HP1-driven phase separation recapitulates the thermodynamics and kinetics of heterochromatin condensate formation.

The spatial segregation of pericentromeric heterochromatin (PCH) into distinct, membrane-less nuclear compartments involves the binding of Heterochromatin Protein 1 (HP1) to H3K9me2/3-rich genomic regions. While HP1 exhibits liquid-liquid phase separation properties in vitro, its mechanistic impact on the structure and dynamics of PCH condensate formation in vivo remains largely unresolved. Here, using a minimal theoretical framework, we systematically investigate the mutual coupling between self-interacting HP1-like molecules and the chromatin polymer. We reveal that the specific affinity of HP1 for H3K9me2/3 loci facilitates coacervation in nucleo and promotes the formation of stable PCH condensates at HP1 levels far below the concentration required to observe phase separation in purified protein assays in vitro. These heterotypic HP1-chromatin interactions give rise to a strong dependence of the nucleoplasmic HP1 density on HP1-H3K9me2/3 stoichiometry, consistent with the thermodynamics of multicomponent phase separation. The dynamical cross talk between HP1 and the viscoelastic chromatin scaffold also leads to anomalously slow equilibration kinetics, which strongly depend on the genomic distribution of H3K9me2/3 domains and result in the coexistence of multiple long-lived, microphase-separated PCH compartments. The morphology of these complex coacervates is further found to be governed by the dynamic establishment of the underlying H3K9me2/3 landscape, which may drive their increasingly abnormal, aspherical shapes during cell development. These findings compare favorably to 4D microscopy measurements of HP1 condensate formation in live Drosophila embryos and suggest a general quantitative model of PCH formation based on the interplay between HP1-based phase separation and chromatin polymer mechanics.

HP1 proteins compact DNA into mechanically and positionally stable phase separated domains.

In mammals, HP1-mediated heterochromatin forms positionally and mechanically stable genomic domains even though the component HP1 paralogs, HP1α, HP1ß, and HP1γ, display rapid on-off dynamics. Here, we investigate whether phase-separation by HP1 proteins can explain these biological observations. Using bulk and single-molecule methods, we show that, within phase-separated HP1α-DNA condensates, HP1α acts as a dynamic liquid, while compacted DNA molecules are constrained in local territories. These condensates are resistant to large forces yet can be readily dissolved by HP1ß. Finally, we find that differences in each HP1 paralog's DNA compaction and phase-separation properties arise from their respective disordered regions. Our findings suggest a generalizable model for genome organization in which a pool of weakly bound proteins collectively capitalize on the polymer properties of DNA to produce self-organizing domains that are simultaneously resistant to large forces at the mesoscale and susceptible to competition at the molecular scale.

Sleep-disordered breathing and comorbidities: role of the upper airway and craniofacial skeleton.

Obstructive sleep-disordered breathing (SDB), which includes primary snoring through to obstructive sleep apnea syndrome (OSAS), may cause compromise of respiratory gas exchange during sleep, related to transient upper airway narrowing disrupting ventilation, and causing oxyhemoglobin desaturation and poor sleep quality. SDB is common in chronic disorders and has significant implications for health. With prevalence rates globally increasing, this condition is causing a substantial burden on health care costs. Certain populations, including people with sickle cell disease (SCD), exhibit a greater prevalence of OSAS. A review of the literature provides the available normal polysomnography and oximetry data for reference and documents the structural upper airway differences between those with and without OSAS, as well as between ethnicities and disease states. There may be differences in craniofacial development due to atypical growth trajectories or extramedullary hematopoiesis in anemias such as SCD. Studies involving MRI of the upper airway illustrated that OSAS populations tend to have a greater amount of lymphoid tissue, smaller airways, and smaller lower facial skeletons from measurements of the mandible and linear mental spine to clivus. Understanding the potential relationship between these anatomical landmarks and OSAS could help to stratify treatments, guiding choice towards those which most effectively resolve the obstruction. OSAS is relatively common in SCD populations, with hypoxia as a key manifestation, and sequelae including increased risk of stroke. Combatting any structural defects with appropriate interventions could reduce hypoxic exposure and consequently reduce the risk of comorbidities in those with SDB, warranting early treatment interventions.

Pulmonary function deficits in newborn screened infants with cystic fibrosis managed with standard UK care are mild and transient.

With the advent of novel designer molecules for cystic fibrosis (CF) treatment, there is huge need for early-life clinical trial outcomes, such as infant lung function (ILF). We investigated the degree and tracking of ILF abnormality during the first 2 years of life in CF newborn screened infants.Forced expiratory volume in 0.5 s (FEV0.5), lung clearance index (LCI) and plethysmographic functional residual capacity were measured at ∼3 months, 1 year and 2 years in 62 infants with CF and 34 controls.By 2 years there was no significant difference in FEV0.5 z-score between CF and controls, whereas mean LCI z-score was 0.81 (95% CI 0.45-1.17) higher in CF. However, there was no significant association between LCI z-score at 2 years with either 3-month or 1-year results. Despite minimal average group changes in any ILF outcome during the second year of life, marked within-subject changes occurred. No child had abnormal LCI or FEV0.5 on all test occasions, precluding the ability to identify "high-risk" infants in early life.In conclusion, changes in lung function are mild and transient during the first 2 years of life in newborn screened infants with CF when managed according to a standardised UK treatment protocol. Their potential role in tracking disease to later childhood will be ascertained by ongoing follow-up.

DNA looping mediates nucleosome transfer.

Proper cell function requires preservation of the spatial organization of chromatin modifications. Maintenance of this epigenetic landscape necessitates the transfer of parental nucleosomes to newly replicated DNA, a process that is stringently regulated and intrinsically linked to replication fork dynamics. This creates a formidable setting from which to isolate the central mechanism of transfer. Here we utilized a minimal experimental system to track the fate of a single nucleosome following its displacement, and examined whether DNA mechanics itself, in the absence of any chaperones or assembly factors, may serve as a platform for the transfer process. We found that the nucleosome is passively transferred to available dsDNA as predicted by a simple physical model of DNA loop formation. These results demonstrate a fundamental role for DNA mechanics in mediating nucleosome transfer and preserving epigenetic integrity during replication.

Small molecule injection into single-cell C. elegans embryos via carbon-reinforced nanopipettes.

The introduction of chemical inhibitors into living cells at specific times in development is a useful method for investigating the roles of specific proteins or cytoskeletal components in developmental processes. Some embryos, such as those of Caenorhabditis elegans, however, possess a tough eggshell that makes introducing drugs and other molecules into embryonic cells challenging. We have developed a procedure using carbon-reinforced nanopipettes (CRNPs) to deliver molecules into C. elegans embryos with high temporal control. The use of CRNPs allows for cellular manipulation to occur just subsequent to meiosis II with minimal damage to the embryo. We have used our technique to replicate classical experiments using latrunculin A to inhibit microfilaments and assess its effects on early polarity establishment. Our injections of latrunculin A confirm the necessity of microfilaments in establishing anterior-posterior polarity at this early stage, even when microtubules remain intact. Further, we find that latrunculin A treatment does not prevent association of PAR-2 or PAR-6 with the cell cortex. Our experiments demonstrate the application of carbon-reinforced nanopipettes to the study of one temporally-confined developmental event. The use of CRNPs to introduce molecules into the embryo should be applicable to investigations at later developmental stages as well as other cells with tough outer coverings.

Structural basis for substrate recognition by a unique Legionella phosphoinositide phosphatase.

Legionella pneumophila is an opportunistic intracellular pathogen that causes sporadic and epidemic cases of Legionnaires' disease. Emerging data suggest that Legionella infection involves the subversion of host phosphoinositide (PI) metabolism. However, how this bacterium actively manipulates PI lipids to benefit its infection is still an enigma. Here, we report that the L. pneumophila virulence factor SidF is a phosphatidylinositol polyphosphate 3-phosphatase that specifically hydrolyzes the D3 phosphate of PI(3,4)P(2) and PI(3,4,5)P(3). This activity is necessary for anchoring of PI(4)P-binding effectors to bacterial phagosomes. Crystal structures of SidF and its complex with its substrate PI(3,4)P(2) reveal striking conformational rearrangement of residues at the catalytic site to form a cationic pocket that specifically accommodates the D4 phosphate group of the substrate. Thus, our findings unveil a unique Legionella PI phosphatase essential for the establishment of lipid identity of bacterial phagosomes.

Diapocynin and apocynin administration fails to significantly extend survival in G93A SOD1 ALS mice.

NADPH oxidase has recently been identified as a promising new therapeutic target in ALS. Genetic deletion of NADPH oxidase (Nox2) in the transgenic SOD1(G93A) mutant mouse model of ALS was reported to increase survival remarkably by 97 days. Furthermore, apocynin, a widely used inhibitor of NADPH oxidase, was observed to dramatically extend the survival of the SOD1(G93A) ALS mice even longer to 113 days (Harraz et al. J Clin Invest 118: 474, 2008). Diapocynin, the covalent dimer of apocynin, has been reported to be a more potent inhibitor of NADPH oxidase. We compared the protection of diapocynin to apocynin in primary cultures of SOD1(G93A)-expressing motor neurons against nitric oxide-mediated death. Diapocynin, 10 µM, provided significantly greater protection compared to apocynin, 200 µM, at the lowest statistically significant concentrations. However, administration of diapocynin starting at 21 days of age in the SOD1(G93A)-ALS mouse model did not extend lifespan. Repeated parallel experiments with apocynin failed to yield protection greater than a 5-day life extension in multiple trials conducted at two separate institutions. The maximum protection observed was an 8-day extension in survival when diapocynin was administered at 100 days of age at disease onset. HPLC with selective ion monitoring by mass spectrometry revealed that both apocynin and diapocynin accumulated in the brain and spinal cord tissue to low micromolar concentrations. Diapocynin was also detected in the CNS of apocynin-treated mice. The failure to achieve significant protection with either apocynin or diapocynin raises questions about the utility for treating ALS patients.

Cognitive impairment predicts functional capacity in dementia-free patients with cardiovascular disease.

BACKGROUND AND RESEARCH OBJECTIVE: A high proportion of elderly people with cardiovascular diseases and risk factors have mild forms of cognitive impairment, the functional impact of which is poorly understood. The aim of this study was to determine whether subtle cognitive impairment contributes to limitations in instrumental activities of daily living in this group and whether this association is independent of physical comorbidity and other potentially confounding factors. SUBJECTS AND METHODS: Two hundred and nineteen nondemented patients were recruited from cardiovascular and diabetic hospital outpatient clinics. Functional dependence was assessed using the self-report version of the instrumental activities of daily living scale. Cognitive ability was assessed using the Montreal Cognitive Assessment (MoCA). Demographic and clinical information was collected via interview and a review of hospital records. Standard logistic regression was performed to identify factors independently associated with functional status. RESULTS: Five variables (sex, cardiovascular disease burden, non-cardiovascular disease burden, cognitive status, and age) were independently associated with an increased likelihood of requiring assistance with 1 or more everyday activities. The likelihood of needing assistance increased 2.05 times (95% confidence interval [CI], 1.59-2.79) for each additional cardiovascular diagnosis present and 1.12 times (95% CI, 1.01-1.27) for every point lower on MoCA. Thus, in comparison to a person with a perfect MoCA score, a person who scored in the cognitively impaired range (<23) was 7.7 (CI, 7.07-8.89) times more likely to report that he/she required assistance with an everyday activity. CONCLUSION: Cognitive impairments appear to reduce the ability to independently carry out routine daily tasks in patients with cardiovascular diseases and risk factors. Cognition should therefore be considered along with physical symptoms when assessing and responding to the support needs of this group.

Prolonged effects of a home-based intervention in patients with chronic illness.

BACKGROUND: Data on the long-term benefits of nonspecific disease management programs are limited. We performed a long-term follow-up of a previously published randomized trial. METHODS: We compared all-cause mortality and recurrent hospitalization during median follow-up of 7.5 years in a heterogeneous cohort of patients with chronic illness initially exposed to a multidisciplinary, home-based intervention (HBI) (n = 260) or to usual postdischarge care (n = 268). RESULTS: During follow-up, HBI had no impact on all-cause mortality (relative risk, 1.04; 95% confidence interval, 0.80-1.35) or event-free survival from death or unplanned hospitalization (relative risk, 1.03; 95% confidence interval, 0.86-1.24). Initial analysis suggested that HBI had only a marginal impact in reducing unplanned hospitalization, with 677 readmissions vs 824 for the usual care group (mean +/- SD rate, 0.72 +/- 0.96 vs 0.84 +/- 1.20 readmissions/patient per year; P = .08). When accounting for increased hospital activity in HBI patients with chronic obstructive pulmonary disease during follow-up for 2 years, post hoc analyses showed that HBI reduced readmissions by 14% within 2 years in patients without this condition (mean +/- SD rate, 0.54 +/- 0.72 vs 0.63 +/- 0.88 readmission/patient per year; P = .04) and by 21% in all surviving patients within 3 to 8 years (mean +/- SD rate, 0.64 +/- 1.26 vs 0.81 +/- 1.61 readmissions/patient per year; P = .03). Overall, recurrent hospital costs were significantly lower (14%) in the HBI group (mean +/- SD, 823 dollars +/- 1642 dollars vs 960 dollars +/- 1376 dollars per patient per year; P = .045). CONCLUSION: This unique study suggests that a nonspecific HBI provides long-term cost benefits in a range of chronic illnesses, except for chronic obstructive pulmonary disease.