MeCP2 binds to methylated DNA independently of phase separation and heterochromatin organisation.

Correlative evidence has suggested that the methyl-CpG-binding protein MeCP2 contributes to the formation of heterochromatin condensates via liquid-liquid phase separation. This interpretation has been reinforced by the observation that heterochromatin, DNA methylation and MeCP2 co-localise within prominent foci in mouse cells. The findings presented here revise this view. MeCP2 localisation is independent of heterochromatin as MeCP2 foci persist even when heterochromatin organisation is disrupted. Additionally, MeCP2 foci fail to show hallmarks of phase separation in live cells. Importantly, we find that mouse cellular models are highly atypical as MeCP2 distribution is diffuse in most mammalian species, including humans. Notably, MeCP2 foci are absent in Mus spretus which is a mouse subspecies lacking methylated satellite DNA repeats. We conclude that MeCP2 has no intrinsic tendency to form condensates and its localisation is independent of heterochromatin. Instead, the distribution of MeCP2 in the nucleus is primarily determined by global DNA methylation patterns.

Rapid response of thyroid eye disease, peripheral edema, and acropathy to teprotumumab infusion.

Purpose: We present a case of rapid improvement in symptoms of thyroid eye disease and amelioration of worsening peripheral edema and acropathy with infusion of teprotumumab, a monoclonal antibody targeting the insulin-like growth factor-1 receptor. Observations: A 66 year old female with history of Hashimoto thyroiditis developed progressive thyroid eye disease (TED), peripheral edema, and acropathy attributable to acute Graves disease. Her signs and symptoms, refractory to oral steroid and diuretic therapy, rapidly improved following a standard dosing regimen of teprotumumab (one infusion 10 mg/kg then seven infusions 20 mg/kg) to resolution. Conclusions & importance: Teprotumumab, a monoclonal antibody targeting the insulin-like growth factor-1 receptor, is the first medication approved by the FDA for use in TED. Teprotumumab may contribute to the treatment of extraocular manifestations of Graves disease, chief among these peripheral soft tissue manifestations.

Age-dependent loss of cohesion protection in human oocytes.

Aneuploid human eggs (oocytes) are a major cause of infertility, miscarriage, and chromosomal disorders. Such aneuploidies increase greatly as women age, with defective linkages between sister chromatids (cohesion) in meiosis as a common cause. We found that loss of a specific pool of the cohesin protector protein, shugoshin 2 (SGO2), may contribute to this phenomenon. Our data indicate that SGO2 preserves sister chromatid cohesion in meiosis by protecting a "cohesin bridge" between sister chromatids. In human oocytes, SGO2 localizes to both sub-centromere cups and the pericentromeric bridge, which spans the sister chromatid junction. SGO2 normally colocalizes with cohesin; however, in meiosis II oocytes from older women, SGO2 is frequently lost from the pericentromeric bridge and sister chromatid cohesion is weakened. MPS1 and BUB1 kinase activities maintain SGO2 at sub-centromeres and the pericentromeric bridge. Removal of SGO2 throughout meiosis I by MPS1 inhibition reduces cohesion protection, increasing the incidence of single chromatids at meiosis II. Therefore, SGO2 deficiency in human oocytes can exacerbate the effects of maternal age by rendering residual cohesin at pericentromeres vulnerable to loss in anaphase I. Our data show that impaired SGO2 localization weakens cohesion integrity and may contribute to the increased incidence of aneuploidy observed in human oocytes with advanced maternal age.

Mechanistic basis for Sgo1-mediated centromere localization and function of the CPC.

Centromere association of the chromosomal passenger complex (CPC; Borealin-Survivin-INCENP-Aurora B) and Sgo1 is crucial for chromosome biorientation, a process essential for error-free chromosome segregation. Phosphorylated histone H3 Thr3 (H3T3ph; directly recognized by Survivin) and histone H2A Thr120 (H2AT120ph; indirectly recognized via Sgo1), together with CPC's intrinsic nucleosome-binding ability, facilitate CPC centromere recruitment. However, the molecular basis for CPC-Sgo1 binding and how their physical interaction influences CPC centromere localization are lacking. Here, using an integrative structure-function approach, we show that the "histone H3-like" Sgo1 N-terminal tail-Survivin BIR domain interaction acts as a hotspot essential for CPC-Sgo1 assembly, while downstream Sgo1 residues and Borealin contribute for high-affinity binding. Disrupting Sgo1-Survivin interaction abolished CPC-Sgo1 assembly and perturbed CPC centromere localization and function. Our findings reveal that Sgo1 and H3T3ph use the same surface on Survivin to bind CPC. Hence, it is likely that these interactions take place in a spatiotemporally restricted manner, providing a rationale for the Sgo1-mediated "kinetochore-proximal" CPC centromere pool.

Establishment of centromere identity is dependent on nuclear spatial organization.

The establishment of centromere-specific CENP-A chromatin is influenced by epigenetic and genetic processes. Central domain sequences from fission yeast centromeres are preferred substrates for CENP-ACnp1 incorporation, but their use is context dependent, requiring adjacent heterochromatin. CENP-ACnp1 overexpression bypasses heterochromatin dependency, suggesting that heterochromatin ensures exposure to conditions or locations permissive for CENP-ACnp1 assembly. Centromeres cluster around spindle-pole bodies (SPBs). We show that heterochromatin-bearing minichromosomes localize close to SPBs, consistent with this location promoting CENP-ACnp1 incorporation. We demonstrate that heterochromatin-independent de novo CENP-ACnp1 chromatin assembly occurs when central domain DNA is placed near, but not far from, endogenous centromeres or neocentromeres. Moreover, direct tethering of central domain DNA at SPBs permits CENP-ACnp1 assembly, suggesting that the nuclear compartment surrounding SPBs is permissive for CENP-ACnp1 incorporation because target sequences are exposed to high levels of CENP-ACnp1 and associated assembly factors. Thus, nuclear spatial organization is a key epigenetic factor that influences centromere identity.

STING nuclear partners contribute to innate immune signaling responses.

STimulator of INterferon Genes (STING) is an adaptor for cytoplasmic DNA sensing by cGAMP/cGAS that helps trigger innate immune responses (IIRs). Although STING is mostly localized in the ER, we find a separate inner nuclear membrane pool of STING that increases mobility and redistributes to the outer nuclear membrane upon IIR stimulation by transfected dsDNA or dsRNA mimic poly(I:C). Immunoprecipitation of STING from isolated nuclear envelopes coupled with mass spectrometry revealed a distinct nuclear envelope-STING proteome consisting of known nuclear membrane proteins and enriched in DNA- and RNA-binding proteins. Seventeen of these nuclear envelope STING partners are known to bind direct interactors of IRF3/7 transcription factors, and testing a subset of these revealed STING partners SYNCRIP, MEN1, DDX5, snRNP70, RPS27a, and AATF as novel modulators of dsDNA-triggered IIRs. Moreover, we find that SYNCRIP is a novel antagonist of the RNA virus, influenza A, potentially shedding light on reports of STING inhibition of RNA viruses.

SUMOylation stabilizes sister kinetochore biorientation to allow timely anaphase.

During mitosis, sister chromatids attach to microtubules from opposite poles, called biorientation. Sister chromatid cohesion resists microtubule forces, generating tension, which provides the signal that biorientation has occurred. How tension silences the surveillance pathways that prevent cell cycle progression and correct erroneous kinetochore-microtubule attachments remains unclear. Here we show that SUMOylation dampens error correction to allow stable sister kinetochore biorientation and timely anaphase onset. The Siz1/Siz2 SUMO ligases modify the pericentromere-localized shugoshin (Sgo1) protein before its tension-dependent release from chromatin. Sgo1 SUMOylation reduces its binding to protein phosphatase 2A (PP2A), and weakening of this interaction is important for stable biorientation. Unstable biorientation in SUMO-deficient cells is associated with persistence of the chromosome passenger complex (CPC) at centromeres, and SUMOylation of CPC subunit Bir1 also contributes to timely anaphase onset. We propose that SUMOylation acts in a combinatorial manner to facilitate dismantling of the error correction machinery within pericentromeres and thereby sharpen the metaphase-anaphase transition.

Aerosol and bioaerosol particle size and dynamics from defective sanitary plumbing systems.

Aerosols are readily transported on airstreams through building sanitary plumbing and sewer systems, and those containing microbial pathogens (known as bioaerosols) are recognized as contributors to infection spread within buildings. When a defect occurs in the sanitary plumbing system that affects the system integrity, a cross-transmission route is created that can enable the emission of bioaerosols from the system into the building. These emission occurrences are characterized as short-burst events (typically <1 min in duration) which make them difficult to detect and predict. The characterization of these emission events is the focus of this research. Two methods were used to characterize bioaerosol emission events in a full-scale test rig: (a) an Aerodynamic Particle Sizer (APS) for particle size distribution and concentrations; and (b) a slit-to-agar sampler to enumerate the ingress of a viable tracer microorganism (Pseudomonas putida). The APS data confirmed that most particles (>99.5%) were <5 µm and were therefore considered aerosols. Particles generated within the sanitary plumbing system as a result of a toilet flush leads to emissions into the building during system defect conditions with an equivalence of someone talking loudly for over 6 and a half minutes. There were no particles detected of a size >11 µm anywhere in the system. Particle count was influenced by toilet flush volume, but it was not possible to determine if there was any direct influence from airflow rate since both particle and biological data showed no correlation with upward airflow rates and velocities. Typical emissions resulting from a 6 L toilet flush were in the range of 280-400 particles per second at a concentration of typically 9-12 number per cm3 and a total particle count in the region of 3000 to 4000 particles, whereas the peak emissions from a 1.2 L toilet flush were 60-80 particles per second at a concentration of 2.4-3 number per cm3 and a total particle count in the region of 886 to 1045 particles. The reduction in particles is in direct proportion to the reduction in toilet flush volume. The slit-to-agar sampler was able to provide viable time course CFU data and confirmed the origin of the particles to be the tracer microorganism flushed into the system. The time course data also have characteristics consistent with the unsteady nature of a toilet flush.

Microtubule-independent movement of the fission yeast nucleus.

Movement of the cell nucleus typically involves the cytoskeleton and either polymerization-based pushing forces or motor-based pulling forces. In the fission yeast Schizosaccharomyces pombe, nuclear movement and positioning are thought to depend on microtubule polymerization-based pushing forces. Here, we describe a novel, microtubule-independent, form of nuclear movement in fission yeast. Microtubule-independent nuclear movement is directed towards growing cell tips, and it is strongest when the nucleus is close to a growing cell tip, and weakest when the nucleus is far from that tip. Microtubule-independent nuclear movement requires actin cables but does not depend on actin polymerization-based pushing or myosin V-based pulling forces. The vesicle-associated membrane protein (VAMP)-associated proteins (VAPs) Scs2 and Scs22, which are critical for endoplasmic reticulum-plasma membrane contact sites in fission yeast, are also required for microtubule-independent nuclear movement. We also find that in cells in which microtubule-based pushing forces are present, disruption of actin cables leads to increased fluctuations in interphase nuclear positioning and subsequent altered septation. Our results suggest two non-exclusive mechanisms for microtubule-independent nuclear movement, which may help illuminate aspects of nuclear positioning in other cells.

Neuronal non-CG methylation is an essential target for MeCP2 function.

DNA methylation is implicated in neuronal biology via the protein MeCP2, the mutation of which causes Rett syndrome. MeCP2 recruits the NCOR1/2 co-repressor complexes to methylated cytosine in the CG dinucleotide, but also to sites of non-CG methylation, which are abundant in neurons. To test the biological significance of the dual-binding specificity of MeCP2, we replaced its DNA binding domain with an orthologous domain from MBD2, which can only bind mCG motifs. Knockin mice expressing the domain-swap protein displayed severe Rett-syndrome-like phenotypes, indicating that normal brain function requires the interaction of MeCP2 with sites of non-CG methylation, specifically mCAC. The results support the notion that the delayed onset of Rett syndrome is due to the simultaneous post-natal accumulation of mCAC and its reader MeCP2. Intriguingly, genes dysregulated in both Mecp2 null and domain-swap mice are implicated in other neurological disorders, potentially highlighting targets of relevance to the Rett syndrome phenotype.

The Proteomic Landscape of Centromeric Chromatin Reveals an Essential Role for the Ctf19CCAN Complex in Meiotic Kinetochore Assembly.

Kinetochores direct chromosome segregation in mitosis and meiosis. Faithful gamete formation through meiosis requires that kinetochores take on new functions that impact homolog pairing, recombination, and the orientation of kinetochore attachment to microtubules in meiosis I. Using an unbiased proteomics pipeline, we determined the composition of centromeric chromatin and kinetochores at distinct cell-cycle stages, revealing extensive reorganization of kinetochores during meiosis. The data uncover a network of meiotic chromosome axis and recombination proteins that bind to centromeres in the absence of the microtubule-binding outer kinetochore sub-complexes during meiotic prophase. We show that the Ctf19cCCAN inner kinetochore complex is essential for kinetochore organization in meiosis. Our functional analyses identify a Ctf19cCCAN-dependent kinetochore assembly pathway that is dispensable for mitotic growth but becomes critical upon meiotic entry. Therefore, changes in kinetochore composition and a distinct assembly pathway specialize meiotic kinetochores for successful gametogenesis.

Convergent genes shape budding yeast pericentromeres.

The three-dimensional architecture of the genome governs its maintenance, expression and transmission. The cohesin protein complex organizes the genome by topologically linking distant loci, and is highly enriched in specialized chromosomal domains surrounding centromeres, called pericentromeres1-6. Here we report the three-dimensional structure of pericentromeres in budding yeast (Saccharomyces cerevisiae) and establish the relationship between genome organization and function. We find that convergent genes mark pericentromere borders and, together with core centromeres, define their structure and function by positioning cohesin. Centromeres load cohesin, and convergent genes at pericentromere borders trap it. Each side of the pericentromere is organized into a looped conformation, with border convergent genes at the base. Microtubule attachment extends a single pericentromere loop, size-limited by convergent genes at its borders. Reorienting genes at borders into a tandem configuration repositions cohesin, enlarges the pericentromere and impairs chromosome biorientation during mitosis. Thus, the linear arrangement of transcriptional units together with targeted cohesin loading shapes pericentromeres into a structure that is competent for chromosome segregation. Our results reveal the architecture of the chromosomal region within which kinetochores are embedded, as well as the restructuring caused by microtubule attachment. Furthermore, we establish a direct, causal relationship between the three-dimensional genome organization of a specific chromosomal domain and cellular function.

Borealin-nucleosome interaction secures chromosome association of the chromosomal passenger complex.

Chromosome association of the chromosomal passenger complex (CPC; consisting of Borealin, Survivin, INCENP, and the Aurora B kinase) is essential to achieve error-free chromosome segregation during cell division. Hence, understanding the mechanisms driving the chromosome association of the CPC is of paramount importance. Here using a multifaceted approach, we show that the CPC binds nucleosomes through a multivalent interaction predominantly involving Borealin. Strikingly, Survivin, previously suggested to target the CPC to centromeres, failed to bind nucleosomes on its own and requires Borealin and INCENP for its binding. Disrupting Borealin-nucleosome interactions excluded the CPC from chromosomes and caused chromosome congression defects. We also show that Borealin-mediated chromosome association of the CPC is critical for Haspin- and Bub1-mediated centromere enrichment of the CPC and works upstream of the latter. Our work thus establishes Borealin as a master regulator determining the chromosome association and function of the CPC.

Spo13 prevents premature cohesin cleavage during meiosis.

Background: Meiosis produces gametes through two successive nuclear divisions, meiosis I and meiosis II. In contrast to mitosis and meiosis II, where sister chromatids are segregated, during meiosis I, homologous chromosomes are segregated. This requires the monopolar attachment of sister kinetochores and the loss of cohesion from chromosome arms, but not centromeres, during meiosis I. The establishment of both sister kinetochore mono-orientation and cohesion protection rely on the budding yeast meiosis I-specific Spo13 protein, the functional homolog of fission yeast Moa1 and mouse MEIKIN. Methods: Here we investigate the effects of loss of SPO13 on cohesion during meiosis I using a live-cell imaging approach. Results: Unlike wild type, cells lacking SPO13 fail to maintain the meiosis-specific cohesin subunit, Rec8, at centromeres and segregate sister chromatids to opposite poles during anaphase I. We show that the cohesin-destabilizing factor, Wpl1, is not primarily responsible for the loss of cohesion during meiosis I. Instead, premature loss of centromeric cohesin during anaphase I in spo13 Δ cells relies on separase-dependent cohesin cleavage. Further, cohesin loss in spo13 Δ anaphase I cells is blocked by forcibly tethering the regulatory subunit of protein phosphatase 2A, Rts1, to Rec8. Conclusions: Our findings indicate that separase-dependent cleavage of phosphorylated Rec8 causes premature cohesin loss in spo13 Δ cells.

Reductional Meiosis I Chromosome Segregation Is Established by Coordination of Key Meiotic Kinases.

Meiosis produces gametes through a specialized, two-step cell division, which is highly error prone in humans. Reductional meiosis I, where maternal and paternal chromosomes (homologs) segregate, is followed by equational meiosis II, where sister chromatids separate. Uniquely during meiosis I, sister kinetochores are monooriented and pericentromeric cohesin is protected. Here, we demonstrate that these key adaptations for reductional chromosome segregation are achieved through separable control of multiple kinases by the meiosis-I-specific budding yeast Spo13 protein. Recruitment of Polo kinase to kinetochores directs monoorientation, while independently, cohesin protection is achieved by containing the effects of cohesin kinases. Therefore, reductional chromosome segregation, the defining feature of meiosis, is established by multifaceted kinase control by a master regulator. The recent identification of Spo13 orthologs, fission yeast Moa1 and mouse MEIKIN, suggests that kinase coordination by a meiosis I regulator may be a general feature in the establishment of reductional chromosome segregation.

Host Vesicle Fusion Protein VAPB Contributes to the Nuclear Egress Stage of Herpes Simplex Virus Type-1 (HSV-1) Replication.

The primary envelopment/de-envelopment of Herpes viruses during nuclear exit is poorly understood. In Herpes simplex virus type-1 (HSV-1), proteins pUL31 and pUL34 are critical, while pUS3 and some others contribute; however, efficient membrane fusion may require additional host proteins. We postulated that vesicle fusion proteins present in the nuclear envelope might facilitate primary envelopment and/or de-envelopment fusion with the outer nuclear membrane. Indeed, a subpopulation of vesicle-associated membrane protein-associated protein B (VAPB), a known vesicle trafficking protein, was present in the nuclear membrane co-locating with pUL34. VAPB knockdown significantly reduced both cell-associated and supernatant virus titers. Moreover, VAPB depletion reduced cytoplasmic accumulation of virus particles and increased levels of nuclear encapsidated viral DNA. These results suggest that VAPB is an important player in the exit of primary enveloped HSV-1 virions from the nucleus. Importantly, VAPB knockdown did not alter pUL34, calnexin or GM-130 localization during infection, arguing against an indirect effect of VAPB on cellular vesicles and trafficking. Immunogold-labelling electron microscopy confirmed VAPB presence in nuclear membranes and moreover associated with primary enveloped HSV-1 particles. These data suggest that VAPB could be a cellular component of a complex that facilitates UL31/UL34/US3-mediated HSV-1 nuclear egress.

Use of Integra in the Management of Complex Hand Wounds From Cancer Resection and Nonburn Trauma.

BACKGROUND: Despite extensive use of Integra in burn reconstruction, little has been published regarding its utility in complex hand wounds from nonburn trauma or cancer resection. This study aimed to review outcomes following Integra use for hand reconstruction following cancer resection or nonburn trauma with exposed bone, joints, and/or tendons. METHODS: Retrospective review was performed of patients undergoing hand reconstruction with Integra for exposed bones, joints, or tendons over a 6-year period at a single institution. RESULTS: Fourteen patients underwent hand reconstruction using Integra, 8 following cancer resection and 6 following acute nonburn trauma. The mean defect size was 19 cm2, 79% had exposed tendon without peritenon, 43% had exposed bone without periosteum, and 28% had exposed joint capsule. Mean time from Integra to skin graft was 11.3 days, and negative-pressure wound therapy did not significantly decrease the mean time from Integra to skin graft placement ( P = .76). Overall, 13 patients achieved successful reconstruction with mean skin graft take of 97%, and 1 required revision amputation at the proximal interphalangeal (PIP) joint. Six months postoperative, 92% patients had return of preoperative hand function. Without any surgical revision, 85% of patients were extremely satisfied with the aesthetic result and 15% were fairly satisfied. CONCLUSIONS: Integra is an effective method to treat complex hand wounds with exposed bone, joints, and/or tendons. This technique can be used in the office, lessens the need for local or free flap coverage, and provides an excellent aesthetic outcome. Integra should be considered a viable option in hand reconstruction algorithm.

Major Lower Limb Amputation: Outcomes are Improving.

BACKGROUND: Outcomes following major lower limb amputation (MLLA) between 2000 and 2002 from the Department of Vascular Surgery at Royal Perth Hospital have been published; mean postoperative length of stay 20 days, inpatient complication rate 54%, and 30-day mortality 10%. The last decade has seen increasing endovascular revascularization techniques, increased focus on MLLA patients, and general improvements in the model of care. The aim of this study is to compare outcomes between 2000-2002 and 2010-2012. METHODS: Data on all patients undergoing MLLA, transtibial or proximal, in the 2 time periods were extracted from the department of vascular surgery database. Medical records, government registries, and phone calls to primary care providers were used to clarify mortality. RESULTS: Limb ischemia remains the most common indication for MLLA with smoking, hypertension, and diabetes being the main comorbid diseases. The rates of wound infections have fallen from 26.4% to 12.4% (P = 0.023), rate of admission to ICU has fallen from 48.3% to 17.5% (P = 0.001), and revision amputation to a higher level has fallen from 11.5% to 7.2% (P = 0.043). Acute hospital, postoperative length of stay has trended down from 15.74 to 20.29 days (P = 0.075). Mortality overall has fallen from 60.92% to 46.39% (P = 0.049). Thirty-day mortality fallen from 10.34% to 5.15% (P = 0.185), 6-month 28.76% to 16.5% (P = 0.046), and 1-year 40.22% to 21.65% (P = 0.006). CONCLUSIONS: Patients undergoing MLLA still carry a high burden of comorbid disease. With changes in revascularization technique, consultant supervision, and multidisciplinary model of care, we have seen the rate of complications fall, length of stay trend down, and overall mortality reduce. Despite improvements, outcomes remain sobering and more can be done.

Pathogen cross-transmission via building sanitary plumbing systems in a full scale pilot test-rig.

The WHO Consensus Document on the epidemiology of the SARS epidemic in 2003, included a report on a concentrated outbreak in one Hong Kong housing block which was considered a 'super-spreading event'. The WHO report conjectured that the sanitary plumbing system was one transmission route for the virus. Empty U-traps allowed the aerosolised virus to enter households from the sewerage system. No biological evidence was presented. This research reports evidence that pathogens can be aerosolised and transported on airstreams within sanitary plumbing systems and enter buildings via empty U-traps. A sanitary plumbing system was built, representing two floors of a building, with simulated toilet flushes on the lower floor and a sterile chamber with extractor fan on the floor above. Cultures of a model organism, Pseudomonas putida at 106-109 cfu ml-1 in 0·85% NaCl were flushed into the system in volumes of 6 to 20 litres to represent single or multiple toilet flushes. Air and surface samples were cultured on agar plates and assessed qualitatively and semi-quantitatively. Flushing from a toilet into a sanitary plumbing system generated enough turbulence to aerosolise pathogens. Typical sanitary plumbing system airflows (between 20-30 ls-1) were sufficient to carry aerosolised pathogens between different floors of a building. Empty U-traps allowed aerosolised pathogens to enter the chamber, encouraging cross-transmission. All parts of the system were found to be contaminated post-flush. Empty U-traps have been observed in many buildings and a risk assessment indicates the potential for high risk cross-transmission under defect conditions in buildings with high pathogen loading such as hospitals. Under defective conditions (which are not uncommon) aerosolised pathogens can be carried on the airflows within sanitary plumbing systems. Our findings show that greater consideration should be given to this mode of pathogen transmission.