RNF212B E3 ligase is essential for crossover designation and maturation during male and female meiosis in the mouse.

Meiosis, a reductional cell division, relies on precise initiation, maturation, and resolution of crossovers (COs) during prophase I to ensure the accurate segregation of homologous chromosomes during metaphase I. This process is regulated by the interplay of RING-E3 ligases such as RNF212 and HEI10 in mammals. In this study, we functionally characterized a recently identified RING-E3 ligase, RNF212B. RNF212B colocalizes and interacts with RNF212, forming foci along chromosomes from zygonema onward in a synapsis-dependent and DSB-independent manner. These consolidate into larger foci at maturing COs, colocalizing with HEI10, CNTD1, and MLH1 by late pachynema. Genetically, RNF212B foci formation depends on Rnf212 but not on Msh4, Hei10, and Cntd1, while the unloading of RNF212B at the end of pachynema is dependent on Hei10 and Cntd1. Mice lacking RNF212B, or expressing an inactive RNF212B protein, exhibit modest synapsis defects, a reduction in the localization of pro-CO factors (MSH4, TEX11, RPA, MZIP2) and absence of late CO-intermediates (MLH1). This loss of most COs by diakinesis results in mostly univalent chromosomes. Double mutants for Rnf212b and Rnf212 exhibit an identical phenotype to that of Rnf212b single mutants, while double heterozygous demonstrate a dosage-dependent reduction in CO number, indicating a functional interplay between paralogs. SUMOylome analysis of testes from Rnf212b mutants and pull-down analysis of Sumo- and Ubiquitin-tagged HeLa cells, suggest that RNF212B is an E3-ligase with Ubiquitin activity, serving as a crucial factor for CO maturation. Thus, RNF212 and RNF212B play vital, yet overlapping roles, in ensuring CO homeostasis through their distinct E3 ligase activities.

A Human Hereditary Cardiomyopathy Shares a Genetic Substrate With Bicuspid Aortic Valve.

BACKGROUND: The complex genetics underlying human cardiac disease is evidenced by its heterogenous manifestation, multigenic basis, and sporadic occurrence. These features have hampered disease modeling and mechanistic understanding. Here, we show that 2 structural cardiac diseases, left ventricular noncompaction (LVNC) and bicuspid aortic valve, can be caused by a set of inherited heterozygous gene mutations affecting the NOTCH ligand regulator MIB1 (MINDBOMB1) and cosegregating genes. METHODS: We used CRISPR-Cas9 gene editing to generate mice harboring a nonsense or a missense MIB1 mutation that are both found in LVNC families. We also generated mice separately carrying these MIB1 mutations plus 5 additional cosegregating variants in the ASXL3, APCDD1, TMX3, CEP192, and BCL7A genes identified in these LVNC families by whole exome sequencing. Histological, developmental, and functional analyses of these mouse models were carried out by echocardiography and cardiac magnetic resonance imaging, together with gene expression profiling by RNA sequencing of both selected engineered mouse models and human induced pluripotent stem cell-derived cardiomyocytes. Potential biochemical interactions were assayed in vitro by coimmunoprecipitation and Western blot. RESULTS: Mice homozygous for the MIB1 nonsense mutation did not survive, and the mutation caused LVNC only in heteroallelic combination with a conditional allele inactivated in the myocardium. The heterozygous MIB1 missense allele leads to bicuspid aortic valve in a NOTCH-sensitized genetic background. These data suggest that development of LVNC is influenced by genetic modifiers present in affected families, whereas valve defects are highly sensitive to NOTCH haploinsufficiency. Whole exome sequencing of LVNC families revealed single-nucleotide gene variants of ASXL3, APCDD1, TMX3, CEP192, and BCL7A cosegregating with the MIB1 mutations and LVNC. In experiments with mice harboring the orthologous variants on the corresponding Mib1 backgrounds, triple heterozygous Mib1 Apcdd1 Asxl3 mice showed LVNC, whereas quadruple heterozygous Mib1 Cep192 Tmx3;Bcl7a mice developed bicuspid aortic valve and other valve-associated defects. Biochemical analysis suggested interactions between CEP192, BCL7A, and NOTCH. Gene expression profiling of mutant mouse hearts and human induced pluripotent stem cell-derived cardiomyocytes revealed increased cardiomyocyte proliferation and defective morphological and metabolic maturation. CONCLUSIONS: These findings reveal a shared genetic substrate underlying LVNC and bicuspid aortic valve in which MIB1-NOTCH variants plays a crucial role in heterozygous combination with cosegregating genetic modifiers.

Doctor trustworthiness influences pain and its neural correlates in virtual medical interactions.

Trust is an important component of the doctor-patient relationship and is associated with improved patient satisfaction and health outcomes. Previously, we reported that patient feelings of trust and similarity toward their clinician predicted reductions in evoked pain in response to painful heat stimulations. In the present study, we investigated the brain mechanisms underlying this effect. We used face stimuli previously developed using a data-driven computational modeling approach that differ in perceived trustworthiness and superimposed them on bodies dressed in doctors' attire. During functional magnetic resonance imaging, participants (n = 42) underwent a series of virtual medical interactions with these doctors during which they received painful heat stimulation as an analogue of a painful diagnostic procedure. Participants reported increased pain when receiving painful heat stimulations from low-trust doctors, which was accompanied by increased activity in pain-related brain regions and a multivariate pain-predictive neuromarker. Findings suggest that patient trust in their doctor may have tangible impacts on pain and point to a potential brain basis for trust-related reductions in pain through the modulation of brain circuitry associated with the sensory-discriminative and affective-motivational dimensions of pain.

The PSMA8 subunit of the spermatoproteasome is essential for proper meiotic exit and mouse fertility.

The ubiquitin proteasome system regulates meiotic recombination in yeast through its association with the synaptonemal complex, a 'zipper'-like structure that holds homologous chromosome pairs in synapsis during meiotic prophase I. In mammals, the proteasome activator subunit PA200 targets acetylated histones for degradation during somatic DNA double strand break repair and during histone replacement during spermiogenesis. We investigated the role of the testis-specific proteasomal subunit α4s (PSMA8) during spermatogenesis, and found that PSMA8 was localized to and dependent on the central region of the synaptonemal complex. Accordingly, synapsis-deficient mice show delocalization of PSMA8. Moreover, though Psma8-deficient mice are proficient in meiotic homologous recombination, there are alterations in the proteostasis of several key meiotic players that, in addition to the known substrate acetylated histones, have been shown by a proteomic approach to interact with PSMA8, such as SYCP3, SYCP1, CDK1 and TRIP13. These alterations lead to an accumulation of spermatocytes in metaphase I and II which either enter massively into apoptosis or give rise to a low number of aberrant round spermatids that apoptose before histone replacement takes place.

Ubiquitin-specific protease 26 (USP26) is not essential for mouse gametogenesis and fertility.

Ubiquitin-specific protease 26 (USP26) is a deubiquitylating enzyme belonging to the USPs family with a transcription pattern restricted to the male germline. Since protein ubiquitination is an essential regulatory mechanism during meiosis, many efforts have been focused on elucidating the function of USP26 and its relationship with fertility. During the last decade, several studies have reported the presence of different polymorphisms in USP26 in patients with non-obstructive azoospermia (NOA) or severe oligozoospermia suggesting that this gene may be associated with human infertility. However, other studies have revealed the presence of these and novel polymorphisms, including nonsense mutations, in men with normal spermatogenesis as well. Thus, the results remain controversial and its function is unknown. In the present study, we describe the in vivo functional analysis of mice lacking USP26. The phenotypic analysis of two different Usp26-null mutants showed no overt-phenotype with both males and females being fertile. Cytological analysis of spermatocytes showed no defects in synapsis, chromosome dynamics, DNA repair, or recombination. Histopathological analysis revealed a normal distribution and number of the different cell types in both male and female mice. Finally, normal counts were observed in fertility assessments. These results represent the first in vivo evidence showing that USP26 is not essential for mouse gametogenesis.

Orthopedic surgery residents' perception of online education in their programs during the COVID-19 pandemic: should it be maintained after the crisis?

Background and purpose - During the COVID-19 pandemic, most of the teaching centers in Chile have shifted to online resources. We decided to do a survey on orthopedic residents regarding this type of education to assess for strengths and weaknesses of digital education in orthopedic programs.Methods - A survey was performed targeting 110 orthopedic residents belonging to different training programs around the country. 100 residents completed the survey.Results - 86% stated that their programs are using online education. When asked in detail, 86% had been involved in webinars, 28% had received online presentations, 12% had participated in online tests, and 7% had evaluated patients. Webinars were rated (1 = very unsatisfactory, 10 = very satisfactory) with a mean grade of 8.1 (1-10), online presentations 7.3 (1-10), online tests 3.8 (1-8), and online patient evaluations 2.9 (1-9). When asked if, after the end of the pandemic, they would continue using the online modalities, 82% would continue attending webinars, 72% would continue watching online presentations, 27% would continue performing online tests, and 33% of the residents would continue performing online evaluations of patients.Interpretation - Even though resident evaluation of online activities is positive, face-to-face theoretical activities are still valued as a necessary complement for orthopedic residency education.

Sororin loads to the synaptonemal complex central region independently of meiotic cohesin complexes.

The distribution and regulation of the cohesin complexes have been extensively studied during mitosis. However, the dynamics of their different regulators in vertebrate meiosis is largely unknown. In this work, we have analyzed the distribution of the regulatory factor Sororin during male mouse meiosis. Sororin is detected at the central region of the synaptonemal complex during prophase I, in contrast with the previously reported localization of other cohesin components in the lateral elements. This localization of Sororin depends on the transverse filaments protein SYCP1, but not on meiosis-specific cohesin subunits REC8 and SMC1ß. By late prophase I, Sororin accumulates at centromeres and remains there up to anaphase II The phosphatase activity of PP2A seems to be required for this accumulation. We hypothesize that Sororin function at the central region of the synaptonemal complex could be independent on meiotic cohesin complexes. In addition, we suggest that Sororin participates in the regulation of centromeric cohesion during meiosis in collaboration with SGO2-PP2A.

Organizational design for strengthening community-based tourism: Empowering stakeholders for self-organization and networking.

The San Pablo community in El Triunfo, Ecuador, is emerging as a promising community-based tourism destination. Despite its potential, the lack of knowledge about self-organization and its implementation has hindered effective tourism management. To address this challenge, a participatory approach was employed, involving the community and key stakeholders, such as the local government of El Triunfo. Through the utilization of Design Thinking and both online and in-person interviews, it was identified that an organizational structure based on networks and a culture of self-organization can drive local tourism. These aspects were incorporated into a Design Thinking-guided process, contributing to the understanding of how to forge an appropriate organizational framework for the community. Furthermore, this study aims for broader impact. The goal is not only to strengthen tourism in San Pablo, but also to inform the management of strategies and policies in other entities. The findings offer valuable insights to similar communities in Ecuador and the region. Collectively, this research enhances the comprehension of community-based tourism and proposes practical solutions for optimizing its management in emerging contexts.

Cory's Shearwater (Calonectris borealis): Exploring Normal Head Anatomy through Cross-Sectional Anatomy, Computed Tomography and Magnetic Resonance Imaging.

Cory's shearwater, or Calonectris borealis, stands out as a symbolic figure in the world of seabirds, playing a crucial role in marine ecosystems globally. Belonging to the Procellariidae family, it is singularized by its imposing wingspan and intricate migration patterns connecting it to various regions from the North Atlantic to the Pacific. Its role in the marine food chain, specialized diet and adaptation for nesting in the Canary Archipelago underscore its ecological importance. However, Cory's shearwater also faces important threats, such as the invasion of foreign species, highlighting the need for its conservation. Among the conservation issues, studies on its biology, the main threats it faces and its normal anatomy are essential to preserve marine biodiversity. Additionally, a variety of imaging techniques, such as computed tomography and magnetic resonance, facilitates the understanding of the bird's neuroanatomy and opens future research possibilities in comparative neuroscience. Moreover, this approach proves particularly relevant given the increasing attention these seabirds receive in environments such as zoos, rehabilitation centers and their natural habitat, where veterinarians play a crucial role in their care and well-being.

The Psychological Impact of the Widespread Availability of Palatable Foods Predicts Uncontrolled and Emotional Eating in Adults.

This study aimed to investigate the role of the psychological impact of environments rich in palatable foods on three aspects of eating behavior: cognitive restraint (CR), uncontrolled eating (UE), and emotional eating (EE). The hypotheses were as follows: (a) The psychological impact (i.e., motivation to eat) of an environment rich in palatable foods will positively predict CR, UE, and EE; (b) dieting will predict CR, UE, and EE; and (c) CR, UE, and EE will positively predict body mass index (BMI). This study had a cross-sectional design in which data were collected online from 413 subjects. The psychological impact of food-rich environments (food available, food present, and food tasted) was assessed using the Power of Food Scale (PFS), and CR, UE, and EE were assessed using the Three-Factor Eating Questionnaire (TFEQ-R18). Both instruments were tested for confirmatory factor analysis. The relationship between constructs was measured using partial least-square structural equation modeling (PLS-SEM). "Food available" positively predicted all TFEQ-R18 factors (p < 0.01). "Food present" positively predicted UE (p < 0.001) and EE (p = 0.01). People currently on a diet showed higher levels of CR (p < 0.001) and EE (p = 0.02). UE and EE positively predicted BMI. Thus, CR, UE, and EE were positively predicted by the motivation to consume palatable foods in varying proximity, suggesting that the presence of food and, more importantly, its general availability may be important determinants of eating behavior, particularly UE and EE. Health strategies should consider the influence of the food environment to prevent and better manage impairments in eating behavior. Sex differences suggest that special attention should be paid to women. Furthermore, dieting was associated with higher levels of EE, which in turn was associated with higher BMI. Weight loss interventions should consider this vulnerability.

The Oncogenic FOXL2 C134W Mutation Is a Key Driver of Granulosa Cell Tumors.

Adult-type granulosa cell tumors (AGCT) are the most common type of malignant ovarian sex cord-stromal tumors. Most AGCTs carry the somatic variant c.402C>G (p.C134W) affecting the transcription factor FOXL2. Germline dominant variants in FOXL2 are responsible for blepharophimosis syndrome, which is characterized by underdevelopment of the eyelid. In this work, we generated a mouse model harboring the C134W variant of FOXL2 to evaluate in vivo the poorly understood oncogenic role of FOXL2. The mutation was dominant regarding eyelid hypoplasia, reminiscent of blepharophimosis syndrome. Interestingly, Foxl2+/C134W female mice had reduced fertility and developed AGCTs through a progression from abnormal ovaries with aberrant granulosa cells to ovaries with stromal hyperplasia and atypia and on to tumors in adut mice. The genes dysregulated in mouse AGCTs exhibited the hallmarks of cancer and were consistent with a gain-of-function of the mutated allele affecting TGFß signaling. A comparison of these data with previous results on human AGCTs indicated similar deregulated pathways. Finally, a mutational analysis of mouse AGCT transcriptomic data suggested the absence of additional driver mutations apart from FOXL2-C134W. These results provide a clear in vivo example in which a single mutational hit triggers tumor development associated with profound transcriptomic alterations. SIGNIFICANCE: A newly generated mouse model carrying a FOXL2 mutation characteristic of adult-type granulosa cell tumors shows that FOXL2 C134W shifts the transcriptome towards a signature of granulosa cell cancer and drives tumorigenesis.

[Primary bone lymphoma with multicentric involvement]. / Linfoma primario de hueso con afectación multicéntrica.

Primary bone lymphoma is a rare disease, which usually has a different presentation and evolution than lymphomas of other locations. We are presenting a case of primary bone lymphoma of rapid growth, in cranial and sternal locations. In its evolution, once the excision of the primary lesion of the skull was performed, the patient presented new lesions of rapid growth at the skull and femur level, and progression of pre-sternal lesion. With large B-cell diffuse non-Hodgkin lymphoma pathology, the patient initiated R-CHOPP (cyclophosphamide, doxorubicin, vincristine, prednisone and rituximab) with rapid reduction of all lesions without evidence of progression after the six cycles.

Comparative genomic analysis of vertebrate mitochondrial reveals a differential of rearrangements rate between taxonomic class.

Vertebrate mitochondrial genomes have been extensively studied for genetic and evolutionary purposes, these are normally believed to be extremely conserved, however, different cases of gene rearrangements have been reported. To verify the level of rearrangement and the mitogenome evolution, we performed a comparative genomic analysis of the 2831 vertebrate mitochondrial genomes representing 12 classes available in the NCBI database. Using a combination of bioinformatics methods, we determined there is a high number of errors in the annotation of mitochondrial genes, especially in tRNAs. We determined there is a large variation in the proportion of rearrangements per gene and per taxonomic class, with higher values observed in Actinopteri, Amphibia and Reptilia. We highlight that these are results for currently available vertebrate sequences, so an increase in sequence representativeness in some groups may alter the rearrangement rates, so in a few years it would be interesting to see if these rates are maintained or altered with the new mitogenome sequences. In addition, within each vertebrate class, different patterns in rearrangement proportion with distinct hotspots in the mitochondrial genome were found. We also determined that there are eleven convergence events in gene rearrangement, nine of which are new reports to the scientific community.

Language Intervention in Down Syndrome: A Systematic Literature Review.

Language is one of the most affected areas in people with Down syndrome and is one of the most influential throughout their development. That is why the linguistic difficulties presented by this group are susceptible to treatment through different specific interventions. However, little emphasis has been placed on the effectiveness and importance of this type of intervention in improving their language skills. Therefore, this work aimed to carry out a systemic literature review of language intervention programs that have been carried out in the last 20 years. To this end, a total of 18 articles were analyzed in which the effectiveness of different types of treatment related to oral language, written language and communication, in general, was studied, using the guidelines of the PRISMA Statement and the COSMIN methodology. The results highlight that language intervention improves linguistic levels in people with Down Syndrome. Most of the research focuses on early interventions and interventions carried out through individual sessions. Nevertheless, the data are unanimous in considering the efficacy and effectiveness of the proposed treatments for improving the language skills of people with Down syndrome. Thus, linguistic intervention is a fundamental area of work throughout the lives of people with Down syndrome.

The Oocyte-Specific Linker Histone H1FOO Is Not Essential for Mouse Oogenesis and Fertility.

Meiosis is a highly conserved specialized cell division process that generates haploid gametes. Many of its events are associated with dynamically regulated chromosomal structures and chromatin remodeling, which are mainly modulated by histone modifications. Histone H1 is a linker histone essential for packing the nucleosome into higher-order structures, and H1FOO (H1 histone family, member O, oocyte-specific) is a H1 variant whose expression pattern is restricted to growing oocytes and zygotes. To further explore the function of H1FOO, we generated mice lacking the H1foo gene by the CRISPR/Cas9 technique. Herein, we combine mouse genetics and cellular studies to show that H1foo-null mutants have no overt phenotype, with both males and females being fertile and presenting no gross defects in meiosis progression nor in synapsis dynamics. Accordingly, the histological sections show a normal development of gametes in both male and female mice. Considering the important role of oocyte constituents in enhancing mammalian somatic cell reprogramming, we analyzed iPSCs generation in H1foo mutant MEFs and observed no differences in the absence of H1FOO. Taken all together, in this work we present the first in vivo evidence of H1FOO dispensability for mouse fertility, clarifying the debate in the field surrounding its essentiality in meiosis.

Dextran-Mimetic Quantum Dots for Multimodal Macrophage Imaging In Vivo, Ex Vivo, and In Situ.

Macrophages are white blood cells with diverse functions contributing to a healthy immune response as well as the pathogenesis of cancer, osteoarthritis, atherosclerosis, and obesity. Due to their pleiotropic and dynamic nature, tools for imaging and tracking these cells at scales spanning the whole body down to microns could help to understand their role in disease states. Here we report fluorescent and radioisotopic quantum dots (QDs) for multimodal imaging of macrophage cells in vivo, ex vivo, and in situ. Macrophage specificity is imparted by click-conjugation to dextran, a biocompatible polysaccharide that natively targets these cell types. The emission spectral band of the crystalline semiconductor core was tuned to the near-infrared for optical imaging deep in tissue, and probes were covalently conjugated to radioactive iodine for nuclear imaging. The performance of these probes was compared with all-organic dextran probe analogues in terms of their capacity to target macrophages in visceral adipose tissue using in vivo positron emission tomography/computed tomography (PET/CT) imaging, in vivo fluorescence imaging, ex vivo fluorescence, post-mortem isotopic analyses, and optical microscopy. All probe classes exhibited equivalent physicochemical characteristics in aqueous solution and similar in vivo targeting specificity. However, dextran-mimetic QDs provided enhanced signal-to-noise ratio for improved optical quantification, long-term photostability, and resistance to chemical fixation. In addition, the vascular circulation time for the QD-based probes was extended 9-fold compared with dextran, likely due to differences in conformational flexibility. The enhanced photophysical and photochemical properties of dextran-mimetic QDs may accelerate applications in macrophage targeting, tracking, and imaging across broad resolution scales, particularly advancing capabilities in single-cell and single-molecule imaging and quantification.

A truncating variant of RAD51B associated with primary ovarian insufficiency provides insights into its meiotic and somatic functions.

Primary ovarian insufficiency (POI) causes female infertility by abolishing normal ovarian function. Although its genetic etiology has been extensively investigated, most POI cases remain unexplained. Using whole-exome sequencing, we identified a homozygous variant in RAD51B -(c.92delT) in two sisters with POI. In vitro studies revealed that this variant leads to translation reinitiation at methionine 64. Here, we show that this is a pathogenic hypomorphic variant in a mouse model. Rad51bc.92delT/c.92delT mice exhibited meiotic DNA repair defects due to RAD51 and HSF2BP/BMRE1 accumulation in the chromosome axes leading to a reduction in the number of crossovers. Interestingly, the interaction of RAD51B-c.92delT with RAD51C and with its newly identified interactors RAD51 and HELQ was abrogated or diminished. Repair of mitomycin-C-induced chromosomal aberrations was impaired in RAD51B/Rad51b-c.92delT human and mouse somatic cells in vitro and in explanted mouse bone marrow cells. Accordingly, Rad51b-c.92delT variant reduced replication fork progression of patient-derived lymphoblastoid cell lines and pluripotent reprogramming efficiency of primary mouse embryonic fibroblasts. Finally, Rad51bc.92delT/c.92delT mice displayed increased incidence of pituitary gland hyperplasia. These results provide new mechanistic insights into the role of RAD51B not only in meiosis but in the maintenance of somatic genome stability.

Nanocarriers targeting adipose macrophages increase glucocorticoid anti-inflammatory potency to ameliorate metabolic dysfunction.

Obesity is associated with systemic inflammation due to macrophage accumulation in adipose tissue (AT). AT macrophages are, therefore, a target for therapeutics to modulate inflammation and prevent comorbidities. Because inflammatory processes have pleiotropic effects throughout the body and are intertwined with metabolic axes, systemic anti-inflammatory therapies are often harmful. We report that targeting AT macrophages using dextran nanocarriers radically alters the pharmacology of anti-inflammatory glucocorticoids, uncoupling the metabolic axis in obese mice. Following a single treatment, expression of inflammatory mediators and markers of inflammatory macrophages decreased with a nearly 20-fold higher potency compared with free drug. As a result, long-term treatment resulted in potent fat mobilization, AT reduction, weight loss, improved glucose tolerance, and altered AT gene expression profiles that led to elevated liver stress. Two weeks after treatment ceased, gene expression of inflammatory mediators in AT remained lower than obese controls, while gene expression related to metabolic function improved. These data demonstrate that nanocarriers show potential for amelioration of obesity-related AT inflammation and metabolic dysfunction, highlighting an important opportunity for nanomedicine to impact chronic metabolic disorders with complex and poorly understood etiology.

BRCA2 binding through a cryptic repeated motif to HSF2BP oligomers does not impact meiotic recombination.

BRCA2 and its interactors are required for meiotic homologous recombination (HR) and fertility. Loss of HSF2BP, a BRCA2 interactor, disrupts HR during spermatogenesis. We test the model postulating that HSF2BP localizes BRCA2 to meiotic HR sites, by solving the crystal structure of the BRCA2 fragment in complex with dimeric armadillo domain (ARM) of HSF2BP and disrupting this interaction in a mouse model. This reveals a repeated 23 amino acid motif in BRCA2, each binding the same conserved surface of one ARM domain. In the complex, two BRCA2 fragments hold together two ARM dimers, through a large interface responsible for the nanomolar affinity - the strongest interaction involving BRCA2 measured so far. Deleting exon 12, encoding the first repeat, from mBrca2 disrupts BRCA2 binding to HSF2BP, but does not phenocopy HSF2BP loss. Thus, results herein suggest that the high-affinity oligomerization-inducing BRCA2-HSF2BP interaction is not required for RAD51 and DMC1 recombinase localization in meiotic HR.

Author Correction: Neural and sociocultural mediators of ethnic differences in pain.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.