Somatic mutations and cell identity linked by Genotyping of Transcriptomes.

Defining the transcriptomic identity of malignant cells is challenging in the absence of surface markers that distinguish cancer clones from one another, or from admixed non-neoplastic cells. To address this challenge, here we developed Genotyping of Transcriptomes (GoT), a method to integrate genotyping with high-throughput droplet-based single-cell RNA sequencing. We apply GoT to profile 38,290 CD34+ cells from patients with CALR-mutated myeloproliferative neoplasms to study how somatic mutations corrupt the complex process of human haematopoiesis. High-resolution mapping of malignant versus normal haematopoietic progenitors revealed an increasing fitness advantage with myeloid differentiation of cells with mutated CALR. We identified the unfolded protein response as a predominant outcome of CALR mutations, with a considerable dependency on cell identity, as well as upregulation of the NF-κB pathway specifically in uncommitted stem cells. We further extended the GoT toolkit to genotype multiple targets and loci that are distant from transcript ends. Together, these findings reveal that the transcriptional output of somatic mutations in myeloproliferative neoplasms is dependent on the native cell identity.

Epigenetic evolution and lineage histories of chronic lymphocytic leukaemia.

Genetic and epigenetic intra-tumoral heterogeneity cooperate to shape the evolutionary course of cancer1. Chronic lymphocytic leukaemia (CLL) is a highly informative model for cancer evolution as it undergoes substantial genetic diversification and evolution after therapy2,3. The CLL epigenome is also an important disease-defining feature4,5, and growing populations of cells in CLL diversify by stochastic changes in DNA methylation known as epimutations6. However, previous studies using bulk sequencing methods to analyse the patterns of DNA methylation were unable to determine whether epimutations affect CLL populations homogeneously. Here, to measure the epimutation rate at single-cell resolution, we applied multiplexed single-cell reduced-representation bisulfite sequencing to B cells from healthy donors and patients with CLL. We observed that the common clonal origin of CLL results in a consistently increased epimutation rate, with low variability in the cell-to-cell epimutation rate. By contrast, variable epimutation rates across healthy B cells reflect diverse evolutionary ages across the trajectory of B cell differentiation, consistent with epimutations serving as a molecular clock. Heritable epimutation information allowed us to reconstruct lineages at high-resolution with single-cell data, and to apply this directly to patient samples. The CLL lineage tree shape revealed earlier branching and longer branch lengths than in normal B cells, reflecting rapid drift after the initial malignant transformation and a greater proliferative history. Integration of single-cell bisulfite sequencing analysis with single-cell transcriptomes and genotyping confirmed that genetic subclones mapped to distinct clades, as inferred solely on the basis of epimutation information. Finally, to examine potential lineage biases during therapy, we profiled serial samples during ibrutinib-associated lymphocytosis, and identified clades of cells that were preferentially expelled from the lymph node after treatment, marked by distinct transcriptional profiles. The single-cell integration of genetic, epigenetic and transcriptional information thus charts the lineage history of CLL and its evolution with therapy.

SIDR: simultaneous isolation and parallel sequencing of genomic DNA and total RNA from single cells.

Simultaneous sequencing of the genome and transcriptome at the single-cell level is a powerful tool for characterizing genomic and transcriptomic variation and revealing correlative relationships. However, it remains technically challenging to analyze both the genome and transcriptome in the same cell. Here, we report a novel method for simultaneous isolation of genomic DNA and total RNA (SIDR) from single cells, achieving high recovery rates with minimal cross-contamination, as is crucial for accurate description and integration of the single-cell genome and transcriptome. For reliable and efficient separation of genomic DNA and total RNA from single cells, the method uses hypotonic lysis to preserve nuclear lamina integrity and subsequently captures the cell lysate using antibody-conjugated magnetic microbeads. Evaluating the performance of this method using real-time PCR demonstrated that it efficiently recovered genomic DNA and total RNA. Thorough data quality assessments showed that DNA and RNA simultaneously fractionated by the SIDR method were suitable for genome and transcriptome sequencing analysis at the single-cell level. The integration of single-cell genome and transcriptome sequencing by SIDR (SIDR-seq) showed that genetic alterations, such as copy-number and single-nucleotide variations, were more accurately captured by single-cell SIDR-seq compared with conventional single-cell RNA-seq, although copy-number variations positively correlated with the corresponding gene expression levels. These results suggest that SIDR-seq is potentially a powerful tool to reveal genetic heterogeneity and phenotypic information inferred from gene expression patterns at the single-cell level.

Evaluation of factors affecting external tibial torsion in patients with cerebral palsy.

BACKGROUND: Gait deviation and associated torsional problems are common in patients with cerebral palsy (CP). Although femoral anteversion in CP has been extensively reviewed in previous studies, only a few studies have focused on tibial torsion. Therefore, this study aimed to evaluate tibial torsion in patients with CP and investigate the affecting factors. METHODS: Consecutive patients with cerebral palsy who underwent 3-dimensional computed tomography for the assessment of rotational profiles were reviewed. Femoral anteversion and tibial torsion were measured, and the demographic characteristics of the patients were recorded. A linear mixed model was implemented to overcome the retrospective nature of the study. RESULTS: After the implementation of inclusion and exclusion criteria, 472 patients were enrolled for this study. With age, external tibial torsion increased, while femoral anteversion decreased. The factors affecting external tibial torsion were increased femoral anteversion (p = 0.0057), increased age (p < 0.0001), higher Gross Motor Function Classification System (GMFCS) level (p < 0.0001), and involved/uninvolved limbs of hemiplegia (p = 0.0471/p = 0.0047). CONCLUSIONS: Older age, GMFCS level IV/V, hemiplegia, and increased femoral anteversion were the independent risk factors of increased external tibial torsion; therefore, performing an imaging study is recommended for assessing the extent of tibial torsion in patients with such characteristics.

Mutual synchronization of two flame-driven thermoacoustic oscillators: Dissipative and time-delayed coupling effects.

Low-emissions can-annular gas turbines are prone to develop low-frequency self-excited thermoacoustic oscillations. Such oscillations arise from the coupling between adjacent combustors and can increase wear and thermal stresses. In this experimental study, we explore the mutual synchronization of two thermoacoustic oscillators (i.e., two model combustors) interacting via dissipative and time-delayed coupling, as introduced via a cross-talk section. Unlike most previous studies, our study makes use of a turbulent lean-premixed flame in each combustor, bringing the system configuration closer to that of practical gas turbines. Using stationary and transient measurements, we examine the effect of the cross-talk diameter and length so as to gain insight into the effect of dissipative and time-delayed coupling. We find that strengthening the dissipative coupling promotes mutual synchronization, but that weakening the dissipative coupling leads to weakly coupled or desynchronized oscillations. On operating the two combustors at different conditions, we find a significant reduction in their overall oscillation amplitude for some coupling conditions. On varying the combustor length and examining the transient response, we find elaborate changes in the pressure-heat-release-rate coupling, spontaneous mode transitions between coupled thermoacoustic modes, and the emergence of a rhomboid structure in the phase plane owing to the coexistence of in-phase and out-of-phase synchronization. In the combustion community, these two types of synchronization are known to be associated with push-push modes and push-pull modes. These findings offer new insight into the mutual synchronization of low-frequency, self-excited thermoacoustic oscillations in can-annular gas turbines, paving the way for the development of improved control strategies.

Chaos, synchronization, and desynchronization in a liquid-fueled diffusion-flame combustor with an intrinsic hydrodynamic mode.

We experimentally investigate the nonlinear dynamics of a thermoacoustically self-excited aero-engine combustion system featuring a turbulent swirling liquid-fueled diffusion flame in a variable-length combustor. We focus on the steady-state dynamics via simultaneous measurements of the acoustic pressure in the combustor and the heat release rate (HRR) from the flame. When the combustor length is increased following the onset of thermoacoustic instability, we find that the pressure signal transitions from a period-1 limit cycle to chaos, whereas the HRR signal remains chaotic owing to the presence of an intrinsic hydrodynamic mode in the flame. When the hydrodynamic mode is filtered out of the data, we find that the pressure and HRR signals are in generalized synchronization. However, when the hydrodynamic mode is retained in the data, we find that the pressure and HRR signals are either weakly phase synchronized or desynchronized. This study has two main contributions: (i) it shows that a liquid-fueled diffusion-flame combustor can exhibit dynamics as complex as those of its gaseous-fueled premixed-flame counterparts and (ii) it highlights the need to be exceptionally careful when selecting a diagnostic signal from which to calculate nonlinear measures of self-excited thermoacoustic oscillations, because our experiments show that the pressure and HRR signals can be desynchronized by the presence of a hydrodynamic mode in the flame at a frequency different from that of the dominant thermoacoustic mode.

Clinical Application of Targeted Deep Sequencing in Solid-Cancer Patients and Utility for Biomarker-Selected Clinical Trials.

Molecular profiling of actionable mutations in refractory cancer patients has the potential to enable "precision medicine," wherein individualized therapies are guided based on genomic profiling. The molecular-screening program was intended to route participants to different candidate drugs in trials based on clinical-sequencing reports. In this screening program, we used a custom target-enrichment panel consisting of cancer-related genes to interrogate single-nucleotide variants, insertions and deletions, copy number variants, and a subset of gene fusions. From August 2014 through April 2015, 654 patients consented to participate in the program at Samsung Medical Center. Of these patients, 588 passed the quality control process for the 381-gene cancer-panel test, and 418 patients were included in the final analysis as being eligible for any anticancer treatment (127 gastric cancer, 122 colorectal cancer, 62 pancreatic/biliary tract cancer, 67 sarcoma/other cancer, and 40 genitourinary cancer patients). Of the 418 patients, 55 (12%) harbored a biomarker that guided them to a biomarker-selected clinical trial, and 184 (44%) patients harbored at least one genomic alteration that was potentially targetable. This study demonstrated that the panel-based sequencing program resulted in an increased rate of trial enrollment of metastatic cancer patients into biomarker-selected clinical trials. Given the expanding list of biomarker-selected trials, the guidance percentage to matched trials is anticipated to increase. IMPLICATIONS FOR PRACTICE: This study demonstrated that the panel-based sequencing program resulted in an increased rate of trial enrollment of metastatic cancer patients into biomarker-selected clinical trials. Given the expanding list of biomarker-selected trials, the guidance percentage to matched trials is anticipated to increase.

Deciphering intratumor heterogeneity using cancer genome analysis.

Intratumor heterogeneity within individual cancer tissues underlies the numerous phenotypes of cancer. Tumor subclones ultimately affect therapeutic outcomes due to their distinct molecular features. Drug-resistant subclones are present at a low frequency in tissues at the time of biopsy, but can also arise as a result of acquired somatic mutations. A number of different approaches have been utilized to understand the nature of intratumor heterogeneity. Clonal analysis using whole exome or genome sequencing data can help monitor subclones in the context of tumor progression. Multiregional biopsies permit the molecular characterization of subclones within tumors. Deep sequencing has also provided researchers with the ability to measure the low allele fraction variant within a small number of cells. Ultimately, single-cell sequencing will enable the identification of every minor population within a tumor microenvironment. In the clinical context, the ability to identify and monitor the subclonal architecture of a tumor is valuable for the development of precise cancer therapeutic methods.

Volume Dependence of Hydrogen Diffusion for Sorption and Desorption Processes in Cylindrical-Shaped Polymers.

In the actual application of gas transport properties under high pressure, the important factors are sample size dependence and permeation efficiency, related to gas sorption. With a modified volumetric analysis technique, we firstly measured the overall diffusion properties and equilibrium times for reaching the saturation of hydrogen content in both hydrogen sorption and desorption processes. The measured parameters of total uptake (C∞), total desorbed content (C0), diffusion coefficient in sorption (Ds), diffusion coefficient in desorption (Dd), sorption equilibrium time (ts) and desorption equilibrium time (td) of hydrogen in two polymers were determined relative to the diameter and thickness of the cylindrical-shaped polymers in the two processes. C∞ and C0 did not demonstrate an appreciable volume dependence for all polymers. The identical values of C∞ and C0 indicate the reversibility between sorption and desorption, which is interpreted by the occurrence of physisorption by sorbed hydrogen molecules. However, the measured diffusivity of the polymers was found to be increased with increasing thickness above 5 mm. Moreover, the larger Dd values measured in the desorption process compared to Ds may be attributed to an increased amorphous phase and volume swelling caused by increased hydrogen voids and polymer chain scission after decompression. The ts and td were found to be linearly proportional to the square of the thickness above an aspect ratio of 3.7, which was consistent with the numerical simulations based on the solution of Fick's law. This finding could be used to predict the ts in a polymer without any measurement, depending on the sample size.

Characterizing the Diffusion Property of Hydrogen Sorption and Desorption Processes in Several Spherical-Shaped Polymers.

We developed a method for characterizing permeation parameters in hydrogen sorption and desorption processes in polymers using the volumetric measurement technique. The technique was utilized for three polymers: nitrile butadiene rubber (NBR), ethylene propylene diene monomer (EPDM), and fluoroelastomer (FKM). The total uptake (C∞), total desorbed content (C0), diffusivity in sorption (Ds), and diffusivity in desorption (Dd) of hydrogen in the polymers were determined versus the sample diameter used in both processes. For all the polymers, the diameter dependence was not detected for C∞ and C0. The average C∞ and C0 at 5.75 MPa were 316 wt∙ppm and 291 wt∙ppm for NBR, 270 wt∙ppm and 279 wt∙ppm for EPDM, and 102 wt∙ppm and 93 wt∙ppm for FKM. The coincidence of C∞ and C0 in the sorption and desorption process indicated physisorption upon introducing hydrogen molecules into the polymers. The larger Dd in the desorption process than Ds could be attributed to an increased amorphous phase and volume swelling after decompression. The equilibrium time to reach the saturation of the hydrogen content in both processes was experimentally confirmed as proportional to the squared radius and consistent with the COMSOL simulation. This method could be used to predict the equilibrium time of the sorption time, depending on the radius of the polymers without any measurement.

Comparison of the Radiologic, Morphometric, and Clinical Outcomes between Kinematically and Mechanically Aligned Total Knee Arthroplasty: A Propensity Matching Study.

The purpose of this study was to compare radiologic, morphometric, and clinical outcomes between kinematically aligned (KA) and mechanically aligned (MA) total knee arthroplasty (TKA) in Korean patients. Overall, 168 patients who underwent primary TKA were retrospectively reviewed, and propensity matching (age, sex, and body mass index) was performed as 1:3 ration (KA TKAs [n = 42]: MA TKAs [n = 126]). Joint-line orientation angle (JLOA), coronal and axial alignments of implants, hip-knee-ankle (HKA) angle, and patellar tilt angle were assessed using full-length standing radiograph, axial computed tomography (CT) scan, and plain radiographs. Morphometric assessment was performed by analyzing the intraoperative measurement of the femoral cut surface and femoral components fitting in five zones. Clinical outcomes more than 2 years of follow-up were evaluated with the Knee Society (KS) knee and functional scores, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) scores, and the Short-Form Health Survey (SF-36). In radiologic results, JLOA was more parallel to the floor in KA TKAs (KA: medial tilt 0.9 ± 1.5 degrees; MA: lateral tilt 1.7 ± 1.5 degrees, p < 0.05), and patellar tilt angle was closer to preoperative status after KA TKA (KA: 2.0 ± 1.6 degrees; MA;0.3 ± 1.2 degrees, p < 0.05). HKA angle and rotational mismatch were similar between two groups. In morphometric analysis, entire overhang of anterior femoral cutting surface was reduced in KA TKA compared with MA TKA (KA: 11.7 ± 6.2 mm; MA: 14.4 ± 5.9 mm, p < 0.05). However, both of MA and KA TKAs showed underhang in mediolateral dimension without difference. There were no significant differences in clinical scores between two groups. KA TKA showed more parallel JLOA to floor, closer patellar tilt to preoperative status, and better anterior flange fitting that can reproduce more natural knee kinematics compared with MA TKA. Although clinical outcomes assessed by conventional evaluating tools were similar between two groups, further evaluation focusing on the patellofemoral symptoms or unawareness of TKA is necessary to clarify the clinical benefit of KA TKA.

Phase II study of ceralasertib (AZD6738) in combination with durvalumab in patients with advanced gastric cancer.

BACKGROUND: Targeting the DNA damage repair (DDR) pathways is an attractive strategy for boosting cancer immunotherapy. Ceralasertib (AZD6738) is an oral kinase inhibitor of ataxia telangiectasia and Rad3 related protein, which is a master regulator of DDR. We conducted a phase II trial of ceralasertib plus durvalumab in patients with previously treated advanced gastric cancer (AGC) to demonstrate the safety, tolerability, and clinical activity of the combination. METHODS: This phase II, open-label, single-center, non-randomized study was designed to evaluate the efficacy and safety of ceralasertib in combination with durvalumab in patients with AGC. The study drug regimen was ceralasertib (240 mg two times a day) days 15-28 in a 28-day cycle in combination with durvalumab (1500 mg) at day 1 every 4 weeks. The primary end point was overall response rate (ORR) by Response Evaluation Criteria in Solid Tumors (V.1.1). Exploratory biomarker analysis was performed using fresh tumor biopsies in all enrolled patients. RESULTS: Among 31 patients, the ORR, disease control rate, median progression-free survival (PFS), and overall survival were 22.6% (95% CI 9.6% to 41.1%), 58.1% (95% CI 39.1% to 75.5%), 3.0 (95% CI 2.1 to 3.9) months, and 6.7 (95% CI 3.8 to 9.6) months, respectively. Common adverse events were manageable with dose modification. A subgroup of patients with a loss of ataxia telangiectasia mutated (ATM) expression and/or high proportion of mutational signature attributable to homologous repair deficiency (sig. HRD) demonstrated a significantly longer PFS than those with intact ATM and low sig. HRD (5.60 vs 1.65 months; HR 0.13, 95% CI 0.045 to 0.39; long-rank p<0.001). During the study treatment, upregulation of the innate immune response by cytosolic DNA, activation of intratumoral lymphocytes, and expansion of circulating tumor-reactive CD8 +T cell clones were identified in responders. Enrichment of the tumor vasculature signature was associated with treatment resistance. CONCLUSIONS: Ceralasertib plus durvalumab has promising antitumor activity, with durable responses in patients with refractory AGC. Thus, a biomarker-driven trial is required. TRIAL REGISTRATION: NCT03780608.

Low-order modeling of the mutual synchronization between two turbulent thermoacoustic oscillators.

We use a low-order oscillator model to investigate the mutual synchronization of a thermoacoustic system consisting of two turbulent lean-premixed combustors coupled via a cross-talk tube. The model consists of two Van der Pol (VDP) oscillators coupled via dissipative and time-delay terms. We show that, despite its simplicity, the model can reproduce many of the synchronization phenomena observed experimentally, such as amplitude death, desynchronization (quasiperiodicity), synchronization (phase locking), and nonlinear energy pumping from a limit-cycle mode to a damped mode. This study shows that the mutual synchronization dynamics of a turbulent thermoacoustic system can be reproduced with just a simple coupled VDP model. This suggests that such a model could be used to identify new strategies for quenching limit-cycle oscillations in turbulent thermoacoustic systems, such as gas turbines and rocket engines.

Blood Loss and Related Laboratory Changes after Single-Event Multilevel Surgery and Hip Reconstructive Surgery in Patients with Cerebral Palsy.

BACKGROUD: Single-event multilevel surgery (SEMLS) and hip reconstructive surgery (HRS) often cause intraoperative bleeding, consequently increasing the probability of transfusion and postoperative laboratory changes. Therefore, it is important to assess risk factors to predict the amount of blood loss. This study aimed to evaluate blood loss, its influencing factors, and the related laboratory changes during SEMLS and HRS in patients with cerebral palsy (CP). METHODS: We retrospectively examined consecutive CP patients who underwent SEMLS and HRS. Surrogate markers of blood loss, including preoperative and postoperative hemoglobin (Hb), hematocrit, and changes in Hb concentration, were assessed. Albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatine levels were also analyzed for related laboratory changes. Risk factors were analyzed using multiple regression and logistic regression models. RESULTS: The overall cohort comprised 1,188 patients. Of them, 1,007 and 181 underwent SEMLS and HRS, respectively. Furthermore, 72 of 181 patients underwent a concomitant Dega osteotomy. The regression model showed that low preoperative Hb concentration (p < 0.001), high albumin level (p = 0.007), low body mass index (BMI) (p = 0.002), and bilateral HRS (p < 0.001) were significant risk factors of postoperative anemia. Valproate medication was associated with Hb drop, and the risk factors for Hb level < 8 g/dL on postoperative day 2 were bilateral HRS and Dega osteotomy in the HRS subgroup. In total, 21.6% had elevated AST levels on postoperative day 2, and bilateral HRS (p < 0.001), Gross Motor Function Classification System (GMFCS) level V (p = 0.041), Dega osteotomy (p < 0.001), and high preoperative AST level (p < 0.001) increased the risk of AST elevation. CONCLUSIONS: We have summarized the estimated blood loss and related laboratory changes after SEMLS and HRS in patients with CP and identified the risk factors. Clinical guidelines should be accordingly developed to include assessment of these risk factors and their impact in the outcomes of CP patients undergoing SEMLS and HRS.

Bispecific Antibody Molecule Inhibits Tumor Cell Proliferation More Efficiently Than the Two-Molecule Combination.

BACKGROUND: Monoclonal antibodies (mAbs) have proved to be a valuable tool for the treatment of different cancer types. However, clinical use of an increasing number of mAbs, have also highlighted limitations with monotherapy for cancers, in particular for such with more complex mechanisms, requiring action on additional molecules or pathways, or for cancers quickly acquiring resistance following monotherapy. An example for the latter is the mAb trastuzumab, FDA approved for treatment of metastatic gastric carcinoma. To circumvent this, researchers have reported synergistic, anti-proliferative effects by combination targeting of HER2 and EGFR by trastuzumab and the EGFR-targeting mAb Cetuximab overcoming trastuzumab resistance. METHODS: Maintaining the proven functionality of trastuzumab, we have designed bi-specific antibody molecules, called AffiMabs, by fusing an EGFR-targeting Affibody molecule to trastuzumab's heavy or light chains. Having confirmed binding to EGFR and Her2 and cytotoxicity of our AffiMabs, we analyzed apoptosis rate, receptor surface levels, phosphorylation levels of receptors and associated signaling pathways as well as differentially expressed genes on transcriptome level with the aim to elucidate the mode of action of our AffiMabs. RESULTS: The AffiMabs are able to simultaneously bind HER2 and EGFR and show increased cytotoxic effect compared to the original trastuzumab therapeutic molecule and, more importantly, even to the combination of trastuzumab and EGFR-targeting Affibody molecule. Analyzing the mode of action, we could show that bi-specific AffiMabs lead to reduced surface receptor levels and a downregulation of cell cycle associated genes on transcriptome level. CONCLUSION: Our study shows that transcriptome analysis can be used to validate the choice of receptor targets and guide the design of novel multi-specific molecules. The inherent modularity of the AffiMab format renders it readily applicable to other receptor targets.

Determinants of Response and Intrinsic Resistance to PD-1 Blockade in Microsatellite Instability-High Gastric Cancer.

Sequence alterations in microsatellites and an elevated mutational burden are observed in 20% of gastric cancers and associated with clinical response to anti-PD-1 antibodies. However, 50% of microsatellite instability-high (MSI-H) cancers are intrinsically resistant to PD-1 therapies. We conducted a phase II trial of pembrolizumab in patients with advanced MSI-H gastric cancer and included serial and multi-region tissue samples in addition to serial peripheral blood analyses. The number of whole-exome sequencing (WES)-derived nonsynonymous mutations correlated with antitumor activity and prolonged progression-free survival (PFS). Coupling WES to single-cell RNA sequencing, we identified dynamic tumor evolution with greater on-treatment collapse of mutational architecture in responders. Diverse T-cell receptor repertoire was associated with longer PFS to pembrolizumab. In addition, an increase in PD-1+ CD8+ T cells correlated with durable clinical benefit. Our findings highlight the genomic, immunologic, and clinical outcome heterogeneity within MSI-H gastric cancer and may inform development of strategies to enhance responsiveness. SIGNIFICANCE: This study highlights response heterogeneity within MSI-H gastric cancer treated with pembrolizumab monotherapy and underscores the potential for extended baseline and early on-treatment biomarker analyses to identify responders. The observed markers of intrinsic resistance have implications for patient stratification to inform novel combinations among patients with intrinsically resistant features.See related commentary by Fontana and Smyth, p. 2126.This article is highlighted in the In This Issue feature, p. 2113.

Demethylation by 5-aza-2'-deoxycytidine in colorectal cancer cells targets genomic DNA whilst promoter CpG island methylation persists.

BACKGROUND: DNA methylation and histone acetylation are epigenetic modifications that act as regulators of gene expression. Aberrant epigenetic gene silencing in tumours is a frequent event, yet the factors which dictate which genes are targeted for inactivation are unknown. DNA methylation and histone acetylation can be modified with the chemical agents 5-aza-2'-deoxycytidine (5-aza-dC) and Trichostatin A (TSA) respectively. The aim of this study was to analyse de-methylation and re-methylation and its affect on gene expression in colorectal cancer cell lines treated with 5-aza-dC alone and in combination with TSA. We also sought to identify methylation patterns associated with long term reactivation of previously silenced genes. METHOD: Colorectal cancer cell lines were treated with 5-aza-dC, with and without TSA, to analyse global methylation decreases by High Performance Liquid Chromatography (HPLC). Re-methylation was observed with removal of drug treatments. Expression arrays identified silenced genes with differing patterns of expression after treatment, such as short term reactivation or long term reactivation. Sodium bisulfite sequencing was performed on the CpG island associated with these genes and expression was verified with real time PCR. RESULTS: Treatment with 5-aza-dC was found to affect genomic methylation and to a lesser extent gene specific methylation. Reactivated genes which remained expressed 10 days post 5-aza-dC treatment featured hypomethylated CpG sites adjacent to the transcription start site (TSS). In contrast, genes with uniformly hypermethylated CpG islands were only temporarily reactivated. CONCLUSION: These results imply that 5-aza-dC induces strong de-methylation of the genome and initiates reactivation of transcriptionally inactive genes, but this does not require gene associated CpG island de-methylation to occur. In addition, for three of our selected genes, hypomethylation at the TSS of an epigenetically silenced gene is associated with the long term reversion of gene expression level brought about by alterations in the epigenetic status following 5-aza-dC treatment.

DNA methylation disruption reshapes the hematopoietic differentiation landscape.

Mutations in genes involved in DNA methylation (DNAme; for example, TET2 and DNMT3A) are frequently observed in hematological malignancies1-3 and clonal hematopoiesis4,5. Applying single-cell sequencing to murine hematopoietic stem and progenitor cells, we observed that these mutations disrupt hematopoietic differentiation, causing opposite shifts in the frequencies of erythroid versus myelomonocytic progenitors following Tet2 or Dnmt3a loss. Notably, these shifts trace back to transcriptional priming skews in uncommitted hematopoietic stem cells. To reconcile genome-wide DNAme changes with specific erythroid versus myelomonocytic skews, we provide evidence in support of differential sensitivity of transcription factors due to biases in CpG enrichment in their binding motif. Single-cell transcriptomes with targeted genotyping showed similar skews in transcriptional priming of DNMT3A-mutated human clonal hematopoiesis bone marrow progenitors. These data show that DNAme shapes the topography of hematopoietic differentiation, and support a model in which genome-wide methylation changes are transduced to differentiation skews through biases in CpG enrichment of the transcription factor binding motif.

Impedance spectroscopy for in situ and real-time observations of the effects of hydrogen on nitrile butadiene rubber polymer under high pressure.

Nondestructive impedance spectroscopy (IS) was developed and demonstrated to detect the effects of hydrogen on nitrile butadiene rubber exposed to hydrogen gas (H2) at high pressures up to 10 MPa. IS was applied to obtain an in situ and real-time quantification of H2 penetration into and its desorption out of rubber under high pressure. The diffusion coefficients of H2 were also obtained from the time evolution of the capacitance, which were compared with those obtained by thermal desorption gas analysis. The in situ measurements of the capacitance and the dissipation factor under various pressures during cyclic stepwise pressurization and decompression demonstrated the diffusion behaviour of H2, the phase of the rubber under high pressure, the transport properties of H2 gas, and the physicochemical interaction between H2 and the rubber. These phenomena were supported by a COMSOL simulation based on the electric current conservation equation and scanning electron microscopy (SEM) observations.

EpiMethylTag: simultaneous detection of ATAC-seq or ChIP-seq signals with DNA methylation.

Activation of regulatory elements is thought to be inversely correlated with DNA methylation levels. However, it is difficult to determine whether DNA methylation is compatible with chromatin accessibility or transcription factor (TF) binding if assays are performed separately. We developed a fast, low-input, low sequencing depth method, EpiMethylTag, that combines ATAC-seq or ChIP-seq (M-ATAC or M-ChIP) with bisulfite conversion, to simultaneously examine accessibility/TF binding and methylation on the same DNA. Here we demonstrate that EpiMethylTag can be used to study the functional interplay between chromatin accessibility and TF binding (CTCF and KLF4) at methylated sites.