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Studies of mammalian development have advanced our understanding of the genetic, epigenetic, and cellular processes that orchestrate embryogenesis and have uncovered new insights into the unique aspects of human embryogenesis. Recent studies have now produced the first epigenetic maps of early human embryogenesis, stimulating new ideas about epigenetic reprogramming, cell fate control, and the potential mechanisms underpinning developmental plasticity in human embryos. In this review, we discuss these new insights into the epigenetic regulation of early human development and the importance of these processes for safeguarding development. We also highlight unanswered questions and key challenges that remain to be addressed.

Water fluoridation is a public health measure to reduce levels of dental caries in populations. A report of the recently completed CATFISH study has been published. This was the first UK evaluation of fluoridation introduction for many years; it was carefully designed and executed and is welcomed. The purpose of this article is to highlight the 180-page report of the study and comment on some aspects to aid interpretation of the findings. Significant features were that two cohorts, from birth and from five years, were followed for six years in a fluoridated and a non-fluoridated area, and clinical and cost analyses were reported. Areas of the report which deserve comment are: a) interruption of fluoridation for a year for half the children in the intervention area and its effect; b) clinical results were reported as absolute reduction in caries prevalence, with the preventive fraction for caries severity downplayed; c) the power of the study was diminished by an unexpected lower caries increment; and d) control for differences in diet was not possible. Nevertheless, this major UK study showed clinical and cost benefits of water fluoridation. The risk of cessation of water fluoridation is highlighted in examples of three recently published studies.

IMPORTANCE: Presenting with diabetic ketoacidosis (DKA) at onset of type 1 diabetes (T1D) remains a risk. Following a 2011 systematic review, considerable additional articles have been published, and the review required updating. OBJECTIVE: To evaluate factors associated with DKA at the onset of T1D among pediatric patients. EVIDENCE REVIEW: In this systematic review, PubMed, Embase, Scopus, CINAHL, Web of Science, and article reference lists were searched using the population, intervention, comparison, outcome search strategy for primary research studies on DKA and T1D onset among individuals younger than 18 years that were published from January 2011 to November 2021. These studies were combined with a 2011 systematic review on the same topic. Data were pooled using a random-effects model. FINDINGS: A total of 2565 articles were identified; 149 were included, along with 46 from the previous review (total 195 articles). Thirty-eight factors were identified and examined for their association with DKA at T1D onset. Factors associated with increased risk of DKA were younger age at T1D onset (<2 years vs ≥2 years; odds ratio [OR], 3.51; 95% CI, 2.85-4.32; P < .001), belonging to an ethnic minority population (OR, 0.40; 95% CI, 0.21-0.74; P = .004), and family history of T1D (OR, 0.46; 95% CI, 0.37-0.57; P < .001), consistent with the 2011 systematic review. Some factors that were not associated with DKA in the 2011 systematic review were associated with DKA in the present review (eg, delayed diagnosis: OR, 2.27; 95% CI, 1.72-3.01; P < .001). Additional factors associated with risk of DKA among patients with new-onset T1D included participation in screening programs (OR, 0.35; 95% CI, 0.21-0.59; P < .001) and presentation during the COVID-19 pandemic (OR, 2.32; 95% CI, 1.76-3.06; P < .001). CONCLUSIONS AND RELEVANCE: In this study, age younger than 2 years at T1D onset, belonging to an ethnic minority population, delayed diagnosis or misdiagnosis, and presenting during the COVID-19 pandemic were associated with increased risk of DKA. Factors associated with decreased risk of DKA included greater knowledge of key signs or symptoms of DKA, such as a family history of T1D or participation in screening programs. Future work should focus on identifying and implementing strategies related to these factors to reduce risk of DKA among new patients with T1D.

Human naive pluripotent stem cells have unrestricted lineage potential. Underpinning this property, naive cells are thought to lack chromatin-based lineage barriers. However, this assumption has not been tested. Here we define the chromatin-associated proteome, histone post-translational modifications and transcriptome of human naive and primed pluripotent stem cells. Our integrated analysis reveals differences in the relative abundance and activities of distinct chromatin modules. We identify a strong enrichment of polycomb repressive complex 2 (PRC2)-associated H3K27me3 in the chromatin of naive pluripotent stem cells and H3K27me3 enrichment at promoters of lineage-determining genes, including trophoblast regulators. PRC2 activity acts as a chromatin barrier restricting the differentiation of naive cells towards the trophoblast lineage, whereas inhibition of PRC2 promotes trophoblast-fate induction and cavity formation in human blastoids. Together, our results establish that human naive pluripotent stem cells are not epigenetically unrestricted, but instead possess chromatin mechanisms that oppose the induction of alternative cell fates.

Dissociative seizures are paroxysmal events resembling epilepsy or syncope with characteristic features that allow them to be distinguished from other medical conditions. We aimed to compare the effectiveness of cognitive behavioural therapy (CBT) plus standardised medical care with standardised medical care alone for the reduction of dissociative seizure frequency.

Formation of the three primary germ layers during gastrulation is an essential step in the establishment of the vertebrate body plan and is associated with major transcriptional changes1–5. Global epigenetic reprogramming accompanies these changes6–8, but the role of the epigenome in regulating early cell-fate choice remains unresolved, and the coordination between different molecular layers is unclear. Here we describe a single-cell multi-omics map of chromatin accessibility, DNA methylation and RNA expression during the onset of gastrulation in mouse embryos. The initial exit from pluripotency coincides with the establishment of a global repressive epigenetic landscape, followed by the emergence of lineage-specific epigenetic patterns during gastrulation. Notably, cells committed to mesoderm and endoderm undergo widespread coordinated epigenetic rearrangements at enhancer marks, driven by ten-eleven translocation (TET)-mediated demethylation and a concomitant increase of accessibility. By contrast, the methylation and accessibility landscape of ectodermal cells is already established in the early epiblast. Hence, regulatory elements associated with each germ layer are either epigenetically primed or remodelled before cell-fate decisions, providing the molecular framework for a hierarchical emergence of the primary germ layers.

Transcriptional enhancers, including super-enhancers (SEs), form physical interactions with promoters to regulate cell-type-specific gene expression. SEs are characterized by high transcription factor occupancy and large domains of active chromatin, and they are commonly assigned to target promoters using computational predictions. How promoter-SE interactions change upon cell state transitions, and whether transcription factors maintain SE interactions, have not been reported. Here, we used promoter-capture Hi-C to identify promoters that interact with SEs in mouse embryonic stem cells (ESCs). We found that SEs form complex, spatial networks in which individual SEs contact multiple promoters, and a rewiring of promoter-SE interactions occurs between pluripotent states. We also show that long-range promoter-SE interactions are more prevalent in ESCs than in epiblast stem cells (EpiSCs) or Nanog-deficient ESCs. We conclude that SEs form cell-type-specific interaction networks that are partly dependent on core transcription factors, thereby providing insights into the gene regulatory organization of pluripotent cells.

Sex chromosome dosage compensation is essential in most metazoans, but the developmental timing and underlying mechanisms vary significantly, even among placental mammals. Here we identify human-specific mechanisms regulating X chromosome activity in early embryonic development. Single-cell RNA sequencing and imaging revealed co-activation and accumulation of the long noncoding RNAs (lncRNAs) XACTand XISTon active X chromosomes in both early human pre-implantation embryos and naive human embryonic stem cells. In these contexts, the XISTRNA adopts an unusual, highly dispersed organization, which may explain why it does not trigger X chromosome inactivation at this stage. Functional studies in transgenic mouse cells show that XACTinfluences XISTaccumulation in cis. Our findings therefore suggest a mechanism involving antagonistic activity of XISTand XACTin controlling X chromosome activity in early human embryos, and they highlight the contribution of rapidly evolving lncRNAs to species-specific developmental mechanisms.

Through the histone methyltransferase EZH2, the Polycomb complex PRC2 mediates H3K27me3 and is associated with transcriptional repression. PRC2 regulates cell-fate decisions in model organisms; however, its role in regulating cell differentiation during human embryogenesis is unknown. Here, we report the characterization of EZH2-deficient human embryonic stem cells (hESCs). H3K27me3 was lost upon EZH2deletion, identifying an essential requirement for EZH2 in methylating H3K27 in hESCs, in contrast to its non-essential role in mouse ESCs. Developmental regulators were derepressed in EZH2-deficient hESCs, and single-cell analysis revealed an unexpected acquisition of lineage-restricted transcriptional programs. EZH2-deficient hESCs show strongly reduced self-renewal and proliferation, thereby identifying a more severe phenotype compared to mouse ESCs. EZH2-deficient hESCs can initiate differentiation toward developmental lineages; however, they cannot fully differentiate into mature specialized tissues. Thus, EZH2is required for stable ESC self-renewal, regulation of transcriptional programs, and for late-stage differentiation in this model of early human development.

ABSTRACTPluripotent cells are characterized by a globally open and accessible chromatin organization that is thought to contribute to cellular plasticity and developmental decision-making. We recently identified the pluripotency factor Nanogas a key regulator of this form of chromatin architecture in mouse embryonic stem cells. In particular, we demonstrated that the transcription factors Nanogand Sall1co-dependently mediate the epigenetic state of pericentromeric heterochromatin to reinforce a more open and accessible organization in pluripotent cells. Here, we summarize our main findings and place the work into a broader context. We explore how heterochromatin domains could be targets of transcriptional networks in pluripotent cells and are coordinated with cell state. We propose this integration may be to balance the requirement for a dynamic and plastic chromatin organization in pluripotent cells, together with priming for a more restrictive nuclear compartmentalization that is triggered rapidly upon lineage commitment.

Discusses critically a recently published review of the effectiveness of water fluoridation.Highlights the lost opportunity to evaluate the vast majority of recent studies on water fluoridation to answer the research questions.Suggests modern and appropriate methods for systematic evaluation of the effectiveness of water fluoridation.

This paper indicates the need for diet advice to be part of dental practice, the evidence for that advice, and what the advice should be for various age groups

In primates, the amnion emerges through cavitation of the epiblast during implantation, whereas in other species it does so later at gastrulation by the folding of the ectoderm. How the mechanisms of amniogenesis diversified during evolution remains unknown. Unexpectedly, single-cell analysis of primate embryos uncovered two transcriptionally and temporally distinct amniogenesis waves. To study this, we employed the naive-to-primed transition of human pluripotent stem cells (hPSCs) to model peri-implantation epiblast development. Partially primed hPSCs transiently gained the ability to differentiate into cavitating epithelium that transcriptionally and morphologically matched the early amnion, whereas fully primed hPSCs produced cells resembling the late amnion instead, thus recapitulating the two independent differentiation waves. The early wave follows a trophectoderm-like pathway and encompasses cavitation, whereas the late wave resembles an ectoderm-like route during gastrulation. The discovery of two independent waves explains how amniogenesis through cavitation could emerge during evolution via duplication of the pre-existing trophectoderm program.

In order to exploit the exceptional potential of human embryonic stem cells (hESCs) incell-replacement therapies, the genetic and epigenetic factors controlling early humandevelopment must be better defined. Limitations in human embryonic material restrict thescale of studies that can be performed, and therefore an in vitro model in which to studyepigenetic regulation in human pre-implantation cell types would be desirable. HESCscould provide such a model, but since they are derived from a stage in mammaliandevelopment when the genome is undergoing global epigenetic remodelling, it is unclearwhether their epigenetic status would be stable or subject to variation. Herein, we discussrecent work that examines allele-specific imprinted gene expression and methylationpatterns, thereby demonstrating that hESCs maintain a substantial degree of epigeneticstability during culture. Therefore, we suggest that hESCs could provide a model forstudying epigenetic regulation during the early stages of human cellular pluripotency anddifferentiation. Furthermore, we propose specific experiments using such a model toaddress important questions pertaining to epigenetic mechanisms of certain humandisorders.

To examine the cerebral structure of 14 patients with partial seizures and acquired lesions, 20 patients with malformations of cortical development (MCDs), and 45 patients with partial seizures and normal conventional MRI using whole-brain T2 mapping and statistical parametric mapping (SPM).

To test the hypothesis that magnetization transfer imaging (MTI), analyzed on a voxel-by-voxel basis, would identify areas of abnormal magnetization transfer ratio (MTR) in patients with focal epilepsy.

Band heterotopia is a malformation of cortical development characterized by bands of gray matter in the white matter parallel to the surface of the neocortex. Histopathological studies have suggested that small white matter tracts pass through the heterotopia, and functional magnetic resonance imaging studies have shown activation in the malformation. We used diffusion tractography to explore the anatomical connectivity of band heterotopia and, in particular, whether in vivo white matter tracts traverse the heterotopic gray matter. Five patients with band heterotopia and five control subjects were scanned with whole brain diffusion tensor imaging. Anisotropy maps were calculated. Using fast marching tractography, we produced maps of connectivity and tract traces from two seed points, in the splenium of the corpus callosum and the right parietal lobe. Eigenvectors were found to pass through the band heterotopia in an aligned fashion. Patterns for maps of connectivity were similar in patients and control subjects. Areas of high connectivity were found in the band heterotopia and in cortical areas on the far side of the malformation from the seed point. The tracts hence appeared to traverse or end within the band heterotopia. The results are in agreement with previous histopathological studies and indicate the structural basis of the functional connectivity and absence of focal deficits in these patients.

The investigation and management of patients after head injury must include the accurate and complete identification of cerebral damage. Using diffusion tensor imaging, abnormalities of diffusion in patients with head injuries and unremarkable MRI have been shown for the first time.

Current optimal MRI identifies a relevant structural abnormality in up to 80% of patients with refractory partial seizures. Identification of a structural lesion is fundamental to pre-surgical evaluation. We used diffusion tensor imaging (DTI) and statistical parametric mapping (SPM) to examine objectively the diffusion properties, and hence structural organization, of cerebral tissue in 10 patients with partial seizures and acquired lesions and 30 patients with partial seizures and normal MRI. Fractional anisotropy and mean diffusivity maps were calculated and, using SPM, individual patients were compared with a group of 30 control subjects. Diffusion tensor imaging and voxel-by-voxel statistical comparisons identified significant increases in diffusivity and significant reductions of anisotropy in all patients with acquired non-progressive cerebral lesions and partial seizures. In all of these patients, the areas of increased diffusivity, and in nine patients the areas of decreased anisotropy, concurred with abnormalities identified on visual inspection of conventional MRI. In addition, there were 10 areas which were normal on conventional imaging which exhibited abnormal anisotropy or diffusivity. Individual analyses of the 30 patients with partial seizures and normal optimal MRI identified a significant increase in diffusivity in eight of the subjects. In six of these, the areas of increased diffusivity concurred with the localization of epileptiform EEG abnormality. Analysis of anisotropy in the MRI-negative patients revealed significant differences in two patients, one of which concurred with electroclinical seizure localization. Group analysis of nine patients with normal conventional MRI and electroclinical seizure onset localizing to the left temporal region revealed a significant increase in diffusivity and a significant reduction in anisotropy within the white matter of the left temporal lobe. DTI analysed using SPM was sensitive in patients with acquired cerebral damage. Significant differences in the diffusion indices in individual MRI negative patients and the group effect in patients with left temporal lobe epilepsy suggest that minor structural disorganization exists in occult epileptogenic cerebral lesions. These techniques are promising, non-invasive imaging methods for identifying the cause of partial seizures, and can contribute to pre-surgical evaluation.

Malformations of cortical development (MCD) are a common cause of epilepsy. Studies of structural MRIs and PET data in patients with MCD have found widespread changes outside the visually identified lesions. The aim of this study was to investigate diffusion changes interictally in patients with MCD and to test the hypothesis that MCD would be associated with widespread abnormalities of diffusion. We used diffusion tensor imaging (DTI) and statistical parametric mapping (SPM) to compare objectively tissue organization in 22 patients with partial seizures and MCD, with 30 control subjects. Whole-brain DTI was acquired using echo planar imaging. Rotationally invariant anisotropy and diffusivity maps were calculated and, after normalization to Talairach space, each patient was compared with the group of control subjects using SPM. Areas of reduced anisotropy were found in 17 patients and areas of increased diffusivity in 10. Two patients had areas of increased anisotropy. There were no patients with reduced diffusivity. Areas of increased diffusivity were in general more extensive than areas of reduced anisotropy. Changes in tissue beyond the MCD, that appeared normal on conventional MRI, were found in six patients for anisotropy and nine patients for diffusivity. Both measurements showed widespread changes in tissue beyond the MCD, i.e. adding information to conventional MRI. Increased or abnormally located grey matter or pathological white matter with abnormal myelination or ectopic neurones could cause reduced anisotropy. Increased diffusivity could be caused by a defect of neurogenesis or cell loss resulting in increased extracellular space. The widespread nature of abnormalities should be considered if surgical treatment is contemplated.

Frontal lobe seizures can be difficult to distinguish from non-epileptic seizures or sleep disorders. Addition- ally, the ictal EEG is often normal. To illustrate the challenging semiology and frequent misdiagnosis of FLE we discuss two patients, accompanied by video-EEG from the Chalfont Centre for Epilepsy.Case 1, a 41-year-old man with right frontal lobe focal cortical dysplasia presented with stereotyped nocturnal focal motor seizures that were associated with right frontal epileptic discharges. Seizures manifested as contralateral head version and tonic posturing of the upper limbs; the characteristic “fencing posture” described in seizures involving the supplementary motor area. In some, these evolved to hyperkinetic seizures.Case 2, a 23-year-old woman with multiple cavernous haemangiomas secondary to KRIT1 gene mutation, presented with stereotyped bipedal cycling movements of the lower limbs and loss of awareness, without associated ictal EEG change. These episodes were previously mis-diagnosed as non-epileptic but were confirmed as epileptic seizures from a right frontal lobe cavernoma.The unusual semiology of FLE represents a diagnostic challenge, which cannot always be resolved with ictal EEG recordings. FLE should be considered with episodes which are nocturnal, brief, stereotyped, motor predominant with, or without, awareness, and often with bilateral upper limb posturing or hyper- kinetic movements.rowangurney1@nhs.net30

The dental health of 457 5-year-old children who have lived continuously in fluoridated (at 1.0 mg F/litre) Newcastle and 370 children of the same age in non-fluoridated (less than 0.1 mg F/litre) South Northumberland has been reported. This paper examines in detail the caries prevalence in social class groups I + II, III, IV + V, and the social class/fluoridation relationship in 1987. The prevalence of dental caries in the three social class groupings I + II, III, and IV + V (and the mean dmft), respectively, was 35% (1.1), 46% (1.7) and 67% (2.4) in the fluoridated area, and 59% (2.2), 67% (3.7) and 77% (5.0) in the non-fluoridated area. Fluoridation was effective in all social class groupings and, because caries levels were higher in social classes IV + V, fluoridation brought about greater savings for these children than for those in social classes I + II. Fluoridation reduces but does not eliminate social inequalities, leaving social disadvantage/social background/social class as the major factors in caries prevalence for this age group. Further research into the fluoridation/social class relationship is required, particularly since the understanding of occupational class, now considered an imperfect representation of social class, is being superseded by other criteria which have stronger associations with measures of poor health

This report is of a two-year longitudinal study of dietary intake and growth in height and weight of 405 children, initially aged 11–12 years. Between 1979 and 1981 they each recorded their food intake on five occasions for three consecutive days using a diet diary with a 20 min private interview on the fourth day. Computerized food tables were used. Social class was recorded.The usual differences in height, weight and growth increments between the social classes were found. There were no significant differences in nutrient intake between social classes. Within a social class there were few statistically significant correlations between energy or protein intake and height or weight increments. It was concluded that a very high reliability of food intake measurements would be required in order to relate diet to growth and many other factors (e.g. energy expenditure, body composition and puberty) may also mask any such relationship. Social class alone would seem an inadequate means of allowing for such factors.