Spatial organization of chromosomes leads to heterogeneous chromatin motion and drives the liquid- or gel-like dynamical behavior of chromatin.

Chromosome organization and dynamics are involved in regulating many fundamental processes such as gene transcription and DNA repair. Experiments unveiled that chromatin motion is highly heterogeneous inside cell nuclei, ranging from a liquid-like, mobile state to a gel-like, rigid regime. Using polymer modeling, we investigate how these different physical states and dynamical heterogeneities may emerge from the same structural mechanisms. We found that the formation of topologically associating domains (TADs) is a key driver of chromatin motion heterogeneity. In particular, we showed that the local degree of compaction of the TAD regulates the transition from a weakly compact, fluid state of chromatin to a more compact, gel state exhibiting anomalous diffusion and coherent motion. Our work provides a comprehensive study of chromosome dynamics and a unified view of chromatin motion enabling interpretation of the wide variety of dynamical behaviors observed experimentally across different biological conditions, suggesting that the "liquid" or "solid" state of chromatin are in fact two sides of the same coin.

Randomised trial of genetic testing and targeted intervention to prevent the development and progression of Paget's disease of bone.

INTRODUCTION: Paget's disease of bone (PDB) frequently presents at an advanced stage with irreversible skeletal damage. Clinical outcomes might be improved by earlier diagnosis and prophylactic treatment. METHODS: We randomised 222 individuals at increased risk of PDB because of pathogenic SQSTM1 variants to receive 5 mg zoledronic acid (ZA) or placebo. The primary outcome was new bone lesions assessed by radionuclide bone scan. Secondary outcomes included change in existing lesions, biochemical markers of bone turnover and skeletal events related to PDB. RESULTS: The median duration of follow-up was 84 months (range 0-127) and 180 participants (81%) completed the study. At baseline, 9 (8.1%) of the ZA group had PDB lesions vs 12 (10.8%) of the placebo group. Two of the placebo group developed new lesions versus none in the ZA group (OR 0.41, 95% CI 0.00 to 3.43, p=0.25). Eight of the placebo group had a poor outcome (lesions which were new, unchanged or progressing) compared with none of the ZA group (OR 0.08, 95% CI 0.00 to 0.42, p=0.003). At the study end, 1 participant in the ZA group had lesions compared with 11 in the placebo group. Biochemical markers of bone turnover were significantly reduced in the ZA group. One participant allocated to placebo required rescue therapy with ZA because of symptomatic disease. The number and severity of adverse events did not differ between groups. CONCLUSIONS: Genetic testing for pathogenic SQSTM1 variants coupled with intervention with ZA is well tolerated and has favourable effects on the progression of early PDB. TRIAL REGISTRATION NUMBER: ISRCTN11616770.

Polymer modelling unveils the roles of heterochromatin and nucleolar organizing regions in shaping 3D genome organization in Arabidopsis thaliana.

The 3D genome is characterized by a complex organization made of genomic and epigenomic layers with profound implications on gene regulation and cell function. However, the understanding of the fundamental mechanisms driving the crosstalk between nuclear architecture and (epi)genomic information is still lacking. The plant Arabidopsis thaliana is a powerful model organism to address these questions owing to its compact genome for which we have a rich collection of microscopy, chromosome conformation capture (Hi-C) and ChIP-seq experiments. Using polymer modelling, we investigate the roles of nucleolus formation and epigenomics-driven interactions in shaping the 3D genome of A. thaliana. By validation of several predictions with published data, we demonstrate that self-attracting nucleolar organizing regions and repulsive constitutive heterochromatin are major mechanisms to regulate the organization of chromosomes. Simulations also suggest that interphase chromosomes maintain a partial structural memory of the V-shapes, typical of (sub)metacentric chromosomes in anaphase. Additionally, self-attraction between facultative heterochromatin regions facilitates the formation of Polycomb bodies hosting H3K27me3-enriched gene-clusters. Since nucleolus and heterochromatin are highly-conserved in eukaryotic cells, our findings pave the way for a comprehensive characterization of the generic principles that are likely to shape and regulate the 3D genome in many species.

Active and repressed biosynthetic gene clusters have spatially distinct chromosome states.

While colocalization within a bacterial operon enables coexpression of the constituent genes, the mechanistic logic of clustering of nonhomologous monocistronic genes in eukaryotes is not immediately obvious. Biosynthetic gene clusters that encode pathways for specialized metabolites are an exception to the classical eukaryote rule of random gene location and provide paradigmatic exemplars with which to understand eukaryotic cluster dynamics and regulation. Here, using 3C, Hi-C, and Capture Hi-C (CHi-C) organ-specific chromosome conformation capture techniques along with high-resolution microscopy, we investigate how chromosome topology relates to transcriptional activity of clustered biosynthetic pathway genes in Arabidopsis thaliana Our analyses reveal that biosynthetic gene clusters are embedded in local hot spots of 3D contacts that segregate cluster regions from the surrounding chromosome environment. The spatial conformation of these cluster-associated domains differs between transcriptionally active and silenced clusters. We further show that silenced clusters associate with heterochromatic chromosomal domains toward the periphery of the nucleus, while transcriptionally active clusters relocate away from the nuclear periphery. Examination of chromosome structure at unrelated clusters in maize, rice, and tomato indicates that integration of clustered pathway genes into distinct topological domains is a common feature in plant genomes. Our results shed light on the potential mechanisms that constrain coexpression within clusters of nonhomologous eukaryotic genes and suggest that gene clustering in the one-dimensional chromosome is accompanied by compartmentalization of the 3D chromosome.

essHi-C: essential component analysis of Hi-C matrices.

MOTIVATION: Hi-C matrices are cornerstones for qualitative and quantitative studies of genome folding, from its territorial organization to compartments and topological domains. The high dynamic range of genomic distances probed in Hi-C assays reflects in an inherent stochastic background of the interactions matrices, which inevitably convolve the features of interest with largely non-specific ones. RESULTS: Here, we introduce and discuss essHi-C, a method to isolate the specific or essential component of Hi-C matrices from the non-specific portion of the spectrum compatible with random matrices. Systematic comparisons show that essHi-C improves the clarity of the interaction patterns, enhances the robustness against sequencing depth of topologically associating domains identification, allows the unsupervised clustering of experiments in different cell lines and recovers the cell-cycle phasing of single-cells based on Hi-C data. Thus, essHi-C provides means for isolating significant biological and physical features from Hi-C matrices. AVAILABILITY AND IMPLEMENTATION: The essHi-C software package is available at https://github.com/stefanofranzini/essHIC. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Hierarchical chromatin organization detected by TADpole.

The rapid development of Chromosome Conformation Capture (3C-based techniques), as well as imaging together with bioinformatics analyses, has been fundamental for unveiling that chromosomes are organized into the so-called topologically associating domains or TADs. While TADs appear as nested patterns in the 3C-based interaction matrices, the vast majority of available TAD callers are based on the hypothesis that TADs are individual and unrelated chromatin structures. Here we introduce TADpole, a computational tool designed to identify and analyze the entire hierarchy of TADs in intra-chromosomal interaction matrices. TADpole combines principal component analysis and constrained hierarchical clustering to provide a set of significant hierarchical chromatin levels in a genomic region of interest. TADpole is robust to data resolution, normalization strategy and sequencing depth. Domain borders defined by TADpole are enriched in main architectural proteins (CTCF and cohesin complex subunits) and in the histone mark H3K4me3, while their domain bodies, depending on their activation-state, are enriched in either H3K36me3 or H3K27me3, highlighting that TADpole is able to distinguish functional TAD units. Additionally, we demonstrate that TADpole's hierarchical annotation, together with the new DiffT score, allows for detecting significant topological differences on Capture Hi-C maps between wild-type and genetically engineered mouse.

Acute distal biceps tendon rupture: retrospective analysis of two different approaches and fixation techniques.

PURPOSE: The aim of our study is to compare the modified double incision (DI) with bone tunnel reinsertion with the single-incision (SI) double tension slide technique in terms of clinical and functional outcomes and complication rates. METHODS: A retrospective comparative analysis was performed on 65 patients treated for total distal biceps tendon rupture. The surgical technique adopted for each patient was based on the preference of two experienced elbow surgeons. The DASH and MAYO questionnaires, functional outcome and ROM were recorded in all subjects. RESULTS: Of 65 patients, we collected data of a cohort of 54 distal biceps tendon ruptures that satisfied inclusion criteria. Twenty-five were treated by modified DI and 29 SI techniques. The recovery of the complete ROM in terms of flexion/extension and prono-supination occurred in the 79.6% of the patients, without statistical significant difference between the adopted technique. We reported a complication rate of 12% and 20.7% for DI and SI techniques, respectively, without statistical correlation (P = 0.84). The average DASH score was similar for DI and SI techniques without significant differences (P = 0,848). The Mayo score results were excellent in the majority of the patients. No significant difference in MAYO results was reported comparing the surgical techniques (P = 1). CONCLUSION: Both techniques provide a reliable and strong repair with an optimal recovery of ROM returning to preinjury activity with substantially overlapping timelines.

Factors Enhancing Treatment of Hepatitis C Virus-Infected Italian People Who Use Drugs: The CLEO-GRECAS Experience.

INTRODUCTION: We assessed the performance of direct-acting antivirals (DAAs) in hepatitis C virus (HCV)-infected people who use drugs (PWUDs) in terms of sustained virological response (SVR) and adherence rates in comparison to a location-matched cohort of non-PWUD HCV patients. METHODS: All consecutive HCV RNA-positive PWUDs were enrolled between 2015 and 2019. All subjects underwent DAA treatment according to international guidelines and then followed, at least, up to 12 weeks after the end of treatment (SVR12). The SVR and adherence to treatment was compared with that of non-PWUD HCV patients observed at hepatological units of the CLEO platform. Intention-to-treat analysis was performed. RESULTS: A total of 1,786 PWUDs who were followed up were available for assessment. Most PWUDs (85.4%) were managed inside the specialized outpatient addiction clinics (SerDs). The overall SVR rate was 95.4%. The SerDs group achieved an SVR rate of 96.2% compared with 91.6% of the non-SerDs group (P < 0.001). Comparison with the non-SerDs group and the control HCV group showed a significant difference in the dropout rate (0.6% in the SerDs group versus 2.8% in the non-SerDs group and 1.2% in the control group; P < 0.001). At multivariate analysis, factors independently associated with SVR were use of the most recent regimens (elbasvir/grazoprevir, glecaprevir/pibrentasvir, and sofosbuvir/velpatasvir; odds ratio: 3.126; P = 0.000) and belonging to the SerDs group (odds ratio: 2.356; P = 0.002). DISCUSSION: The performance of DAAs in PWUD is excellent, if 2 conditions are met: (i) that the latest generation drugs are used and (ii) that the patients are managed within the SerDs.

Dietary calcium intake in a cohort of individuals evaluated for low bone mineral density: a multicenter Italian study.

BACKGROUND: A low calcium intake is a well-known factor that influences the bone mineral density (BMD) maintenance. In the presence of inadequate calcium intake, secondary hyperparathyroidism develops, leading to an increased bone turnover and fracture risk. AIMS: To assess the dietary calcium intake in relation with osteoporosis and fragility fracture in a cohort of Italian individuals evaluated for low BMD. METHODS: A 7-day food-frequency questionnaire was administered to 1793 individuals, who were consecutively referred at the Centers of the Italian Society for Osteoporosis, Mineral Metabolism and Skeletal Diseases (SIOMMMS) for low BMD. RESULTS: In 30.3% and 20.9% of subjects, the calcium intake was inadequate (< 700 mg/day) and adequate (> 1200 mg/day), respectively. As compared with patients with adequate calcium intake, those with inadequate calcium intake were younger (65.5 ± 10.8 vs 63.9 ± 11.5 years, p = 0.03) and they more frequently reported adverse reactions to food (3.2% vs 7.2% p = 0.01) and previous major fragility fractures (20.8% vs 27.0%, p = 0.03). Patients with calcium intake < 700 mg/day showed a higher prevalence of diabetes mellitus, idiopathic hypercalciuria and food allergy/intolerance (8.1%, 5.1%, 7.2%, respectively) than patients with calcium intake > 700 mg/day (5.3%, 3.0%, 4.1%, respectively, p < 0.04 for all comparisons), also after adjusting for age, gender and body mass index. In 30.3% of fractured subjects, the calcium intake was < 700 mg/day. DISCUSSION: In Italy, a low calcium intake is highly prevalent in individuals at risk for low BMD. Importantly, an inadequate calcium intake is highly prevalent even in patients with history of fragility fractures. CONCLUSIONS: Only about a fifth of patients being assessed for low BMD in an Italian SIOMMMS referral Centre have an adequate calcium intake.

Vitamin D Status in Paget Disease of Bone and Efficacy-Safety Profile of Cholecalciferol Treatment in Pagetic Patients with Hypovitaminosis D.

Adequate vitamin D status is essential for skeletal health. Paget's disease of bone (PDB) is a common metabolic skeletal disorder, but data regarding the vitamin D status in PDB patients are lacking. We performed a case-control study to estimate vitamin D status in 708 PDB patients and in 1803 healthy controls from Italy and an observational prospective study to evaluate the efficacy-safety profile of oral cholecalciferol treatment [400.000 International Units (UI) of cholecalciferol administered in cycles of 8 weeks until 25OHD levels reaches 70 nmol/L as primary therapy and 50.000 UI of cholecalciferol administered every 2 weeks for 52 weeks for the maintenance therapy] in 82 PDB patients with hypovitaminosis D, i.e., 25OHD < 50 nmol/L. The main outcome measures for the prospective study were 25OHD levels, metabolic risk factors (RF) for nephrolithiasis, bone pain score (BPS), and pain medication score (PMS). Over half of PDB patients had hypovitaminosis D. Among PDB patients treated with cholecalciferol, 76 patients reached 25OHD levels ≥ 70 nmol/L after the first cycle of primary therapy and the remaining six patients after a second cycle. The maintenance therapy guaranteed 25OHD levels ≥ 70 nmol/L during the entire follow-up. The increase in 25OHD levels reduced PTH, BPS, and PMS levels, without changes in RF for nephrolithiasis. We can conclude that (i) hypovitaminosis D is frequent in PDB patients, (ii) cholecalciferol significantly increased 25OHD levels in PDB patients, and (iii) the correction of hypovitaminosis D improves the quality of life of PDB patients without inducing significant changes in RF for nephrolithiasis.

Type 2 diabetes affects bone cells precursors and bone turnover.

BACKGROUND: Here we study the effect of type 2 diabetes (T2DM) on bone cell precursors, turnover and cytokines involved in the control of bone cell formation and activity. METHODS: We enrolled in the study 21 T2DM women and 21 non diabetic controls matched for age and body mass index (BMI). In each subject we measured bone cell precursors, Receptor Activator of Nuclear Factor κB (RANKL), Osteoprotegerin (OPG), Sclerostin (SCL) and Dickoppf-1 (DKK-1) as cytokines involved in the control of osteoblast and osteoclast formation and activity, bone density (BMD) and quality trough trabecular bone score (TBS) and bone turnover. T2DM patients and controls were compared for the analyzed variables by one way ANOVA for Gaussian ones and by Mann-Whitney or Kruskal-Wallis test for non-Gaussian variables. RESULTS: RANKL was decreased and DKK-1 increased in T2DM. Accordingly, patients with T2DM have lower bone turnover compared to controls. BMD and TBS were not significantly different from healthy controls. Bone precursor cells were more immature in T2DM. However the number of osteoclast precursors was increased and that of osteoblasts decreased. CONCLUSIONS: Patients with T2DM have more immature bone cells precursors, with increased number of osteoclasts and decreased osteoblasts, confirming low bone turnover and reduced cytokines such as RANKL and DKK-1. BMD and TBS are not significantly altered in T2DM although, in contrast with other studies, this may be due to the match of patients and controls for BMI rather than age.

Absence of knots in known RNA structures.

The ongoing effort to detect and characterize physical entanglement in biopolymers has so far established that knots are present in many globular proteins and also, abound in viral DNA packaged inside bacteriophages. RNA molecules, however, have not yet been systematically screened for the occurrence of physical knots. We have accordingly undertaken the systematic profiling of the several thousand RNA structures present in the Protein Data Bank (PDB). The search identified no more than three deeply knotted RNA molecules. These entries are rRNAs of about 3,000 nt solved by cryo-EM. Their genuine knotted state is, however, doubtful based on the detailed structural comparison with homologs of higher resolution, which are all unknotted. Compared with the case of proteins and viral DNA, the observed incidence of knots in available RNA structures is, therefore, practically negligible. This fact suggests that either evolutionary selection or thermodynamic and kinetic folding mechanisms act toward minimizing the entanglement of RNA to an extent that is unparalleled by other types of biomolecules. A possible general strategy for designing synthetic RNA sequences capable of self-tying in a twist-knot fold is finally proposed.

The elusive quest for RNA knots.

Physical entanglement, and particularly knots arise spontaneously in equilibrated polymers that are sufficiently long and densely packed. Biopolymers are no exceptions: knots have long been known to occur in proteins as well as in encapsidated viral DNA. The rapidly growing number of RNA structures has recently made it possible to investigate the incidence of physical knots in this type of biomolecule, too. Strikingly, no knots have been found to date in the known RNA structures. In this Point of View Article we discuss the absence of knots in currently available RNAs and consider the reasons why knots in RNA have not yet been found, despite the expectation that they should exist in Nature. We conclude by singling out a number of RNA sequences that, based on the properties of their predicted secondary structures, are good candidates for knotted RNAs.

X-ray, CT and DXA study of bone loss on medieval remains from North-West Italy.

PURPOSE: The aim of this study was to investigate whether the population differences in osteoporosis observed nowadays is a reflection of the times and modern lifestyle factors, or whether they were also present in the past. MATERIALS AND METHODS: The study was performed on the skeletal remains of medieval and post-medieval populations from a burial ground in the North-West of Italy. Some individuals had been buried inside the church (privileged subjects), others outside in the parvis (members of rural population), and others still to the north of the church. X-ray, computed tomography and dual-energy X-ray absorptiometry studies were carried out on the lumbar spines and/or femurs of 27 male and 28 female individuals to determine any associations between cortical index, bone mineral density (BMD), gender, age and social status. RESULTS: No statistically significant differences were observed in cortical index values according to gender, age or place of burial. Conversely, statistically significant differences in average BMD values were observed according to place of burial; in particular, among those buried inside the church, a lower BMD was observed compared to the parvis group (1.09 vs. 1.42, p < 0.001) and the north group (1.09 vs. 1.49, p < 0.001). CONCLUSIONS: The differences observed in the BMD values may be related to the different lifestyle of the rural population, i.e. more dietary calcium intake, more sun exposure and vigorous physical activity, compared to that of the privileged individuals.

Colocalization of coregulated genes: a steered molecular dynamics study of human chromosome 19.

The connection between chromatin nuclear organization and gene activity is vividly illustrated by the observation that transcriptional coregulation of certain genes appears to be directly influenced by their spatial proximity. This fact poses the more general question of whether it is at all feasible that the numerous genes that are coregulated on a given chromosome, especially those at large genomic distances, might become proximate inside the nucleus. This problem is studied here using steered molecular dynamics simulations in order to enforce the colocalization of thousands of knowledge-based gene sequences on a model for the gene-rich human chromosome 19. Remarkably, it is found that most (≈ 88%) gene pairs can be brought simultaneously into contact. This is made possible by the low degree of intra-chromosome entanglement and the large number of cliques in the gene coregulatory network. A clique is a set of genes coregulated all together as a group. The constrained conformations for the model chromosome 19 are further shown to be organized in spatial macrodomains that are similar to those inferred from recent HiC measurements. The findings indicate that gene coregulation and colocalization are largely compatible and that this relationship can be exploited to draft the overall spatial organization of the chromosome in vivo. The more general validity and implications of these findings could be investigated by applying to other eukaryotic chromosomes the general and transferable computational strategy introduced here.

Driving knots on DNA with AC/DC electric fields: topological friction and memory effects.

The dynamical properties of entangled polyelectrolytes are investigated theoretically and computationally for a proposed novel micromanipulation setup. Specifically, we investigate the effects of DC and AC electric fields acting longitudinally on knotted DNA chains, modelled as semiflexible chains of charged beads, under mechanical tension. We consider various experimentally accessible values of the field amplitude and frequency as well as several of the simplest knot types. In particular, we consider both torus and twist knots because they are respectively known to be able or unable to slide along macroscopic threads and ropes. Strikingly, this qualitative distinction disappears in this microscopic context because all the considered knot types acquire a systematic drift in the direction of the electric force. Notably, the knot drift velocity and diffusion coefficient in zero field (both measurable also experimentally) can be used to define a characteristic "frictional" lengthscale for the various knot types. This previously unexplored length provides valuable information on the extent of self-interactions in the nominal knotted region. It is finally observed that the motion of a knot can effectively follow the AC field only if the driving period is larger than the knot relaxation time (for which the self-diffusion time provides an upper bound). These results suggest that salient aspects of the intrinsic dynamics of knots in DNA chains could be probed experimentally by means of external, time-dependent electric fields.

Bone mineral density at diagnosis of celiac disease and after 1 year of gluten-free diet.

Atypical or silent celiac disease may go undiagnosed for many years and can frequently lead to loss of bone mineral density, with evolution to osteopenia or osteoporosis. The prevalence of the latter conditions, in case of new diagnosis of celiac disease, has been evaluated in many studies but, due to the variability of epidemiologic data and patient features, the results are contradictory. The aim of this study was to evaluate bone mineral density by dual-energy X-ray absorptiometry in 175 consecutive celiac patients at time of diagnosis (169 per-protocol, 23 males, 146 females; average age 38.9 years). Dual-energy X-ray absorptiometry was repeated after 1 year of gluten-free diet in those with T-score value <-1 at diagnosis. Stratification of patients according to sex and age showed a higher prevalence of low bone mineral density in men older than 30 years and in women of all ages. A 1-year gluten-free diet led to a significant improvement in lumbar spine and femoral neck mean T-score value. We propose that dual-energy X-ray absorptiometry should be performed at diagnosis of celiac disease in all women and in male aged >30 years, taking into account each risk factor in single patients.

Spider Bite Presenting as Fever, Macrophage Activation Syndrome and a Skin Ulcer.

Introduction: Fever of unknown origin (FUO) refers to a condition of prolonged increased body temperature, without identified causes. The most common cause of FUO worldwide are infections; arthropod bites (loxoscelism) should be considered in view of the spread of the fiddleback spider. Loxoscelism can present in a cutaneous form (a necrotic cutaneous ulcer) or in a systemic form with fever, haemolytic anaemia, rhabdomyolysis and, rarely, macrophage activation syndrome (MAS). For this suspicion, it is important to have actually seen the spider. Case description: A 71-year-old man was admitted to our department because of intermittent fever, arthralgia and a necrotic skin lesion on his right forearm that appeared after gardening. Laboratory tests were negative for infectious diseases, and several courses of antibiotics were administered empirically without clinical benefit. Whole-body computed tomography showed multiple colliquative lymphadenomegalies, the largest one in the right axilla, presumably of reactive significance. A shave biopsy of the necrotic lesion was performed: culture tests were negative and histological examination showed non-specific necrotic material, so a second skin and lymph node biopsy was performed. The patient developed MAS for which he received corticosteroid therapy with clinical/laboratory benefit. Cutaneous and systemic loxoscelism complicated by MAS was diagnosed. Subsequently, the second biopsy revealed morphological and immunophenotypic findings consistent with primary cutaneous anaplastic large cell lymphoma (PC-ALCL). Conclusions: Skin lesions and lymphadenomegalies of unknown origin should always be biopsied. It is very common to get indeterminate results, but this does not justify not repeating the procedure to avoid misdiagnosis. LEARNING POINTS: In case of necrotic skin lesions with fever, malignancy (and in particular cutaneous lymphoma) should always be considered.Misdiagnosis of loxoscelism is common. Definitive diagnosis requires the identification of the responsible spider.It is frequent to obtain inconclusive results from biopsies, but this does not justify not repeating the procedure to avoid misdiagnosis.

Integrative studies of 3D genome organization and chromatin structure.

The structural organization of the genome is emerging as a crucial regulator of the cell state, affecting gene transcription, DNA replication, and repair. Over the last twenty years, increasing evidence prompted the development of new experimental techniques to study genome structure. In parallel with the complexity of the novel techniques, computational approaches have become an essential tool in any structural genomics laboratory to analyze and model the data. For biologists to be able to apply the most appropriate modeling approach, it is fundamental to understand the conceptual bases of distinct methods and the insights they can provide. Here, we will discuss recent advances that were possible thanks to 3D genome modeling, discuss their limitations and highlight future perspectives.