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Background and Objectives: To describe a novel, practical, reproducible, and effective preoperative marking technique for accurate localization of the spinal level in a series of patients with tumor lesions., Methods: We retrospectively analyzed patients undergoing minimally invasive (MIS) surgery for spine tumors from 2016 to 2021, in which this marking technique was used. Twenty-one patients, with tumor lesions involving difficult radioscopic visualization (cervicothoracic junction or upper dorsal spine, C6-T8), were included. Tumor lesion level was previously determined with enhanced MRI in all cases. Twenty-four to forty-eight hours before surgery, computed tomography image-guided carbon marking was performed by administration of aqueous suspension of carbon with a 21-gauge needle placed resembling the MIS approach planned trajectory. During surgery, activated carbon marking was followed until reaching the final target on the bone. Next, sequential dilators and an MIS retractor were placed. Then, bone resection and tumor exeresis were performed according to the case., Results: Average age was 60.6 years (26-76 years). Fifteen (71%) patients were women. In most cases (76%), tumor pathology involved intradural lesions (meningiomas and schwannomas). In all cases, the marking described allowed to accurately guide the MIS approach to tumor site. Neither intraoperative fluoroscopy nor approach enlargement was required in any procedure. Postoperative complications were reported in only 4 patients, none related with the marking., Conclusion: Computed tomography image-guided activated carbon marking allows to accurately lead MIS approaches in a practical, reproducible, and effective way in cases of tumors localized in regions of the spine of difficult radioscopic visualization., (Copyright © Congress of Neurological Surgeons 2024. All rights reserved.)

The imprinted Dlk1-Dio3 domain comprises the developmental genes Dlk1 and Rtl1, which are silenced on the maternal chromosome in different cell types. On this parental chromosome, the domain's imprinting control region activates a polycistron that produces the lncRNA Meg3 and many miRNAs (Mirg) and C/D-box snoRNAs (Rian). Although Meg3 lncRNA is nuclear and associates with the maternal chromosome, it is unknown whether it controls gene repression in cis. We created mouse embryonic stem cells (mESCs) that carry an ectopic poly(A) signal, reducing RNA levels along the polycistron, and generated Rian-/- mESCs as well. Upon ESC differentiation, we found that Meg3 lncRNA (but not Rian) is required for Dlk1 repression on the maternal chromosome. Biallelic Meg3 expression acquired through CRISPR-mediated demethylation of the paternal Meg3 promoter led to biallelic Dlk1 repression, and to loss of Rtl1 expression. lncRNA expression also correlated with DNA hypomethylation and CTCF binding at the 5'-side of Meg3. Using Capture Hi-C, we found that this creates a Topologically Associating Domain (TAD) organization that brings Meg3 close to Dlk1 on the maternal chromosome. The requirement of Meg3 for gene repression and TAD structure may explain how aberrant MEG3 expression at the human DLK1-DIO3 locus associates with imprinting disorders., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The last edition of the X-chromosome inactivation (XCI) meeting was held as an EMBO workshop in Berlin on 19-22 June 2023. The conference took place at the Harnack-haus in the Dahlem district, birthplace of the first modern research campus, where notable scientists such as Lise Meitner, Hans Krebs and, briefly, Albert Einstein conducted their research. This special edition, also accessible online, was organized by Rafael Galupa (Centre for Integrative Biology of Toulouse, France), Joost Gribnau (Erasmus MC Rotterdam, The Netherlands), Claire Rougeulle (Université Paris Cité/CNRS, Epigenetics and Cell Fate Center, Paris, France), Edda Schulz (Max Planck Institute for Molecular Genetics, Berlin, Germany) and James Turner (The Francis Crick Institute, London, UK). Originally scheduled for 2021, to commemorate the 60th anniversary of Mary Lyon's hypothesis on X-chromosome inactivation in mammals and the 30th anniversary of XIST/Xist discovery, the meeting had to be postponed because of the COVID-19 pandemic. Seven years after the latest XCI meeting in London, the enthusiasm and expectations of the community were at their highest, bringing together over 160 scientists from around the world to share and discuss their research. Eighty posters and more than 40 talks were presented at this event, in a collegial and collaborative atmosphere. A historical session and several breakout discussions were also organized, as well as the now traditional boat trip, all thanks to great organization. Here, we debrief readers on this fantastic conference., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Background: Recently, a scoring system to grade sacroiliac joint (SIJ) degeneration using computed tomography (CT) scans was described. No independent evaluation has determined the inter- and intra-observer agreement using this scheme., Purpose: To perform an independent inter- and intra-observer agreement assessment using the Eno classification and determining gas in the SIJ., Material and Methods: We studied 64 patients aged ≥60 years who were evaluated with abdominal and pelvic computed tomography scans. Six physicians (three orthopaedic spine surgeons and three musculoskeletal radiologists) assessed axial images to grade SIJ degeneration into grade 0 (normal), grade 1 (mild degeneration), grade 2 (significant degeneration), and grade 3 (ankylosis). We also evaluated the agreement assessing the presence of gas in the SIJ. After a four-week interval, all cases were presented in a random sequence for repeat assessment. We determined the agreement using the kappa (κ) or weighted kappa coefficient (wκ)., Results: The inter-observer agreement was moderate (wκ = 0.50 [0.44-0.56]), without differences among surgeons (wκ = 0.53 [0.45-0.61]) and radiologists (wκ = 0.49 [0.42-0.57]). The agreement evaluating the presence of gas was also moderate (κ = 0.45 [0.35-0.54]), but radiologists obtained better agreement (κ = 0.61 [0.48-0.72]) than surgeons (κ = 0.29 [0.18-0.39]). The intra-observer agreement using the classification was substantial (wκ = 0.79 [0.76-0.82]), without differences comparing surgeons (wκ = 0.75 [0.70-0.80]) and radiologists (wκ = 0.83 [0.79-0.87]). The intra-rater agreement evaluating gas was substantial (κ = 0.77 [0.72-0.82]), without differences between surgeons (κ = 0.71 [0.63-0.78]) and radiologists (κ = 0.84 [0.78-0.90])., Conclusion: Given the only moderate agreement obtained using the Eno classification, it does not seem adequate to be used in clinical practice or in research.

Heterozygous loss-of-function variants in the suppressor of fused protein gene (SUFU) can result in Gorlin syndrome, which is characterized by an increased frequency of basal cell carcinoma, medulloblastoma, odontogenic keratocysts, as well as other tumors. We describe a case of a 5-month-old female who presented with multiple intra-abdominal leiomyomata and was found to have a likely pathogenic splice site variant in the SUFU gene. This is the first reported case of leiomyomatosis secondary to a pathogenic SUFU variant in an infant and may represent an early, atypical presentation of Gorlin syndrome., Competing Interests: The authors declare no conflict of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

The interplay between the topological organization of the genome and the regulation of gene expression remains unclear. Depletion of molecular factors (e.g. CTCF) underlying topologically associating domains (TADs) leads to modest alterations in gene expression, whereas genomic rearrangements involving TAD boundaries disrupt normal gene expression and can lead to pathological phenotypes. Here, we targeted the TAD neighboring that of the noncoding transcript Xist, which controls X-chromosome inactivation. Inverting 245 kb within the TAD led to expected rearrangement of CTCF-based contacts but revealed heterogeneity in the 'contact' potential of different CTCF sites. Expression of most genes therein remained unaffected in mouse embryonic stem cells and during differentiation. Interestingly, expression of Xist was ectopically upregulated. The same inversion in mouse embryos led to biased Xist expression. Smaller inversions and deletions of CTCF clusters led to similar results: rearrangement of contacts and limited changes in local gene expression, but significant changes in Xist expression in embryos. Our study suggests that the wiring of regulatory interactions within a TAD can influence the expression of genes in neighboring TADs, highlighting the existence of mechanisms of inter-TAD communication., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Genomic imprinting refers to the mono-allelic and parent-specific expression of a subset of genes. While long recognized for their role in embryonic development, imprinted genes have recently emerged as important modulators of postnatal physiology, notably through hypothalamus-driven functions. Here, using mouse models of loss, gain and parental inversion of expression, we report that the paternally expressed Zdbf2 gene controls neonatal growth in mice, in a dose-sensitive but parent-of-origin-independent manner. We further found that Zdbf2 -KO neonates failed to fully activate hypothalamic circuits that stimulate appetite, and suffered milk deprivation and diminished circulating Insulin Growth Factor 1 (IGF-1). Consequently, only half of Zdbf2 -KO pups survived the first days after birth and those surviving were smaller. This study demonstrates that precise imprinted gene dosage is essential for vital physiological functions at the transition from intra- to extra-uterine life, here the adaptation to oral feeding and optimized body weight gain., Competing Interests: JG, JI, MB, MM, AG, CJ, AT, CG No competing interests declared, DB Reviewing editor, eLife, (© 2022, Glaser et al.)

Background: Image-guided percutaneous transthoracic lung biopsy has become a widely used and less invasive diagnostic method. Pneumothorax is the most frequent complication after lung biopsy. The aim of the present study is to describe the experience with expectant management of asymptomatic small post-biopsy pneumothorax in order to reduce unnecessary hospital admissions., Methods: A retrospective review was performed analyzing the results of subjects who underwent expectant and conservative treatment after presenting pneumothorax following  percutaneous lung biopsy, in a period of 6 years (January 2013 - December 2019), Results: 160 subjects who underwent diagnostic percutaneous lung biopsy of lung nodules were evaluated. Of these, 46 subjects (29%) presented pneumothorax, of which 36 were small. This group of subjects was managed expectantly, with a therapeutic success of 81% (7 subjects had to undergo percutaneous pleural drainage)., Conclusions: Expectant management in subjects with pneumothorax following percutaneous lung biopsy is a useful tool and should be applied by surgeons in order to avoid hospitalizations and / or unnecessary  and expensive procedures., (Universidad Nacional de Córdoba)

Immunofluorescence and RNA fluorescence in situ hybridization (FISH) methods enable the detection of, respectively, proteins and RNA molecules in single cells. Adapted to preimplantation mouse embryos, these techniques allow the investigation of transcriptional dynamics in the first embryonic and extraembryonic lineages and can circumvent the limited amount of starting material. This can as well be coupled to examination of chromatin modification, i.e., histone marks, by immunofluorescence. Here is outlined an immunofluorescence protocol combined to nascent RNA-FISH after immunosurgery of the mouse inner cell mass of the blastocyst to study early changes in transcription and/or histone marks of both primitive endoderm and epiblast cells. The method describes the different steps from coverslips and FISH probe preparation to inner cell mass isolation and immunofluorescence followed by RNA-FISH. Furthermore, this is applicable to earlier developmental stages and other mammalian species provided little technical adjustments.

Over the past two decades, the development of chromosome conformation capture technologies has allowed to intensively probe the properties of genome folding in various cell types. High-throughput versions of these C-based assays (named Hi-C) have released the mapping of 3D chromosome folding for the entire genomes. Applied to mammalian preimplantation embryos, it has revealed a unique chromosome organization after fertilization when a new individual is being formed. However, the questions of whether specific structures could arise depending on their parental origins or of their transcriptional status remain open. Our method chapter is dedicated to the technical description on how applying scHi-C to mouse embryos at different stages of preimplantation development. This approach capitalized with the limited amount of material available at these developmental stages. It also provides new research avenues, such as the study of mutant embryos for further functional studies.

Paternal and maternal epigenomes undergo marked changes after fertilization 1 . Recent epigenomic studies have revealed the unusual chromatin landscapes that are present in oocytes, sperm and early preimplantation embryos, including atypical patterns of histone modifications 2-4 and differences in chromosome organization and accessibility, both in gametes 5-8 and after fertilization 5,8-10 . However, these studies have led to very different conclusions: the global absence of local topological-associated domains (TADs) in gametes and their appearance in the embryo 8,9 versus the pre-existence of TADs and loops in the zygote 5,11 . The questions of whether parental structures can be inherited in the newly formed embryo and how these structures might relate to allele-specific gene regulation remain open. Here we map genomic interactions for each parental genome (including the X chromosome), using an optimized single-cell high-throughput chromosome conformation capture (HiC) protocol 12,13 , during preimplantation in the mouse. We integrate chromosome organization with allelic expression states and chromatin marks, and reveal that higher-order chromatin structure after fertilization coincides with an allele-specific enrichment of methylation of histone H3 at lysine 27. These early parental-specific domains correlate with gene repression and participate in parentally biased gene expression-including in recently described, transiently imprinted loci 14 . We also find TADs that arise in a non-parental-specific manner during a second wave of genome assembly. These de novo domains are associated with active chromatin. Finally, we obtain insights into the relationship between TADs and gene expression by investigating structural changes to the paternal X chromosome before and during X chromosome inactivation in preimplantation female embryos 15 . We find that TADs are lost as genes become silenced on the paternal X chromosome but linger in regions that escape X chromosome inactivation. These findings demonstrate the complex dynamics of three-dimensional genome organization and gene expression during early development.

Study Design: Retrospective analysis. Level of evidence III., Objectives: Low-energy vertebral compression fractures are an increasing socioeconomic problem among elderly patients. Percutaneous vertebroplasty has been extensively used for the treatment of painful fractures because of its effectiveness. However, some complications have been described; among them, new vertebral compression fractures, whether adjacent or not to the treated vertebra, are commonly reported complications (8% to 52%)., Methods: We retrospectively analyzed epidemiological and technical variables presumably associated with new vertebral compression fractures. To determine the relationship between new vertebral compression fracture and percutaneous vertebroplasty, 30 patients (study group) with this complication were compared with 60 patients treated with percutaneous vertebroplasty without this condition (control group)., Results: A higher cement percentage was found in the study group (40.3%) compared with the control group (30.5%). Initial vertebral kyphosis was significantly higher in the first group (15°) compared with the control group (9°). Epidemiological factors were similar in both groups., Conclusions: In our study, increased cement percentage injected and a higher kyphosis were associated with new vertebral compression fractures., Competing Interests: Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Early mouse development is regulated and accompanied by dynamic changes in chromatin modifications, including G9a-mediated histone H3 lysine 9 dimethylation (H3K9me2). Previously, we provided insights into its role in post-implantation development (Zylicz et al., 2015). Here we explore the impact of depleting the maternally inherited G9a in oocytes on development shortly after fertilisation. We show that G9a accumulates typically at 4 to 8 cell stage to promote timely repression of a subset of 4 cell stage-specific genes. Loss of maternal inheritance of G9a disrupts the gene regulatory network resulting in developmental delay and destabilisation of inner cell mass lineages by the late blastocyst stage. Our results indicate a vital role of this maternally inherited epigenetic regulator in creating conducive conditions for developmental progression and on cell fate choices., Competing Interests: JZ, MB, FW, YH, CL, SD, MS No competing interests declared, (© 2018, Zylicz et al.)

Single-cell RNA-sequencing (scRNAseq) enables the detection and quantification of mature RNAs in an individual cell. Assessing single cell transcriptomes can circumvent the limited amount of starting material obtained in oocytes or embryos, in particular when working with mutant mice. Here we outline our scRNAseq protocol to study mouse oocyte transcriptomes, derived from Tang et al., Nat Methods 6(5):377-382, 2009 . The method describes the different steps from single cell isolation and cDNA amplification to high-throughput sequencing. The bioinformatics pipeline used to analyze and compare genome-wide gene expression between individual oocytes is then described.

X-chromosome inactivation is established during early development. In mice, transcriptional repression of the paternal X-chromosome (Xp) and enrichment in epigenetic marks such as H3K27me3 is achieved by the early blastocyst stage. X-chromosome inactivation is then reversed in the inner cell mass. The mechanisms underlying Xp reactivation remain enigmatic. Using in vivo single-cell approaches (allele-specific RNAseq, nascent RNA-fluorescent in situ hybridization and immunofluorescence), we show here that different genes are reactivated at different stages, with more slowly reactivated genes tending to be enriched in H3meK27. We further show that in UTX H3K27 histone demethylase mutant embryos, these genes are even more slowly reactivated, suggesting that these genes carry an epigenetic memory that may be actively lost. On the other hand, expression of rapidly reactivated genes may be driven by transcription factors. Thus, some X-linked genes have minimal epigenetic memory in the inner cell mass, whereas others may require active erasure of chromatin marks.

The long noncoding RNA Xist is expressed from only the paternal X chromosome in mouse preimplantation female embryos and mediates transcriptional silencing of that chromosome. In females, absence of Xist leads to postimplantation lethality. Here, through single-cell RNA sequencing of early preimplantation mouse embryos, we found that the initiation of imprinted X-chromosome inactivation absolutely requires Xist. Lack of paternal Xist leads to genome-wide transcriptional misregulation in the early blastocyst and to failure to activate the extraembryonic pathway that is essential for postimplantation development. We also demonstrate that the expression dynamics of X-linked genes depends on the strain and parent of origin as well as on the location along the X chromosome, particularly at the first 'entry' sites of Xist. This study demonstrates that dosage-compensation failure has an effect as early as the blastocyst stage and reveals genetic and epigenetic contributions to orchestrating transcriptional silencing of the X chromosome during early embryogenesis.

Upon fertilization, the highly specialised sperm and oocyte genomes are remodelled to confer totipotency. The mechanisms of the dramatic reprogramming events that occur have remained unknown, and presumed roles of histone modifying enzymes are just starting to be elucidated. Here, we explore the function of the oocyte-inherited pool of a histone H3K4 and K9 demethylase, LSD1/KDM1A during early mouse development. KDM1A deficiency results in developmental arrest by the two-cell stage, accompanied by dramatic and stepwise alterations in H3K9 and H3K4 methylation patterns. At the transcriptional level, the switch of the maternal-to-zygotic transition fails to be induced properly and LINE-1 retrotransposons are not properly silenced. We propose that KDM1A plays critical roles in establishing the correct epigenetic landscape of the zygote upon fertilization, in preserving genome integrity and in initiating new patterns of genome expression that drive early mouse development.

During early development of female mouse embryos, both X chromosomes are transiently active. X gene dosage is then equalized between the sexes through the process of X chromosome inactivation (XCI). Whether the double dose of X-linked genes in females compared with males leads to sex-specific developmental differences has remained unclear. Using embryonic stem cells with distinct sex chromosome compositions as a model system, we show that two X chromosomes stabilize the naive pluripotent state by inhibiting MAPK and Gsk3 signaling and stimulating the Akt pathway. Since MAPK signaling is required to exit the pluripotent state, differentiation is paused in female cells as long as both X chromosomes are active. By preventing XCI or triggering it precociously, we demonstrate that this differentiation block is released once XX cells have undergone X inactivation. We propose that double X dosage interferes with differentiation, thus ensuring a tight coupling between X chromosome dosage compensation and development., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The myogenic regulatory factor Myod and insulin-like growth factor 2 (Igf2) have been shown to interact in vitro during myogenic differentiation. In order to understand how they interact in vivo, we produced double-mutant mice lacking both the Myod and Igf2 genes. Surprisingly, these mice display neonatal lethality due to severe diaphragm atrophy. Alteration of diaphragm muscle development occurs as early as 15.5 days post-coitum in the double-mutant embryos and leads to a defect in the terminal differentiation of muscle progenitor cells. A negative-feedback loop was detected between Myod and Igf2 in embryonic muscles. Igf2 belongs to the imprinted H19-Igf2 locus. Molecular analyses show binding of Myod on a mesodermal enhancer (CS9) of the H19 gene. Chromatin conformation capture experiments reveal direct interaction of CS9 with the H19 promoter, leading to increased H19 expression in the presence of Myod. In turn, the non-coding H19 RNA represses Igf2 expression in trans. In addition, Igf2 also negatively regulates Myod expression, possibly by reducing the expression of the Srf transcription factor, a known Myod activator. In conclusion, Igf2 and Myod are tightly co-regulated in skeletal muscles and act in parallel pathways in the diaphragm, where they affect the progression of myogenic differentiation. Igf2 is therefore an essential player in the formation of a functional diaphragm in the absence of Myod.

Brown fat or brown adipose tissue (BAT), found in newborn mammals as small depots localized in the interscapular region, plays a prominent role in regulating thermogenesis perinatally. The physiological importance of functional BAT has been recently reasserted in human adults. Because myoblasts and adipoblasts emerge from a common mesodermal precursor, we investigated developmental determination and the reciprocal relationship between muscle and adipocyte commitment. Here we show that a mutant mouse defective for both Igf2 and Myod genes exhibits massive BAT hypertrophy compared with wild-type and single-mutant newborns. The increased adipocyte proliferation in BAT of double-mutant newborns was associated with overexpression of the brown fat-specific marker Ucp1. More strikingly, expression of the master key gene Prdm16 involved in the switch between myogenic and brown adipogenic lineages was drastically enhanced. We further demonstrate that concomitant Myod and Igf2 inactivation accelerates differentiation of a brown preadipocyte cell line and induces lipid accumulation and increased Ucp1 and Prdm16 expression. This in vitro approach brings additional support for the implication of both Myod and Igf2 in BAT development. These results provide the first in vivo evidence that a myogenic regulator together with a growth factor act simultaneously but through independent pathways to repress Prdm16, which opens potential therapeutic perspectives for human metabolic disorders.