Effect of loops on the mean-square displacement of Rouse-model chromatin.

Chromatin polymer dynamics are commonly described using the classical Rouse model. The subsequent discovery, however, of intermediate-scale chromatin organization known as topologically associating domains (TADs) in experimental Hi-C contact maps for chromosomes across the tree of life, together with the success of loop extrusion factor (LEF) model in explaining TAD formation, motivates efforts to understand the effect of loops and loop extrusion on chromatin dynamics. This paper seeks to fulfill this need by combining LEF-model simulations with extended Rouse-model polymer simulations to investigate the dynamics of chromatin with loops and dynamic loop extrusion. We show that loops significantly suppress the averaged mean-square displacement (MSD) of a gene locus, consistent with recent experiments that track fluorescently labeled chromatin loci. We also find that loops reduce the MSD's stretching exponent from the classical Rouse-model value of 1/2 to a loop-density-dependent value in the 0.45-0.40 range. Remarkably, stretching exponent values in this range have also been observed in recent experiments [Weber et al., Phys. Rev. Lett. 104, 238102 (2010)0031-900710.1103/PhysRevLett.104.238102; Bailey et al., Mol. Biol. Cell 34, ar78 (2023)1059-152410.1091/mbc.E23-04-0119]. We also show that the dynamics of loop extrusion itself negligibly affects chromatin mobility. By studying static "rosette" loop configurations, we also demonstrate that chromatin MSDs and stretching exponents depend on the location of the locus in question relative to the position of the loops and on the local friction environment.

Loops and the activity of loop extrusion factors constrain chromatin dynamics.

The chromosomes-DNA polymers and their binding proteins-are compacted into a spatially organized, yet dynamic, three-dimensional structure. Recent genome-wide chromatin conformation capture experiments reveal a hierarchical organization of the DNA structure that is imposed, at least in part, by looping interactions arising from the activity of loop extrusion factors. The dynamics of chromatin reflects the response of the polymer to a combination of thermal fluctuations and active processes. However, how chromosome structure and enzymes acting on chromatin together define its dynamics remains poorly understood. To gain insight into the structure-dynamics relationship of chromatin, we combine high-precision microscopy in living Schizosaccharomyces pombe cells with systematic genetic perturbations and Rouse model polymer simulations. We first investigated how the activity of two loop extrusion factors, the cohesin and condensin complexes, influences chromatin dynamics. We observed that deactivating cohesin, or to a lesser extent condensin, increased chromatin mobility, suggesting that loop extrusion constrains rather than agitates chromatin motion. Our corresponding simulations reveal that the introduction of loops is sufficient to explain the constraining activity of loop extrusion factors, highlighting that the conformation adopted by the polymer plays a key role in defining its dynamics. Moreover, we find that the number of loops or residence times of loop extrusion factors influence the dynamic behavior of the chromatin polymer. Last, we observe that the activity of the INO80 chromatin remodeler, but not the SWI/SNF or RSC complexes, is critical for ATP-dependent chromatin mobility in fission yeast. Taking the data together, we suggest that thermal and INO80-dependent activities exert forces that drive chromatin fluctuations, which are constrained by the organization of the chromosome into loops.

Diagnostic criteria and evolving molecular characterisation of pulmonary neuroendocrine carcinomas.

Neuroendocrine carcinomas of the lung are currently classified into two categories: small-cell lung carcinoma and large-cell neuroendocrine carcinoma. Diagnostic criteria for small-cell and large-cell neuroendocrine carcinoma are based solely on tumour morphology; however, overlap in histologic and immunophenotypic features between the two types of carcinomas can potentially make their classification challenging. Accurate diagnosis of pulmonary neuroendocrine carcinomas is paramount for patient management, as clinical course and treatment differ between small-cell and large-cell neuroendocrine carcinoma. Molecular-genetic, transcriptomic, and proteomic data published over the past decade suggest that small-cell and large-cell neuroendocrine carcinomas are not homogeneous categories but rather comprise multiple groups of distinctive malignancies. Nuances in the susceptibility of small-cell lung carcinoma subtypes to different chemotherapeutic regimens and the discovery of targetable mutations in large-cell neuroendocrine carcinoma suggest that classification and treatment of neuroendocrine carcinomas may be informed by ancillary molecular and protein expression testing going forward. This review summarizes the current diagnostic criteria, prognostic and predictive correlates of classification, and evidence of previously unrecognised subtypes of small-cell and large-cell neuroendocrine carcinoma.

Grading of Medullary Thyroid Carcinoma: an Interobserver Reproducibility Study.

Grade, based on proliferative activity and tumor necrosis, has recently been shown to be prognostic in medullary thyroid carcinoma (MTC) in multivariate analysis. The aim of this study was to evaluate the interobserver reproducibility of assessed grade in MTC. Three groups (each group included one resident/fellow and one attending pathologist) independently evaluated a cohort of 44 sporadic MTC. For each case, all available tumor slides were reviewed, and mitotic count and the presence of tumor necrosis were recorded. Ki-67 was performed, and the Ki-67 proliferative index was determined in the area of highest proliferative activity. Tumors were graded according to the recently published International Medullary Thyroid Carcinoma Grading System (IMTCGS). Kappa statistics were calculated for each individual criterion (mitotic count, Ki-67 proliferative index, and necrosis) and for assigned IMTCGS grade. For our cohort of 44 MTCs, the kappa statistic for mitotic count, Ki-67 proliferative index, and necrosis was 0.68, 0.86, and 0.89, respectively. The kappa statistic for assigned IMTCGS grade was 0.87. Our findings indicate that there was a strong level of agreement for assessment of grade in our cohort of MTC, indicating that grade as assessed by the IMTCGS is not only prognostic but also reproducible.

Extrusion of chromatin loops by a composite loop extrusion factor.

Chromatin loop extrusion by structural maintenance of chromosome (SMC) complexes is thought to underlie intermediate-scale chromatin organization inside cells. Motivated by a number of experiments suggesting that nucleosomes may block loop extrusion by SMCs, such as cohesin and condensin complexes, we introduce and characterize theoretically a composite loop extrusion factor (composite LEF) model. In addition to an SMC complex that creates a chromatin loop by encircling two threads of DNA, this model includes a remodeling complex that relocates or removes nucleosomes as it progresses along the chromatin, and nucleosomes that block SMC translocation along the DNA. Loop extrusion is enabled by SMC motion along nucleosome-free DNA, created in the wake of the remodeling complex, while nucleosome rebinding behind the SMC acts as a ratchet, holding the SMC close to the remodeling complex. We show that, for a wide range of parameter values, this collection of factors constitutes a composite LEF that extrudes loops with a velocity, comparable to the velocity of remodeling complex translocation on chromatin in the absence of SMC, and much faster than loop extrusion by an isolated SMC that is blocked by nucleosomes.

Covariance distributions in single particle tracking.

Several recent experiments, including our own experiments in the fission yeast, Schizosaccharomyces pombe, have characterized the motions of gene loci within living nuclei by measuring the locus position over time, then proceeding to obtain the statistical properties of this motion. To address the question of whether a population of such single-particle tracks, obtained from many different cells, corresponds to a single mode of diffusion, we derive theoretical equations describing the probability distribution of the displacement covariance, assuming the displacement itself is a zero-mean multivariate Gaussian random variable. We also determine the corresponding theoretical means, variances, and third central moments. Bolstering the theory is good agreement between its predictions and the results obtained for various simulated and measured data sets, including simulated particle trajectories undergoing simple and anomalous diffusion, and the measured trajectories of an optically trapped bead in water, and in a viscoelastic polymer solution. We also show that, for sufficiently long tracks, each covariance distribution in all of these examples is well-described by a skew-normal distribution with mean, variance, and skewness given by the theory. However, for the experimentally measured motion of a gene locus in S. pombe, we find that the first two covariance distributions are wider than predicted, although the third and subsequent covariance distributions are well-described by theory. This observation suggests that the origin of the theory-experiment discrepancy in this case is associated with localization noise, which influences only the first two covariances. Thus, we hypothesized that the discrepancy is caused by locus-to-locus heterogeneity in the localization noise, of independent measurements of the same tagged site. Indeed, simulations implementing heterogeneous localization noise revealed that the excess covariance widths can be largely recreated on the basis of heterogeneous noise. Thus, we conclude that the motion of gene loci in fission yeast is consistent with a single mode of diffusion.

Acceptance and Commitment Therapy for perinatal mood and anxiety disorders: A feasibility and proof of concept study.

OBJECTIVES: The aim of the current study was to assess the feasibility, safety, and effectiveness of a newly developed Acceptance and Commitment Therapy (ACT) intervention developed specifically to address the unique context of pregnancy and parenthood. The intervention was delivered to women accessing a specialist Perinatal Community Mental Health Service (PCMHS). DESIGN: An open-label pilot study was conducted of an 8-week, group-delivered ACT intervention targeting women with moderate-to-severe mood and/or anxiety disorders during pregnancy and/or postpartum. METHODS: Outcomes included session attendance rates, dropout rates, crisis/inpatient service use, and standardized symptom scales. Participant's responses to open-ended questions contained in an end of therapy questionnaire were analysed using thematic analysis. RESULTS: Seventy-four women were referred to the intervention with 65 (88%) completing treatment. The median number of sessions attended was 7. No women required input from crisis/inpatient services. All reported finding the intervention helpful. The implementation of ACT in daily life, therapist support, and group processes were cited as helpful aspects of the intervention. At post-treatment, there was a significant reduction in global distress (d = 0.99) and depressive symptoms (d = 1.05), and an increase in psychological flexibility (d = 0.93). On the secondary outcome of global distress, 38% of women were classified as recovered, 31% had reliably improved, 27% remained the same, and 4% had reliably deteriorated. CONCLUSIONS: The delivery of ACT in a routine practice setting is feasible, safe, and effective. A randomized control trial (RCT) is needed to establish the efficacy and cost-effectiveness of this group-delivered ACT intervention. PRACTITIONER POINTS: Group-delivered Acceptance and Commitment Therapy (ACT) is acceptable for women with moderate-to-severe perinatal mood and/or anxiety disorders and can be feasibly delivered in a routine practice setting. The trans-diagnostic nature of ACT enables the concurrent treatment of depressive and anxiety symptoms within the same intervention which is particularly helpful in the perinatal context given the comorbidity of mood and anxiety disorders. With training and supervision, mental health practitioners without extensive education in the delivery of psychological therapies can facilitate the ACT group programme. As this was a feasibility study, there was no control group, adherence to the manual was not assessed, and the absence of a follow-up period limits our knowledge of the longer-term benefits of the ACT group programme.

Modulation of flagellar rotation in surface-attached bacteria: A pathway for rapid surface-sensing after flagellar attachment.

Attachment is a necessary first step in bacterial commitment to surface-associated behaviors that include colonization, biofilm formation, and host-directed virulence. The Gram-negative opportunistic pathogen Pseudomonas aeruginosa can initially attach to surfaces via its single polar flagellum. Although many bacteria quickly detach, some become irreversibly attached and express surface-associated structures, such as Type IV pili, and behaviors, including twitching motility and biofilm initiation. P. aeruginosa that lack the GTPase FlhF assemble a randomly placed flagellum that is motile; however, we observed that these mutant bacteria show defects in biofilm formation comparable to those seen for non-motile, aflagellate bacteria. This phenotype was associated with altered behavior of ΔflhF bacteria immediately following surface-attachment. Forward and reverse genetic screens led to the discovery that FlhF interacts with FimV to control flagellar rotation at a surface, and implicated cAMP signaling in this pathway. Although cAMP controls many transcriptional programs in P. aeruginosa, known targets of this second messenger were not required to modulate flagellar rotation in surface-attached bacteria. Instead, alterations in switching behavior of the motor appeared to result from direct or indirect effects of cAMP on switch complex proteins and/or the stators associated with them.

Suppressing Aneuploidy-Associated Phenotypes Improves the Fitness of Trisomy 21 Cells.

An abnormal number of chromosomes, or aneuploidy, accounts for most spontaneous abortions, causes developmental defects, and is associated with aging and cancer. The molecular mechanisms by which aneuploidy disrupts cellular function remain largely unknown. Here, we show that aneuploidy disrupts the morphology of the nucleus. Mutations that increase the levels of long-chain bases suppress nuclear abnormalities of aneuploid yeast independent of karyotype identity. Quantitative lipidomics indicates that long-chain bases are integral components of the nuclear membrane in yeast. Cells isolated from patients with Down syndrome also show that abnormal nuclear morphologies and increases in long-chain bases not only suppress these abnormalities but also improve their fitness. We obtained similar results with cells isolated from patients with Patau or Edward syndrome, indicating that increases in long-chain bases improve the fitness of aneuploid cells in yeast and humans. Targeting lipid biosynthesis pathways represents an important strategy to suppress nuclear abnormalities in aneuploidy-associated diseases.

RNA-DNA Hybrids Support Recombination-Based Telomere Maintenance in Fission Yeast.

A subset of cancers rely on telomerase-independent mechanisms to maintain their chromosome ends. The predominant "alternative lengthening of telomeres" pathway appears dependent on homology-directed repair (HDR) to maintain telomeric DNA. However, the molecular changes needed for cells to productively engage in telomeric HDR are poorly understood. To gain new insights into this transition, we monitored the state of telomeres during serial culture of fission yeast (Schizosaccharomyces pombe) lacking the telomerase recruitment factor Ccq1. Rad52 is loaded onto critically short telomeres shortly after germination despite continued telomere erosion, suggesting that recruitment of recombination factors is not sufficient to maintain telomeres in the absence of telomerase function. Instead, survivor formation coincides with the derepression of telomeric repeat-containing RNA (TERRA). In this context, degradation of TERRA associated with the telomere in the form of R-loops drives a severe growth crisis, ultimately leading to a novel type of survivor with linear chromosomes and altered cytological telomere characteristics, including the loss of the shelterin component Rap1 (but not the TRF1/TRF2 ortholog, Taz1) from the telomere. We demonstrate that deletion of Rap1 is protective in this context, preventing the growth crisis that is otherwise triggered by degradation of telomeric R-loops in survivors with linear chromosomes. These findings suggest that upregulation of telomere-engaged TERRA, or altered recruitment of shelterin components, can support telomerase-independent telomere maintenance.

A versatile image analysis platform for three-dimensional nuclear reconstruction.

Nuclear morphology is indicative of cellular health in many contexts. In order to robustly and quantitatively measure nuclear size and shape, numerous experimental methods leveraging fluorescence microscopy have been developed. While these methods are useful for quantifying two-dimensional morphology, they often fail to accurately represent the three-dimensional structure of the nucleus, thus omitting important spatial and volumetric information. To address the need for a more accurate image analysis modality, we have developed a software platform that faithfully reconstructs membrane surfaces in three dimensions with sub-pixel resolution. Here, we showcase its broad applicability across species and nuclear scale, as well as provide information on how to employ this platform for diverse experimental systems.