Wilms' tumour (WT) is a pediatric tumor of the kidney that arises via failure of the fetal developmental program. The absence of identifiable mutations in the majority of WTs suggests the frequent involvement of epigenetic aberrations in WT. We therefore conducted a genome-wide analysis of promoter hypermethylation in WTs and identified hypermethylation at chromosome 5q31 spanning 800 kilobases (kb) and more than 50 genes. The methylated genes all belong to alpha-, beta-, and gamma-protocadherin (PCDH) gene clusters (Human Genome Organization nomenclature PCDHA@, PCDHB@, and PCDHG@, respectively). This demonstrates that long-range epigenetic silencing (LRES) occurs in developmental tumors as well as in adult tumors. Bisulfite polymerase chain reaction analysis showed that PCDH hypermethylation is a frequent event found in all Wilms' tumor subtypes. Hypermethylation is concordant with reduced PCDH expression in tumors. WT precursor lesions showed no PCDH hypermethylation, suggesting that de novo PCDH hypermethylation occurs during malignant progression. Discrete boundaries of the PCDH domain are delimited by abrupt changes in histone modifications; unmethylated genes flanking the LRES are associated with permissive marks which are absent from methylated genes within the domain. Silenced genes are marked with non-permissive histone 3 lysine 9 dimethylation. Expression analysis of embryonic murine kidney and differentiating rat metanephric mesenchymal cells demonstrates that Pcdh expression is developmentally regulated and that Pcdhg@ genes are expressed in blastemal cells. Importantly, we show that PCDHs negatively regulate canonical Wnt signalling, as short-interfering RNA-induced reduction of PCDHG@ encoded proteins leads to elevated beta-catenin protein, increased beta-catenin/T-cell factor (TCF) reporter activity, and induction of Wnt target genes. Conversely, over-expression of PCDHs suppresses beta-catenin/TCF-reporter activity and also inhibits colony formation and growth of cancer cells in soft agar. Thus PCDHs are candidate tumor suppressors that modulate regulatory pathways critical in development and disease, such as canonical Wnt signaling.

The conversion of auditory and vestibular stimuli into electrical signals is initiated by force transmitted to a mechanotransduction channel through the tip link, a double stranded protein filament held together by two adhesion bonds in the middle. Although thought to form a relatively static structure, the dynamics of the tip-link connection has not been measured. Here, we biophysically characterize the strength of the tip-link connection at single-molecule resolution. We show that a single tip-link bond is more mechanically stable relative to classic cadherins, and our data indicate that the double stranded tip-link connection is stabilized by single strand rebinding facilitated by strong cis-dimerization domains. The measured lifetime of seconds suggests the tip-link is far more dynamic than previously thought. We also show how Ca2+ alters tip-link lifetime through elastic modulation and reveal the mechanical phenotype of a hereditary deafness mutation. Together, these data show how the tip link is likely to function during mechanical stimuli.

The brain is protected against invading organisms and other unwanted substances by tightly regulated barriers. However, these central nervous system (CNS) barriers impede the delivery of drugs into the brain via the blood circulation and are therefore considered major hurdles in the treatment of neurological disorders. Consequently, there is a high need for efficient delivery systems that are able to cross these strict barriers. While most research focuses on the blood-brain barrier (BBB), the design of drug delivery platforms that are able to cross the blood-cerebrospinal fluid (CSF) barrier, formed by a single layer of choroid plexus epithelial cells, remains a largely unexplored domain. The discovery that extracellular vesicles (EVs) make up a natural mechanism for information transfer between cells and across cell layers, has stimulated interest in their potential use as drug delivery platform. Here, we report that choroid plexus epithelial cell-derived EVs exhibit the capacity to home to the brain after peripheral administration. Moreover, these vesicles are able to functionally deliver cargo into the brain. Our findings underline the therapeutic potential of choroid plexus-derived EVs as a brain drug delivery vehicle via targeting of the blood-CSF interface.

Extracellular matrix (ECM) deposition after central nervous system (CNS) injury leads to inhibitory scarring in humans and other mammals, whereas it facilitates axon regeneration in the zebrafish. However, the molecular basis of these different fates is not understood. Here, we identify small leucine-rich proteoglycans (SLRPs) as a contributing factor to regeneration failure in mammals. We demonstrate that the SLRPs chondroadherin, fibromodulin, lumican, and prolargin are enriched in rodent and human but not zebrafish CNS lesions. Targeting SLRPs to the zebrafish injury ECM inhibits axon regeneration and functional recovery. Mechanistically, we find that SLRPs confer mechano-structural properties to the lesion environment that are adverse to axon growth. Our study reveals SLRPs as inhibitory ECM factors that impair axon regeneration by modifying tissue mechanics and structure, and identifies their enrichment as a feature of human brain and spinal cord lesions. These findings imply that SLRPs may be targets for therapeutic strategies to promote CNS regeneration.

Developmental programming links growth in early life with health status in adulthood. Although environmental factors such as maternal diet can influence the growth and adult health status of offspring, the genetic influences on this process are poorly understood. Using the mouse as a model, we identify the imprinted gene Grb10 as a mediator of nutrient supply and demand in the postnatal period. The combined actions of Grb10 expressed in the mother, controlling supply, and Grb10 expressed in the offspring, controlling demand, jointly regulate offspring growth. Furthermore, Grb10 determines the proportions of lean and fat tissue during development, thereby influencing energy homeostasis in the adult. Most strikingly, we show that the development of normal lean/fat proportions depends on the combined effects of Grb10 expressed in the mother, which has the greater effect on offspring adiposity, and Grb10 expressed in the offspring, which influences lean mass. These distinct functions of Grb10 in mother and pup act complementarily, which is consistent with a coadaptation model of imprinting evolution, a model predicted but for which there is limited experimental evidence. In addition, our findings identify Grb10 as a key genetic component of developmental programming, and highlight the need for a better understanding of mother-offspring interactions at the genetic level in predicting adult disease risk.

Time spent sitting is associated with negative health outcomes, motivating some individuals to adopt standing desk workstations. This study represents the first investigation of the effects of standing desk use on reading comprehension and creativity. In a counterbalanced, within-subjects design, 96 participants completed reading comprehension and creativity tasks while both sitting and standing. Participants self-reported their mood during the tasks and also responded to measures of expended effort and task difficulty. In addition, participants indicated whether they expected that they would perform better on work-relevant tasks while sitting or standing. Despite participants' beliefs that they would perform worse on most tasks while standing, body position did not affect reading comprehension or creativity performance, nor did it affect perceptions of effort or difficulty. Mood was also unaffected by position, with a few exceptions: Participants exhibited greater task engagement (i.e., interest, enthusiasm, and alertness) and less comfort while standing rather than sitting. In sum, performance and psychological experience as related to task completion were nearly entirely uninfluenced by acute (~30-min) standing desk use.

Imprinted genes, defined by their preferential expression of a single parental allele, represent a subset of the mammalian genome and often have key roles in embryonic development, but also postnatal functions including energy homeostasis and behaviour. When the two parental alleles are unequally represented within a social group (when there is sex bias in dispersal and/or variance in reproductive success), imprinted genes may evolve to modulate social behaviour, although so far no such instance is known. Predominantly expressed from the maternal allele during embryogenesis, Grb10 encodes an intracellular adaptor protein that can interact with several receptor tyrosine kinases and downstream signalling molecules. Here we demonstrate that within the brain Grb10 is expressed from the paternal allele from fetal life into adulthood and that ablation of this expression engenders increased social dominance specifically among other aspects of social behaviour, a finding supported by the observed increase in allogrooming by paternal Grb10-deficient animals. Grb10 is, therefore, the first example of an imprinted gene that regulates social behaviour. It is also currently alone in exhibiting imprinted expression from each of the parental alleles in a tissue-specific manner, as loss of the peripherally expressed maternal allele leads to significant fetal and placental overgrowth. Thus Grb10 is, so far, a unique imprinted gene, able to influence distinct physiological processes, fetal growth and adult behaviour, owing to actions of the two parental alleles in different tissues.

Since Haldane first noticed an excess of paternally derived mutations, it has been considered that most mutations derive from errors during germ line replication. Miyata et al. (1987) proposed that differences in the rate of neutral evolution on X, Y, and autosome can be employed to measure the extent of this male bias. This commonly applied method assumes replication to be the sole source of between-chromosome variation in substitution rates. We propose a simple test of this assumption: If true, estimates of the male bias should be independent of which two chromosomal classes are compared. Prior evidence from rodents suggested that this might not be true, but conclusions were limited by a lack of rat Y-linked sequence. We therefore sequenced two rat Y-linked bacterial artificial chromosomes and determined evolutionary rate by comparison with mouse. For estimation of rates we consider both introns and synonymous rates. Surprisingly, for both data sets the prediction of congruent estimates of alpha is strongly rejected. Indeed, some comparisons suggest a female bias with autosomes evolving faster than Y-linked sequence. We conclude that the method of Miyata et al. (1987) has the potential to provide incorrect estimates. Correcting the method requires understanding of the other causes of substitution that might differ between chromosomal classes. One possible cause is recombination-associated substitution bias for which we find some evidence. We note that if, as some suggest, this association is dominantly owing to male recombination, the high estimates of alpha seen in birds is to be expected as Z chromosomes recombine in males.

The pooled cohort equations (PCE) predict atherosclerotic cardiovascular disease (ASCVD) risk in patients with characteristics within prespecified ranges and has uncertain performance among Asians or Hispanics. It is unknown if machine learning (ML) models can improve ASCVD risk prediction across broader diverse, real-world populations. We developed ML models for ASCVD risk prediction for multi-ethnic patients using an electronic health record (EHR) database from Northern California. Our cohort included patients aged 18 years or older with no prior CVD and not on statins at baseline (n = 262,923), stratified by PCE-eligible (n = 131,721) or PCE-ineligible patients based on missing or out-of-range variables. We trained ML models [logistic regression with L2 penalty and L1 lasso penalty, random forest, gradient boosting machine (GBM), extreme gradient boosting] and determined 5-year ASCVD risk prediction, including with and without incorporation of additional EHR variables, and in Asian and Hispanic subgroups. A total of 4309 patients had ASCVD events, with 2077 in PCE-ineligible patients. GBM performance in the full cohort, including PCE-ineligible patients (area under receiver-operating characteristic curve (AUC) 0.835, 95% confidence interval (CI): 0.825-0.846), was significantly better than that of the PCE in the PCE-eligible cohort (AUC 0.775, 95% CI: 0.755-0.794). Among patients aged 40-79, GBM performed similarly before (AUC 0.784, 95% CI: 0.759-0.808) and after (AUC 0.790, 95% CI: 0.765-0.814) incorporating additional EHR data. Overall, ML models achieved comparable or improved performance compared to the PCE while allowing risk discrimination in a larger group of patients including PCE-ineligible patients. EHR-trained ML models may help bridge important gaps in ASCVD risk prediction.

The dimeric two-pore OSCA/TMEM63 family has recently been identified as mechanically activated ion channels. Previously, based on the unique features of the structure of OSCA1.2, we postulated the potential involvement of several structural elements in sensing membrane tension 1 . Interestingly, while OSCA1, 2, and 3 clades are activated by membrane stretch in cell-attached patches (i.e., they are stretch-activated channels), they differ in their ability to transduce membrane deformation induced by a blunt probe (poking). In an effort to understand the domains contributing to mechanical signal transduction, we used cryo-electron microscopy to solve the structure of Arabidopsis thaliana (At) OSCA3.1, which, unlike AtOSCA1.2, only produced stretch- but not poke-activated currents in our initial characterization 2 . Mutagenesis and electrophysiological assessment of conserved and divergent putative mechanosensitive features of OSCA1.2 reveal a selective disruption of the macroscopic currents elicited by poking without considerable effects on stretch-activated currents (SAC). Our results support the involvement of the amphipathic helix and lipid-interacting residues in the membrane fenestration in the response to poking. Our findings position these two structural elements as potential sources of functional diversity within the family.

N-cadherin (Ncad) is a classical cadherin that is implicated in several aspects of vertebrate embryonic development, including somitogenesis, heart morphogenesis, neural tube formation and establishment of left-right asymmetry. However, genetic in vivo analyses of its role during neural development have been rather limited. We report the isolation and characterization of the zebrafish parachute (pac) mutations. By mapping and candidate gene analysis, we demonstrate that pac corresponds to a zebrafish n-cadherin (ncad) homolog. Three mutant alleles were sequenced and each is likely to encode a non-functional Ncad protein. All result in a similar neural tube phenotype that is most prominent in the midbrain, hindbrain and the posterior spinal cord. Neuroectodermal cell adhesion is altered, and convergent cell movements during neurulation are severely compromised. In addition, many neurons become progressively displaced along the dorsoventral and the anteroposterior axes. At the cellular level, loss of Ncad affects beta-catenin stabilization/localization and causes mispositioned and increased mitoses in the dorsal midbrain and hindbrain, a phenotype later correlated with enhanced apoptosis and the appearance of ectopic neurons in these areas. Our results thus highlight novel and crucial in vivo roles for Ncad in the control of cell convergence, maintenance of neuronal positioning and dorsal cell proliferation during vertebrate neural tube development.

The mitotic spindle is an essential molecular machine involved in cell division, whose composition has been studied extensively by detailed cellular biology, high-throughput proteomics, and RNA interference experiments. However, because of its dynamic organization and complex regulation it is difficult to obtain a complete description of its molecular composition. We have implemented an integrated computational approach to characterize novel human spindle components and have analysed in detail the individual candidates predicted to be spindle proteins, as well as the network of predicted relations connecting known and putative spindle proteins. The subsequent experimental validation of a number of predicted novel proteins confirmed not only their association with the spindle apparatus but also their role in mitosis. We found that 75% of our tested proteins are localizing to the spindle apparatus compared to a success rate of 35% when expert knowledge alone was used. We compare our results to the previously published MitoCheck study and see that our approach does validate some findings by this consortium. Further, we predict so-called "hidden spindle hub", proteins whose network of interactions is still poorly characterised by experimental means and which are thought to influence the functionality of the mitotic spindle on a large scale. Our analyses suggest that we are still far from knowing the complete repertoire of functionally important components of the human spindle network. Combining integrated bio-computational approaches and single gene experimental follow-ups could be key to exploring the still hidden regions of the human spindle system.

RASSF7, a member of the N-terminal Ras association domain family, has increased expression in various cancers and, on the basis of our previous work in Xenopus embryos, may be a regulator of mitosis. In the present study, we address, for the first time, the role of human RASSF7 in mitosis. We demonstrate that RASSF7 is expressed in a broad range of different cell types and that this expression could be enhanced following exposure to hypoxia. Knocking down RASSF7 in human cell lines inhibited cell growth and induced defects in mitosis, including aberrant spindle formation and a failure in chromosomal congression. In order to understand the molecular basis of the defects in more detail, we analysed the activity of mitotic signalling proteins and found that activation of Aurora B did not occur in cells in which RASSF7 was knocked down. We also show that endogenous RASSF7 protein localizes to the centrosome and demonstrate using microtubule-regrowth assays that RASSF7 is an important regulator of microtubule dynamics. On the basis of these observations, we propose that, owing to its key role in regulating the microtubule cytoskeleton, RASSF7 is required for mitosis in human cells.

A major challenge lies in understanding the complexities of gene regulation. Mutation of the transcription factor SOX10 is associated with several human diseases. The disease phenotypes reflect the function of SOX10 in diverse tissues including the neural crest, central nervous system and otic vesicle. As expected, the SOX10 expression pattern is complex and highly dynamic, but little is known of the underlying mechanisms regulating its spatiotemporal pattern. SOX10 expression is highly conserved between all vertebrates characterised.

A fundamental problem in developmental biology concerns how multipotent precursors choose specific fates. Neural crest cells (NCCs) are multipotent, yet the mechanisms driving specific fate choices remain incompletely understood. Sox10 is required for specification of neural cells and melanocytes from NCCs. Like sox10 mutants, zebrafish shady mutants lack iridophores; we have proposed that sox10 and shady are required for iridophore specification from NCCs. We show using diverse approaches that shady encodes zebrafish leukocyte tyrosine kinase (Ltk). Cell transplantation studies show that Ltk acts cell-autonomously within the iridophore lineage. Consistent with this, ltk is expressed in a subset of NCCs, before becoming restricted to the iridophore lineage. Marker analysis reveals a primary defect in iridophore specification in ltk mutants. We saw no evidence for a fate-shift of neural crest cells into other pigment cell fates and some NCCs were subsequently lost by apoptosis. These features are also characteristic of the neural crest cell phenotype in sox10 mutants, leading us to examine iridophores in sox10 mutants. As expected, sox10 mutants largely lacked iridophore markers at late stages. In addition, sox10 mutants unexpectedly showed more ltk-expressing cells than wild-type siblings. These cells remained in a premigratory position and expressed sox10 but not the earliest neural crest markers and may represent multipotent, but partially-restricted, progenitors. In summary, we have discovered a novel signalling pathway in NCC development and demonstrate fate specification of iridophores as the first identified role for Ltk.

Imprinted genes are highly expressed in monoaminergic regions of the midbrain and their functions in this area are thought to have an impact on mammalian social behaviors. One such imprinted gene is Grb10, of which the paternal allele is generally recognized as mediating social dominance behavior. However, there has been no detailed study of social dominance in Grb10 +/p mice. Moreover, the original study examined tube-test behavior in isolated mice 10 months of age. Isolation testing favors more territorial and aggressive behaviors, and does not address social dominance strategies employed in group housing contexts. Furthermore, isolation stress impacts midbrain function and dominance related behavior, often through alterations in monoaminergic signaling. Thus, we undertook a systematic study of Grb10 +/p social rank and dominance behavior within the cage group, using a number of convergent behavioral tests. We examined both male and female mice to account for sex differences and tested cohorts aged 2, 6 and 10 months to examine any developments related to age. We found group-housed Grb10 +/p mice do not show evidence of enhanced social dominance, but cages containing Grb10 +/p and wild-type mice lacked the normal correlation between three different measures of social rank. Moreover, a separate study indicated isolation stress induced inconsistent changes in tube test behavior. Taken together, these data suggest future research on Grb10 +/p mice should focus on the stability of social behaviors, rather than dominance per se.

Identifying high-risk patients is crucial for effective cardiovascular disease (CVD) prevention. It is not known whether electronic health record (EHR)-based machine-learning (ML) models can improve CVD risk stratification compared with a secondary prevention risk score developed from randomised clinical trials (Thrombolysis in Myocardial Infarction Risk Score for Secondary Prevention, TRS 2°P).

Lepidopteran stemborers are among the most damaging agricultural pests worldwide, able to reduce crop yields by up to 40%. Sugarcane is the world's most prolific crop, and several stemborer species from the families Noctuidae, Tortricidae, Crambidae and Pyralidae attack sugarcane. Australia is currently free of the most damaging stemborers, but biosecurity efforts are hampered by the difficulty in morphologically distinguishing stemborer species. Here we assess the utility of DNA barcoding in identifying stemborer pest species. We review the current state of the COI barcode sequence library for sugarcane stemborers, assembling a dataset of 1297 sequences from 64 species. Sequences were from specimens collected and identified in this study, downloaded from BOLD or requested from other authors. We performed species delimitation analyses to assess species diversity and the effectiveness of barcoding in this group. Seven species exhibited <0.03 K2P interspecific diversity, indicating that diagnostic barcoding will work well in most of the studied taxa. We identified 24 instances of identification errors in the online database, which has hampered unambiguous stemborer identification using barcodes. Instances of very high within-species diversity indicate that nuclear markers (e.g. 18S, 28S) and additional morphological data (genitalia dissection of all lineages) are needed to confirm species boundaries.

There is a significant drive to identify alternative materials that exhibit room temperature phosphorescence for technologies including bio-imaging, photodynamic therapy and organic light-emitting diodes. Ideally, these materials should be non-toxic and cheap, and it will be possible to control their photoluminescent properties. This was achieved here by embedding carbon nanodots within crystalline particles of alkaline earth carbonates, sulphates and oxalates. The resultant nanocomposites are luminescent and exhibit a bright, sub-second lifetime afterglow. Importantly, the excited state lifetimes, and steady-state and afterglow colours can all be systematically controlled by varying the cations and anions in the host inorganic phase, due to the influence of the cation size and material density on emissive and non-emissive electronic transitions. This simple strategy provides a flexible route for generating materials with specific, phosphorescent properties and is an exciting alternative to approaches relying on the synthesis of custom-made luminescent organic molecules.

The analysis of cross-frequency coupling (CFC) has become popular in studies involving intracranial and scalp EEG recordings in humans. It has been argued that some cases where CFC is mathematically present may not reflect an interaction of two distinct yet functionally coupled neural sources with different frequencies. Here we provide two empirical examples from intracranial recordings where CFC can be shown to be driven by the shape of a periodic waveform rather than by a functional interaction between distinct sources. Using simulations, we also present a generalized and realistic scenario where such coupling may arise. This scenario, which we term waveform-dependent CFC, arises when sharp waveforms (e.g., cortical potentials) occur throughout parts of the data, in particular if they occur rhythmically. Since the waveforms contain both low- and high-frequency components, these components can be inherently phase-aligned as long as the waveforms are spaced with appropriate intervals. We submit that such behavior of the data, which seems to be present in various cortical signals, cannot be interpreted as reflecting functional modulation between distinct neural sources without additional evidence. In addition, we show that even low amplitude periodic potentials that cannot be readily observed or controlled for, are sufficient for significant CFC to occur.