Validation of the Russian Version of the Prolapse Quality-of-life Questionnaire and its Application to Assess the Impact of Pelvic Organ Prolapse on Quality of Life and the Effect of Treatment in Women Undergoing Reconstructive Surgery.

INTRODUCTION AND HYPOTHESIS: The objective was to validate the translated Russian version of the prolapse quality-of-life (P-QoL) questionnaire and test its applicability to assess the impact of pelvic organ prolapse (POP) on QoL and the effect of treatment in women undergoing reconstructive surgery. METHODS: Following a forward- and back-translation of the original English P-QOL questionnaire into Russian, the translated questionnaire was reviewed by a group of patients as well as an expert panel. Women with POP who were admitted to a university hospital for reconstructive surgery were recruited. All the women completed the P-QoL questionnaire, Pelvic Floor Distress Inventory (PFDI-20) and 36-Item Short Form Survey (SF-36) questionnaires before surgery. Clinical data and POP Quantification (POP-Q) Index according to the International Continence Society were obtained. Psychometric properties of the questionnaire were assessed. RESULTS: A total of 303 women with POP were included in the study. Most patients presented with POP-Q >2. The P-QoL questionnaire demonstrated good psychometric properties. High internal consistency was shown in all domains (Cronbach's alpha coefficient from 0.65 to 0.92). The test-retest reliability confirmed a highly significant stability between the total scores for each domain. Significant correlations of the P-QoL domains with the PFDI-20 and SF-36 scales (p < 0.05) were obtained, demonstrating satisfactory convergent validity. Discriminative construct validity was proved by the differences in the mean scores for P-QoL domains across POP-Q stages (p < 0.05): general health perceptions, role limitations, physical limitations, social limitations and severity measures were significantly higher for POP-Q stages 3 and 4 than for POP-Q stage 2 (p < 0.01); general health perceptions and severity measures were higher for POP-Q stage 4 than for POP-Q stage 3 (p < 0.05); sleep/energy was higher for POP-Q stage 3 than for POP-Q stage 2 (p < 0.05). Significant improvement of QoL in the 2 months after surgery (p < 0.05) indicated that the P-QoL questionnaire is sensitive to change. CONCLUSIONS: The Russian version of the P-QoL questionnaire is characterized by appropriate psychometric properties. The P-QoL questionnaire is a useful tool for describing the QoL profile in women with POP before reconstructive surgery and evaluating treatment outcomes after the procedure.

Design, synthesis and antiviral evaluation of novel conjugates of the 1,7,7-trimethylbicyclo[2.2.1]heptane scaffold and saturated N-heterocycles via 1,2,3-triazole linker.

A new series of heterocyclic derivatives with a 1,7,7-trimethylbicyclo[2.2.1]heptane fragment was designed, synthesised and biologically evaluated. Synthesis of the target compounds was performed using the Cu(I) catalysed cycloaddition reaction. The key starting substances in the click reaction were an alkyne containing a 1,7,7-trimethylbicyclo[2.2.1]heptane fragment and a series of azides with saturated nitrogen-containing heterocycles. Some of the derivatives were found to exhibit strong antiviral activity against Marburg and Ebola pseudotype viruses. Lysosomal trapping assays revealed the derivatives to possess lysosomotropic properties. The molecular modelling study demonstrated the binding affinity between the compounds investigated and the possible active site to be mainly due to hydrophobic interactions. Thus, combining a natural hydrophobic structural fragment and a lysosome-targetable heterocycle may be an effective strategy for designing antiviral agents.

Predictors of Perioperative Vasoactive Drug Requirement During Retroperitoneal Adrenalectomy for Pheochromocytoma: A Retrospective Exploratory Study.

The aim of the present study was to test a hypothesis that baseline systemic vascular resistance index (SVRI) assessed by method of transpulmonary thermodilution predicts perioperative requirement for vasoactive drugs. The primary outcomes were: (1) peak vasoactive-inotropic score (VIS) and (2) peak dose of hypotensive drugs at any stage of surgery. The main exposure variable was baseline SVRI. Hemodynamics were retrospectively assessed by transpulmonary thermodilution in 50 adults who had undergone posterior retroperitoneal surgery for pheochromocytoma. Univariate linear regression analysis showed predictive value of SVRI on VIS [regression coefficient, 95% CI; 0.024 (0.005, 0.4), p=0.015]. Other significant factors were the history of peak diastolic pressure, baseline MAP, baseline betablocker therapy, and history of coronary artery disease (CAD). After adjustment of SVRI for the history of CAD, its prognostic value became non-significant [0.018 (0.008, 0.03), p=0.063 and 29.6 (19, 40.2), p=0.007 for SVRI and history of CAD, respectively]. Requirements of vasodilators were predicted by baseline adrenergic activity [0.37 (0.005, 0.74), p=0.047]. In conclusion, baseline SVRI is associated with perioperative requirement of vasopressor drugs, but history of CAD is a stronger prognostic factor for vasopressor support. Perioperative requirement in vasodilators is associated with baseline adrenergic activity.

Nucleosome spacing periodically modulates nucleosome chain folding and DNA topology in circular nucleosome arrays.

The length of linker DNA that separates nucleosomes is highly variable, but its mechanistic role in modulating chromatin structure and functions remains unknown. Here, we established an experimental system using circular arrays of positioned nucleosomes to investigate whether variations in nucleosome linker length could affect nucleosome folding, self-association, and interactions. We conducted EM, DNA topology, native electrophoretic assays, and Mg2+-dependent self-association assays to study intrinsic folding of linear and circular nucleosome arrays with linker DNA length of 36 bp and 41 bp (3.5 turns and 4 turns of DNA double helix, respectively). These experiments revealed that potential artifacts arising from open DNA ends and full DNA relaxation in the linear arrays do not significantly affect overall chromatin compaction and self-association. We observed that the 0.5 DNA helical turn difference between the two DNA linker lengths significantly affects DNA topology and nucleosome interactions. In particular, the 41-bp linkers promoted interactions between any two nucleosome beads separated by one bead as expected for a zigzag fiber, whereas the 36-bp linkers promoted interactions between two nucleosome beads separated by two other beads and also reduced negative superhelicity. Monte Carlo simulations accurately reproduce periodic modulations of chromatin compaction, DNA topology, and internucleosomal interactions with a 10-bp periodicity. We propose that the nucleosome spacing and associated chromatin structure modulations may play an important role in formation of different chromatin epigenetic states, thus suggesting implications for how chromatin accessibility to DNA-binding factors and the RNA transcription machinery is regulated.

A mosaic intragenic microduplication of LAMA1 and a constitutional 18p11.32 microduplication in a patient with keratosis pilaris and intellectual disability.

The application of array-based comparative genomic hybridization and next-generation sequencing has identified many chromosomal microdeletions and microduplications in patients with different pathological phenotypes. Different copy number variations are described within the short arm of chromosome 18 in patients with skin diseases. In particular, full or partial monosomy 18p has also been associated with keratosis pilaris. Here, for the first time, we report a young male patient with intellectual disability, diabetes mellitus (type I), and keratosis pilaris, who exhibited a de novo 45-kb microduplication of exons 4-22 of LAMA1, located at 18p11.31, and a 432-kb 18p11.32 microduplication of paternal origin containing the genes METTL4, NDC80, and CBX3P2 and exons 1-15 of the SMCHD1 gene. The microduplication of LAMA1 was identified in skin fibroblasts but not in lymphocytes, whereas the larger microduplication was present in both tissues. We propose LAMA1 as a novel candidate gene for keratosis pilaris. Although inherited from a healthy father, the 18p11.32 microduplication, which included relevant genes, could also contribute to phenotype manifestation.

Novel nucleosomal particles containing core histones and linker DNA but no histone H1.

Eukaryotic chromosomal DNA is assembled into regularly spaced nucleosomes, which play a central role in gene regulation by determining accessibility of control regions. The nucleosome contains ∼147 bp of DNA wrapped ∼1.7 times around a central core histone octamer. The linker histone, H1, binds both to the nucleosome, sealing the DNA coils, and to the linker DNA between nucleosomes, directing chromatin folding. Micrococcal nuclease (MNase) digests the linker to yield the chromatosome, containing H1 and ∼160 bp, and then converts it to a core particle, containing ∼147 bp and no H1. Sequencing of nucleosomal DNA obtained after MNase digestion (MNase-seq) generates genome-wide nucleosome maps that are important for understanding gene regulation. We present an improved MNase-seq method involving simultaneous digestion with exonuclease III, which removes linker DNA. Remarkably, we discovered two novel intermediate particles containing 154 or 161 bp, corresponding to 7 bp protruding from one or both sides of the nucleosome core. These particles are detected in yeast lacking H1 and in H1-depleted mouse chromatin. They can be reconstituted in vitro using purified core histones and DNA. We propose that these 'proto-chromatosomes' are fundamental chromatin subunits, which include the H1 binding site and influence nucleosome spacing independently of H1.

Methotrexate treatment may prevent uveitis onset in patients with juvenile idiopathic arthritis: experiences and subgroup analysis in a cohort with frequent methotrexate use.

OBJECTIVES: To re-evaluate the ability of methotrexate (MTX) to prevent the onset of uveitis in Russian children with juvenile idiopathic arthritis (JIA). METHODS: The clinical charts for all consecutive patients who received a stable management for at least 2 years with or without MTX were reviewed. Patients who were given systemic medications other than MTX (except NSAID) and patients with systemic arthritis, rheumatoid factor-positive arthritis, or enthesitis-related arthritis were excluded. Each patient was examined after at least a 2-year follow-up period after the first visit to establish whether uveitis had occurred. RESULTS: A total of 281 patients with a median disease duration of 3.8 years were included. 191 patients (68%) were treated with MTX. During the observation period, 64 patients (22.8%) developed uveitis, a median of 1.6 year after disease onset. The frequency of uveitis was lower in MTX-treated than in MTX-untreated patients (11.5% vs. 46.7%, respectively, OR=6.7 (95%CI:3.7-12.3), p=0.0000001). Survival analysis confirmed that patients treated with MTX had a lower probability of developing uveitis (HR=4.35, p=0.000001). In subgroup analysis it was shown that MTX was more preventive in boys than in girls, and in patients with JIA onset age of over 5 years compared to those with disease onset less than 5 years. The data of survival analysis of MTX prevention has shown that benefits do not depend on the number of active joints and ANA status. CONCLUSIONS: MTX therapy may prevent the onset of uveitis in children with JIA. Further randomised controlled trials are required to confirm our results.

Iodinated contrast media cause direct tubular cell damage, leading to oxidative stress, low nitric oxide, and impairment of tubuloglomerular feedback.

Iodinated contrast media (CM) have adverse effects that may result in contrast-induced acute kidney injury. Oxidative stress is believed to play a role in CM-induced kidney injury. We test the hypothesis that oxidative stress and reduced nitric oxide in tubules are consequences of CM-induced direct cell damage and that increased local oxidative stress may increase tubuloglomerular feedback. Rat thick ascending limbs (TAL) were isolated and perfused. Superoxide and nitric oxide were quantified using fluorescence techniques. Cell death rate was estimated using propidium iodide and trypan blue. The function of macula densa and tubuloglomerular feedback responsiveness were measured in isolated, perfused juxtaglomerular apparatuses (JGA) of rabbits. The expression of genes related to oxidative stress and the activity of superoxide dismutase (SOD) were investigated in the renal medulla of rats that received CM. CM increased superoxide concentration and reduced nitric oxide bioavailability in TAL. Propidium iodide fluorescence and trypan blue uptake increased more in CM-perfused TAL than in controls, indicating increased rate of cell death. There were no marked acute changes in the expression of genes related to oxidative stress in medullary segments of Henle's loop. SOD activity did not differ between CM and control groups. The tubuloglomerular feedback in isolated JGA was increased by CM. Tubular cell damage and accompanying oxidative stress in our model are consequences of CM-induced direct cell damage, which also modifies the tubulovascular interaction at the macula densa, and may therefore contribute to disturbances of renal perfusion and filtration.

(+)-fenchol and (-)-isopinocampheol derivatives targeting the entry process of filoviruses.

The increasing frequency of filovirus outbreaks in African countries has led to a pressing need for the development of effective antifilovirus agents. In continuation of our previous research on the antifilovirus activity of monoterpenoid derivatives, we synthesized a series of (+)-fenchol and (-)-isopinocampheol derivatives by varying the type of heterocycle and linker length. Derivatives with an N-alkylpiperazine cycle proved to be the most potent antiviral compounds, with half-maximal inhibitory concentration (IC50) 1.4-20 µÐœ against Lenti-EboV-GP infection and 11.3-47 µÐœ against Lenti-MarV-GP infection. Mechanism-of-action experiments revealed that the compounds may exert their action by binding to surface glycoproteins (GPs). It was demonstrated that the binding of the synthesized compounds to the Marburg virus GP is less efficient as compared to the Ebola virus GP. Furthermore, it was shown that the compounds possess lysosomotropic properties. Thus, the antiviral activity may be due to dual effects. This study offers new antiviral agents that are worthy of further exploration.

The Methyltransferases METTL7A and METTL7B Confer Resistance to Thiol-Based Histone Deacetylase Inhibitors.

Histone deacetylase inhibitors (HDACi) are part of a growing class of epigenetic therapies used for the treatment of cancer. Although HDACis are effective in the treatment of T-cell lymphomas, treatment of solid tumors with this class of drugs has not been successful. Overexpression of the multidrug resistance protein P-glycoprotein (P-gp), encoded by ABCB1, is known to confer resistance to the HDACi romidepsin in vitro, yet increased ABCB1 expression has not been associated with resistance in patients, suggesting that other mechanisms of resistance arise in the clinic. To identify alternative mechanisms of resistance to romidepsin, we selected MCF-7 breast cancer cells with romidepsin in the presence of the P-gp inhibitor verapamil to reduce the likelihood of P-gp-mediated resistance. The resulting cell line, MCF-7 DpVp300, does not express P-gp and was found to be selectively resistant to romidepsin but not to other HDACis such as belinostat, panobinostat, or vorinostat. RNA-sequencing analysis revealed upregulation of the mRNA coding for the putative methyltransferase, METTL7A, whose paralog, METTL7B, was previously shown to methylate thiol groups on hydrogen sulfide and captopril. As romidepsin has a thiol as the zinc-binding moiety, we hypothesized that METTL7A could inactivate romidepsin and other thiol-based HDACis via methylation of the thiol group. We demonstrate that expression of METTL7A or METTL7B confers resistance to thiol-based HDACis and that both enzymes are capable of methylating thiol-containing HDACis. We thus propose that METTL7A and METTL7B confer resistance to thiol-based HDACis by methylating and inactivating the zinc-binding thiol.

Histone N-tails modulate sequence-specific positioning of nucleosomes.

The precise mechanisms governing sequence-dependent positioning of nucleosomes on DNA remain unknown in detail. Existing algorithms, taking into account the sequence-dependent deformability of DNA and its interactions with the histone globular domains, predict rotational setting of only 65% of human nucleosomes mapped in vivo. To uncover novel factors responsible for the nucleosome positioning, we analyzed potential involvement of the histone N-tails in this process. To this aim, we reconstituted the H2A/H4 N-tailless nucleosomes on human BRCA1 DNA (~100 kb) and compared their positions and sequences with those of the wild-type nucleosomes. In the case of H2A tailless nucleosomes, the AT content of DNA sequences is changed locally at superhelical location (SHL) ±4, while maintaining the same rotational setting as their wild-type counterparts. Conversely, the H4 tailless nucleosomes display widespread changes of the AT content near SHL ±1 and SHL ±2, where the H4 N-tails interact with DNA. Furthermore, a substantial number of H4 tailless nucleosomes exhibit rotational setting opposite to that of the wild-type nucleosomes. Thus, our findings strongly suggest that the histone N-tails are operative in selection of nucleosome positions, which may have wide-ranging implications for epigenetic modulation of chromatin states.

Ultrastructural Abnormalities in Induced Pluripotent Stem Cell-Derived Neural Stem Cells and Neurons of Two Cohen Syndrome Patients.

Cohen syndrome is an autosomal recessive disorder caused by VPS13B (COH1) gene mutations. This syndrome is significantly underdiagnosed and is characterized by intellectual disability, microcephaly, autistic symptoms, hypotension, myopia, retinal dystrophy, neutropenia, and obesity. VPS13B regulates intracellular membrane transport and supports the Golgi apparatus structure, which is critical for neuron formation. We generated induced pluripotent stem cells from two patients with pronounced manifestations of Cohen syndrome and differentiated them into neural stem cells and neurons. Using transmission electron microscopy, we documented multiple new ultrastructural changes associated with Cohen syndrome in the neuronal cells. We discovered considerable disturbances in the structure of some organelles: Golgi apparatus fragmentation and swelling, endoplasmic reticulum structural reorganization, mitochondrial defects, and the accumulation of large autophagosomes with undigested contents. These abnormalities underline the ultrastructural similarity of Cohen syndrome to many neurodegenerative diseases. The cell models that we developed based on patient-specific induced pluripotent stem cells can serve to uncover not only neurodegenerative processes, but the causes of intellectual disability in general.

Stem Cell-Based Trophoblast Models to Unravel the Genetic Causes of Human Miscarriages.

Miscarriage affects approximately 15% of clinically recognized pregnancies, and 1-3% of couples experience pregnancy loss recurrently. Approximately 50-60% of miscarriages result from chromosomal abnormalities, whereas up to 60% of euploid recurrent abortions harbor variants in candidate genes. The growing number of detected genetic variants requires an investigation into their role in adverse pregnancy outcomes. Since placental defects are the main cause of first-trimester miscarriages, the purpose of this review is to provide a survey of state-of-the-art human in vitro trophoblast models that can be used for the functional assessment of specific abnormalities/variants implicated in pregnancy loss. Since 2018, when primary human trophoblast stem cells were first derived, there has been rapid growth in models of trophoblast lineage. It has been found that a proper balance between self-renewal and differentiation in trophoblast progenitors is crucial for the maintenance of pregnancy. Different responses to aneuploidy have been shown in human embryonic and extra-embryonic lineages. Stem cell-based models provide a powerful tool to explore the effect of a specific aneuploidy/variant on the fetus through placental development, which is important, from a clinical point of view, for deciding on the suitability of embryos for transfer after preimplantation genetic testing for aneuploidy.

Identification of differentially methylated genes in first-trimester placentas with trisomy 16.

The presence of an extra chromosome in the embryo karyotype often dramatically affects the fate of pregnancy. Trisomy 16 is the most common aneuploidy in first-trimester miscarriages. The present study identified changes in DNA methylation in chorionic villi of miscarriages with trisomy 16. Ninety-seven differentially methylated sites in 91 genes were identified (false discovery rate (FDR) < 0.05 and Δß > 0.15) using DNA methylation arrays. Most of the differentially methylated genes encoded secreted proteins, signaling peptides, and receptors with disulfide bonds. Subsequent analysis using targeted bisulfite massive parallel sequencing showed hypermethylation of the promoters of specific genes in miscarriages with trisomy 16 but not miscarriages with other aneuploidies. Some of the genes were responsible for the development of the placenta and embryo (GATA3-AS1, TRPV6, SCL13A4, and CALCB) and the formation of the mitotic spindle (ANKRD53). Hypermethylation of GATA3-AS1 was associated with reduced expression of GATA3 protein in chorionic villi of miscarriages with trisomy 16. Aberrant hypermethylation of genes may lead to a decrease in expression, impaired trophoblast differentiation and invasion, mitotic disorders, chromosomal mosaicism and karyotype self-correction via trisomy rescue mechanisms.

Uveitis Is a Risk Factor for Juvenile Idiopathic Arthritis' Significant Flare in Patients Treated With Biologics.

Objectives: Uveitis is the most frequent extra-articular manifestation of juvenile idiopathic arthritis (JIA). Our study is aimed to evaluate the possible difference in arthritis course depending on uveitis presence in patients with JIA, treated with biologics. Methods: From our database of patients with JIA treated with biologics, we extracted patients to whom the first agent was administrated with or without MTX. The exclusion criteria included treatment with current systemic corticosteroids, infliximab, rituximab, observation period <3 years, and no missing data. After selection, 175 patients were eligible for analysis. We evaluated clinically significant flare with joint involvement (which required change of biologic or non-biologic DMARD) and time to flare. We compared two groups: (i) patients with uveitis (n = 32) and (ii) patients without uveitis (n = 143). For statistical analysis, we used Cox's regression models, the log-Rank test, x 2 test, and the Mann-Whitney test. Results: There was no difference in gender distribution and achievement of arthritis remission between groups. Patients in the non-uveitis group predominantly received etanercept (64.3%). In the uveitis group, the most prescribed biologic agent was adalimumab (71.9%). The presence of uveitis increased the risk of JIA flare, OR = 3.8 (95% CI: 1.7; 8.7), and the cumulative probability of joint flare, RR = 4.5 (95% CI: 1.7; 12.1), p =.003, after adjustment on methotrexate, RR = 3.1 (1.6; 6.), p =.0008. In the subgroup of patients treated with adalimumab, the absence of methotrexate increased the cumulative probability of flare [RR = 6.5 (95% CI: 1.4; 31.1), p = 0.02]. Conclusion: The presence of uveitis proved to be a risk factor in JIA flare. Methotrexate can decrease the cumulative flare probability. Further trials are required.

Impact of malnutrition on survival in adult patients after elective cardiac surgery: Long-term follow up data.

The data article refers to the paper titles "Impact of malnutrition on long-term survival in adult patients after elective cardiac surgery" [1]. The data refer to the analysis of the relationship between baseline malnutrition and long-term mortality after cardiac surgery. Baseline demographic, nutritional, and medical history data were collected for each enrolled patient. Baseline serum albumin and C-reactive (CRP) protein levels were also obtained. Surgical risk was assessed in accordance with the logistic EuroSCORE. Intraoperative data including cardiopulmonary bypass (CPB) time and postoperative characteristics, such as postoperative complications, number of days in the ICU, and hospitalization duration, were also collected. Data on nutritional status were collected using four nutritional screening tools: (1) malnutrition universal screening tool (MUST), (2) short nutritional assessment questionnaire (SNAQ), (3) mini-nutritional assessment (MNA), and (4) nutritional risk screening 2002 (NRS-2002). Both electronic medical records and phone interviews were used for survival data collection. ROC analysis was performed to analyze prognostic value of baseline and perioperative variables on long-term mortality. Univariate and multivariate logistic regression analysis of predictors of 3- and 8-year mortality were performed. Kaplan-Meyer curves, describing the impact of baseline and perioperative characteristics on 3- and 8-year survival were also performed.

Effects of malnutrition on long-term survival in adult patients after elective cardiac surgery.

OBJECTIVES: The aim of this study was to investigate the relationship between malnutrition and long-term survival in patients who underwent cardiopulmonary bypass (CPB). METHODS: This study analyzed the long-term survival data of a mixed cohort of 1187 cardiac patients previously enrolled in a prospective observational study of nutritional screening in cardiac surgery. Nutritional status was assessed using the Malnutrition Universal Screening Tool (MUST). The mean age of patients was 58.86 ± 10.07 y (95% confidence interval [CI], 58.2-59.4). The median time of follow-up was 73.4 mo (25th-75th percentiles, 18.3-101.3). RESULTS: In all, 449 patients (37.8%) were lost to follow-up after hospitalization. For the remaining participants, the overall 8-y survival was 68% (95% CI, 59-76) and 77% (95% CI, 73-80; log-rank, P = 0.12) in patients with and without malnutrition risk, respectively. Statistically significant differences in survival were found during the 3-y follow-up of patients with heart valve disease: 83% (95% CI, 74-92) with malnutrition versus 93% (95% CI, 90-96) without malnutrition (log-rank, P = 0.03). The final multivariate Cox regression model revealed logistic EuroSCORE (hazard ratio (HR), 1.337; 95% CI, 1.110-1.612), cardiopulmonary bypass time <110.5 min (HR 0.463, 95% CI 0.255-0.842), preoperative albumin (HR 0.799, 95% CI 0.691-0.924), and C-reactive protein (HR, 1.106; 95% CI, 1.018-1.202) as independent predictors of 3-y survival. CONCLUSION: Preoperative malnutrition is not associated with 8-y mortality in a mixed cardiac surgery cohort. However, it may be associated with worse 3-y outcomes in patients with heart valve disease.

Unique physical properties and interactions of the domains of methylated DNA binding protein 2.

Methylated DNA binding protein 2 (MeCP2) is a methyl CpG binding protein whose key role is the recognition of epigenetic information encoded in DNA methylation patterns. Mutation or misregulation of MeCP2 function leads to Rett syndrome as well as a variety of other autism spectrum disorders. Here, we have analyzed in detail the properties of six individually expressed human MeCP2 domains spanning the entire protein with emphasis on their interactions with each other, with DNA, and with nucleosomal arrays. Each domain contributes uniquely to the structure and function of the full-length protein. MeCP2 is approximately 60% unstructured, with nine interspersed alpha-molecular recognition features (alpha-MoRFs), which are polypeptide segments predicted to acquire secondary structure upon forming complexes with binding partners. Large increases in secondary structure content are induced in some of the isolated MeCP2 domains and in the full-length protein by binding to DNA. Interactions between some MeCP2 domains in cis and trans seen in our assays likely contribute to the structure and function of the intact protein. We also show that MeCP2 has two functional halves. The N-terminal portion contains the methylated DNA binding domain (MBD) and two highly disordered flanking domains that modulate MBD-mediated DNA binding. One of these flanking domains is also capable of autonomous DNA binding. In contrast, the C-terminal portion of the protein that harbors at least two independent DNA binding domains and a chromatin-specific binding domain is largely responsible for mediating nucleosomal array compaction and oligomerization. These findings led to new mechanistic and biochemical insights regarding the conformational modulations of this intrinsically disordered protein, and its context-dependent in vivo roles.

Multiple modes of interaction between the methylated DNA binding protein MeCP2 and chromatin.

Mutations of the methylated DNA binding protein MeCP2, a multifunctional protein that is thought to transmit epigenetic information encoded as methylated CpG dinucleotides to the transcriptional machinery, give rise to the debilitating neurodevelopmental disease Rett syndrome (RTT). In this in vitro study, the methylation-dependent and -independent interactions of wild-type and mutant human MeCP2 with defined DNA and chromatin substrates were investigated. A combination of electrophoretic mobility shift assays and visualization by electron microscopy made it possible to understand the different conformational changes underlying the gel shifts. MeCP2 is shown to have, in addition to its well-established methylated DNA binding domain, a methylation-independent DNA binding site (or sites) in the first 294 residues, while the C-terminal portion of MeCP2 (residues 295 to 486) contains one or more essential chromatin interaction regions. All of the RTT-inducing mutants tested were quantitatively bound to chromatin under our conditions, but those that tend to be associated with the more severe RTT symptoms failed to induce the extensive compaction observed with wild-type MeCP2. Two modes of MeCP2-driven compaction were observed, one promoting nucleosome clustering and the other forming DNA-MeCP2-DNA complexes. MeCP2 binding to DNA and chromatin involves a number of different molecular interactions, some of which result in compaction and oligomerization. The multifunctional roles of MeCP2 may be reflected in these different interactions.

Closed chromatin loops at the ends of chromosomes.

The termini of eukaryotic chromosomes contain specialized protective structures, the telomeres, composed of TTAGGG repeats and associated proteins which, together with telomerase, control telomere length. Telomere shortening is associated with senescence and inappropriate telomerase activity may lead to cancer. Little is known about the chromatin context of telomeres, because, in most cells, telomere chromatin is tightly anchored within the nucleus. We now report the successful release of telomere chromatin from chicken erythrocyte and mouse lymphocyte nuclei, both of which have a reduced karyoskeleton. Electron microscopy reveals telomere chromatin fibers in the form of closed terminal loops, which correspond to the "t-loop" structures adopted by telomere DNA. The ability to recognize isolated telomeres in their native chromatin conformation opens the way for detailed structural and compositional studies.