Repression and 3D-restructuring resolves regulatory conflicts in evolutionarily rearranged genomes.

Regulatory landscapes drive complex developmental gene expression, but it remains unclear how their integrity is maintained when incorporating novel genes and functions during evolution. Here, we investigated how a placental mammal-specific gene, Zfp42, emerged in an ancient vertebrate topologically associated domain (TAD) without adopting or disrupting the conserved expression of its gene, Fat1. In ESCs, physical TAD partitioning separates Zfp42 and Fat1 with distinct local enhancers that drive their independent expression. This separation is driven by chromatin activity and not CTCF/cohesin. In contrast, in embryonic limbs, inactive Zfp42 shares Fat1's intact TAD without responding to active Fat1 enhancers. However, neither Fat1 enhancer-incompatibility nor nuclear envelope-attachment account for Zfp42's unresponsiveness. Rather, Zfp42's promoter is rendered inert to enhancers by context-dependent DNA methylation. Thus, diverse mechanisms enabled the integration of independent Zfp42 regulation in the Fat1 locus. Critically, such regulatory complexity appears common in evolution as, genome wide, most TADs contain multiple independently expressed genes.

Comparison of the Hi-C, GAM and SPRITE methods using polymer models of chromatin.

Hi-C, split-pool recognition of interactions by tag extension (SPRITE) and genome architecture mapping (GAM) are powerful technologies utilized to probe chromatin interactions genome wide, but how faithfully they capture three-dimensional (3D) contacts and how they perform relative to each other is unclear, as no benchmark exists. Here, we compare these methods in silico in a simplified, yet controlled, framework against known 3D structures of polymer models of murine and human loci, which can recapitulate Hi-C, GAM and SPRITE experiments and multiplexed fluorescence in situ hybridization (FISH) single-molecule conformations. We find that in silico Hi-C, GAM and SPRITE bulk data are faithful to the reference 3D structures whereas single-cell data reflect strong variability among single molecules. The minimal number of cells required in replicate experiments to return statistically similar contacts is different across the technologies, being lowest in SPRITE and highest in GAM under the same conditions. Noise-to-signal levels follow an inverse power law with detection efficiency and grow with genomic distance differently among the three methods, being lowest in GAM for genomic separations >1 Mb.

Oncological treatment administration at end of life: a retrospective study.

Background: This work evaluated the proportion of patients who continue therapy until their last month of life or initiate a new therapy in the last 3 months of life (end of life [EOL]). Methods: Data for 486 patients were retrospectively collected. Results: In EOL, 205 (42.3%) received systemic therapy. Better performance status (last month overall response [OR]: 0.39; 95% CI: 0.25-0.60; p < 0.001; last 3 months OR: 0.47; 95% CI: 0.34-0.65; p < 0.001) and lack of activation of palliative care (last month OR: 0.26; 95% CI: 0.13-0.54; p < 0.001; last 3 months OR: 0.18; 95% CI: 0.10-0.32; p < 0.001) were associated with higher probability of EOL therapy. Conclusion: A non-negligible proportion of patients in real-life settings continue to receive systemic treatment in EOL.

Adjunctive techniques in endovascular repair of postcarotid endarterectomy pseudoaneurysm: Case report and literature review.

Pseudoaneurysm (PA) following carotid endarterectomy (CEA) is a rare and dangerous complication. In recent years endovascular approach has been preferred to open surgery as it is less invasive and reduces complications in an already operated neck, especially cranial nerve injuries. We report a case of large post-CEA PA causing dysphagia, successfully treated by deployment of two balloon-expandable covered stents and coil embolization of the external carotid artery. A literature review dealing with all cases of post-CEA PAs since 2000 treated by endovascular means is also reported. The research was conducted on Pubmed database using keywords "carotid pseudoaneurysm after carotid endarterectomy," "false aneurysm after carotid endarterectomy," "postcarotid endarterectomy pseudoaneurysm," and "carotid pseudoaneurysm."

Hmga2 protein loss alters nuclear envelope and 3D chromatin structure.

BACKGROUND: The high-mobility group Hmga family of proteins are non-histone chromatin-interacting proteins which have been associated with a number of nuclear functions, including heterochromatin formation, replication, recombination, DNA repair, transcription, and formation of enhanceosomes. Due to its role based on dynamic interaction with chromatin, Hmga2 has a pathogenic role in diverse tumors and has been mainly studied in a cancer context; however, whether Hmga2 has similar physiological functions in normal cells remains less explored. Hmga2 was additionally shown to be required during the exit of embryonic stem cells (ESCs) from the ground state of pluripotency, to allow their transition into epiblast-like cells (EpiLCs), and here, we use that system to gain further understanding of normal Hmga2 function. RESULTS: We demonstrated that Hmga2 KO pluripotent stem cells fail to develop into EpiLCs. By using this experimental system, we studied the chromatin changes that take place upon the induction of EpiLCs and we observed that the loss of Hmga2 affects the histone mark H3K27me3, whose levels are higher in Hmga2 KO cells. Accordingly, a sustained expression of polycomb repressive complex 2 (PRC2), responsible for H3K27me3 deposition, was observed in KO cells. However, gene expression differences between differentiating wt vs Hmga2 KO cells did not show any significant enrichments of PRC2 targets. Similarly, endogenous Hmga2 association to chromatin in epiblast stem cells did not show any clear relationships with gene expression modification observed in Hmga2 KO. Hmga2 ChIP-seq confirmed that this protein preferentially binds to the chromatin regions associated with nuclear lamina. Starting from this observation, we demonstrated that nuclear lamina underwent severe alterations when Hmga2 KO or KD cells were induced to exit from the naïve state and this phenomenon is accompanied by a mislocalization of the heterochromatin mark H3K9me3 within the nucleus. As nuclear lamina (NL) is involved in the organization of 3D chromatin structure, we explored the possible effects of Hmga2 loss on this phenomenon. The analysis of Hi-C data in wt and Hmga2 KO cells allowed us to observe that inter-TAD (topologically associated domains) interactions in Hmga2 KO cells are different from those observed in wt cells. These differences clearly show a peculiar compartmentalization of inter-TAD interactions in chromatin regions associated or not to nuclear lamina. CONCLUSIONS: Overall, our results indicate that Hmga2 interacts with heterochromatic lamin-associated domains, and highlight a role for Hmga2 in the crosstalk between chromatin and nuclear lamina, affecting the establishment of inter-TAD interactions.

Unveiling the Machinery behind Chromosome Folding by Polymer Physics Modeling.

Understanding the mechanisms underlying the complex 3D architecture of mammalian genomes poses, at a more fundamental level, the problem of how two or multiple genomic sites can establish physical contacts in the nucleus of the cells. Beyond stochastic and fleeting encounters related to the polymeric nature of chromatin, experiments have revealed specific, privileged patterns of interactions that suggest the existence of basic organizing principles of folding. In this review, we focus on two major and recently proposed physical processes of chromatin organization: loop-extrusion and polymer phase-separation, both supported by increasing experimental evidence. We discuss their implementation into polymer physics models, which we test against available single-cell super-resolution imaging data, showing that both mechanisms can cooperate to shape chromatin structure at the single-molecule level. Next, by exploiting the comprehension of the underlying molecular mechanisms, we illustrate how such polymer models can be used as powerful tools to make predictions in silico that can complement experiments in understanding genome folding. To this aim, we focus on recent key applications, such as the prediction of chromatin structure rearrangements upon disease-associated mutations and the identification of the putative chromatin organizing factors that orchestrate the specificity of DNA regulatory contacts genome-wide.

A novel complex genomic rearrangement affecting the KCNJ2 regulatory region causes a variant of Cooks syndrome.

Cooks syndrome (CS) is an ultrarare limb malformation due to in tandem microduplications involving KCNJ2 and extending to the 5' regulatory element of SOX9. To date, six CS families were resolved at the molecular level. Subsequent studies explored the evolutionary and pathological complexities of the SOX9-KCNJ2/Sox9-Kcnj2 locus, and suggested a key role for the formation of novel topologically associating domain (TAD) by inter-TAD duplications in causing CS. Here, we report a unique case of CS associated with a de novo 1;17 translocation affecting the KCNJ2 locus. On chromosome 17, the breakpoint mapped between KCNJ16 and KCNJ2, and combined with a ~ 5 kb deletion in the 5' of KCNJ2. Based on available capture Hi-C data, the breakpoint on chromosome 17 separated KCNJ2 from a putative enhancer. Gene expression analysis demonstrated downregulation of KCNJ2 in both patient's blood cells and cultured skin fibroblasts. Our findings suggest that a complex rearrangement falling in the 5' of KCNJ2 may mimic the developmental consequences of in tandem duplications affecting the SOX9-KCNJ2/Sox9-Kcnj2 locus. This finding adds weight to the notion of an intricate role of gene regulatory regions and, presumably, the related three-dimensional chromatin structure in normal and abnormal human morphology.

Preformed chromatin topology assists transcriptional robustness of Shh during limb development.

Long-range gene regulation involves physical proximity between enhancers and promoters to generate precise patterns of gene expression in space and time. However, in some cases, proximity coincides with gene activation, whereas, in others, preformed topologies already exist before activation. In this study, we investigate the preformed configuration underlying the regulation of the Shh gene by its unique limb enhancer, the ZRS, in vivo during mouse development. Abrogating the constitutive transcription covering the ZRS region led to a shift within the Shh-ZRS contacts and a moderate reduction in Shh transcription. Deletion of the CTCF binding sites around the ZRS resulted in the loss of the Shh-ZRS preformed interaction and a 50% decrease in Shh expression but no phenotype, suggesting an additional, CTCF-independent mechanism of promoter-enhancer communication. This residual activity, however, was diminished by combining the loss of CTCF binding with a hypomorphic ZRS allele, resulting in severe Shh loss of function and digit agenesis. Our results indicate that the preformed chromatin structure of the Shh locus is sustained by multiple components and acts to reinforce enhancer-promoter communication for robust transcription.

Interobserver variability in the evaluation of primary graft dysfunction after lung transplantation: impact of radiological training and analysis of discordant cases.

PURPOSE: Radiologic criteria for the diagnosis of primary graft dysfunction (PGD) after lung transplantation are nonspecific and can lead to misinterpretation. The primary aim of our study was to assess the interobserver agreement in the evaluation of chest X-rays (CXRs) for PGD diagnosis and to establish whether a specific training could have an impact on concordance rates. Secondary aim was to analyze causes of interobserver discordances. MATERIAL AND METHODS: We retrospectively enrolled 164 patients who received bilateral lung transplantation at our institution, between February 2013 and December 2019. Three radiologists independently reviewed postoperative CXRs and classified them as suggestive or not for PGD. Two of the Raters performed a specific training before the beginning of the study. A senior thoracic radiologist subsequently analyzed all discordant cases among the Raters with the best agreement. Statistical analysis to calculate interobserver variability was percent agreement, Cohen's kappa and intraclass correlation coefficient. RESULTS: A total of 473 CXRs were evaluated. A very high concordance among the two trained Raters, 1 and 2, was found (K = 0.90, ICC = 0.90), while a poorer agreement was found in the other two pairings (Raters 1 and 3: K = 0.34, ICC = 0.40; Raters 2 and 3: K = 0.35, ICC = 0.40). The main cause of disagreement (52.4% of discordant cases) between Raters 1 and 2 was the overestimation of peribronchial thickening in the absence of unequivocal bilateral lung opacities or the incorrect assessment of unilateral alterations. CONCLUSION: To properly identify PGD, it is recommended for radiologists to receive an adequate specific training.

An approach to evaluate the quality of radiological reports in Head and Neck cancer loco-regional staging: experience of two Academic Hospitals.

OBJECTIVES: To evaluate the quality of the reports of loco-regional staging computed tomography (CT) or magnetic resonance imaging (MRI) in head and neck (H&N) cancer. METHODS: Consecutive reports of staging CT and MRI of all H&N cancer cases from 2018 to 2020 were collected. We created lists of quality indicators for tumor (T) for each district and for node (N). We marked these as 0 or 1 in the report calculating a report score (RS) and a maximum sum (MS) of each list. Two radiologists and two otolaryngologists in consensus classified reports as low quality (LQ) if the RS fell in the percentage range 0-59% of MS and as high quality (HQ) if it fell in the range 60-100%, annotating technique and district. We evaluated the distribution of reports in these categories. RESULTS: Two hundred thirty-seven reports (97 CT and 140 MRI) of 95 oral cavity, 52 laryngeal, 47 oropharyngeal, 19 hypo-pharyngeal, 14 parotid, and 10 nasopharyngeal cancers were included. Sixty-six percent of all the reports were LQ for T, 66% out of all the MRI reports, and 65% out of all CT reports were LQ. Eight-five percent of reports were HQ for N, 85% out of all the MRI reports, and 82% out of all CT reports were HQ. Reports of oral cavity, oro-nasopharynx, and parotid were LQ, respectively, in 76%, 73%, 100% and 92 out of cases. CONCLUSION: Reports of staging CT/MRI in H&N cancer were LQ for T description and HQ for N description.

Polymer models are a versatile tool to study chromatin 3D organization.

The development of new experimental technologies is opening the way to a deeper investigation of the three-dimensional organization of chromosomes inside the cell nucleus. Genome architecture is linked to vital functional purposes, yet a full comprehension of the mechanisms behind DNA folding is still far from being accomplished. Theoretical approaches based on polymer physics have been employed to understand the complexity of chromatin architecture data and to unveil the basic mechanisms shaping its structure. Here, we review some recent advances in the field to discuss how Polymer Physics, combined with numerical Molecular Dynamics simulation and Machine Learning based inference, can capture important aspects of genome organization, including the description of tissue-specific structural rearrangements, the detection of novel, regulatory-linked architectural elements and the structural variability of chromatin at the single-cell level.

Inference of chromosome 3D structures from GAM data by a physics computational approach.

The combination of modelling and experimental advances can provide deep insights for understanding chromatin 3D organization and ultimately its underlying mechanisms. In particular, models of polymer physics can help comprehend the complexity of genomic contact maps, as those emerging from technologies such as Hi-C, GAM or SPRITE. Here we discuss a method to reconstruct 3D structures from Genome Architecture Mapping (GAM) data, based on PRISMR, a computational approach introduced to find the minimal polymer model best describing Hi-C input data from only polymer physics. After recapitulating the PRISMR procedure, we describe how we extended it for treating GAM data. We successfully test the method on a 6 Mb region around the Sox9 gene and, at a lower resolution, on the whole chromosome 7 in mouse embryonic stem cells. The PRISMR derived 3D structures from GAM co-segregation data are finally validated against independent Hi-C contact maps. The method results to be versatile and robust, hinting that it can be similarly applied to different experimental data, such as SPRITE or microscopy distance data.

Pseudo-pneumatosis of the gastrointestinal tract: its incidence and the accuracy of a checklist supported by artificial intelligence (AI) techniques to reduce the misinterpretation of pneumatosis.

PURPOSE: To assess the incidence of erroneous diagnosis of pneumatosis (pseudo-pneumatosis) in patients who underwent an emergency abdominal CT and to verify the performance of imaging features, supported by artificial intelligence (AI) techniques, to reduce this misinterpretation. METHODS: We selected 71 radiological reports where the presence of pneumatosis was considered definitive or suspected. Surgical findings, clinical outcomes, and reevaluation of the CT scans were used to assess the correct diagnosis of pneumatosis. We identified four imaging signs from literature, to differentiate pneumatosis from pseudo-pneumatosis: gas location, dissecting gas in the bowel wall, a circumferential gas pattern, and intramural gas beyond a gas-fluid/faecal level. Two radiologists reevaluated in consensus all the CT scans, assessing the four above-mentioned variables. Variable discriminative importance was assessed using the Fisher exact test. Accurate and statistically significant variables (p-value < 0.05, accuracy > 75%) were pooled using boosted Random Forests (RFs) executed using a Leave-One-Out cross-validation (LOO cv) strategy to obtain unbiased estimates of individual variable importance by permutation analysis. After the LOO cv, the comparison of the variable importance distribution was validated by one-sided Wilcoxon test. RESULTS: Twenty-seven patients proved to have pseudo-pneumatosis (error: 38%). The most significant features to diagnose pneumatosis were presence of dissecting gas in the bowel wall (accuracy: 94%), presence of intramural gas beyond a gas-fluid/faecal level (accuracy: 86%), and a circumferential gas pattern (accuracy: 78%). CONCLUSION: The incidence of pseudo-pneumatosis can be high. The use of a checklist which includes three imaging signs can be useful to reduce this overestimation.

Eversion Endarterectomy of the Femoral Bifurcation: Technique, Results and Potential Advantages.

BACKGROUND: Despite recent advances in endovascular techniques, surgical endarterectomy remains the "gold standard" for treatment of atherosclerotic lesions of the femoral bifurcation. Eversion endarterectomy (EE) of the femoral bifurcation is a well-known technique that ensures an extensive plaque removal; furthermore, EE can be performed to avoid the use of prosthetic material. The aim of this study is to evaluate the efficacy and safety of the EE of the femoral bifurcation in a contemporary prospective series from a single-center experience. MATERIALS AND METHODS: All patients undergoing EE at our institution between January 2014 and December 2016 were retrospectively reviewed. EE was performed as an isolated procedure or in a hybrid fashion. Clinical presentation was defined according to Rutherford's classification. End points included major complications and patency rates. RESULTS: Thirty-three EEs were performed on 31 patients during the study period. Thirteen procedures (39%) were performed in a hybrid fashion with concurrent endovascular interventions. Technical success was achieved in 100%. Thirty-day mortality was null, whereas 5 overall complications (15%) were recorded; among those, 2 (6%) were major. In both cases, an early thrombosis of the femoral bifurcation occurred, successfully treated by a short Dacron replacement of the common femoral artery. During follow-up, no femoral pseudoaneurysm or groin infections were observed. Two restenosis occurred at 7 and 10 months after EE, respectively. Two-year primary patency and assisted primary patency rates were 87% and 100%, respectively. During follow-up, two patients underwent percutaneous revascularization of the contralateral femoropopliteal axis at 5 and 8 months after EE, respectively. In both of them, the procedure was successfully performed through direct puncture of the endoarterectomized common femoral artery, without any access-site complications. CONCLUSIONS: Endarterectomy remains the gold standard in the treatment of atherosclerotic lesions of the femoral bifurcation with excellent long-term patency rates. Furthermore, EE adds the advantages of avoiding the use of prosthetic materials in the groin and the possibility to use the treated vessels as access for further percutaneous procedures.

Characterization of liver nodules in patients with chronic liver disease by MRI: performance of the Liver Imaging Reporting and Data System (LI-RADS v.2018) scale and its comparison with the Likert scale.

OBJECTIVES: To evaluate the performance of the LI-RADS v.2018 scale by comparing it with the Likert scale, in the characterization of liver lesions. METHODS: A total of 39 patients with chronic liver disease underwent MR examination for characterization of 44 liver lesions. Images were independently analyzed by two radiologists using the LI-RADS scale and by another two radiologists using the Likert scale. The reference standard used was either histopathological evaluation or a 4-year MRI follow-up. Receiver operating characteristic analysis was performed. RESULTS: The LI-RADS scale obtained an accuracy of 80%, a sensitivity of 72%, a specificity of 93%, a positive predictive value (PPV) of 93% and a negative predictive value (NPV) of 70%, while the Likert scale achieved an accuracy of 79%, a sensitivity of 73%, a specificity of 87%, a PPV of 89% and a NPV of 70%. The area under the curve (AUC) was 85% for the LI-RADS scale and 83% for the Likert scale. The inter-observer agreement was strong (k = 0.89) between the LI-RADS evaluators and moderate (k = 0.69) between the Likert evaluators. CONCLUSIONS: There was no statistically significant difference between the performances of the two scales; nevertheless, we suggest that the LI-RADS scale be used, as it appeared more objective and consistent.

Molecular Dynamics simulations of the Strings and Binders Switch model of chromatin.

In recent years interest has grown on the applications of polymer physics to model chromatin folding in order to try to make sense of the complexity of experimental data emerging from new technologies such as Hi-C or GAM, in a principled way. Here we review the methods employed to efficiently implement Molecular Dynamics computer simulations of polymer models, focusing in particular on the String&Binders Switch (SBS) model. The constant improvement of such methods and computer power is returning increasingly more accurate insights on the structure and molecular mechanisms underlying the spatial organization of chromosomes in the cell nucleus. We aim to provide an account of the state of the art of computational techniques employed in this type of investigations and to review recent applications of such methods to the description of real genomic loci, such as the Sox9 locus in mESC.

Predicting chromatin architecture from models of polymer physics.

We review the picture of chromatin large-scale 3D organization emerging from the analysis of Hi-C data and polymer modeling. In higher mammals, Hi-C contact maps reveal a complex higher-order organization, extending from the sub-Mb to chromosomal scales, hierarchically folded in a structure of domains-within-domains (metaTADs). The domain folding hierarchy is partially conserved throughout differentiation, and deeply correlated to epigenomic features. Rearrangements in the metaTAD topology relate to gene expression modifications: in particular, in neuronal differentiation models, topologically associated domains (TADs) tend to have coherent expression changes within architecturally conserved metaTAD niches. To identify the nature of architectural domains and their molecular determinants within a principled approach, we discuss models based on polymer physics. We show that basic concepts of interacting polymer physics explain chromatin spatial organization across chromosomal scales and cell types. The 3D structure of genomic loci can be derived with high accuracy and its molecular determinants identified by crossing information with epigenomic databases. In particular, we illustrate the case of the Sox9 locus, linked to human congenital disorders. The model in-silico predictions on the effects of genomic rearrangements are confirmed by available 5C data. That can help establishing new diagnostic tools for diseases linked to chromatin mis-folding, such as congenital disorders and cancer.

First Case of 2 Synchronous Gluteal Arteries Aneurysms Treated by Endovascular Plug Embolization: Case Report and Literature Review.

INTRODUCTION: Gluteal artery aneurysms (GAAs) are rare, accounting for less than 1% of all arterial aneurysms. Most of them are post-traumatic in nature and involve the superior gluteal artery (SGA), while injuries of the inferior gluteal artery (IGA) have been reported less frequently. We report an unusual case of a patient with double saccular GAA of unknown etiology, involving both the SGA and IGA, successfully treated by endovascular embolization. CASE REPORT: A 80-year-old man referred to our hospital complaining of the progressive onset of left buttock pain and swelling exacerbated by sitting position in the last 4 months. His past medical history was positive for hypertension, prostatic adenocarcinoma treated by brachytherapy, and endocarditis diagnosed about 30 years before and treated by cardiac surgical valve replacement; no history of trauma was reported. After ultrasonography was carried out, an enhanced computed tomography (CT) scan confirmed the presence of 2 large GAAs involving both the SGA and IGA, with maximum transverse diameter of 38 and 84 mm, respectively. The patient was referred for endovascular treatment after informed consent was provided. After sequential selective catheterization of SGA and IGA, 3 Amplatzer Plugs II (St. Jude Medical, Zaventem, Belgium) were deployed inside the aneurysms. Postoperative course was uneventful as buttock pain completely disappeared on the second postoperative day. The patient was discharged to home on the third postoperative day. One-month CT scan confirmed the complete thrombosis of the aneurysms without any endoleak. CONCLUSIONS: GAAs represent a rare pathology, and for that reason, the correct timing and choice of treatment are not clearly defined. Endovascular techniques are the first step in the approach to GAAs. In case of complex anatomy, GAAs embolization by the use of vascular plugs can be successfully performed.