Mechanical Interlocking to Unlock the Reinforcing Potential of Carbon Nanotubes.

Single-walled carbon nanotubes (SWNTs) present extraordinary mechanical properties, with Youngs' modulus>1 TPa and tensile strength>50 GPa; this makes them ideal candidates as fillers for the reinforcement of polymers. However, the performance of SWNTs in this field has fallen behind expectations. This is due to a combination of imperfect individualization of the SWNTs and poor load transfer from the polymer to the SWNTs. Here, we study the reinforcement of polymers of different chemical nature using mechanically interlocked derivatives of single-walled carbon nanotubes (MINTs). We compare the mechanical properties of fibers made of poly (methyl methacrylate) (PMMA) and polysulfone (PSU) and their composites made with pristine SWNTs, MINTs, and the corresponding supramolecular models. With very low loading of MINTs (0.01 % w/w), improvements of more than 100 % on Youngs Modulus and the tensile strength are observed for both the nonpolar aliphatic PMMA and the very polar aromatic PSU polymers, while pristine carbon nanotubes and the supramolecular nanofillers showed smaller reinforcement. These data, together with our previous report on the reinforcement of polystyrene (nonpolar and aromatic), indicate that derivatization of SWNTs as MINTs is a valid general strategy to optimize the interaction between SWNT fillers and the polymer matrix.

BRCA1 recruitment to transcriptional pause sites is required for R-loop-driven DNA damage repair.

The mechanisms contributing to transcription-associated genomic instability are both complex and incompletely understood. Although R-loops are normal transcriptional intermediates, they are also associated with genomic instability. Here, we show that BRCA1 is recruited to R-loops that form normally over a subset of transcription termination regions. There it mediates the recruitment of a specific, physiological binding partner, senataxin (SETX). Disruption of this complex led to R-loop-driven DNA damage at those loci as reflected by adjacent γ-H2AX accumulation and ssDNA breaks within the untranscribed strand of relevant R-loop structures. Genome-wide analysis revealed widespread BRCA1 binding enrichment at R-loop-rich termination regions (TRs) of actively transcribed genes. Strikingly, within some of these genes in BRCA1 null breast tumors, there are specific insertion/deletion mutations located close to R-loop-mediated BRCA1 binding sites within TRs. Thus, BRCA1/SETX complexes support a DNA repair mechanism that addresses R-loop-based DNA damage at transcriptional pause sites.

Integrative analysis of 111 reference human epigenomes.

The reference human genome sequence set the stage for studies of genetic variation and its association with human disease, but epigenomic studies lack a similar reference. To address this need, the NIH Roadmap Epigenomics Consortium generated the largest collection so far of human epigenomes for primary cells and tissues. Here we describe the integrative analysis of 111 reference human epigenomes generated as part of the programme, profiled for histone modification patterns, DNA accessibility, DNA methylation and RNA expression. We establish global maps of regulatory elements, define regulatory modules of coordinated activity, and their likely activators and repressors. We show that disease- and trait-associated genetic variants are enriched in tissue-specific epigenomic marks, revealing biologically relevant cell types for diverse human traits, and providing a resource for interpreting the molecular basis of human disease. Our results demonstrate the central role of epigenomic information for understanding gene regulation, cellular differentiation and human disease.

Conserved nucleosome positioning defines replication origins.

The origin recognition complex (ORC) specifies replication origin location. The Saccharomyces cerevisiae ORC recognizes the ARS (autonomously replicating sequence) consensus sequence (ACS), but only a subset of potential genomic sites are bound, suggesting other chromosomal features influence ORC binding. Using high-throughput sequencing to map ORC binding and nucleosome positioning, we show that yeast origins are characterized by an asymmetric pattern of positioned nucleosomes flanking the ACS. The origin sequences are sufficient to maintain a nucleosome-free origin; however, ORC is required for the precise positioning of nucleosomes flanking the origin. These findings identify local nucleosomes as an important determinant for origin selection and function.

The conserved bromo-adjacent homology domain of yeast Orc1 functions in the selection of DNA replication origins within chromatin.

The origin recognition complex (ORC) binds to the specific positions on chromosomes that serve as DNA replication origins. Although ORC is conserved from yeast to humans, the DNA sequence elements that specify ORC binding are not. In particular, metazoan ORC shows no obvious DNA sequence specificity, whereas yeast ORC binds to a specific DNA sequence within all yeast origins. Thus, whereas chromatin must play an important role in metazoan ORC's ability to recognize origins, it is unclear whether chromatin plays a role in yeast ORC's recognition of origins. This study focused on the role of the conserved N-terminal bromo-adjacent homology domain of yeast Orc1 (Orc1BAH). Recent studies indicate that BAH domains are chromatin-binding modules. We show that the Orc1BAH domain was necessary for ORC's stable association with yeast chromosomes, and was physiologically relevant to DNA replication in vivo. This replication role was separable from the Orc1BAH domain's previously defined role in transcriptional silencing. Genome-wide analyses of ORC binding in ORC1 and orc1bahDelta cells revealed that the Orc1BAH domain contributed to ORC's association with most yeast origins, including a class of origins highly dependent on the Orc1BAH domain for ORC association (orc1bahDelta-sensitive origins). Orc1bahDelta-sensitive origins required the Orc1BAH domain for normal activity on chromosomes and plasmids, and were associated with a distinct local nucleosome structure. These data provide molecular insights into how the Orc1BAH domain contributes to ORC's selection of replication origins, as well as new tools for examining conserved mechanisms governing ORC's selection of origins within eukaryotic chromosomes.

A cis-regulatory map of the Drosophila genome.

Systematic annotation of gene regulatory elements is a major challenge in genome science. Direct mapping of chromatin modification marks and transcriptional factor binding sites genome-wide has successfully identified specific subtypes of regulatory elements. In Drosophila several pioneering studies have provided genome-wide identification of Polycomb response elements, chromatin states, transcription factor binding sites, RNA polymerase II regulation and insulator elements; however, comprehensive annotation of the regulatory genome remains a significant challenge. Here we describe results from the modENCODE cis-regulatory annotation project. We produced a map of the Drosophila melanogaster regulatory genome on the basis of more than 300 chromatin immunoprecipitation data sets for eight chromatin features, five histone deacetylases and thirty-eight site-specific transcription factors at different stages of development. Using these data we inferred more than 20,000 candidate regulatory elements and validated a subset of predictions for promoters, enhancers and insulators in vivo. We identified also nearly 2,000 genomic regions of dense transcription factor binding associated with chromatin activity and accessibility. We discovered hundreds of new transcription factor co-binding relationships and defined a transcription factor network with over 800 potential regulatory relationships.

Developmental control of gene copy number by repression of replication initiation and fork progression.

Precise DNA replication is crucial for genome maintenance, yet this process has been inherently difficult to study on a genome-wide level in untransformed differentiated metazoan cells. To determine how metazoan DNA replication can be repressed, we examined regions selectively under-replicated in Drosophila polytene salivary glands, and found they are transcriptionally silent and enriched for the repressive H3K27me3 mark. In the first genome-wide analysis of binding of the origin recognition complex (ORC) in a differentiated metazoan tissue, we find that ORC binding is dramatically reduced within these large domains, suggesting reduced initiation as one mechanism leading to under-replication. Inhibition of replication fork progression by the chromatin protein SUUR is an additional repression mechanism to reduce copy number. Although repressive histone marks are removed when SUUR is mutated and copy number restored, neither transcription nor ORC binding is reinstated. Tethering of the SUUR protein to a specific site is insufficient to block replication, however. These results establish that developmental control of DNA replication, at both the initiation and elongation stages, is a mechanism to change gene copy number during differentiation.

Chromatin signatures of the Drosophila replication program.

DNA replication initiates from thousands of start sites throughout the Drosophila genome and must be coordinated with other ongoing nuclear processes such as transcription to ensure genetic and epigenetic inheritance. Considerable progress has been made toward understanding how chromatin modifications regulate the transcription program; in contrast, we know relatively little about the role of the chromatin landscape in defining how start sites of DNA replication are selected and regulated. Here, we describe the Drosophila replication program in the context of the chromatin and transcription landscape for multiple cell lines using data generated by the modENCODE consortium. We find that while the cell lines exhibit similar replication programs, there are numerous cell line-specific differences that correlate with changes in the chromatin architecture. We identify chromatin features that are associated with replication timing, early origin usage, and ORC binding. Primary sequence, activating chromatin marks, and DNA-binding proteins (including chromatin remodelers) contribute in an additive manner to specify ORC-binding sites. We also generate accurate and predictive models from the chromatin data to describe origin usage and strength between cell lines. Multiple activating chromatin modifications contribute to the function and relative strength of replication origins, suggesting that the chromatin environment does not regulate origins of replication as a simple binary switch, but rather acts as a tunable rheostat to regulate replication initiation events.

Automated statistical analysis of raman spectra of nanomaterials.

Both at the academic and the industrial level, material scientists are exploring routes for mass production and functionalization of graphene, carbon nanotubes (CNT), carbon dots, 2D materials, and heterostructures of these. Proper application of the novel materials requires fast and thorough characterization of the samples. Raman spectroscopy stands out as a standard non-invasive technique capable of giving key information on the structure and electronic properties of nanomaterials, including the presence of defects, degree of functionalization, diameter (in the case of CNT), different polytypes, doping, etc. Here, we present a computational tool to automatically analyze the Raman spectral features of nanomaterials, which we illustrate with the example of CNT and graphene. The algorithm manages hundreds of spectra simultaneously and provides statistical information (distribution of Raman shifts, average values of shifts and relative intensities, standard deviations, correlation between different peaks, etc.) of the main spectral features defining the structure and electronic properties of the samples, as well as publication-ready graphical material.

Targeting RARA overexpression with tamibarotene, a potent and selective RARα agonist, is a novel approach in AML.

A superenhancer at the retinoic acid receptor alpha (RARA) gene is associated with RARA mRNA overexpression in ∼30% of non-acute promyelocytic leukemia acute myeloid leukemia (AML) and in ∼50% of myelodysplastic syndromes (MDS). RARA overexpression is an actionable target for treatment with tamibarotene, an oral potent and selective RARα agonist. Sensitivity to the RARα agonist tamibarotene was demonstrated in RARA-high but not RARA-low preclinical AML models. The combination of oral tamibarotene plus azacitidine was evaluated in a phase 2 clinical study in 51 newly diagnosed unfit patients with AML identified as RARA-positive (n = 22) or RARA-negative (n = 29) for RARA mRNA overexpression in peripheral blasts using a blood-based biomarker test. In 18 response-evaluable RARA-positive patients, complete remission (CR)/CR with incomplete hematologic recovery rate was 61%, CR rate was 50%, and time to initial composite CR was rapid at 1.2 months. Transfusion independence was attained by 72% of RARA-positive patients. In contrast, 28 response-evaluable RARA-negative patients had response rates that were consistent with azacitidine monotherapy. Tamibarotene in combination with azacitidine was well tolerated. The majority of nonhematologic adverse events were low grade and hematologic adverse events were comparable to single-agent azacitidine, demonstrating that there was no additional myelosuppression when tamibarotene was combined with azacitidine. These results support further evaluation of tamibarotene-based treatment strategies in patients with AML or MDS with RARA overexpression to provide a targeted approach with the goal of improving patient outcomes. This trial was registered at www.clinicaltrials.gov as #NCT02807558.

ΔNp63/p73 drive metastatic colonization by controlling a regenerative epithelial stem cell program in quasi-mesenchymal cancer stem cells.

Cancer stem cells (CSCs) may serve as the cellular seeds of tumor recurrence and metastasis, and they can be generated via epithelial-mesenchymal transitions (EMTs). Isolating pure populations of CSCs is difficult because EMT programs generate multiple alternative cell states, and phenotypic plasticity permits frequent interconversions between these states. Here, we used cell-surface expression of integrin ß4 (ITGB4) to isolate highly enriched populations of human breast CSCs, and we identified the gene regulatory network operating in ITGB4+ CSCs. Specifically, we identified ΔNp63 and p73, the latter of which transactivates ΔNp63, as centrally important transcriptional regulators of quasi-mesenchymal CSCs that reside in an intermediate EMT state. We found that the transcriptional program controlled by ΔNp63 in CSCs is largely distinct from the one that it orchestrates in normal basal mammary stem cells and, instead, it more closely resembles a regenerative epithelial stem cell response to wounding. Moreover, quasi-mesenchymal CSCs repurpose this program to drive metastatic colonization via autocrine EGFR signaling.

Functionalized epoxy with adjustable fluorescence and UV-shielding enabled by reactive addition of 9-anthracenemethoxyl glycidyl ether.

Functionalized epoxies show unique advantages in some special applications. Herein, a fluorescent and UV-shielding dual-functional epoxy resin, epoxy-EAn, is prepared via synthesizing and incorporating a reactive additive, 9-anthracenemethoxyl glycidyl ether (EAn), into the resin system. The results show that EAn performs an outstanding migration resistance. The addition of a small amount of EAn (0.1%) enables epoxy-EAn to emit bright blue-violet fluorescence under UV light, making epoxy-EAn a feasible fluorescent adhesive for reconstruction and repair. The fluorescence emission intensity is adjustable by the treatment with UV light and heat. Correspondingly, the epoxy-EAn can serve as an information storage material to realize information input and decryption. In addition, epoxy-EAn also possesses the ability to shield UV radiation, and transmittance in the near ultraviolet and middle ultraviolet region (200-400 nm) can decrease to zero when the EAn concentration is 0.8%. Meanwhile, epoxy-EAn also shows excellent mechanical properties similar to that of the pure epoxy. This work paves a facile way to fabricate a multi-functional epoxy suitable for various occasions.

Exploring the effect of humidity on thermoplastic starch films using the quartz crystal microbalance.

The quartz crystal microbalance (QCM) is used as a non-destructive and efficient characterization tool for thin thermoplastic starch (TPS) films. Thin TPS films (1-2 µm) were prepared with 30% (w/w starch) plasticizers using either glycerol or an ionic liquid, 1-ethyl-3-methylimidiazolium acetate ([emim+][Ac-]), as the plasticizer. The differences in the mechanical properties and environmental effects on the plasticized TPS films were explored. The modulus of starch-glycerol films was higher than starch-[emim+][Ac-], consistent with literature data and bulk AFM measurements, likely due to superior plasticization by the ionic liquid. The starch-[emim+][Ac-] films were shown to have relative stable properties at low humidity that may be due to some antiplasticization effects at low water content despite absorbing more water than starch-glycerol films at higher humidity.

The use of oxygen hoods in patients failing on conventional high-flow oxygen delivery systems, the effects on oxygenation, mechanical ventilation and mortality rates in hypoxic patients with COVID-19. A Prospective Controlled Cohort Study.

INTRODUCTION: Efforts to meet increased oxygen demands in COVID-19 patients are a priority in averting mechanical ventilation (MV), associated with high mortality approaching 76.4-97.2%. Novel methods of oxygen delivery could mitigate that risk. Oxygen hoods/helmets may improve: O2-saturation (SaO2), reduce in-hospital mechanical ventilation and mortality rates, and reduce length of hospitalization in hypoxic Covid-19 patients failing on conventional high-flow oxygen delivery systems. METHODS: DesignProspective Controlled Cohort Study. SettingSingle Center. ParticipantsAll patients admitted with a diagnosis of COVID-19 were reviewed and 136/347 patients met inclusion criteria. Study period3/6/2020 to 5/1/2020. 136 participants completed the study with known status for all outcome measures. Intervention or exposureOxygen hoods/helmets as compared to conventional high-flow oxygen delivery systems. MAIN OUTCOME(S) AND MEASURE(S): 1) Pre and post change in oxygen saturation (SaO2). 2) In-hospital Mechanical Ventilation (MV). 3) In-hospital Mortality. 4) Length of hospitalization. RESULTS: 136 patients including 58-intervention and 78-control patients were studied. Age, gender, and other demographics/prognostic indicators were comparable between cohorts. Oxygen hoods averted imminent or immediate intubation/MV in all 58 COVID-19 patients failing on conventional high-flow oxygen delivery systems with a mean improvement in SaO2 of 8.8%, p < 0.001. MV rates were observed to be higher in the control 37/78 (47.4%) as compared to the intervention cohort 23/58 (39.7%), a difference of 7.7%, a 27% risk reduction, not statistically significant, OR 95%CI 0.73 (0.37-1.5). Mortality rates were observed higher in the control 54/78 (69.2%) as compared to the intervention cohort 36/58 (62.1%), a difference of 7.1%, a 27% risk reduction, not statistically significant OR 95%CI 0.73 (0.36-1.5). CONCLUSION: Oxygen hoods demonstrate improvement in SaO2 for patients failing on conventional high-flow oxygen-delivery systems and prevented imminent mechanical ventilation. In-hospital mechanical ventilation and mortality rates were reduced with the use of oxygen hoods but not found to be statistically significant. The oxygen hood is a safe, effective oxygen-delivery system which may reduce intubation/MV and mortality rates. Their use should be considered in treating hypoxic COVID-19 patients. Further research is warranted. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04407260.

Correction to: DNA copy number evolution in Drosophila cell lines.

Following publication of the original article [1], the authors reported the following errors.

Obstructive Sleep Apnea in a Patient with Cornelia de Lange Syndrome.

Cornelia de Lange syndrome (CdLS) is a rare genetic disorder that is characterized by specific facial, skeletal, and behavioral features associated with variable degrees of intellectual disabilities. Sleep disturbances have been reported in patients with CdLS including insomnia, sleep-disordered breathing, intrinsic sleep disorders, and circadian rhythm disorders. The prevalence of sleep-related breathing disorders, in particular, obstructive sleep apnea (OSA), was conducted using validated questionnaires. We report the first case of CdLS that confirmed a moderate to severe degree of OSA using overnight polysomnography, which is the gold standard diagnostic test for OSA.

Superenhancer Analysis Defines Novel Epigenomic Subtypes of Non-APL AML, Including an RARα Dependency Targetable by SY-1425, a Potent and Selective RARα Agonist.

We characterized the enhancer landscape of 66 patients with acute myeloid leukemia (AML), identifying 6 novel subgroups and their associated regulatory loci. These subgroups are defined by their superenhancer (SE) maps, orthogonal to somatic mutations, and are associated with distinct leukemic cell states. Examination of transcriptional drivers for these epigenomic subtypes uncovers a subset of patients with a particularly strong SE at the retinoic acid receptor alpha (RARA) gene locus. The presence of a RARA SE and concomitant high levels of RARA mRNA predisposes cell lines and ex vivo models to exquisite sensitivity to a selective agonist of RARα, SY-1425 (tamibarotene). Furthermore, only AML patient-derived xenograft (PDX) models with high RARA mRNA were found to respond to SY-1425. Mechanistically, we show that the response to SY-1425 in RARA-high AML cells is similar to that of acute promyelocytic leukemia treated with retinoids, characterized by the induction of known retinoic acid response genes, increased differentiation, and loss of proliferation.Significance: We use the SE landscape of primary human AML to elucidate transcriptional circuitry and identify novel cancer vulnerabilities. A subset of patients were found to have an SE at RARA, which is predictive for response to SY-1425, a potent and selective RARα agonist, in preclinical models, forming the rationale for its clinical investigation in biomarker-selected patients. Cancer Discov; 7(10); 1136-53. ©2017 AACR.See related commentary by Wang and Aifantis, p. 1065.This article is highlighted in the In This Issue feature, p. 1047.

Noncoding Transcription Is a Driving Force for Nucleosome Instability in spt16 Mutant Cells.

FACT (facilitates chromatin transcription) consists of two essential subunits, Spt16 and Pob3, and functions as a histone chaperone. Mutation of spt16 results in a global loss of nucleosomes as well as aberrant transcription. Here, we show that the majority of nucleosome changes upon Spt16 depletion are alterations in nucleosome fuzziness and position shift. Most nucleosomal changes are suppressed by the inhibition of RNA polymerase II (Pol II) activity. Surprisingly, a small subgroup of nucleosome changes is resistant to transcriptional inhibition. Notably, Spt16 and distinct histone modifications are enriched at this subgroup of nucleosomes. We also report 1,037 Spt16-suppressed noncoding transcripts (SNTs) and found that the SNT start sites are enriched with the subgroup of nucleosomes resistant to Pol II inhibition. Finally, the nucleosomes at genes overlapping SNTs are more susceptible to changes upon Spt16 depletion than those without SNTs. Taken together, our results support a model in which Spt16 has a role in maintaining local nucleosome stability to inhibit initiation of SNT transcription, which once initiated drives additional nucleosome loss upon Spt16 depletion.

Alzheimer's loci: epigenetic associations and interaction with genetic factors.

OBJECTIVE: We explore the role of DNA methylation in Alzheimer's disease (AD). To elucidate where DNA methylation falls along the causal pathway linking risk factors to disease, we examine causal models to assess its role in the pathology of AD. METHODS: DNA methylation profiles were generated in 740 brain samples using the Illumina HumanMet450K beadset. We focused our analysis on CpG sites from 11 AD susceptibility gene regions. The primary outcome was a quantitative measure of neuritic amyloid plaque (NP), a key early element of AD pathology. We tested four causal models: (1) independent associations, (2) CpG mediating the association of a variant, (3) reverse causality, and (4) genetic variant by CpG interaction. RESULTS: Six genes regions (17 CpGs) showed evidence of CpG associations with NP, independent of genetic variation - BIN1 (5), CLU (5), MS4A6A (3), ABCA7 (2), CD2AP (1), and APOE (1). Together they explained 16.8% of the variability in NP. An interaction effect was seen in the CR1 region for two CpGs, cg10021878 (P = 0.01) and cg05922028 (P = 0.001), in relation to NP. In both cases, subjects with the risk allele rs6656401(AT) (/) (AA) display more methylation being associated with more NP burden, whereas subjects with the rs6656401(TT) protective genotype have an inverse association with more methylation being associated with less NP. INTERPRETATION: These observations suggest that, within known AD susceptibility loci, methylation is related to pathologic processes of AD and may play a largely independent role by influencing gene expression in AD susceptibility loci.

DNA copy number evolution in Drosophila cell lines.

BACKGROUND: Structural rearrangements of the genome resulting in genic imbalance due to copy number change are often deleterious at the organismal level, but are common in immortalized cell lines and tumors, where they may be an advantage to cells. In order to explore the biological consequences of copy number changes in the Drosophila genome, we resequenced the genomes of 19 tissue-culture cell lines and generated RNA-Seq profiles. RESULTS: Our work revealed dramatic duplications and deletions in all cell lines. We found three lines of evidence indicating that copy number changes were due to selection during tissue culture. First, we found that copy numbers correlated to maintain stoichiometric balance in protein complexes and biochemical pathways, consistent with the gene balance hypothesis. Second, while most copy number changes were cell line-specific, we identified some copy number changes shared by many of the independent cell lines. These included dramatic recurrence of increased copy number of the PDGF/VEGF receptor, which is also over-expressed in many cancer cells, and of bantam, an anti-apoptosis miRNA. Third, even when copy number changes seemed distinct between lines, there was strong evidence that they supported a common phenotypic outcome. For example, we found that proto-oncogenes were over-represented in one cell line (S2-DRSC), whereas tumor suppressor genes were under-represented in another (Kc167). CONCLUSION: Our study illustrates how genome structure changes may contribute to selection of cell lines in vitro. This has implications for other cell-level natural selection progressions, including tumorigenesis.