MCM complexes are barriers that restrict cohesin-mediated loop extrusion.

Eukaryotic genomes are compacted into loops and topologically associating domains (TADs)1-3, which contribute to transcription, recombination and genomic stability4,5. Cohesin extrudes DNA into loops that are thought to lengthen until CTCF boundaries are encountered6-12. Little is known about whether loop extrusion is impeded by DNA-bound machines. Here we show that the minichromosome maintenance (MCM) complex is a barrier that restricts loop extrusion in G1 phase. Single-nucleus Hi-C (high-resolution chromosome conformation capture) of mouse zygotes reveals that MCM loading reduces CTCF-anchored loops and decreases TAD boundary insulation, which suggests that loop extrusion is impeded before reaching CTCF. This effect extends to HCT116 cells, in which MCMs affect the number of CTCF-anchored loops and gene expression. Simulations suggest that MCMs are abundant, randomly positioned and partially permeable barriers. Single-molecule imaging shows that MCMs are physical barriers that frequently constrain cohesin translocation in vitro. Notably, chimeric yeast MCMs that contain a cohesin-interaction motif from human MCM3 induce cohesin pausing, indicating that MCMs are 'active' barriers with binding sites. These findings raise the possibility that cohesin can arrive by loop extrusion at MCMs, which determine the genomic sites at which sister chromatid cohesion is established. On the basis of in vivo, in silico and in vitro data, we conclude that distinct loop extrusion barriers shape the three-dimensional genome.

Polycomb Group Proteins Regulate Chromatin Architecture in Mouse Oocytes and Early Embryos.

In mammals, chromatin organization undergoes drastic reorganization during oocyte development. However, the dynamics of three-dimensional chromatin structure in this process is poorly characterized. Using low-input Hi-C (genome-wide chromatin conformation capture), we found that a unique chromatin organization gradually appears during mouse oocyte growth. Oocytes at late stages show self-interacting, cohesin-independent compartmental domains marked by H3K27me3, therefore termed Polycomb-associating domains (PADs). PADs and inter-PAD (iPAD) regions form compartment-like structures with strong inter-domain interactions among nearby PADs. PADs disassemble upon meiotic resumption from diplotene arrest but briefly reappear on the maternal genome after fertilization. Upon maternal depletion of Eed, PADs are largely intact in oocytes, but their reestablishment after fertilization is compromised. By contrast, depletion of Polycomb repressive complex 1 (PRC1) proteins attenuates PADs in oocytes, which is associated with substantial gene de-repression in PADs. These data reveal a critical role of Polycomb in regulating chromatin architecture during mammalian oocyte growth and early development.

The genome of Schmidtea mediterranea and the evolution of core cellular mechanisms.

The planarian Schmidtea mediterranea is an important model for stem cell research and regeneration, but adequate genome resources for this species have been lacking. Here we report a highly contiguous genome assembly of S. mediterranea, using long-read sequencing and a de novo assembler (MARVEL) enhanced for low-complexity reads. The S. mediterranea genome is highly polymorphic and repetitive, and harbours a novel class of giant retroelements. Furthermore, the genome assembly lacks a number of highly conserved genes, including critical components of the mitotic spindle assembly checkpoint, but planarians maintain checkpoint function. Our genome assembly provides a key model system resource that will be useful for studying regeneration and the evolutionary plasticity of core cell biological mechanisms.

The axolotl genome and the evolution of key tissue formation regulators.

Salamanders serve as important tetrapod models for developmental, regeneration and evolutionary studies. An extensive molecular toolkit makes the Mexican axolotl (Ambystoma mexicanum) a key representative salamander for molecular investigations. Here we report the sequencing and assembly of the 32-gigabase-pair axolotl genome using an approach that combined long-read sequencing, optical mapping and development of a new genome assembler (MARVEL). We observed a size expansion of introns and intergenic regions, largely attributable to multiplication of long terminal repeat retroelements. We provide evidence that intron size in developmental genes is under constraint and that species-restricted genes may contribute to limb regeneration. The axolotl genome assembly does not contain the essential developmental gene Pax3. However, mutation of the axolotl Pax3 paralogue Pax7 resulted in an axolotl phenotype that was similar to those seen in Pax3-/- and Pax7-/- mutant mice. The axolotl genome provides a rich biological resource for developmental and evolutionary studies.

Author Correction: The axolotl genome and the evolution of key tissue formation regulators.

In the originally published version of this Article, the sequenced axolotl strain (the homozygous white mutant) was denoted as 'D/D' rather than 'd/d' in Fig. 1a and the accompanying legend, the main text and the Methods section. The original Article has been corrected online.

Qualitative evaluation of the implementation and future sustainability of an e-referral system for smoking cessation at a US NCI-designated comprehensive cancer center: lessons learned.

BACKGROUND: Promoting smoking cessation is recognized as an essential part of cancer care. Moffitt Cancer Center, supported by the National Cancer Institute Cancer Moonshot Cancer Center Cessation Initiative, developed and implemented an opt-out-based automatic electronic health record (EHR)-mediated referral (e-referral) system for Tobacco Quitline services along with options for local group cessation support and an in-house tobacco treatment specialist. This study evaluated barriers and facilitators for implementation of the e-referral system. METHOD: Steering committee members (N=12) responsible for developing and implementing the new clinical workflow and nurses (N=12) who were expected to use the new e-referral system completed semi-structured interviews. Qualitative thematic content analyses were conducted. RESULTS: Interviewees perceived the e-referral system as an effective strategy for identifying and referring smokers to cessation services. However, barriers were noted including competing demands and perceptions that smoking cessation was a low priority and that some patients were likely to have low motivation to quit smoking. Suggestions to improve future implementation and sustainability included providing regular trainings and e-referral outcome reports and increasing the visibility of the e-referral system within the EHR. CONCLUSION: Initial implementation of the e-referral system was perceived as successful; however, additional implementation strategies are needed to ensure sustainability at both the clinician and system levels. Recommendations for future modifications include providing regular clinician trainings and developing a fully closed-loop system. Implications for cancer survivors Initial implementation of an e-referral system for smoking cessation for cancer patients revealed opportunities to improve the smoking cessation referral process at cancer centers.

A Method for Economical Smartphone-Based Clinical 3D Facial Scanning.

PURPOSE: Three-dimensional (3D) facial scanning is an emerging clinical tool to capture external anatomical features for quantitative assessment and treatment in a wide range of clinical settings. MATERIALS AND METHODS: In this study, an economical approach for rapid scanning of faces in the clinic was developed and validated to record valuable 3D patient data using smartphone cameras and photogrammetry software. Five novice operators were recruited to watch an instructional video developed on the technique before scanning 20 healthy adult participants. RESULTS: The smartphone-based photogrammetry approach produced scans with 1.3 mm (±0.3 mm) accuracy in comparison to a metrology-rated gold standard device and were 88% (±14%) complete, with no significant difference observed between operators. A moderate to strong intrarater reliability was determined for all novice operators, suggesting that first-use operators can capture consistent scans based on watching an instructional video. CONCLUSION: Smartphone photogrammetry could provide a rapid, noninvasive and economical method to capture patient morphological data for clinical assessment and personalized device manufacture. Inexperienced operators can quickly learn and utilize smartphone photogrammetry to provide accurate and reliable facial scans, essential for future clinical translation.

eggNOG v4.0: nested orthology inference across 3686 organisms.

With the increasing availability of various 'omics data, high-quality orthology assignment is crucial for evolutionary and functional genomics studies. We here present the fourth version of the eggNOG database (available at http://eggnog.embl.de) that derives nonsupervised orthologous groups (NOGs) from complete genomes, and then applies a comprehensive characterization and analysis pipeline to the resulting gene families. Compared with the previous version, we have more than tripled the underlying species set to cover 3686 organisms, keeping track with genome project completions while prioritizing the inclusion of high-quality genomes to minimize error propagation from incomplete proteome sets. Major technological advances include (i) a robust and scalable procedure for the identification and inclusion of high-quality genomes, (ii) provision of orthologous groups for 107 different taxonomic levels compared with 41 in eggNOGv3, (iii) identification and annotation of particularly closely related orthologous groups, facilitating analysis of related gene families, (iv) improvements of the clustering and functional annotation approach, (v) adoption of a revised tree building procedure based on the multiple alignments generated during the process and (vi) implementation of quality control procedures throughout the entire pipeline. As in previous versions, eggNOGv4 provides multiple sequence alignments and maximum-likelihood trees, as well as broad functional annotation. Users can access the complete database of orthologous groups via a web interface, as well as through bulk download.

An integrated approach for genome annotation of the eukaryotic thermophile Chaetomium thermophilum.

The thermophilic fungus Chaetomium thermophilum holds great promise for structural biology. To increase the efficiency of its biochemical and structural characterization and to explore its thermophilic properties beyond those of individual proteins, we obtained transcriptomics and proteomics data, and integrated them with computational annotation methods and a multitude of biochemical experiments conducted by the structural biology community. We considerably improved the genome annotation of Chaetomium thermophilum and characterized the transcripts and expression of thousands of genes. We furthermore show that the composition and structure of the expressed proteome of Chaetomium thermophilum is similar to its mesophilic relatives. Data were deposited in a publicly available repository and provide a rich source to the structural biology community.

eggNOG v3.0: orthologous groups covering 1133 organisms at 41 different taxonomic ranges.

Orthologous relationships form the basis of most comparative genomic and metagenomic studies and are essential for proper phylogenetic and functional analyses. The third version of the eggNOG database (http://eggnog.embl.de) contains non-supervised orthologous groups constructed from 1133 organisms, doubling the number of genes with orthology assignment compared to eggNOG v2. The new release is the result of a number of improvements and expansions: (i) the underlying homology searches are now based on the SIMAP database; (ii) the orthologous groups have been extended to 41 levels of selected taxonomic ranges enabling much more fine-grained orthology assignments; and (iii) the newly designed web page is considerably faster with more functionality. In total, eggNOG v3 contains 721,801 orthologous groups, encompassing a total of 4,396,591 genes. Additionally, we updated 4873 and 4850 original COGs and KOGs, respectively, to include all 1133 organisms. At the universal level, covering all three domains of life, 101,208 orthologous groups are available, while the others are applicable at 40 more limited taxonomic ranges. Each group is amended by multiple sequence alignments and maximum-likelihood trees and broad functional descriptions are provided for 450,904 orthologous groups (62.5%).

Suicide Risk Screening for Head and Neck Cancer Patients: An Implementation Study.

OBJECTIVES: There is limited research on suicide risk screening (SRS) among head and neck cancer (HNC) patients, a population at increased risk for suicide. To address this gap, this single-site mixed methods study assessed oncology professionals' perspectives about the feasibility, acceptability, and appropriateness of an electronic SRS program that was implemented as a part of routine care for HNC patients. METHODS: Staff who assisted with SRS implementation completed (e.g., nurses, medical assistants, advanced practice providers, physicians, social workers) a one-time survey (N = 29) and interview (N = 25). Quantitative outcomes were assessed using previously validated feasibility, acceptability, and appropriateness measures. Additional qualitative data were collected to provide context for interpreting the scores. RESULTS: Nurses and medical assistants, who were directly responsible for implementing SRS, reported low feasibility, acceptability, and appropriateness, compared with other team members (e.g., physicians, social workers, advanced practice providers). Team members identified potential improvements needed to optimize SRS, such as hiring additional staff, improving staff training, providing different modalities for screening completion among individuals with disabilities, and revising the patient-reported outcomes to improve suicide risk prediction. CONCLUSION: Staff perspectives about implementing SRS as a part of routine cancer care for HNC patients varied widely. Before screening can be implemented on a larger scale for HNC and other cancer patients, additional implementation strategies may be needed that optimize workflow and reduce staff burden, such as staff training, multiple modalities for completion, and refined tools for identifying which patients are at greatest risk for suicide.

Orthology prediction methods: a quality assessment using curated protein families.

The increasing number of sequenced genomes has prompted the development of several automated orthology prediction methods. Tests to evaluate the accuracy of predictions and to explore biases caused by biological and technical factors are therefore required. We used 70 manually curated families to analyze the performance of five public methods in Metazoa. We analyzed the strengths and weaknesses of the methods and quantified the impact of biological and technical challenges. From the latter part of the analysis, genome annotation emerged as the largest single influencer, affecting up to 30% of the performance. Generally, most methods did well in assigning orthologous group but they failed to assign the exact number of genes for half of the groups. The publicly available benchmark set (http://eggnog.embl.de/orthobench/) should facilitate the improvement of current orthology assignment protocols, which is of utmost importance for many fields of biology and should be tackled by a broad scientific community.

An automated parametric ear model to improve frugal 3D scanning methods for the advanced manufacturing of high-quality prosthetic ears.

Ear prostheses are commonly used for restoring aesthetics to those suffering missing or malformed external ears. Traditional fabrication of these prostheses is labour intensive and requires expert skill from a prosthetist. Advanced manufacturing including 3D scanning, modelling and 3D printing has the potential to improve this process, although more work is required before it is ready for routine clinical use. In this paper, we introduce a parametric modelling technique capable of producing high quality 3D models of the human ear from low-fidelity, frugal, patient scans; significantly reducing time, complexity and cost. Our ear model can be tuned to fit the frugal low-fidelity 3D scan through; (a) manual tuning, or (b) our automated particle filter approach. This potentially enables low-cost smartphone photogrammetry-based 3D scanning for high quality personalised 3D printed ear prosthesis. In comparison to standard photogrammetry, our parametric model improves completeness, from (81 ± 5)% to (87 ± 4)%, with only a modest reduction in accuracy, with root mean square error (RMSE) increasing from (1.0 ± 0.2) mm to (1.5 ± 0.2) mm (relative to metrology rated reference 3D scans, n = 14). Despite this reduction in the RMS accuracy, our parametric model improves the overall quality, realism, and smoothness. Our automated particle filter method differs only modestly compared to manual adjustments. Overall, our parametric ear model can significantly improve quality, smoothness and completeness of 3D models produced from 30-photograph photogrammetry. This enables frugal high-quality 3D ear models to be produced for use in the advanced manufacturing of ear prostheses.

Oncology Providers' and Professionals' Experiences With Suicide Risk Screening Among Patients With Head and Neck Cancer: A Qualitative Study.

PURPOSE: There has been limited study of the implementation of suicide risk screening for patients with head and neck cancer (HNC) as a part of routine care. To address this gap, this study assessed oncology providers' and professionals' perspectives about barriers and facilitators of implementing a suicide risk screening among patients with HNC. MATERIALS AND METHODS: All patients with HNC with an in-person visit completed a suicide risk screening on an electronic tablet. Patients reporting passive death wish were then screened for active suicidal ideation and referred for appropriate intervention. Interviews were conducted with 25 oncology providers and professionals who played a key role in implementation including nurses, medical assistants, patient access representatives, advanced practice providers, physicians, social workers, and informatics staff. The interview guide was based on the Consolidated Framework for Implementation Research. Interviews were transcribed and analyzed for themes. RESULTS: Participants identified multilevel implementation barriers, such as intervention level (eg, patient difficulty with using a tablet), process level (eg, limited nursing engagement), organizational level (eg, limited clinic Wi-Fi connectivity), and individual level (eg, low clinician self-efficacy for interpreting and acting upon patient-reported outcome scores). Participants noted facilitators, such as effective care coordination across nursing and social work staff and the opportunity for patients to be screened multiple times. Participants recommended strengthening patient and clinician education and providing patients with other modalities for data entry (eg, desktop computer in the waiting room). CONCLUSION: Participants identified important intervention modifications that may be needed to optimize suicide risk screening in cancer care settings.

Ultrasound Imaging Offers Promising Alternative to Create 3-D Models for Personalised Auricular Implants.

Three-dimensional imaging and advanced manufacturing are being applied in health care research to create novel diagnostic and surgical planning methods, as well as personalised treatments and implants. For ear reconstruction, where a cartilage-shaped implant is embedded underneath the skin to re-create shape and form, volumetric imaging and segmentation processing to capture patient anatomy are particularly challenging. Here, we introduce 3-D ultrasound (US) as an available option for imaging the external ear and underlying auricular cartilage structure, and compare it with computed tomography (CT) and magnetic resonance imaging (MRI) against micro-CT (µCT) as a high-resolution reference (gold standard). US images were segmented to create 3-D models of the auricular cartilage and compared against models generated from µCT to assess accuracy. We found that CT was significantly less accurate than the other methods (root mean square [RMS]: 1.30 ± 0.5 mm) and had the least contrast between tissues. There was no significant difference between MRI (RMS: 0.69 ± 0.2 mm) and US (0.55 ± 0.1 mm). US was also the least expensive imaging method at half the cost of MRI. These results unveil a novel use of ultrasound imaging that has not been presented before, as well as support its more widespread use in biofabrication as a low-cost imaging technique to create patient-specific 3D models and implants.

Frugal 3D scanning using smartphones provides an accessible framework for capturing the external ear.

Three-dimensional (3D) scanning technologies, such as medical imaging and surface scanning, have important applications for capturing patient anatomy to create personalised prosthetics. Digital approaches for capturing anatomical detail as opposed to traditional, invasive impression techniques significantly reduces turnaround times and lower production costs while still maintaining the high aesthetic quality of the end product. While previous case studies utilise expensive 3D scanning and modelling frameworks, their clinical translation is limited due to high equipment costs. In this study, we develop and validate a low-cost framework for clinical 3D scanning of the external ear using photogrammetry and a smartphone camera. We recruited five novice operators who watched an instructional video before scanning 20 healthy adult participant ears who did not have microtia. Our results show that the smartphone-based photogrammetry methodology produces 3D scans of the external ear that were accurate to (1.5 ± 0.4) mm and were (71 ± 14) % complete compared with those from a gold standard reference scanner, with no significant difference observed between operators. A moderate to strong interrater reliability was determined for all novice operators, suggesting that all novice operators were able to capture repeatable scans. The development of this smartphone photogrammetry approach has the potential to provide a non-invasive, inexpensive and accessible means to capture patient morphology for use in clinical assessment and personalised device manufacture, specifically for ear prostheses. We also demonstrate that inexperienced operators can rapidly learn and apply smartphone photogrammetry for accurate and reliable scans of the external ear with important applications for future clinical translation.

Ovulation suppression protects against chromosomal abnormalities in mouse eggs at advanced maternal age.

The frequency of egg aneuploidy and trisomic pregnancies increases with maternal age. To what extent individual approaches can delay the "maternal age effect" is unclear because multiple causes contribute to chromosomal abnormalities in mammalian eggs. We propose that ovulation frequency determines the physiological aging of oocytes, a key aspect of which is the ability to accurately segregate chromosomes and produce euploid eggs. To test this hypothesis, ovulations were reduced using successive pregnancies, hormonal contraception, and a pre-pubertal knockout mouse model, and the effects on chromosome segregation and egg ploidy were examined. We show that each intervention reduces chromosomal abnormalities in eggs of aged mice, suggesting that ovulation reduction delays oocyte aging. The protective effect can be partly explained by retention of chromosomal Rec8-cohesin that maintains sister chromatid cohesion in meiosis. In addition, single-nucleus Hi-C (snHi-C) revealed deterioration in the 3D chromatin structure including an increase in extruded loop sizes in long-lived oocytes. Artificial cleavage of Rec8 is sufficient to increase extruded loop sizes, suggesting that cohesin complexes maintaining cohesion restrict loop extrusion. These findings suggest that ovulation suppression protects against Rec8 loss, thereby maintaining both sister chromatid cohesion and 3D chromatin structure and promoting production of euploid eggs. We conclude that the maternal age effect can be delayed in mice. An implication of this work is that long-term ovulation-suppressing conditions can potentially reduce the risk of aneuploid pregnancies at advanced maternal age.

Past, Present, and Future of Soft-Tissue Prosthetics: Advanced Polymers and Advanced Manufacturing.

Millions of people worldwide experience disfigurement due to cancers, congenital defects, or trauma, leading to significant psychological, social, and economic disadvantage. Prosthetics aim to reduce their suffering by restoring aesthetics and function using synthetic materials that mimic the characteristics of native tissue. In the 1900s, natural materials used for thousands of years in prosthetics were replaced by synthetic polymers bringing about significant improvements in fabrication and greater realism and utility. These traditional methods have now been disrupted by the advanced manufacturing revolution, radically changing the materials, methods, and nature of prosthetics. In this report, traditional synthetic polymers and advanced prosthetic materials and manufacturing techniques are discussed, including a focus on prosthetic material degradation. New manufacturing approaches and future technological developments are also discussed in the context of specific tissues requiring aesthetic restoration, such as ear, nose, face, eye, breast, and hand. As advanced manufacturing moves from research into clinical practice, prosthetics can begin new age to significantly improve the quality of life for those suffering tissue loss or disfigurement.

Advancements in Soft-Tissue Prosthetics Part A: The Art of Imitating Life.

Physical disfigurement due to congenital defects, trauma, or cancer causes considerable distress and physical impairment for millions of people worldwide; impacting their economic, psychological and social wellbeing. Since 3000 B.C., prosthetic devices have been used to address these issues by restoring both aesthetics and utility to those with disfigurement. Internationally, academic and industry researchers are constantly developing new materials and manufacturing techniques to provide higher quality and lower cost prostheses to those people who need them. New advanced technologies including 3D imaging, modeling, and printing are revolutionizing the way prostheses are now made. These new approaches are disrupting the traditional and manual art form of prosthetic production which are laborious and costly and are being replaced by more precise and quantitative processes which enable the rapid, low cost production of patient-specific prostheses. In this two part review, we provide a comprehensive report of past, present and emerging soft-tissue prosthetic materials and manufacturing techniques. In this review, part A, we examine, historically, the ideal properts of a polymeric material when applied in soft-tissue prosthetics. We also detail new research approaches to target specific tissues which commonly require aesthetic restoration (e.g. ear, nose and eyes) and discuss both traditional and advanced fabrication methods, from hand-crafted impression based approaches to advanced manufactured prosthetics. We discuss the chemistry and related details of most significant synthetic polymers used in soft-tissue prosthetics in Part B. As advanced manufacturing transitions from research into practice, the five millennia history of prosthetics enters a new age of economic, personalized, advanced soft tissue prosthetics and with this comes significantly improved quality of life for the people affected by tissue loss.

Advancements in Soft-Tissue Prosthetics Part B: The Chemistry of Imitating Life.

Each year, congenital defects, trauma or cancer often results in considerable physical disfigurement for many people worldwide. This adversely impacts their psychological, social and economic outlook, leading to poor life experiences and negative health outcomes. In many cases of soft tissue disfigurement, highly personalized prostheses are available to restore both aesthetics and function. As discussed in part A of this review, key to the success of any soft tissue prosthetic is the fundamental properties of the materials. This determines the maximum attainable level of aesthetics, attachment mechanisms, fabrication complexity, cost, and robustness. Since the early-mid 20th century, polymers have completely replaced natural materials in prosthetics, with advances in both material properties and fabrication techniques leading to significantly improved capabilities. In part A, we discussed the history of polymers in prosthetics, their ideal properties, and the application of polymers in prostheses for the ear, nose, eye, breast and finger. We also reviewed the latest developments in advanced manufacturing and 3D printing, including different fabrication technologies and new and upcoming materials. In this review, Part B, we detail the chemistry of the most commonly used synthetic polymers in soft tissue prosthetics; silicone, acrylic resin, vinyl polymer, and polyurethane elastomer. For each polymer, we briefly discuss their history before detailing their chemistry and fabrication processes. We also discuss degradation of the polymer in the context of their application in prosthetics, including time and weathering, the impact of skin secretions, microbial growth and cleaning and disinfecting. Although advanced manufacturing promises new fabrication capabilities using exotic synthetic polymers with programmable material properties, silicones and acrylics remain the most commonly used materials in prosthetics today. As research in this field progresses, development of new variations and fabrication techniques based on these synthetic polymers will lead to even better and more robust soft tissue prosthetics, with improved life-like aesthetics and lower cost manufacturing.