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Wildlife biodiversity is essential for healthy, resilient and sustainable ecosystems. For biologists, this diversity also represents a treasure trove of genetic, molecular and developmental mechanisms that deepen our understanding of the origins and rules of life. However, the rapid decline in biodiversity reported recently foreshadows a potentially catastrophic collapse of many important ecosystems and the associated irreversible loss of many forms of life on our planet. Immediate action by conservationists of all stripes is required to avert this disaster. In this Spotlight, we draw together insights and proposals discussed at a recent workshop hosted by Revive & Restore, which gathered experts to discuss how stem cell technologies can support traditional conservation techniques and help protect animal biodiversity. We discuss reprogramming, in vitro gametogenesis, disease modelling and embryo modelling, and we highlight the prospects for leveraging stem cell technologies beyond mammalian species.

Firefly flashes are well-known visual signals used by these insects to find, identify, and choose mates. However, many firefly species have lost the ability to produce light as adults. These "unlighted" species generally lack developed adult light organs, are diurnal rather than nocturnal, and are believed to use volatile pheromones acting over a distance to locate mates. While cuticular hydrocarbons, which may function in mate recognition at close range, have been examined for a handful of the over 2000 extant firefly species, no volatile pheromone has ever been identified. In this study, using coupled gas chromatography - electroantennographic detection, we detected a single female-emitted compound that elicited antennal responses from wild-caught male winter fireflies, Photinus corruscus. The compound was identified as (1S)-exo-3-hydroxycamphor (hydroxycamphor). In field trials at two sites across the species' eastern North American range, large numbers of male P. corruscus were attracted to synthesized hydroxycamphor, verifying its function as a volatile sex attractant pheromone. Males spent more time in contact with lures treated with synthesized hydroxycamphor than those treated with solvent only in laboratory two-choice assays. Further, using single sensillum recordings, we characterized a pheromone-sensitive odorant receptor neuron in a specific olfactory sensillum on male P. corruscus antennae and demonstrated its sensitivity to hydroxycamphor. Thus, this study has identified the first volatile pheromone and its corresponding sensory neuron for any firefly species, and provides a tool for monitoring P. corruscus populations for conservation and further inquiry into the chemical and cellular bases for sexual communication among fireflies.

Abstract The eastern quoll (Dasyurus viverrinus) is an endangered marsupial native to Australia. Since the extirpation of its mainland populations in the 20th century, wild eastern quolls have been restricted to two islands at the southern end of their historical range. Eastern quolls are the subject of captive breeding programs and attempts have been made to re-establish a population in mainland Australia. However, few resources currently exist to guide the genetic management of this species. Here, we generated a reference genome for the eastern quoll with gene annotations supported by multi-tissue transcriptomes. Our assembly is among the most complete marsupial genomes currently available. Using this assembly, we infer the species’ demographic history, identifying potential evidence of a long-term decline beginning in the late Pleistocene. Finally, we identify a deletion at the ASIP locus that likely underpins pelage color differences between the eastern quoll and the closely related Tasmanian devil (Sarcophilus harrisii).

Abstract Erectile dysfunction (ED) is an extremely prevalent condition which significantly impacts quality of life. The rapid increase of ED in recent decades suggests the existence of unidentified environmental risk factors contributing to this condition. Endocrine Disrupting Chemicals (EDCs) are one likely candidate, given that development and function of the erectile tissues are hormonally dependent. We use the estrogenic-EDC diethylstilbestrol (DES) to model how widespread estrogenic-EDC exposure may impact erectile function in humans. Here we show that male mice chronically exposed to DES exhibit abnormal contractility of the erectile tissue, indicative of ED. The treatment did not affect systemic testosterone production yet significantly increased estrogen receptor α (Esr1) expression in the primary erectile tissue, suggesting EDCs directly impact erectile function. In response, we isolated the erectile tissue from mice and briefly incubated them with the estrogenic-EDCs DES or genistein (a phytoestrogen). These acute-direct exposures similarly caused a significant reduction in erectile tissue contractility, again indicative of ED. Overall, these findings demonstrate a direct link between estrogenic EDCs and erectile dysfunction and show that both chronic and acute estrogenic exposures are likely risk factors for this condition.

Background Specialist palliative care (SPC) services address the needs of people with advanced illness. Meta-analyses to date have been challenged by heterogeneity in SPC service models and outcome measures and have failed to produce an overall effect. The best service models are unknown. We aimed to estimate the summary effect of SPC across settings on quality of life and emotional wellbeing and identify the optimum service delivery model. Methods and findings We conducted a systematic review with meta-analysis and meta-regression. Databases (Cochrane, MEDLINE, CINAHL, ICTRP, clinicaltrials.gov) were searched (January 1, 2000; December 28, 2023), supplemented with further hand searches (i.e., conference abstracts). Two researchers independently screened identified studies. We included randomized controlled trials (RCTs) testing SPC intervention versus usual care in adults with life-limiting disease and including patient or proxy reported outcomes as primary or secondary endpoints. The meta-analysis used, to our knowledge, novel methodology to convert outcomes into minimally clinically important difference (MID) units and the number needed to treat (NNT). Bias/quality was assessed via the Cochrane Risk of Bias 2 tool and certainty of evidence was assessed using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) tool. Random-effects meta-analyses and meta-regressions were used to synthesize endpoints between 2 weeks and 12 months for effect on quality of life and emotional wellbeing expressed and combined in units of MID. From 42,787 records, 39 international RCTs (n = 38 from high- and middle-income countries) were included. For quality of life (33 trials) and emotional wellbeing (22 trials), statistically and clinically significant benefit was seen from 3 months' follow-up for quality of life, standardized mean difference (SMD in MID units) effect size of 0.40 at 13 to 36 weeks, 95% confidence interval (CI) [0.21, 0.59], p < 0.001, I.sup.2 = 60%). For quality of life at 13 to 36 weeks, 13% of the SPC intervention group experienced an effect of at least 1 MID unit change (relative risk (RR) = 1.13, 95% CI [1.06, 1.20], p < 0.001, I.sup.2 = 0%). For emotional wellbeing, 16% experienced an effect of at least 1 MID unit change at 13 to 36 weeks (95% CI [1.08, 1.24], p < 0.001, I.sup.2 = 0%). For quality of life, the NNT improved from 69 to 15; for emotional wellbeing from 46 to 28, from 2 weeks and 3 months, respectively. Higher effect sizes were associated with multidisciplinary and multicomponent interventions, across settings. Sensitivity analyses using robust MID estimates showed substantial (quality of life) and moderate (emotional wellbeing) benefits, and lower number-needed-to-treat, even with shorter follow-up. As the main limitation, MID effect sizes may be biased by relying on derivation in non-palliative care samples. Conclusions Using, to our knowledge, novel methods to combine different outcomes, we found clear evidence of moderate overall effect size for both quality of life and emotional wellbeing benefits from SPC, regardless of underlying condition, with multidisciplinary, multicomponent, and multi-setting models being most effective. Our data seriously challenge the current practice of referral to SPC close to death. Policy and service commissioning should drive needs-based referral at least 3 to 6 months before death as the optimal standard of care., Author(s): Miriam J. Johnson 1, Leah Rutterford 2, Anisha Sunny 3, Sophie Pask 1, Susanne de Wolf-Linder 1,4, Fliss E. M. Murtagh 1, Christina Ramsenthaler 1,4,* Introduction Palliative and end-of-life [...]

BackgroundSpecialist palliative care (SPC) services address the needs of people with advanced illness. Meta-analyses to date have been challenged by heterogeneity in SPC service models and outcome measures and have failed to produce an overall effect. The best service models are unknown. We aimed to estimate the summary effect of SPC across settings on quality of life and emotional wellbeing and identify the optimum service delivery model.Methods and findingsWe conducted a systematic review with meta-analysis and meta-regression. Databases (Cochrane, MEDLINE, CINAHL, ICTRP, clinicaltrials.gov) were searched (January 1, 2000; December 28, 2023), supplemented with further hand searches (i.e., conference abstracts). Two researchers independently screened identified studies. We included randomized controlled trials (RCTs) testing SPC intervention versus usual care in adults with life-limiting disease and including patient or proxy reported outcomes as primary or secondary endpoints. The meta-analysis used, to our knowledge, novel methodology to convert outcomes into minimally clinically important difference (MID) units and the number needed to treat (NNT). Bias/quality was assessed via the Cochrane Risk of Bias 2 tool and certainty of evidence was assessed using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) tool. Random-effects meta-analyses and meta-regressions were used to synthesize endpoints between 2 weeks and 12 months for effect on quality of life and emotional wellbeing expressed and combined in units of MID. From 42,787 records, 39 international RCTs (n = 38 from high- and middle-income countries) were included. For quality of life (33 trials) and emotional wellbeing (22 trials), statistically and clinically significant benefit was seen from 3 months' follow-up for quality of life, standardized mean difference (SMD in MID units) effect size of 0.40 at 13 to 36 weeks, 95% confidence interval (CI) [0.21, 0.59], p < 0.001, I2 = 60%). For quality of life at 13 to 36 weeks, 13% of the SPC intervention group experienced an effect of at least 1 MID unit change (relative risk (RR) = 1.13, 95% CI [1.06, 1.20], p < 0.001, I2 = 0%). For emotional wellbeing, 16% experienced an effect of at least 1 MID unit change at 13 to 36 weeks (95% CI [1.08, 1.24], p < 0.001, I2 = 0%). For quality of life, the NNT improved from 69 to 15; for emotional wellbeing from 46 to 28, from 2 weeks and 3 months, respectively. Higher effect sizes were associated with multidisciplinary and multicomponent interventions, across settings. Sensitivity analyses using robust MID estimates showed substantial (quality of life) and moderate (emotional wellbeing) benefits, and lower number-needed-to-treat, even with shorter follow-up. As the main limitation, MID effect sizes may be biased by relying on derivation in non-palliative care samples.ConclusionsUsing, to our knowledge, novel methods to combine different outcomes, we found clear evidence of moderate overall effect size for both quality of life and emotional wellbeing benefits from SPC, regardless of underlying condition, with multidisciplinary, multicomponent, and multi-setting models being most effective. Our data seriously challenge the current practice of referral to SPC close to death. Policy and service commissioning should drive needs-based referral at least 3 to 6 months before death as the optimal standard of care.

Abstract Introduction Many genome projects were underway before the advent of high-throughput sequencing and have thus been supported by a wealth of genome information from other technologies. Such information frequently takes the form of linkage and physical maps, both of which can provide a substantial amount of data useful in de novo sequencing projects. Furthermore, the recent abundance of genome resources enables the use of conserved synteny maps identified in related species to further enhance genome assemblies. Methods The tammar wallaby (Macropus eugenii) is a model marsupial mammal with a low coverage genome. However, we have access to extensive comparative maps containing over 14,000 markers constructed through the physical mapping of conserved loci, chromosome painting and comprehensive linkage maps. Using a custom Bioperl pipeline, information from the maps was aligned to assembled tammar wallaby contigs using BLAT. This data was used to construct pseudo paired-end libraries with intervals ranging from 5-10 MB. We then used Bambus (a program designed to scaffold eukaryotic genomes by ordering and orienting contigs through the use of paired-end data) to scaffold our libraries. To determine how map data compares to sequence based approaches to enhance assemblies, we repeated the experiment using a 0.5× coverage of unique reads from 4 KB and 8 KB Illumina paired-end libraries. Finally, we combined both the sequence and non-sequence-based data to determine how a combined approach could further enhance the quality of the low coverage de novo reconstruction of the tammar wallaby genome. Results Using the map data alone, we were able order 2.2% of the initial contigs into scaffolds, and increase the N50 scaffold size to 39 KB (36 KB in the original assembly). Using only the 0.5× paired-end sequence based data, 53% of the initial contigs were assigned to scaffolds. Combining both data sets resulted in a further 2% increase in the number of initial contigs integrated into a scaffold (55% total) but a 35% increase in N50 scaffold size over the use of sequence-based data alone. Conclusions We provide a relatively simple pipeline utilizing existing bioinformatics tools to integrate map data into a genome assembly which is available at http://www.mcb.uconn.edu/fac.php?name=paska. While the map data only contributed minimally to assigning the initial contigs to scaffolds in the new assembly, it greatly increased the N50 size. This process added structure to our low coverage assembly, greatly increasing its utility in further analyses.

Marsupials exhibit distinctive modes of reproduction and early development that set them apart from their eutherian counterparts and render them invaluable for comparative studies. However, marsupial genomic resources still lag far behind those of eutherian mammals. We present a series of novel genomic resources for the fat-tailed dunnart (Sminthopsis crassicaudata), a mouse-like marsupial that, due to its ease of husbandry and ex-utero development, is emerging as a laboratory model. We constructed a highly representative multi-tissue de novo transcriptome assembly of dunnart RNA-seq reads spanning 12 tissues. The transcriptome includes 2,093,982 assembled transcripts and has a mammalian transcriptome BUSCO completeness score of 93.3%, the highest amongst currently published marsupial transcriptomes. This global transcriptome, along with ab initio predictions, supported annotation of the existing dunnart genome, revealing 21,622 protein-coding genes. Altogether, these resources will enable wider use of the dunnart as a model marsupial and deepen our understanding of mammalian genome evolution.

Introduction: During early development in most male mammals the testes move from a position near the kidneys through the abdomen to eventually reside in the scrotum. The transabdominal phase of this migration is driven by insulin-like peptide 3 (INSL3) which stimulates growth of the gubernaculum, a key ligament connecting the testes with the abdominal wall. While all marsupials, except the marsupial mole (Notoryctes typhlops), have a scrotum and fully descended testes, it is unclear if INSL3 drives this process in marsupials especially given that marsupials have a different mechanism of scrotum determination and position relative to the phallus compared to eutherian mammals.Methods: To understand if INSL3 plays a role in marsupial testicular descent we have sequenced and curated the INSL3 gene and its receptor (RXFP2) in a range of marsupials representing every order. Furthermore, we looked at single cell RNA-seq and qPCR analysis of INSL3 in the fat-tailed dunnart testis (Sminthopsis crassicaudata) to understand the location and timing of expression during development.Results: These data show a strong phylogenetic similarity between marsupial and eutherian orthologues, but not with monotreme INSL3s which were more similar to the ancestral RLN3 gene. We have also shown the genomic location of INSL3, and surrounding genes is conserved in a range of marsupials and eutherians. Single cell RNA-seq and qPCR data show that INSL3 mRNA is expressed specifically in Leydig cells and expressed at higher levels during the testicular descent phase in developing marsupials.Discussion: Together, these data argue strongly for a therian origin of INSL3 mediated testicular descent in mammals and suggests that a coordinated movement of the testes to the abdominal wall may have preceded externalization in marsupials and therian mammals.

Abstract Background Small RNAs have proven to be essential regulatory molecules encoded within eukaryotic genomes. These short RNAs participate in a diverse array of cellular processes including gene regulation, chromatin dynamics and genome defense. The tammar wallaby, a marsupial mammal, is a powerful comparative model for studying the evolution of regulatory networks. As part of the genome sequencing initiative for the tammar, we have explored the evolution of each of the major classes of mammalian small RNAs in an Australian marsupial for the first time, including the first genome-scale analysis of the newest class of small RNAs, centromere repeat associated short interacting RNAs (crasiRNAs). Results Using next generation sequencing, we have characterized the major classes of small RNAs, micro (mi) RNAs, piwi interacting (pi) RNAs, and the centromere repeat associated short interacting (crasi) RNAs in the tammar. We examined each of these small RNA classes with respect to the newly assembled tammar wallaby genome for gene and repeat features, salient features that define their canonical sequences, and the constitution of both highly conserved and species-specific members. Using a combination of miRNA hairpin predictions and co-mapping with miRBase entries, we identified a highly conserved cluster of miRNA genes on the X chromosome in the tammar and a total of 94 other predicted miRNA producing genes. Mapping all miRNAs to the tammar genome and comparing target genes among tammar, mouse and human, we identified 163 conserved target genes. An additional nine genes were identified in tammar that do not have an orthologous miRNA target in human and likely represent novel miRNA-regulated genes in the tammar. A survey of the tammar gonadal piRNAs shows that these small RNAs are enriched in retroelements and carry members from both marsupial and tammar-specific repeat classes. Lastly, this study includes the first in-depth analyses of the newly discovered crasiRNAs. These small RNAs are derived largely from centromere-enriched retroelements, including a novel SINE. Conclusions This study encompasses the first analyses of the major classes of small RNAs for the newly completed tammar genome, validates preliminary annotations using deep sequencing and computational approaches, and provides a foundation for future work on tammar-specific as well as conserved, but previously unknown small RNA progenitors and targets identified herein. The characterization of new miRNA target genes and a unique profile for crasiRNAs has allowed for insight into multiple RNA mediated processes in the tammar, including gene regulation, species incompatibilities, centromere and chromosome function.

Abstract Background In marsupials, growth and development of the young occur postnatally, regulated by milk that changes in composition throughout the long lactation. To initiate lactation in mammals, there is an absolute requirement for insulin (INS), a gene known to be imprinted in the placenta. We therefore examined whether INS is imprinted in the mammary gland of the marsupial tammar wallaby (Macropus eugenii) and compared its expression with that of insulin-like growth factor 2 (IGF2). Results INS was expressed in the mammary gland and significantly increased, while IGF2 decreased, during established milk production. Insulin and IGF2 were both detected in the mammary gland macrophage cells during early lactation and in the alveolar cells later in lactation. Surprisingly, INS, which was thought only to be imprinted in the therian yolk sac, was imprinted and paternally expressed in the liver of the developing young, monoallelically expressed in the tammar mammary gland and biallelic in the stomach and intestine. The INS transcription start site used in the liver and mammary gland was differentially methylated. Conclusions This is the first study to identify tissue-specific INS imprinting outside the yolk sac. These data suggest that there may be an advantage of selective monoallelic expression in the mammary gland and that this may influence the growth of the postnatal young. These results are not consistent with the parental conflict hypothesis, but instead provide support for the maternal–infant co-adaptation hypothesis. Thus, imprinting in the mammary gland maybe as critical for postnatal growth and development in mammals as genomic imprinting in the placenta is prenatally.

Abstract Background The HOX gene clusters are thought to be highly conserved amongst mammals and other vertebrates, but the long non-coding RNAs have only been studied in detail in human and mouse. The sequencing of the kangaroo genome provides an opportunity to use comparative analyses to compare the HOX clusters of a mammal with a distinct body plan to those of other mammals. Results Here we report a comparative analysis of HOX gene clusters between an Australian marsupial of the kangaroo family and the eutherians. There was a strikingly high level of conservation of HOX gene sequence and structure and non-protein coding genes including the microRNAs miR-196a, miR-196b, miR-10a and miR-10b and the long non-coding RNAs HOTAIR, HOTAIRM1 and HOXA11AS that play critical roles in regulating gene expression and controlling development. By microRNA deep sequencing and comparative genomic analyses, two conserved microRNAs (miR-10a and miR-10b) were identified and one new candidate microRNA with typical hairpin precursor structure that is expressed in both fibroblasts and testes was found. The prediction of microRNA target analysis showed that several known microRNA targets, such as miR-10, miR-414 and miR-464, were found in the tammar HOX clusters. In addition, several novel and putative miRNAs were identified that originated from elsewhere in the tammar genome and that target the tammar HOXB and HOXD clusters. Conclusions This study confirms that the emergence of known long non-coding RNAs in the HOX clusters clearly predate the marsupial-eutherian divergence 160 Ma ago. It also identified a new potentially functional microRNA as well as conserved miRNAs. These non-coding RNAs may participate in the regulation of HOX genes to influence the body plan of this marsupial.

Abstract Background The placenta is essential for supplying nutrients and gases to the developing mammalian young before birth. While all mammals have a functional placenta, only in therian mammals (marsupials and eutherians) does the placenta closely appose or invade the uterine endometrium. The eutherian placenta secretes hormones that are structurally and functionally similar to pituitary growth hormone (GH), prolactin (PRL) and luteinizing hormone (LH). Marsupial and eutherian mammals diverged from a common ancestor approximately 125 to 148 million years ago and developed distinct reproductive strategies. As in eutherians, marsupials rely on a short-lived but functional placenta for embryogenesis. Results We characterized pituitary GH, GH-R, IGF-2, PRL and LHβ in a macropodid marsupial, the tammar wallaby, Macropus eugenii. These genes were expressed in the tammar placenta during the last third of gestation when most fetal growth occurs and active organogenesis is initiated. The mRNA of key growth genes GH, GH-R, IGF-2 and PRL were expressed during late pregnancy. We found significant up-regulation of GH, GH-R and IGF-2 after the start of the rapid growth phase of organogenesis which suggests that the placental growth hormones regulate the rapid phase of fetal growth. Conclusions This is the first demonstration of the existence of pituitary hormones in the marsupial placenta. Placental expression of these pituitary hormones has clearly been conserved in marsupials as in eutherian mammals, suggesting an ancestral origin of the evolution of placental expression and a critical function of these hormones in growth and development of all therian mammals.

Abstract Background Hormones are critical for early gonadal development in nonmammalian vertebrates, and oestrogen is required for normal ovarian development. In contrast, mammals determine sex by the presence or absence of the SRY gene, and hormones are not thought to play a role in early gonadal development. Despite an XY sex-determining system in marsupial mammals, exposure to oestrogen can override SRY and induce ovarian development of XY gonads if administered early enough. Here we assess the effect of exogenous oestrogen on the molecular pathways of mammalian gonadal development. Results We examined the expression of key testicular (SRY, SOX9, AMH and FGF9) and ovarian (WNT4, RSPO1, FOXL2 and FST) markers during gonadal development in the marsupial tammar wallaby (Macropus eugenii) and used these data to determine the effect of oestrogen exposure on gonadal fate. During normal development, we observed male specific upregulation of AMH and SOX9 as in the mouse and human testis, but this upregulation was initiated before the peak in SRY expression and 4 days before testicular cord formation. Similarly, key genes for ovarian development in mouse and human were also upregulated during ovarian differentiation in the tammar. In particular, there was early sexually dimorphic expression of FOXL2 and WNT4, suggesting that these genes are key regulators of ovarian development in all therian mammals. We next examined the effect of exogenous oestrogen on the development of the mammalian XY gonad. Despite the presence of SRY, exogenous oestrogen blocked the key male transcription factor SOX9 from entering the nuclei of male somatic cells, preventing activation of the testicular pathway and permitting upregulation of key female genes, resulting in ovarian development of the XY gonad. Conclusions We have uncovered a mechanism by which oestrogen can regulate gonadal development through the nucleocytoplasmic shuttling of SOX9. This may represent an underlying ancestral mechanism by which oestrogen promotes ovarian development in the gonads of nonmammalian vertebrates. Furthermore, oestrogen may retain this function in adult female mammals to maintain granulosa cell fate in the differentiated ovary by suppressing nuclear translocation of the SOX9 protein. See commentary: http://www.biomedcentral.com/1741-7007/8/110

Abstract Background WNT4 is a critical signalling molecule in embryogenesis and homeostasis, but the elements that control its transcriptional regulation are largely unknown. This study uses comparative cross species sequence and functional analyses between humans and a marsupial (the tammar wallaby,Macropus eugenii) to refine the mammalian Wnt4 promoter. Results We have defined a highly conserved 89 bp minimal promoter region in human WNT4 by comparative analysis with the tammar wallaby. There are many conserved transcription factor binding sites in the proximal promoter region, including SP1, MyoD, NFκB and AP2, as well as highly conserved CpG islands within the human, mouse and marsupial promoters, suggesting that DNA methylation may play an important role in WNT4 transcriptional regulation. Conclusion Using a marsupial model, we have been able to provide new information on the transcriptional regulators in the promoter of this essential mammalian developmental gene, WNT4. These transcription factor binding sites and CpG islands are highly conserved in two disparate mammals, and are likely key controlling elements in the regulation of this essential developmental gene.

Abstract Background Genomic imprinting occurs in both marsupial and eutherian mammals. The CDKN1C and IGF2 genes are both imprinted and syntenic in the mouse and human, but in marsupials only IGF2 is imprinted. This study examines the evolution of features that, in eutherians, regulate CDKN1C imprinting. Results Despite the absence of imprinting, CDKN1C protein was present in the tammar wallaby placenta. Genomic analysis of the tammar region confirmed that CDKN1C is syntenic with IGF2. However, there are fewer LTR and DNA elements in the region and in intron 9 of KCNQ1. In addition there are fewer LINEs in the tammar compared with human and mouse. While the CpG island in intron 10 of KCNQ1 and promoter elements could not be detected, the antisense transcript KCNQ1OT1 that regulates CDKN1C imprinting in human and mouse is still expressed. Conclusion CDKN1C has a conserved function, likely antagonistic to IGF2, in the mammalian placenta that preceded its acquisition of imprinting. CDKN1C resides in synteny with IGF2, demonstrating that imprinting of the two genes did not occur concurrently to balance maternal and paternal influences on the growth of the placenta. The expression of KCNQ1OT1 in the absence of CDKN1C imprinting suggests that antisense transcription at this locus preceded imprinting of this domain. These findings demonstrate the stepwise accumulation of control mechanisms within imprinted domains and show that CDKN1C imprinting cannot be due to its synteny with IGF2 or with its placental expression in mammals.

Implantable hydrogels should ideally possess mechanical properties matched to the surrounding tissues to enable adequate mechanical function while regeneration occurs. This can be challenging, especially when degradable systems with a high water content and hydrolysable chemical bonds are required in anatomical sites under constant mechanical stimulation, e.g., a foot ulcer cavity. In these circumstances, the design of hydrogel composites is a promising strategy for providing controlled structural features and macroscopic properties over time. To explore this strategy, the synthesis of a new photocurable elastomeric polymer, poly(glycerol-co-sebacic acid-co-lactic acid-co-polyethylene glycol) acrylate (PGSLPA), is investigated, along with its processing into UV-cured hydrogels, electrospun nonwovens and fibre-reinforced variants, without the need for a high temperature curing step or the use of hazardous solvents. The mechanical properties of bioresorbable PGSLPA hydrogels were studied with and without electrospun nonwoven reinforcement and with varied layered configurations, aiming to determine the effects of the microstructure on the bulk compressive strength and elasticity. The nonwoven reinforced PGSLPA hydrogels exhibited a 60% increase in compressive strength and an 80% increase in elastic moduli compared to the fibre-free PGSLPA samples. The mechanical properties of the fibre-reinforced hydrogels could also be modulated by altering the layering arrangement of the nonwoven and hydrogel phase. The nanofibre-reinforced PGSLPA hydrogels also exhibited good elastic recovery, as evidenced by the hysteresis in compression fatigue stress–strain evaluations showing a return to the original dimensions.