Basement membrane remodelling regulates mouse embryogenesis.

Tissue sculpting during development has been attributed mainly to cellular events through processes such as convergent extension or apical constriction1,2. However, recent work has revealed roles for basement membrane remodelling in global tissue morphogenesis3-5. Upon implantation, the epiblast and extraembryonic ectoderm of the mouse embryo become enveloped by a basement membrane. Signalling between the basement membrane and these tissues is critical for cell polarization and the ensuing morphogenesis6,7. However, the mechanical role of the basement membrane in post-implantation embryogenesis remains unknown. Here we demonstrate the importance of spatiotemporally regulated basement membrane remodelling during early embryonic development. Specifically, we show that Nodal signalling directs the generation and dynamic distribution of perforations in the basement membrane by regulating the expression of matrix metalloproteinases. This basement membrane remodelling facilitates embryo growth before gastrulation. The establishment of the anterior-posterior axis8,9 further regulates basement membrane remodelling by localizing Nodal signalling-and therefore the activity of matrix metalloproteinases and basement membrane perforations-to the posterior side of the embryo. Perforations on the posterior side are essential for primitive-streak extension during gastrulation by rendering the basement membrane of the prospective primitive streak more prone to breaching. Thus spatiotemporally regulated basement membrane remodelling contributes to the coordination of embryo growth, morphogenesis and gastrulation.

Chemical modification patterns for microRNA therapeutic mimics: a structure-activity relationship (SAR) case-study on miR-200c.

microRNA (miRNA) mimics are an emerging class of oligonucleotide therapeutics, with a few compounds already in clinical stages. Synthetic miRNAs are able to restore downregulated levels of intrinsic miRNAs, allowing for parallel regulation of multiple genes involved in a particular disease. In this work, we examined the influence of chemical modifications patterns in miR-200c mimics, assessing the regulation of a selection of target messenger RNAs (mRNA) and, subsequently, of the whole transcriptome in A549 cells. We have probed 37 mimics and provided an initial set of instructions for designing miRNA mimics with potency and selectivity similar to an unmodified miRNA duplex. Additionally, we have examined the stability of selected mimics in serum. Finally, the selected two modification patterns were translated to two other miRNAs, miR-34a and miR-155. To differing degrees, these designs acted on target mRNAs in a similar manner to the unmodified mimic. Here, for the first time, we describe a structured overview of 'miRNA mimics modification templates' that are chemically stabilised and optimised for use in an in vitro set up and highlight the need of further sequence specific optimization when mimics are to be used beyond in vitro tool experiments.

Prevention of the foreign body response to implantable medical devices by inflammasome inhibition.

SignificanceImplantable electronic medical devices (IEMDs) are used for some clinical applications, representing an exciting prospect for the transformative treatment of intractable conditions such Parkinson's disease, deafness, and paralysis. The use of IEMDs is limited at the moment because, over time, a foreign body reaction (FBR) develops at the device-neural interface such that ultimately the IEMD fails and needs to be removed. Here, we show that macrophage nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activity drives the FBR in a nerve injury model yet integration of an NLRP3 inhibitor into the device prevents FBR while allowing full healing of damaged neural tissue to occur.

Characterization of primary models of human trophoblast.

Two recently developed models, trophoblast organoids and trophoblast stem cells (TSCs), are useful tools to further the understanding of human placental development. Both differentiate from villous cytotrophoblast (VCT) to either extravillous trophoblast (EVT) or syncytiotrophoblast (SCT). Here, we compare the transcriptomes and miRNA profiles of these models to identify which trophoblast they resemble in vivo. Our findings indicate that TSCs do not readily undergo SCT differentiation and closely resemble cells at the base of the cell columns from where EVT derives. In contrast, organoids are similar to VCT and undergo spontaneous SCT differentiation. A defining feature of human trophoblast is that VCT and SCT are human leukocyte antigen (HLA) null, whereas EVT expresses HLA-C, -G and -E molecules. We find that trophoblast organoids retain these in vivo characteristics. In contrast, TSCs express classical HLA-A and HLA-B molecules, and maintain their expression after EVT differentiation, with upregulation of HLA-G. Furthermore, HLA expression in TSCs differs when grown in 3D rather than in 2D, suggesting that mechanical cues are important. Our results can be used to select the most suitable model for the study of trophoblast development, function and pathology.

Trophoblast organoids as a model for maternal-fetal interactions during human placentation.

The placenta is the extraembryonic organ that supports the fetus during intrauterine life. Although placental dysfunction results in major disorders of pregnancy with immediate and lifelong consequences for the mother and child, our knowledge of the human placenta is limited owing to a lack of functional experimental models1. After implantation, the trophectoderm of the blastocyst rapidly proliferates and generates the trophoblast, the unique cell type of the placenta. In vivo, proliferative villous cytotrophoblast cells differentiate into two main sub-populations: syncytiotrophoblast, the multinucleated epithelium of the villi responsible for nutrient exchange and hormone production, and extravillous trophoblast cells, which anchor the placenta to the maternal decidua and transform the maternal spiral arteries2. Here we describe the generation of long-term, genetically stable organoid cultures of trophoblast that can differentiate into both syncytiotrophoblast and extravillous trophoblast. We used human leukocyte antigen (HLA) typing to confirm that the organoids were derived from the fetus, and verified their identities against four trophoblast-specific criteria3. The cultures organize into villous-like structures, and we detected the secretion of placental-specific peptides and hormones, including human chorionic gonadotropin (hCG), growth differentiation factor 15 (GDF15) and pregnancy-specific glycoprotein (PSG) by mass spectrometry. The organoids also differentiate into HLA-G+ extravillous trophoblast cells, which vigorously invade in three-dimensional cultures. Analysis of the methylome reveals that the organoids closely resemble normal first trimester placentas. This organoid model will be transformative for studying human placental development and for investigating trophoblast interactions with the local and systemic maternal environment.

Evaluation of commercial devices for patient specific QA of stereotactic radiotherapy plans.

Stereotactic radiotherapy (SRT) methods have become common for the treatment of small tumors in various parts of the body. Small field dosimetry has a unique set of challenges when it comes to the pre-treatment validation of a radiotherapy plan that involves film dosimetry or high-resolution detectors. Comparison of commercial quality assurance (QA) devices to the film dosimetry method for pre-treatment evaluation of stereotactic radiosurgery (SRS), fractionated SRT, and stereotactic body radiation therapy treatment plans have been evaluated in this study. Forty stereotactic QA plans were measured using EBT-XD film, IBA Matrixx Resolution, SNC ArcCHECK, Varian aS1200 EPID, SNC SRS MapCHECK, and IBA myQA SRS. The results of the commercial devices are compared to the EBT-XD film dosimetry results for each gamma criteria. Treatment plan characteristics such as modulation factor and target volume were investigated for correlation with the passing rates. It was found that all detectors have greater than 95% passing rates at 3%/3 mm. Passing rates decrease rapidly for ArcCHECK and the Matrixx as criteria became more strict. In contrast, EBT-XD film, SNC SRS MapCHECK, and IBA myQA SRS passing rates do not decline as rapidly when compared to Matrix Resolution, ArcCHECK, and the EPID. EBT-XD film, SNC SRS MapCHECK, and IBA myQA SRS maintain greater than 90% passing rate at 2%/1 mm and greater than 80% at 1%/1 mm. Additionally, the ability of these devices to detect changes in dose distribution due to MLC positioning errors was investigated. Ten VMAT SBRT/SRS treatment plans were created with 6 MV FFF or 10 MV FFF beam energies using Eclipse 15.6. A MATLAB script was used to create two MLC positioning error scenarios from the original treatment plan. It was found that errors in MLC positioning were most reliably detected at 2%/1 mm for high-resolution detectors and that lower-resolution detectors did not consistently detect MLC positioning errors.

Two-year outcomes of episcleral brachytherapy adjunct to anti-VEGF therapy for treatment-resistant nAMD.

PURPOSE: This study was designed to demonstrate the safety and feasibility of episcleral brachytherapy (ESB) for the treatment of anti-vascular endothelial growth factor (anti-VEGF) resistant neovascular age-related macular degeneration (nAMD) in a 6-subject cohort adjunct to anti-VEGF therapy. METHODS: Six eyes of six subjects with anti-VEGF resistant nAMD (persistent fluid or hemorrhage despite frequent anti-VEGF treatment) were treated with ESB between May 2018 and July 2018 as part of a larger early feasibility trial. Baseline and follow-up exams with multi-modal imaging were conducted. RESULTS: In this analysis, six eyes were included. The mean age was 74.7 years; 33% were female; 67% had polypoidal choroidal vasculopathy. The mean number of lifetime anti-VEGF injections received prior to the study enrollment was 33.9 injections and 10 injections in the year prior to the study enrollment. In the first and second years following ESB, the mean number of injections was 8.5 and 8, respectively. No evidence of radiation-induced toxicity through 2 years following ESB was observed. The mean baseline VA was 55.3 letters. At 1 year, the mean VA increased by 3.2 letters and 1.7 letters at year 2. At 2 years, the mean change in vascular complex on ICGA was - 18%, - 43% on OCTA, and - 5% on FA. The subjects also experienced a mean decrease in CRT on OCT of 21% after 2 years. CONCLUSIONS: The results from this six-subject cohort with 2-year data support additional investigations of ESB for nAMD, specifically those with persistent disease activity and treatment resistant nAMD.

Coronary artery aneurysm presenting as acute coronary syndrome: two case reports and a review of the literature.

Coronary artery aneurysm (CAA) is an uncommon coronary disease, with a reported incidence in adults ranging from 0.33 to 4.9%.It is usually considered a variant of coronary artery disease (CAD). CAA is associated with thrombus formation due to abnormal laminar flow, as well as abnormal platelet and endothelial-derived pathophysiologic factors within the CAA. CAA identified in the context of acute coronary syndrome (ACS) poses several unique management challenges. Optimal antiplatelet and anticoagulant therapy is the mainstay of therapy. Percutaneous intervention for CAA is associated with complications including distal embolization of thrombus, no-reflow phenomenon, stent malposition, dissection, and rupture. There are currently no accepted guidelines to direct the management of CAA in patients presenting with ACS. Preference for conservative vs. surgical or catheter-based management is controversial. We review the literature and report different treatment strategies for two cases with both CAA and ACS.

Blastocyst transfer in mice alters the placental transcriptome and growth.

Assisted reproduction technologies (ARTs) are becoming increasingly common. Therefore, how these procedures influence gene regulation and foeto-placental development are important to explore. Here, we assess the effects of blastocyst transfer on mouse placental growth and transcriptome. C57Bl/6 blastocysts were transferred into uteri of B6D2F1 pseudopregnant females and dissected at embryonic day 10.5 for analysis. Compared to non-transferred controls, placentas from transferred conceptuses weighed less even though the embryos were larger on average. This suggested a compensatory increase in placental efficiency. RNA sequencing of whole male placentas revealed 543 differentially expressed genes (DEGs) after blastocyst transfer: 188 and 355 genes were downregulated and upregulated, respectively. DEGs were independently validated in male and female placentas. Bioinformatic analyses revealed that DEGs represented expression in all major placental cell types and included genes that are critical for placenta development and/or function. Furthermore, the direction of transcriptional change in response to blastocyst transfer implied an adaptive response to improve placental function to maintain foetal growth. Our analysis revealed that CpG methylation at regulatory regions of two DEGs was unchanged in female transferred placentas and that DEGs had fewer gene-associated CpG islands (within ~20 kb region) compared to the larger genome. These data suggested that altered methylation at proximal promoter regions might not lead to transcriptional disruption in transferred placentas. Genomic clustering of some DEGs warrants further investigation of long-range, cis-acting epigenetic mechanisms including histone modifications together with DNA methylation. We conclude that embryo transfer, a protocol required for ART, significantly impacts the placental transcriptome and growth.

Stratifying risk of recurrence of atrial fibrillation following balloon cryoablation using the CAAP-AF risk scoring system.

OBJECTIVE: To determine if the CAAP-AF tool could be applied to a cohort of patients in the United States undergoing cryoablation or AF utilizing second-generation cryoballoons. BACKGROUND: Atrial fibrillation (AF) is a major source of morbidity and expense, with over 33 million individuals affected worldwide and over 450 000 hospitalizations annually in the United States. Catheter ablation for AF is a class I indication for patients with symptomatic AF. The ability to predict postablation recurrence would have an enormous impact on both patient outcomes and cost to the health care system. METHODS: Our study was an observational, single-center retrospective study to evaluate the utility of the CAAP-AF risk scoring system in predicting recurrence of AF following second-generation balloon cryoablation for AF. RESULTS: There were a total of 235 patients. From the initial cohort, 30.2% (71) had a recurrence of AF within 1 year of the cryoablation procedure. There was a statistically significant increase in mean age, left atrial diameter, left atrial volume index, CHADS2 , CHADS2 -VASc, and number of antiarrhythmics failed in the group that had recurrence of AF. There was also a statistically significant increase in the CAAP-AF score in patients who had recurrence of AF. CONCLUSIONS: The CAAP-AF score predicted the freedom from AF 1 year following cryoablation for AF. The CAAP-AF score can aid in selecting patients most likely to benefit from cryoablation, which includes patients with a low CAAP-AF score, as they are most likely to remain AF free at 1 year.

A simple knowledge-based tool for stereotactic radiosurgery pre-planning.

We studied the dosimetry of single-isocenter treatment plans generated to treat a solitary intracranial lesion using linac-based stereotactic radiosurgery (SRS). A common metric for evaluating SRS plan quality is the volume of normal brain tissue irradiated by a dose of at least 12 Gy (V12), which is important because multiple studies have shown a strong correlation between V12 and incidence of radiation necrosis. Unrealistic expectations for values of V12 can lead to wasted planning time. We present a model that estimates V12 without having to construct a full treatment plan. This model was derived by retrospectively analyzing 50 SRS treatment plans, each clinically approved for delivery using circular collimator cone arc therapy (CAT). Each case was re-planned for delivery via dynamic conformal arc therapy (DCAT), and then scaling arguments were used to extend dosimetric data to account for different prescription dose (PD) values (15, 18, 21, or 24 Gy). We determined a phenomenological expression for the total volume receiving at least 12 Gy (TV12) as a function of both planning target volume (PTV) and PD: T V 12 / 1 c c = n ∗ P D / 1 G y + d ∗ P T V / 1 c c a ∗ P D / 1 G y c , where a , c , n , d are fit parameters, and a separate set of values is determined for each plan type. In addition, we generated a sequence of plots to clarify how the relationship between conformity index (CI) and TV12 depends on plan type (CAT vs DCAT), PTV, and PD. These results can be used to suggest realistic plan parameters and planning goals before the start of treatment planning. In the absence of access to more sophisticated pre-planning tools, this model can be locally generated and implemented at relatively low cost with respect to time, money, and expertise.

Application of the Strengths and Difficulties Questionnaire Added Value Score in evaluating the effectiveness of Functional Family Therapy within local authority social work services.

BACKGROUND: As 'Blueprints' evidence-based programmes, such as Functional Family Therapy (FFT), originating from the United States, are increasingly implemented in Social Work services, the importance of assessing their effectiveness in a UK context is crucial. To do this, it is not always practical for services to commission randomised control trials or quasi-experimental control trials. The Strengths and Difficulties Questionnaire (SDQ) Added Value Score has been shown to have utility in the evaluation of intervention programmes by controlling for regression to the mean, attenuation and the shifting nature of most childhood psychopathology. METHOD: The SDQ Added Value Score was used to assess the effectiveness of FFT in two local authorities in Scotland. One hundred and sixty-four families who had finished FFT completed the Strengths and Difficulties Questionnaire, the Outcome Questionnaire and the Client Outcome Measure at pre- and postintervention. RESULTS: Both parents' and adolescents' average psychosocial distress scores significantly decreased on all measures after FFT and many of the scores postintervention fell to a range equivalent with the general population. Furthermore, calculation of the SDQ Added Value Score indicated that adolescents' mean total difficulties scores were lower following FFT than what would have been expected had this intervention not been received, producing an effect size that compares favourably to other interventions. CONCLUSIONS: Functional Family Therapy has been identified as an effective intervention for improving the psychosocial functioning of high-risk adolescents and their families.

High hospital research participation and improved colorectal cancer survival outcomes: a population-based study.

OBJECTIVE: In 2001, the National Institute for Health Research Cancer Research Network (NCRN) was established, leading to a rapid increase in clinical research activity across the English NHS. Using colorectal cancer (CRC) as an example, we test the hypothesis that high, sustained hospital-level participation in interventional clinical trials improves outcomes for all patients with CRC managed in those research-intensive hospitals. DESIGN: Data for patients diagnosed with CRC in England in 2001-2008 (n=209 968) were linked with data on accrual to NCRN CRC studies (n=30 998). Hospital Trusts were categorised by the proportion of patients accrued to interventional studies annually. Multivariable models investigated the relationship between 30-day postoperative mortality and 5-year survival and the level and duration of study participation. RESULTS: Most of the Trusts achieving high participation were district general hospitals and the effects were not limited to cancer 'centres of excellence', although such centres do make substantial contributions. Patients treated in Trusts with high research participation (≥16%) in their year of diagnosis had lower postoperative mortality (p<0.001) and improved survival (p<0.001) after adjustment for casemix and hospital-level variables. The effects increased with sustained research participation, with a reduction in postoperative mortality of 1.5% (6.5%-5%, p<2.2×10-6) and an improvement in survival (p<10-19; 5-year difference: 3.8% (41.0%-44.8%)) comparing high participation for ≥4 years with 0 years. CONCLUSIONS: There is a strong independent association between survival and participation in interventional clinical studies for all patients with CRC treated in the hospital study participants. Improvement precedes and increases with the level and years of sustained participation.

Progression of asymptomatic mild carotid artery stenosis: Implications for frequency of surveillance.

We looked retrospectively at the 3- to 5-year progression of mild, asymptomatic carotid artery stenosis (CAS). A random sample of 600 patients who had undergone at least two carotid artery duplex ultrasounds between 31 October 2006 and 1 November 2016 with a second duplex ⩾3 and ⩽5 years following the initial one were screened for inclusion. Internal carotid arteries (ICAs) were included if they had 20-49% stenosis on the initial duplex, with 440 carotid arteries meeting this criteria. Analyses were performed utilizing chi-squared and two-tailed t-tests. Twenty-four (5.45%) of the initial 440 carotid arteries progressed to moderate CAS. There was a statistically significant increase in the prevalence of hypertension (68% vs 47%, p=0.022) and diabetes mellitus (44% vs 22%, p=0.008) in patients with carotids that progressed to moderate CAS. There was a decrease in moderate-intensity statin use (32% vs 58%, p=0.005) and an increase in patients not on statins (36% vs 11%, p=0.001) in the group of carotids that progressed to moderate CAS. One carotid artery (0.2%) progressed from mild CAS to severe CAS. If supported by others, our data may lead to a change in the recommendations regarding appropriate follow-up of asymptomatic CAS.

Drosophila Syncrip modulates the expression of mRNAs encoding key synaptic proteins required for morphology at the neuromuscular junction.

Localized mRNA translation is thought to play a key role in synaptic plasticity, but the identity of the transcripts and the molecular mechanism underlying their function are still poorly understood. Here, we show that Syncrip, a regulator of localized translation in the Drosophila oocyte and a component of mammalian neuronal mRNA granules, is also expressed in the Drosophila larval neuromuscular junction, where it regulates synaptic growth. We use RNA-immunoprecipitation followed by high-throughput sequencing and qRT-PCR to show that Syncrip associates with a number of mRNAs encoding proteins with key synaptic functions, including msp-300, syd-1, neurexin-1, futsch, highwire, discs large, and α-spectrin. The protein levels of MSP-300, Discs large, and a number of others are significantly affected in syncrip null mutants. Furthermore, syncrip mutants show a reduction in MSP-300 protein levels and defects in muscle nuclear distribution characteristic of msp-300 mutants. Our results highlight a number of potential new players in localized translation during synaptic plasticity in the neuromuscular junction. We propose that Syncrip acts as a modulator of synaptic plasticity by regulating the translation of these key mRNAs encoding synaptic scaffolding proteins and other important components involved in synaptic growth and function.

Co-regulated expression of alpha and beta mRNAs encoding HLA-DR surface heterodimers is mediated by the MHCII RNA operon.

Major histocompatibility complex class II (MHCII) molecules are heterodimeric surface proteins involved in the presentation of exogenous antigens during the adaptive immune response. We demonstrate the existence of a fine level of regulation, coupling the transcription and processing of mRNAs encoding α and ß chains of MHCII molecules, mediated through binding of their Untraslated Regions (UTRs) to the same ribonucleoproteic complex (RNP). We propose a dynamic model, in the context of the 'MHCII RNA operon' in which the increasing levels of DRA and DRB mRNAs are docked by the RNP acting as a bridge between 5'- and 3'-UTR of the same messenger, building a loop structure and, at the same time, joining the two chains, thanks to shared common predicted secondary structure motifs. According to cell needs, as during immune surveillance, this RNP machinery guarantees a balanced synthesis of DRA and DRB mRNAs and a consequent balanced surface expression of the heterodimer.

Doppler Echocardiographic Phenotypes in Suspected 'Severe' Aortic Stenosis: Matrix-Based Approach to Diagnosis and Management.

BACKGROUND: Among patients with suspected severe aortic stenosis (AS), Doppler echocardiographic (DE) data are often discordant, and further analysis is required for accurate diagnosis and optimal management. In this study, an automated matrix-based approach was applied to an echocardiographic database of patients with AS that identified 5 discrete echocardiographic data patterns, 1 concordant and 4 discordant, each reflecting a particular pathophysiology/measurement error that guides further workup and management. METHODS: A primary/discovery cohort of consecutive echocardiographic studies with at least 1 DE parameter of severe AS and analogous data from an independent secondary/validation cohort were retrospectively analyzed. Parameter thresholds for inclusion were aortic valve area (AVA) <1.0 cm2, transaortic mean gradient (MG) ≥ 40 mmHg, and/or transaortic peak velocity (PV) ≥ 4.0 m/sec. Doppler velocity index (DVI) was also determined. Logic provided by an in-line SQL query embedded within the database was used to assign each patient to 1 of 5 discrete matrix patterns, each reflecting 1 or more specific pathophysiologies. Feasibility of automated pattern-driven triage of discordant cases was also evaluated. RESULTS: In both cohorts, data from each patient fitted only 1 data pattern. Of the 4,643 primary cohort patients, 39% had concordant parameters for severe AS and DVI <0.30 (pattern 1); 35% had AVA < 1.0 cm2, MG < 40 mm Hg, PV < 4 m/sec, DVI < 0.30 (pattern 2); 9% had MG ≥ 40 mmHg and/or PV ≥ 4 m/sec, DVI > 0.30 (pattern 3); 10% had AVA < 1.0 cm2, MG < 40 mmHg, PV < 4 m/sec, DVI >0.30 (pattern 4); and 7% had MG > 40 mmHg and/or PV ≥ 4 m/sec, AVA > 1.0 cm2, DVI < 0.30 (pattern 5). Findings were validated among the 387 secondary cohort patients in whom pattern distribution was remarkably similar. CONCLUSIONS: Matrix-based pattern recognition permits automated in-line identification of specific pathophysiology and/or measurement error among patients with suspected severe AS and discordant DE data.

The mitosis and neurodevelopment proteins NDE1 and NDEL1 form dimers, tetramers, and polymers with a folded back structure in solution.

Paralogs NDE1 (nuclear distribution element 1) and NDEL1 (NDE-like 1) are essential for mitosis and neurodevelopment. Both proteins are predicted to have similar structures, based upon high sequence similarity, and they co-complex in mammalian cells. X-ray diffraction studies and homology modeling suggest that their N-terminal regions (residues 8-167) adopt continuous, extended α-helical coiled-coil structures, but no experimentally derived information on the structure of their C-terminal regions or the architecture of the full-length proteins is available. In the case of NDE1, no biophysical data exists. Here we characterize the structural architecture of both full-length proteins utilizing negative stain electron microscopy along with our established paradigm of chemical cross-linking followed by tryptic digestion, mass spectrometry, and database searching, which we enhance using isotope labeling for mixed NDE1-NDEL1. We determined that full-length NDE1 forms needle-like dimers and tetramers in solution, similar to crystal structures of NDEL1, as well as chain-like end-to-end polymers. The C-terminal domain of each protein, required for interaction with key protein partners dynein and DISC1 (disrupted-in-schizophrenia 1), includes a predicted disordered region that allows a bent back structure. This facilitates interaction of the C-terminal region with the N-terminal coiled-coil domain and is in agreement with previous results showing N- and C-terminal regions of NDEL1 and NDE1 cooperating in dynein interaction. It sheds light on recently identified mutations in the NDE1 gene that cause truncation of the encoded protein. Additionally, analysis of mixed NDE1-NDEL1 complexes demonstrates that NDE1 and NDEL1 can interact directly.

Reassessment of weak parent-of-origin expression bias shows it rarely exists outside of known imprinted regions.

In mouse and human, genes subjected to genomic imprinting have been shown to function in development, behavior, and post-natal adaptations. Failure to correctly imprint genes in human is associated with developmental syndromes, adaptive, and metabolic disorders during life as well as numerous forms of cancer. In recent years researchers have turned to RNA-seq technologies applied to reciprocal hybrid strains of mice to identify novel imprinted genes, causing a threefold increase in genes reported as having a parental origin-specific expression bias. The functional relevance of parental origin-specific expression bias is not fully appreciated especially since many are reported with only minimal parental bias (e.g. 51:49). Here, we present an in-depth meta-analysis of previously generated RNA-seq data and show that the methods used to generate and analyze libraries greatly influence the calling of allele-specific expression. Validation experiments show that most novel genes called with parental-origin-specific allelic bias are artefactual, with the mouse strain contributing a larger effect on expression biases than parental origin. Of the weak novel genes that do validate, most are located at the periphery of known imprinted domains, suggesting they may be affected by local allele- and tissue-specific conformation. Together these findings highlight the need for robust tools, definitions, and validation of putative imprinted genes to provide meaningful information within imprinting databases and to understand the functional and mechanistic implications of the process.

Primitive haematopoiesis in the human placenta gives rise to macrophages with epigenetically silenced HLA-DR.

The earliest macrophages are generated during embryonic development from erythro-myeloid progenitors (EMPs) via primitive haematopoiesis. Although this process is thought to be spatially restricted to the yolk sac in the mouse, in humans, it remains poorly understood. Human foetal placental macrophages, or Hofbauer cells (HBC), arise during the primitive haematopoietic wave ~18 days post conception and lack expression of human leukocyte antigen (HLA) class II. Here, we identify a population of placental erythro-myeloid progenitors (PEMPs) in the early human placenta that have conserved features of primitive yolk sac EMPs, including the lack of HLF expression. Using in vitro culture experiments we demonstrate that PEMP generate HBC-like cells lacking HLA-DR expression. We find the absence of HLA-DR in primitive macrophages is mediated via epigenetic silencing of class II transactivator, CIITA, the master regulator of HLA class II gene expression. These findings establish the human placenta as an additional site of primitive haematopoiesis.