of displaced damselfishes, but it was anticipated that if damselfishes moved outside of the experimental plot, at least some individuals would be found within the 5 m buffer zone around each plot. If fish suffered mortality with the death of their host colony, this would be apparent from a net decline in the abundance of fish in each plot. If however, fishes moved between coral colonies within each plot, then the total abundance and size structure of fish within each plot would remain constant despite a decline in the number of live coral colonies.To account for localized movement of damselfishes outside of the experimental plot we cleared a 5-m wide buffer zone  visually estimated to the nearest cm. During surveys, two divers would independently count and recount all damselfishes in each successive coral colony, until they arrived at a consensus. For every coral colony, the maximum diameter and perpendicular diameter were recorded, from which we calculated 2-dimensional projected area87. Surveys of corals and coral-dwelling fishes took up to 80 min per plot and all four plots were surveyed within the same day.In order to document responses of coral-dwelling damselfishes to host coral mortality, the distribution and abundance of coral-dwelling damselfishes within each experimental plot and associated buffer zones was recorded immediately before (on day 0), and then every 2–5 days until there were no coral-dwelling damselfishes remaining on coral colonies that had been consumed by A. cf. solaris avoided coral colonies that were occupied by coral-dwelling damselfishes, we compared the ratio of colonies consumed versus those that were not consumed  for colonies that were and were not occupied by one or more species of damselfish, using the Pearson statistic with Yate’s correction. It was not possible to take full account of taxonomic differences in coral hosts and data was pooled across all coral species to maximize cell counts. However, it was recognized that these comparisons may be confounded by inherent feeding preferences of Acanthaster spp.29, especially if starfish preferentially target coral species that are generally not used by coral-dwelling damselfish . Therefore, a separate comparison was also conducted based on the single most abundant coral taxa, Pocillopora cf. damicornis, which was used by all three species of damselfish.To test whether D. aruanus, D. reticulatus, and/or P. moluccensis), the 2-dimensional planar area of live tissue for each coral colony was calculated from measurements of external dimensions less proportional area of partial mortality. Initial habitat preferences were analyzed by comparing the relative use of different coral taxa  by each damselfish species to their proportional availability on day 0. Pearson statistics were used to take account of all coral types that were and were not used. Separate analyses were conducted for each species of damselfish, combining data from all four plots and locations . Changes in habitat use thereafter, were analyzed using the log-likelihood statistic to compare frequencies of each damselfish across the limited range of different coral taxa that were used by each species of damselfish on day 0 versus day 16, following Manly et al.88. These analyses  are potentially confounded by aggregative behavior of coral-dwelling damselfish59, though tests of habitat preferences explicitly account for the areal extent (rather than number of distinct colonies) of each coral type. Moreover, simple comparisons of occupied versus unoccupied colonies would obscure individual differences in recolonization success and subsequent habitat-associations of fishes.To explore the differential availability coral species, and occupation of these potential coral hosts by each of the three coral-dwelling fishes  was non-negative and far from normally distributed . Secondly, we tested for size-specific differences in the fate of displaced fishes, comparing the number of displaced fishes  that did versus did not successfully recolonize other corals within experimental plots using the log-linear statistic. Since no fish were observed within the buffer zone, we assumed that those fish that disappeared from corals consumed by A. cf. solaris, either moved well outside the experimental plots and buffer zones, or died. For frequency analyses, fish were assigned to one of four different size classes .To test for taxonomic and size-based differences in the responses of damselfishes to host coral mortality, we compared time (in days) that individual fishes persisted on dead coral hosts, which had been consumed by 89. Individual host corals were used as the random effect, effectively testing for changes through time in the number of fish per host coral. Models were initially run accounting for the fate of colonies, distinguishing between those individual coral colonies that died versus survived, as well as treating all colonies in the control plot separately. This revealed whether there were temporal changes in the number of fishes on corals that survived, which may be caused by colonization of fishes that vacated dead coral hosts. We also tested for overall changes in the abundance of damselfish (each species separately), thereby assessing whether movement and recolonization of surviving coral provided resilience to coral loss. All analyses were conducted in R 3.3.290.Changes in the overall abundance of each damselfish species on corals, were analyzed through the course of the experiment (16 days) using a repeated-measures linear mixed-effects models in “nlme”


Hepatocellular carcinoma (HCC) incidence has dramatically decreased in patients infected with HCV and HBV due to the widespread use of highly effective antiviral agents. Nevertheless, a substantial proportion of patients with advanced fibrosis or cirrhosis following HCV clearance of in case of HBV control whatever the stage of fibrosis remains at risk of liver cancer development. Cancer predictors in these virus-free patients include routine parameters estimating coexisting comorbidities, persisting liver inflammation or function impairment, and results of non-invasive tests which can be easily combined into HCC risk scoring systems. The latter enables stratification according to various liver cancer incidences and allocation of patients into low, intermediate or high HCC risk probability groups. All international guidelines endorse lifelong surveillance of these patients using semi-annual ultrasound, with known sensibility issues. Refining HCC prediction in this growing population ultimately will trigger personalized management using more effective surveillance tools such as contrast-enhanced imaging techniques or circulating biomarkers while taking into account cost-effectiveness parameters. The widespread implementation of anti-HBV and anti-HCV therapies has deeply modified the course of chronic viral liver diseases, of which hepatocellular carcinoma (HCC) has become the leading cause of death . The maiOver the past decades, numerous HCC risk scoring systems for stratifying HBV- or patients infected with HCV into various HCC risk classes have been proposed and validated . HoweverThe goal of HCC surveillance programmes is the detection of liver tumours at the earliest stage possible, in order to allocate patients to curative procedures which has been shown to provide a survival benefit . AlthougHowever, implementing these new tools into surveillance programmes may not be cost-effective for all patients, particularly for those who have achieved HCV clearance or HBV control, in whom decreased annual HCC incidences are now well established . In thisFollowing SVR, the risk of HCC is highest in patients with cirrhosis and is considered negligible in patients with mild or no fibrosis; HCC surveillance is not recommended for the latter group . Howevern = 1323, median follow-up 58 months), a prospective multicentre study reported a 5-year cumulative HCC incidence of 6.7% [Ageing usually triggers the development of various comorbidities known to impact liver-related outcomes, including liver cancer . Studies of 6.7% .A similar evolution might be expected in patients who achieve SVR by means of DAA therapy. HCC incidence could even be higher in older patients with more pronounced liver disease and comorbidities compared to the available profiles reported for interferon-treated patients. The first available multicentre reports were restricted to the retrospective analysis of registries from the Veterans Affairs system, which reported a 71% lower risk of HCC in patients with HCV clearance ,26,27. TOverall, the magnitude of the decrease in HCC risk is similar regardless of the antiviral treatment regimen. However, due to the relatively short follow-up of patients who received DAAs, longer follow-up of patients and future updates of cohort studies should clarify the pattern of temporal evolution of HCC incidence. Until then, lifelong surveillance for HCC is recommended in patients with documented advanced fibrosis and cirrhosis, as it seems unlikely that the risk of liver cancer would eventually decrease over time to a point at which surveillance becomes unnecessary. Nevertheless, allocation of personalized screening procedures might be triggered by HCC risk stratification.Despite the low HCC incidence following SVR reported above, all patients with advanced fibrosis or cirrhosis do not have the same risk of developing HCC . Until tUntil recently, the stratification of HCC risk has been based only on simple scoring systems that combine routine clinical features without considering if these variable might operate in combination or independently ,47. In tEntecavir (ETV), tenofovir disoproxil fumarate (TDF) and tenofovir alafenamide (TAF) are the three first-line NUCs with a high genetic barrier to resistance that are recommended by international treatment guidelines . ETV andOnce cirrhosis is diagnosed, NUCs are beneficial in preventing cirrhosis progression towards liver decompensation and reduce the risk of HCC development . Most stIn a multicentre, retrospective-prospective cohort study conducted in Taiwan, HCC incidence was 2.4% in the ETV group and 5.2% in the untreated group in the first 2.7 years; this corresponded to an HCC risk reduction of 60%  . In prevPapatheodoridis et al. studied the risk of HCC in a European, multicentre cohort study that included 1951 Caucasian chronic hepatitis B patients who received ETV or TDF . Among tPatients with chronic hepatitis B virus (HBV) infection are at risk of HCC development even in the absence of cirrhosis . ReducinFor noncirrhotic CHB patients, the mean annual incidence of HCC is lower and is reported to be 0.68% in patients undergoing treatment with NUCs and 2.97% in patients without treatment . ConversTwo recent large Asian retrospective studies were the cause of some controversy. Using available adjusted data (multivariate or propensity-matched data), the risk of HCC among patients treated with ETV was 27% higher than that among patients treated with TDF . MoreoveSimilar to the case for patients infected with HCV with advanced fibrosis who reached SVR, numerous HCC risk scoring systems have been developed for CHB patients who are undergoing long-term NUC treatment. Initially, these scoring systems were constructed for untreated patients infected with HBV with chronic active HBV replication, while the more recently developed systems are specifically dedicated to patients under antiviral therapy. Most of these scoring systems were developed in Asians, and they often mix patients with and without cirrhosis ,41,42,43As described above, the global annual HCC incidence in patients with controlled HBV infection or cured HCV in the case of advanced chronic liver disease ranges from 0.2 to 2.5%. These rates are similar to those observed in patients with cirrhosis due to nonviral causes, whether alcohol- or NASH-related ,73, and The availability of different models has enabled so far to define several HCC risk classes that might define new surveillance strategies. The goal is not to identify patients who have “zero risk” following viral eradication or control; in any case, this would only be true at one time point in the patient’s lifetime since the probability of developing HCC typically increases over time: older age, the development of comorbidities and worsening of liver dysfunction are all cancer risk factors, justifying lifelong surveillance until further notice. In contrast, long-term follow-up of longitudinal cohorts has been able to highlight specific subgroups in which, despite their virus-free status, the persisting risk of liver cancer is sufficiently high to trigger personalized management through reinforced surveillance programs. The modelling approaches that have been used to achieve this goal are based on simple routine measurements that are readily available; these algorithms may possibly be enriched by the incorporation of circulating biomarkers  such as


The plant apoplast contains the four hydrophobic polymer, lignin, suberin, cutin, and cutan, that are crucial for stress resistance, controlling solute diffusion, and strengthening the cell wall. Some of these polymers are widely used in industry and daily life products, such as all wood-containing goods (lignin) and wine cork (suberin). Despite the importance of these polymers, several aspects of their formation remain unknown. This mini review highlights technical bottlenecks in the current research and summarizes recent insights into the precursor transmembrane transport, an essential step in the polymer formation. We also briefly discuss how some of the remaining knowledge gaps can be closed and how a better understanding of these biopolymers will benefit other research fields. Plants secrete hydrophobic compounds into the apoplast, where they contribute substantially to the plant’s structural strength and resistance against environmental stresses . Both liPhyscomitrella patens  transporters], (2) transport via cargo vesicles, (3) passive diffusion through the membrane, and/or (4) channel-facilitated transport. Considering that different species and even organs can contain a variety of lignin precursors (Four models for lignin precursor transport into the apoplast have been proposed : (1) actecursors , it is lArabidopsis leaf- and Populus root-derived plasma membrane vesicles that showed an ATP-dependent uptake of monolignols  is somewhat counterintuitive to the widely accepted concept that lignin precursors are synthesized in the cytoplasm  to what extent does vesicle-mediated lignin precursor secretion contribute to total precursor secretion, and (3) what is the physiological significance of utilizing both cytoplasmic- and vesicle-mediated transport routes for precursor secretion? A proton porter may not be the only mechanism for sequestering glucosylated monolignols. Arabidopsis root cells. However, this drug did not affect the same transport mechanisms in differentiating xylem cells of cypress and spruce , which suggest that uncharged monolignols diffuse faster through a Zea mays root membrane than, e.g., glucosylated derivatives (The third model (passive diffusion) is supported by molecular dynamics simulations  lacks support by experimental evidence. Future studies may mine membrane proteomes for candidate channels and additionally test whether any facilitated diffusion mechanisms  support lignin precursor transport.The fourth transport model , which are ester-bonded to the predominant aliphatic domains (16–24) and epoxy derivatives with minor primary fatty alcohols and glycerol   is poorlmponents . Recentlvia a glycosylphosphatidylinositol-anchor, might govern suberin monomer export in seeds because the suberin aliphatic components are significantly decreased in knockout mutants. AtLTPI-4, a non-specific LTP that diffuses freely in the apoplast ; Cutin forms a polyester layer like suberin but contains less phenolics and shorter hydroxy fatty acids [e.g., 10,16-dihydroxyhexadecanoic acid , and the content of most cutin monomers decreased to ∼50% of the wild-type  could be responsible for this ransport . Experimransport . Cutinsoransport .4–33 α,ω-diacids, alkanes, and alkenes  would be an excellent toolset for high-resolution live-cell imaging.So far, only transporters belonging to the ABCG group have been verified as part of the active transport mechanism for lignin, suberin, and cutin precursors. The apparent dominance of these transporters might be due to their membrane topology, which allows for trafficking hydrophobic compounds. To test this hypothesis, crystallization of ABCGs with ligands can be used to qualify the interacting mode. A prominent bottleneck in the field is the lack of sensitive techniques that allow for tracking the movement of precursors in living cells. For example, there is no experimental evidence for the hypothesized passive diffusion of lignin precursors through membranes. To provide direct evidence on how cells transport various lipid-like molecules, trackable precursors (e.g., Overall, elucidating transmembrane export mechanism for hydrophobic molecules is an exciting field and deserves more attention. Gained knowledge will help to assess how plants react to climate change scenarios, as these polymers are fundamental for regulating the plant water household. Future studies may investigate whether plants can adjust their biopolymer compositions through switching between certain precursor transport mechanisms. This would open new opportunities for engineering plants as it would allow for regulating, e.g., the lignin content by modifying transporters rather than amending the lignin biosynthesis machinery, which could interfere with the cellular metabolism. Biomedically, new insights into the intriguing mechanism of cutin transport through the cell wall could inspire new drug delivery systems, such as nanocarriers that do not only pass through both hydrophilic and hydrophobic barriers but also target their destination with high efficiency.AX drafted the manuscript and 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.


Cyp1a1 mRNA levels are induced after irradiation in a dose-dependent manner, and CYP1A1 protein expression increased in the irradiated intestinal tract as well. BNF also reduces DNA strand breaks induced by irradiation. These studies demonstrate that BNF pretreatment prolonged median survival time of mice upon exposure to a lethal dose of radiation and alleviated irradiation-induced toxicity within the bowel.Radiotherapy induced gastrointestinal syndrome results from the acute damage of intestinal stem cells, impaired crypts reconstruction, and subsequent breakdown of the mucosal barrier. The toxicity of ionizing radiation is associated with oxidative stress in the intestinal epithelial cells (IECs). Moreover, the rapid proliferation of IECs is a risk factor for radiation damage. β-naphthoflavone (BNF) is an agonist of the aryl hydrocarbon receptor (AhR) and possesses potential antioxidative activity. We investigated BNF radioprotection in IECs experiencing γ-ray exposure, contributed to mitigation of radiation enteritis. BNF significantly enhanced cell viability and suppressed cell apoptosis in an AhR activation-dependent manner. The mechanism of BNF reducing the IECs radiosensitivity was associated with cell cycle arrest and suppression of cell proliferation. In contrast, AhR antagonist CH-223191 significantly blocked BNF-induced cell cycle arrest.  Irradiation induces gastrointestinal adverse effects in humans (~60% incidence rate) and is one of the primary limitations to delivering tumoricidal doses in abdominal radiation therapy. Although reversal of this toxicity is observed after radiotherapy has finished, 10% of the treated patients develop gastrointestinal syndrome, a disease characterized by intestinal cell death, which results in the destruction of the entire intestine and ultimately patient death carbazole can protect several cell lines  from oxidative DNA damage [2DCFDA by flow cytometry. Ionizing radiation stimulated ROS accumulation in a dose-dependent manner, however, the BNF treatment did not suppress ROS levels compared with vehicle counterparts. Therefore, the primary mechanism of BNF radioprotection of IECs may be associated with its influence on the cell cycle or cell survival signals. To better understand AhR activation potential after irradiation exposure, AhR protein levels were analyzed in the presence or absence of BNF. AhR protein expression was similar in the irradiated IECs, before and after BNF exposure. Nevertheless, ionizing radiation induced ROS may contribute to increased AhR expression directly [Our results reveal that BNF treatment can significantly mitigate apoptosis stimulated by irradiation exposure in IEC-6 cells. In contrast, the regulation is impaired in the cells with attenuated AhR activity, which demonstrates that the impact of apoptosis is AhR activity dependent. Use of the comet assay is another important parameter to evaluate the radiation sensitivity in vitro and in vivo, as ionizing radiation induced double strand breaks is the most lethal form of DNA damage and a primary cause of cell death . In our A damage . In ordedirectly , while adirectly . Perhapsdirectly . NeverthIn our study examining BNF radioprotection in vivo, results show that BNF exhibited a preventative phenotype leading to prolonged survival time of mice exposed with lethal dose irradiation. The histological observation shows alleviated intestinal toxicity, contributing to intestinal function recovery. However, when we treated mice with BNF for five days after irradiation to evaluate its therapeutic effect, there was no obvious beneficial effect observed. This result is consistent with excessive suppression of IECs proliferation after irradiation, which likely would cause impairment of crypt regeneration, which is crucial for the maintenance of intestinal tract integrity .In summary, our study demonstrates that BNF mitigates irradiation-induced intestinal system injury in mice exposed with TBI or LBI, and this protection is due to AhR activation associated with other antioxidative defense mechanism(s). Additionally, AhR activation can induce IECs cell cycle arrest at G2/M stage, consequently, reducing IECs sensitivity to irradiation. Hence, it is essential to maintain proper AhR activity in IECs experiencing radiotherapy; specifically, an AhR ligand-rich diet may be considered as potentially supportive care to benefit tumor therapies in the clinic . However


Mycobacterium tuberculosis (Mtb) culture. Traditional phenotypic drug susceptibility testing for Mtb is costly and time consuming, while PCR-based methods are limited to selected target loci. We herein utilized a portable, USB-powered, long-read sequencing instrument (MinION), to investigate Mtb genomic DNA from clinical isolates to determine the presence of anti-TB drug-resistance conferring mutations. Data analysis platform EPI2ME and antibiotic-resistance analysis using the real time ARMA workflow, identified Mtb species as well as extensive resistance gene profiles. The approach was highly sensitive, being able to detect almost all described drug resistance conferring mutations based on previous whole genome sequencing analysis. Our findings are supportive of the practical use of this system as a suitable method for the detection of antimicrobial resistance genes, and effective in providing Mtb genomic information. Future improvements in the error rate through statistical analysis, drug resistance prediction algorithms and reference databases would make this a platform suited for the clinical setting. The small size, relatively inexpensive cost of the device, as well as its rapid and simple library preparation protocol and analysis, make it an attractive option for settings with limited laboratory infrastructure. Clinical management of tuberculosis (TB) in endemic areas is often challenged by a lack of resources including laboratories for  Mycobacterium tuberculosis (Mtb) complex (MTBC), which has been thought to have low DNA sequence diversity [Tuberculosis (TB) is the number one cause of human death due to an infectious disease, with 1.7 million deaths per year worldwide [iversity . This liiversity . VariousTB surveillance of highly-virulent and multi-drug resistant (MDR) strains is paramount for adequate diagnosis and treatment ,3. TradiDevelopment of a diagnostic assay that can be used at the point of care to rapidly and accurately diagnose TB and to include multidrug-resistant tuberculosis (MDR-TB) or extensively drug-resistant tuberculosis (XDR-TB) should be given a high priority. MDR-TB characterization typically requires costly machinery and handling in a specialized reference laboratory, not to mention the time required for shipping and processing the sample. A portable sequencing system that could be taken to the field, would not only reduce the cost of TB testing, but will also speed up the diagnoses. A rapid direct sample sequencing device would significantly reduce the time to obtain test results.Mycobacterium bovis BCG strain DNA, not direct sputum sample. Despite the advantage of a portable sequencer in MDR-TB testing, so far there is no peer-reviewed published protocol of ONT-WGS based, rapid MDR-TB testing of patient sputum samples. It is unknown if this portable DNA sequencing system would be effective in providing information on Mtb genotype, drug-susceptibility in a sputum sample.The MinION -Oxford Nanopore Technologies Limited (ONT), is a pocket sized (10 × 3 × 2 cm), portable, USB-powered, long-read sequencing instrument . Among tIn this pilot study, we evaluated the performance of this portable sequence system for Mtb species identification and detection of genes related to drug resistance, as a means of MDR-TB testing in a diverse set of samples, including DNA isolated from sputum samples and from clinical microbiological isolates.Homo sapiens in clinical isolates was minimal, and absent in the commercial Mtb genomic DNA.Upon sequencing of a set of DNA obtained from various sources, utilizing the What’s In My Pot (WIMP) app  we were We evaluated molecular genome-based drug resistance mutation analysis by sequencing DNA samples using a portable, long-read sequencing platform. Sequenced and analyzed data from Mtb culture isolates and commercially available Mtb genomic DNA showed numerous drug resistance-conferring mutations .Comparison of the sequencing results of Mtb DNA obtained from direct sputum vs. those of sequencing from Mtb DNA obtained from culture isolates, showed that the amount of reads for Mtb was much higher from the latter samples . The higAs pointed out previously, a limitation imposed by sequencing Mtb DNA from sputum samples was the high proportion of human DNA. Despite this relatively low availability of Mtb DNA for sequencing, it proved to be sufficient for obtaining a read coverage that allowed the identification of drug resistance mutations .katG  but also to inhA and mutations in the 16S rRNA gene were listed for amikacin, streptomycin and kanamycin resistance independently.We then aimed to compare the results obtained for a subset of samples for which whole genome analysis (WGA) data were available. Analyzing the MinION reads in real time with the ARMA pipeline identified a larger number of mutations in genes related to drug resistance, which in some cases included all, or the majority, of those identified by WGS analysis . Most ofIn this study we have evaluated the genomic identification and drug mutation gene profiling of Mtb isolates utilizing the MinION portable sequencer. Our findings endorse the need of further research regarding the practical use of MinION for the detection and characterization of Mtb in clinical isolates and in sputum samples. Our sample set consisted of Mtb genomic DNA obtained by different extraction methods. Recently, low-cost DNA extraction methods for Mtb WGS directly from patient samples have been reported , allowinThe portable WGS-based detection system utilized here proved to be fast, relatively inexpensive, with rapid and simple library preparation, and automated real-time analysis tools . The mosThe MinION has several advantages that make it uniquely suited for TB surveillance . AmongstrpoB, katG and inhA for first line drugs and gyrA, gyrB, rss and eis promoters for second line drugs) and for specific point mutations within them [mtrA or AAC(2’)-IC, which were detected in 5 out of 7 isolates irrespective of their resistance profile and could correspond to polymorphisms.The number of mutations in drug resistance related genes overly surpassed those detected in previous WGA. This may have several explanations. First, a high error rate has been acknowledged as a limitation of Nanopore technology , thus, shin them . Howeverhin them ,22. Almohin them , which cgidB mutation, which confers resistance to streptomycin, is not the most frequent among clinical isolates but is characteristic of this cluster and was acquired four decades ago when the expansion of this cluster began [Nevertheless, the sensitivity of the MinION sequencing for the detection of drug resistance mutations was good. Isolates 410 and 6548 belong to the extensively studied MDR M strain ,24 whicher began . These per began ,27,28,29Overall, our findings indicate that the improvements in the future should focus on: (1) recovering higher number of reads corresponding to Mtb from sputa; (2) lowering MinION sequencing error rates; (3) improving the drug-resistance conferring mutation detection algorithms for automated analysis and (4) curating the reference database to include only those hits that have a strong correlation with Mtb drug resistance phenotype.Although our data relies on a short number of DNA samples, our findings suggest that this portable DNA sequencing system could be effective in reducing time and providing information on Mtb genotype and drug-susceptibility from direct sputum samples. As larger studies—evaluating parameters such as the minimal number of reads for a complete reliable drug susceptibility profiling, optimization in software and database accuracy for the prediction of new drug resistance genes, and reduction in false positive drug detection—are conducted, this system could potentially revolutionize current TB testing procedures, especially in genomic surveillance for MDR-TB in the clinical setting.TM DNA Extraction Kit , as well as inactivation and lysis by sonication protocol [Mtb genomic DNA, strain HN878 was acquired through bei resources (NR-14867). Genomic bacterial DNA extracted from four laboratory cultured Mtb isolates from sputum samples was also utilized . These cprotocol  were useprotocol . Whole genome sequencing (WGS) with Illumina was available for isolates 6548 and 410  and for DNA sample libraries were constructed using Rapid Sequencing Kit , and sequencing was conducted on MinION-compatible R9.4 flow cells . Primary data acquisition was done using MinKNOW, the operating software that drives nanopore sequencing devices. Raw data were processed for basecalling via Albacore. Data were then further processed using the cloud-based data analysis platform EPI2ME . MicrobiIn this study we have evaluated the genomic identification and drug mutation gene profiling of Mtb isolates utilizing the MinION portable sequencer The approach was highly sensitive, being able to detect almost all described drug resistance conferring mutations based on previous whole genome sequencing analysis. Our findings are supportive of the practical use of this system as a suitable method for the detection of antimicrobial resistance genes, and effective in providing Mtb genomic information. Future improvements in the error rate through statistical analysis, drug resistance prediction algorithms and reference databases would make this a platform suited for the clinical setting. The small size, relatively inexpensive cost of the device, as well as its rapid and simple library preparation protocol and analysis, make it an attractive option for settings with limited laboratory infrastructure.


It usually refers to a unique atmosphere, for instance, when enjoying a couple of drinks with your mates in a comfy location like a beergarden. Seemingly, disseminating cancer cells when going on their long and lonesome journey to generate distant metastases prefer to travel via the lymphatics, a somewhat fairly friendly and “gemütlich” environment, rather than the bloodstream , that catalyzes the glutathione (GSH)-dependent reduction of lipid hydroperoxides to their corresponding alcohols. While this enzyme is highly relevant for optimal functioning of normal cells, a growing body of evidence suggests a strong dependence on GPX4 for growth and survival of cancer cells.1 whereby the authors have focused on comparing dissemination of metastasizing melanoma cells through blood versus lymphatic fluid. To this end, the authors injected mice with patient-derived or mouse melanoma cells and found that melanoma cells are by far more efficient in metastasis formation after intranodal injection than intravenous injection. Further experiments, in which melanoma cells were injected subcutaneously, revealed that cancer cells were more abundant in tumor-draining lymph nodes than in tumor-draining blood, suggesting a higher fitness and better survival of cancer cells in lymph than in blood. This apparent difference in the survival of circulating metastasizing cells was associated with an increased level of reactive oxygen species and a lower ratio of reduced versus oxidized GSH in cells disseminating in blood, indicating increased intracellular oxidative stress. This may result from a more oxidative and hostile environment in the blood, characterized by a higher concentration of free iron and a lower concentration of reduced GSH. Interestingly, cells that spontaneously metastasized through the blood also exhibited increased lipid peroxide formation. To ascertain that ferroptosis indeed contributes to impaired metastasis, melanoma cells were treated with a ferroptosis inhibitor, liproxstatin-1, a potent radical trapping antioxidant. While the extent of metastatic disease burden was not affected after intranodal transplantation of liproxstatin-1-pretreated cells, a substantial increase of metastasis was observed after intravenous injection, suggesting that ferroptosis restrains tumor metastasis via the blood. This was corroborated by experiments using mouse melanoma cells genetically engineered to lack Gpx4. The targeted knockout of Gpx4 decreased the percentage of mice with formed metastatic tumors after intravenous injection of Gpx4-deleted melanoma cells, but not after intranodal injection. These results clearly demonstrate that cancer cells are prone to undergo ferroptosis and become dependent on GPX4 activity during metastasis in the blood.One may assume that increased ferroptosis could potentially account for the low survival rates of cancer cells metastasizing via the blood. Evidence supporting the involvement of ferroptosis in tumor metastasis has been provided in this study by Ubellacker et al.,4 This correlates with the finding that the levels of oleic acid-containing lipids were higher in lymph, where oleic acid is overwhelmingly present in triacylglycerols within ApoB-positive vesicles. Consequently, pretreatment of melanoma cells with oleic acid increased tumor metastasis after intravenous injection.1 Incorporation of oleic acid into cellular membranes was further shown to require the activity of a distinct fatty acid ligase, i.e., acyl-CoA synthetase long-chain family member 3. As a final proof-of-concept, the authors addressed whether a lymph environment provides any benefits for cancer cells to form subsequent distal metastases through the blood. To this end, cancer cells derived from lymph-node metastases were intravenously injected and they emerged to be more efficient in metastasis than intravenously injected control cells from the subcutaneous tumor. Besides, those cells were generally more resistant to a ferroptosis inducer erastin in vitro. Interestingly, the transient subcutaneous growth of the lymph-node-derived cells completely abrogated their ability to efficiently metastasize through blood, suggesting a reversible mechanism of the lymph-associated antiferroptotic phenotype.To unravel additional cell-intrinsic mechanisms that might contribute to protection of cancer cells against ferroptosis in the lymph, the authors performed metabolomic and lipidomic analyses of metastasizing melanoma cells, and detected an enrichment of oleic acid in lymph-derived cells, a monounsaturated fatty acid previously described to confer robust antiferroptotic activity.c−, a heterodimeric amino acid transporter supplying cells with cysteine, the building block of GSH, is considered the most upstream component of ferroptosis. Besides its role in cysteine delivery to cells, system xc− was also shown to be the driver of the cystine/cysteine redox cycle that is critically involved in generating a highly reducing environment that might also be favorable to nearby cells. In this context, a recent study showed that the targeted knockout of the substate-specific subunit of system xc−, xCT, in B16F10 melanoma cells almost completely abrogated tumor metastasis and consequently allowed drastically increased survival rates of tumor-bearing mice.5 The findings presented by this and earlier studies thus reinforce the notion that pharmacological targeting of xCT is perhaps one of the most promising strategies to combat disseminating tumor cells.Conclusively, the findings of this exciting study provide compelling evidence supporting the concept that lymph provides a more “cozy” and favorable environment for disseminating cancer cells, and after being exposed to that environment, the cells become more efficient in subsequent metastasis in the blood. This study may have far-fetched implications as the cystine/glutamate antiporter, known as system x


Carica papaya that have proved to show better physiological performance under water deficit stress than the commercial cultivar grown in Mexico. In the present study, plants from a wild Carica papaya genotype and a commercial genotype were subjected to water deficit stress (WDS), and their response was characterized in physiological and molecular terms. The physiological parameters measured  confirmed that the papaya wild genotype showed better physiological responses than the commercial one when exposed to WDS. Subsequently, RNA-Seq was performed for 4 cDNA libraries in both genotypes (susceptible and tolerant) under well-watered conditions, and when they were subjected to WDS for 14 days. Consistently, differential expression analysis revealed that after 14 days of WDS, the wild tolerant genotype had a higher number of up-regulated genes, and a higher number of transcription factors (TF) that were differentially expressed in response to WDS, than the commercial genotype. Thus, six TF genes  were selected for further qRT-PCR analysis as they were highly expressed in response to WDS in the wild papaya genotype. qRT-PCR results confirmed that the wild genotype had higher expression levels (REL) in all 6 TF genes than the commercial genotype. Our transcriptomic analysis should help to unravel candidate genes that may be useful in the development of new drought-tolerant cultivars of this important tropical crop.Most of the commercial papaya genotypes show susceptibility to water deficit stress and require high volumes of irrigation water to yield properly. To tackle this problem, we have collected wild native genotypes of  Merr, 8 NAC genes  related to drought stress, showed greater transcript levels in the drought-tolerant soybean varieties than in the drought-sensitive varieties  re re72] rearieties .Our quantitative results using RT-qPCR are consistent with those predicted from the RNA-Seq analysis. Thus, we confirmed that when plants from both papaya genotypes were subjected to WDS, the expression of important TF genes associated to drought-response was induced, but they were most abundantly expressed in the wild tolerant genotype.We are now investigating if important drought-tolerance genes might have been lost during the papaya domestication process. The knowledge gained from this study, should pave the way to an improved understanding of the molecular processes involved in the papaya response to water deficit stress, and they might set the basis for papaya wild-relative-based plant breeding programs, aiming to develop new papaya cultivars with enhanced drought tolerance.The present study confirmed that the papaya wild genotype is physiologically more drought-tolerant than the commercial papaya genotype. The superior drought tolerance of the wild genotype may have resulted from its ability to maintain lower EL (that might reflect its ability to maintain membrane stability), higher Fv/FM values (that may reflect its higher efficiency of PSII) and higher stomatal conductance (that might reflect that their stomata remained open longer) than its commercial counterpart when subjected to WDS.Furthermore, our detailed transcriptome analysis indicated that the wild genotype has a higher number of up-regulated DEGs than the commercial genotype when exposed to 14 d.p.t. of WDS. Likewise, wild genotype consistently showed higher expression levels of TF genes, and higher expression levels of drought-associated TF, than the commercial genotype. Therefore, that improved performance of the wild genotype under WDS, may be related to its ability to maintain high expression levels of key TF genes that may in turn, regulate other downstream genes involved in triggering protective mechanisms to reduce WDS-related damage and they should be part of the complex molecular mechanism for drought tolerance in this tropical species. Although more research is needed, the high expression levels showed by these 4 TF genes in response to WDS, observed in the transcriptome analysis and confirmed with the qRT-PCR data, suggest that these 4 TF  might be important candidate TF genes involved in drought tolerance in this species, that might be useful in breeding programs aiming to generate cultivars with enhanced drought tolerance.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Although rare, pancreatic neuroendocrine tumours (pNETs) represent the second most common group of pancreatic neoplasms, and associated patient outcomes remain largely poor. Advances in diagnosis and management rely strongly on the ability to accurately model these tumours ex vivo; however, currently available models are limited. Moreover, the importance of the extracellular matrix in disease development and progression should be heavily considered when modelling the disease. This review will outline the most clinically relevant disease models of pNETs and challenges in their use, as well as recent advances and future directions in their modelling.Pancreatic neuroendocrine tumours (pNETs) are a heterogeneous group of epithelial tumours with neuroendocrine differentiation. Although rare , they are the second most common group of pancreatic neoplasms after pancreatic ductal adenocarcinoma (PDAC). pNET incidence is however on the rise and patient outcomes, although variable, have been linked with 5-year survival rates as low as 40%. Improvement of diagnostic and treatment modalities strongly relies on disease models that reconstruct the disease ex vivo. A key constraint in pNET research, however, is the absence of human pNET models that accurately capture the original tumour phenotype. In attempts to more closely mimic the disease in its native environment, three-dimensional culture models as well as in vivo models, such as genetically engineered mouse models (GEMMs), have been developed. Despite adding significant contributions to our understanding of more complex biological processes associated with the development and progression of pNETs, factors such as ethical considerations and low rates of clinical translatability limit their use. Furthermore, a role for the site-specific extracellular matrix (ECM) in disease development and progression has become clear. Advances in tissue engineering have enabled the use of tissue constructs that are designed to establish disease ex vivo within a close to native ECM that can recapitulate tumour-associated tissue remodelling. Yet, such advanced models for studying pNETs remain underdeveloped. This review summarises the most clinically relevant disease models of pNETs currently used, as well as future directions for improved modelling of the disease. Pancreatic neuroendocrine tumours (pNETs) are the second most common subtype of primary pancreatic tumours and represent around 1–2% of pancreatic neoplasms despite their relative rarity  ,4. pNETsPreclinical disease models offer platforms for studying the complex tumour biology and pathophysiology as well as the effects of different drug treatments in a controlled environment. Popular models include cell lines, patient derived xenograft cells and animal models ), while three-dimensional (3D) models have been a recent focus of attempts to better mimic the in vivo like behaviour of cancer cells and allow the study of complex molecular interactions in cancers .Surrogate models for studying diseases are pivotal yet are not easy to construct in a way that completely recapitulates the complex physiology and cell–cell interactions with both cancerous and noncancerous cells of the tumour microenvironment. Models for studying pNETs are scarce, due in part to the rarity of the disease. However, considering the rise in incidence and the often poor outcomes for patients diagnosed with advanced disease, ongoing development of such models to better mimic and reflect the biology of these tumours are warranted. In this review we summarise the most clinically relevant models of pNETs in current use.Available established pNET cell lines include four human, three mouse and two rat cell lines. Animal cell lines have mostly been used for the purpose of studying islet hormone production and their regulation mechanisms in the settings of diabetes mellitus , rather The most widely used human cell lines are BON-1 and QGP-1. The BON-1 cell line was isolated from a pancreatic serotonin-producing metastatic carcinoid present in a lymph node excised from a 28-year-old male patient , while QQGP-1 and BON-1 cells have both been used for the study of molecular processes associated with tumourigenesis  and the effects of their inhibition on disease progression. Both cell lines have been key to studies of current first-line treatments, such as everolimus  and somatostatin analogues for treating gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs), as well as studies aimed at overcoming drug resistance to these using novel PI3K/mTOR pathway inhibitors ,12,13,14Additional human pNET cell lines include the CM and NT-3 lines. The CM cell line was isolated from the ascitic fluid of a patient with a primary pNET; due to the high resemblance to pancreatic β-cells , it has been used for the study of β-cell function . More reRecent studies aimed at mapping the genomic landscape of BON-1 and QGP-1 cells, however, raise certain issues with respect to their true origin and epithelial/pancreatic-endocrine phenotype.P53, TSC2 and NRAS mutations alongside aberrant activation of the mTOR/Akt pathways, while QGP-1 cells harbour mutations in P53, ATRX and SMAD4, both showing only partial resemblance to commonly occurring pNET genetic mutations [BON-1 cells bear utations . With reutations , and thautations . Luley eutations . While autations . Lastly,utations . These futations ,21,22.Two-dimensional (2D) cell culture systems do not take into account the biology and physiology of the original tissue structure. Instead, cells are typically grown in monolayers leading to polarised cell adhesion, as opposed to the three-dimensional (3D) environment of neoplasms. Consequently, gene expression patterns, the complex cell-to-cell interactions and mechanical and biochemical cell signals that occur in vivo are significantly modified ,24. PharFurther attempts to create a more realistic tumour environment have led to the popular use of orthotopic transplant models. Implantation of BON-1 and QGP-1 cells in immunocompromised mice enables their propagation as tumours that retain their neuroendocrine phenotype . An orthRecognising the above discussed shortcomings of cell lines and their questionable predictive value following modification while in long-term culture, the use of primary cells taken from biopsies obtained surgically or by endoscopic sampling has gained popularity among researchers. The culture of primary pNET cells taken from 25 tumours in bovine ECM has been described by Mohamed et al. . This nePDX models require tumour resection or sampling followed by their transplantation into animals for expansion (most commonly immunodeficient mice), and they offer the advantage of preserving the original histological and genetic tumour properties as opposed to the altered genomic landscape of established cell lines ,34. CancPDX models enable the study of cancer biology in a natural microenvironment, reflecting the tumour heterogenic nature . HoweverMouse models provide a productive platform to study pNETs as they allow simultaneous observation of small populations, which have well-characterised genotypes and a short life span, alongside the ease with which malignant lesions and their precursors can be retrieved. Moreover, advances in genetics and gene manipulation allow relatively simple induction of tumourigenesis . The rarThe RIP-Tag2 and RIP-Tag5 models are examples of genetic alteration in which the tissue-specific, transgenic expression of the oncogenic Simian virus 40 large T-antigen is directed by the rat insulin gene-2 promoter (RIP) ,44. The TAM/RIP-Bcl-xL-RIP models, transgenic stimulation and inhibition of the c-Myc oncogene and the apoptosis inhibitor Bcl-xL, respectively, promote the formation of an aggressive insulinoma subtype [RIP-Tag5 mice develop tumours at a later stage, when the host immune response to these lesions can be studied . Similar subtype .Glucagon-producing tumours can be studied using GLU-Tag2 transgenic mice which form tumours not only in the pancreas, but also in the brain . The GLU−/− model, in which glucagon receptor deletion is used to study diabetes-associated glucagon signalling and the oncogenic effects of deletion of the p53 and Rb tumour-suppressor genes in renin-expressing cells. These mice harbour renal and subcutaneous tumours together with glucagonomas [−/− mice usually remain localised, and hence the process of metastasis cannot be studied adequately in this model. To overcome this deficiency, the Gcg gfp/gfp mouse model (GCGKO), homozygous for glucagon-knockout/green fluorescent protein knock-in alleles, was developed [−/− models but also develop more invasive metastatic neuroendocrine tumours. The difference between the two models might be explained by the absence of GLP-1 expression in Gcg gfp/gfp mice, owing to its assumed role in inhibition of α-cell proliferation and subsequent hyperplasia [−/− for studying dissemination and metastasis in pNETs.Several GEMMs that were originally designed to study other disease processes but surprisingly were found to develop pNETs have been reported. These include the Gcgreveloped . Gcg gfperplasia . FurtherPrkar1afl/fl and Pdx1-CRE) is characterised by inactivation of the PRKAR1A gene, a regulator of the cAMP-dependent kinase PKA [The NfpNET GEMM . This model was developed by crossbreeding MEN-1 floxed mice (MEN-1L/L) carrying a tamoxifen-inducible Cre recombinase with mice expressing a modified oestrogen receptor [flox/flox Ptenflox/flox RIP-Cre) and MPM (MEN-1flox/flox Ptenflox/flox MIP-Cre)—characterised by loss of both MEN-1 and PTEN1 using the Cre-LoxP system. Both models developed well-differentiated insulinomas faster than control models with a single MEN-1 gene inactivation. Even though mice with a single PTEN gene loss do not develop pNETs, this gene may cooperate with MEN-1 to suppress pNET tumourigenesis. Consistent with PTEN deletion causing an aberrant activation of the mTOR pathway, treatment with the mTOR inhibitor rapamycin induced delayed tumour growth in these models.The in vivo study of MEN-1-associated pNETs is possible using MEN-1-knockout mice . The devreceptor . Wong etreceptor  subsequeThe use of 3D in vitro models has gained popularity in recent years under the assumption that these better recapitulate the complex cell-to-cell interactions that exist in tumours. Despite their reduced capture of certain features of the physiological microenvironment  that detrimentally alter cancer cell phenotypic properties ,60, a 3D3D cultures also allow control over most experimental variables compared to in vivo animal models and rely on the induction of cellular aggregate (tumour spheroid) formation that can be incorporated into or grown on top of an extracellular substitute, acting as miniature 3D models of the tumour and its immediate environment . MoreoveSpheroids are 3D culture models that are grown under nonadherent conditions, with their use going back to the early 1970s . Tumour Cellular spheroids are concentrically arranged into several layers —an exterSpheroid models of pancreatic ductal adenocarcinomas have previously demonstrated higher chemoresistance compared to conventional monolayer platforms using the MiaPaca2 and PANC-1 cell lines . The useOrganoid and spheroid cultures have gained significant popularity in recent years as disease models for the study of organ development, tumour biology, drug screening and toxicity . The terDespite their advantage over traditional monolayer cultures, spheroid and organoid cultures neglect the role of the tumour microenvironment in disease progression. The ECM is a complex network of structural and signalling protein, also consisting of proteoglycans which serve not only as a support framework in different tissues but also as significant participants in mechanical and biochemical signalling mediated by cell surface receptors ,92. ECM Noncancerous supporting cells promote cancer cell survival and tumour structure and function, and they are associated with the chemoresistance that challenges effective cancer treatment .Cancer-associated stromal cells are also able to modulate the antitumour immune response by interacting with immune cells by secreting chemokines that have an immunosuppressive as well as proapoptotic role against T-cells .The use of animal and 3D culture models has enabled researchers not only to study genetic aberrations leading to pNET formation and the efficacy of novel therapeutic agents, but also to study the close interaction between tumour cells and their microenvironment. In vitro cancer disease models have also been significantly modified to include both artificial and natural ECM components, as well as a variety of cancer-supporting cells, in attempts to study cancer development, progression and response to treatment under more realistic biological conditions.Hpa-Tg), which constitutively overexpress heparinase, and heparanase-knockout mice (−/−Hpse) [Studies looking into the close interaction between NETs and their stroma have revealed that NETs are one of the highly vascularised cancers and are 10-fold more vascularised compared to epithelial-cell-derived carcinomas . NET celHpse−/−) .Products of ECM degradation produced by proteolytic activity of enzymes such as MMPs are collectively called matrikines . ModulatDespite the overall benefit gained so far, the use of animal models raises ethical issues around subjecting animals to discomfort and death. Moreover, such models are limited in their ability to represent complex biological processes around cancer physiology, development and progression in humans due to interspecies differences in genetics and biology, as well as variability in their expression of molecular targets . AlongsiAdvances in proteomics, more specifically ‘bottom-up’ proteomics where samples are enzymatically digested into peptides prior to analysis using mass spectrometry, enable quantitative profiling of the complex in vivo ECM protein composition . DiffereTissue decellularisation can also be applied to construct platforms for in vitro 3D disease modelling of cancers within their native tissue. When designing culture models, it is not only the ability to combine known components of the ECM, such as collagens, fibronectin, proteoglycans and growth factors, that is key. The association, concentrations and ratios of ECM components play an important role in preserving its function and unique characteristics that will support cellular processes, growth and function ,119. TheNeuroendocrine tumours of the pancreas are a rare entity, yet their incidence is on the rise. The often poor outcomes associated with pNETs, however, warrant improvement in their management and therefore support further research of the disease. Disease models are pivotal in ex vivo studies of biological and molecular pathogenetic processes in cancer and have evolved over the last few decades, with technological advances allowing the development of more complex platforms.Traditional 2D monolayer cultures of established cell lines were key in preclinical drug development stages that resulted in approval of current first-line agents for treatment of pNETs such as sunitinib. Such models, however, are insufficient for studying more complex genetic characteristics and overlook the in vivo like effects of the tumour environment on disease progression and chemoresistance. Moreover, they do not align with the current shift in research toward a more personalised treatment approach. Models that consider the in vivo like cell–cell interactions and spatial arrangement, such as spheroids, allow for more realistic studies preserving to some extent oxygen and nutrient gradients that are observed in malignant masses. With respect to neuroendocrine cancer of pancreatic origin, reports of spheroid cultures have mostly focused on model characterisation while observing specific cell processes, investigating molecular signalling  and conducting in vitro drug screening.Organoids that are derived directly from patient biopsies more closely resemble the tumour of origin than 2D cell lines and maintain genetic expression and aberrations while preserving the tumour architecture yet are technically more challenging. Despite a recent trend toward organoid-based research in other cancer types, reports of NET-derived organoids are lacking. This is mostly attributed to the rarity of the disease and the technical challenges (including slow tumour growth rates) involved in culture of pNET primary cells. Since organoids have been used for the study of embryonic tissue development, the study of the role of cancer stem cells, as well as cancer-specific processes, is also likely to prove valuable for studying the development and progression of NETs. A very recent study by April-Monn et al. highlighted the potential of organoid models as a personalised drug screening approach performed on primary pNET cultures .The ability to artificially induce known associated genetic aberrations which lead to tumour formation made GEMMs an attractive platform for studying pNETs , which have been extensively linked with genetic syndromes and various signalling pathway alterations. Transgenic mouse models in which MEN-1 and PTEN gene knockouts are induced (resembling mutations identified in a subset of pNETs) are therefore extremely valuable. Animal models also enable the study of cancer cell–ECM interactions, as tumours are observed within their supporting tissue. However, the low clinical translatability rates of findings in animals could perhaps be improved by using tissue constructs obtained from surgical resections and from transplant and deceased donors. Decellularised tissue depleted of resident cells retains the ECM components that confer important features such as physiological substrate stiffness and protein signatures of the native tissue. In the context of studies involving the pancreatic endocrine system, such existing models mostly focus on tissue regeneration and islet transplantation for the treatment of diabetes. The combination of such tissue constructs with primary patient cancer cells has the potential to provide a more accurate ex vivo platform to study cancer-induced ECM remodelling.


Cases of COVID-19 are rising quickly on the African continent. A critical element of any health system response to such a surge of active cases is the existence of functional emergency care systems. Yet, these systems are markedly underdeveloped in African countries. This short letter reviews the key role emergency medicine plays in epidemic disease response and actions that ministries of health can take now to shore up gaps in emergency care capacity to avoid needless death and suffering of COVID-19 patients. In recent weeks, there has been a large rise in documented coronavirus disease (COVID-19) cases, with over 1 million current cases and 23 000 deaths.3During the current pandemic, emergency departments have been highlighted as mission critical locations to screen for syndromic disease, isolate and protect patients and health care workers, triage, and provide immediate care for emergency conditions associated with COVID-19, such as respiratory failure and shock. While emergency care systems are necessary for a successful health sector response, they remain inadequately supported in low- and middle-income countries. A review of 59 countries highlighted the major limitations of emergency care delivery: markedly higher mortality rates than high-income countries and inadequate training across all cadres of health care providers.4During epidemics, weak emergency care systems can become overwhelmed by increased demand or directly compromised by the impact of the outbreak. When service delivery is undermined, both direct disease mortality and preventable mortality from everyday emergency conditions can increase dramatically. This is especially true of emergent health conditions that rely on skilled health personnel, medicine, and equipment for treatment. During the 2014 Ebola outbreak, strains on the overall health system led to excess mortality from non-Ebola-related conditions, including malaria and emergency obstetric conditions.5Targeted capacity augmentation for emergency departments is necessary now to avoid excess mortality from the expected surge in COVID-19 cases in Africa. As Africa braces for a possible explosion of COVID-19 cases in the coming months, excess mortality is not inevitable. Timely, strategic implementation of targeted emergency care solutions in African countries can help avoid needless human suffering and death.