TAL1 activation in T-cell acute lymphoblastic leukemia: a novel oncogenic 3' neo-enhancer.

T-cell acute lymphocytic leukemia protein 1 (TAL1) is one of the most frequently deregulated oncogenes in T-cell acute lymphoblastic leukemia (T-ALL). Its deregulation can occur through diverse cis-alterations, including SIL-TAL1 microdeletions, translocations with T-cell Receptor loci, and more recently described upstream intergenic non-coding mutations. These mutations consist of recurrent focal microinsertions that create an oncogenic neo-enhancer accompanied by activating epigenetic marks. This observation laid the groundwork for an innovative paradigm concerning the activation of proto-oncogenes via genomic alterations of non-coding intergenic regions. However, for the majority of T-ALL expressing TAL1 (TAL1+), the deregulation mechanism remains 'unresolved'. We took advantage of H3K27ac and H3K4me3 chromatin immunoprecipitation sequencing data of eight cases of T-ALL, including five TAL1+ cases. We identified a putative novel oncogenic neo-enhancer downstream of TAL1 in an unresolved monoallelic TAL1+ case. A rare but recurrent somatic heterozygous microinsertion within this region creates a de novo binding site for MYB transcription factor. Here we demonstrate that this mutation leads to increased enhancer activity, gain of active epigenetic marks, and TAL1 activation via recruitment of MYB. These results highlight the diversity of non-coding mutations that can drive oncogene activation.

Global hypomethylation in childhood asthma identified by genome-wide DNA-methylation sequencing preferentially affects enhancer regions.

BACKGROUND: Childhood asthma is a result of a complex interaction of genetic and environmental components causing epigenetic and immune dysregulation, airway inflammation and impaired lung function. Although different microarray based EWAS studies have been conducted, the impact of epigenetic regulation in asthma development is still widely unknown. We have therefore applied unbiased whole genome bisulfite sequencing (WGBS) to characterize global DNA-methylation profiles of asthmatic children compared to healthy controls. METHODS: Peripheral blood samples of 40 asthmatic and 42 control children aged 5-15 years from three birth cohorts were sequenced together with paired cord blood samples. Identified differentially methylated regions (DMRs) were categorized in genotype-associated, cell-type-dependent, or prenatally primed. Network analysis and subsequent natural language processing of DMR-associated genes was complemented by targeted analysis of functional translation of epigenetic regulation on the transcriptional and protein level. RESULTS: In total, 158 DMRs were identified in asthmatic children compared to controls of which 37% were related to the eosinophil content. A global hypomethylation was identified affecting predominantly enhancer regions and regulating key immune genes such as IL4, IL5RA, and EPX. These DMRs were confirmed in n = 267 samples and could be linked to aberrant gene expression. Out of the 158 DMRs identified in the established phenotype, 56 were perturbed already at birth and linked, at least in part, to prenatal influences such as tobacco smoke exposure or phthalate exposure. CONCLUSION: This is the first epigenetic study based on whole genome sequencing to identify marked dysregulation of enhancer regions as a hallmark of childhood asthma.

Altered DNA Methylation Profiles in SF3B1 Mutated CLL Patients.

Mutations in splicing factor genes have a severe impact on the survival of cancer patients. Splicing factor 3b subunit 1 (SF3B1) is one of the most frequently mutated genes in chronic lymphocytic leukemia (CLL); patients carrying these mutations have a poor prognosis. Since the splicing machinery and the epigenome are closely interconnected, we investigated whether these alterations may affect the epigenomes of CLL patients. While an overall hypomethylation during CLL carcinogenesis has been observed, the interplay between the epigenetic stage of the originating B cells and SF3B1 mutations, and the subsequent effect of the mutations on methylation alterations in CLL, have not been investigated. We profiled the genome-wide DNA methylation patterns of 27 CLL patients with and without SF3B1 mutations and identified local decreases in methylation levels in SF3B1mut CLL patients at 67 genomic regions, mostly in proximity to telomeric regions. These differentially methylated regions (DMRs) were enriched in gene bodies of cancer-related signaling genes, e.g., NOTCH1, HTRA3, and BCL9L. In our study, SF3B1 mutations exclusively emerged in two out of three epigenetic stages of the originating B cells. However, not all the DMRs could be associated with the methylation programming of B cells during development, suggesting that mutations in SF3B1 cause additional epigenetic aberrations during carcinogenesis.

Method for direct observation of Bloch oscillations in semiconductors.

We propose a scheme for real-time observations of Bloch oscillations in semiconductors using time-resolved band gap emission spectroscopy. By solving the time-dependent Schrödinger equation, we find one remarkable band gap emission besides the normal high harmonics generated in the interaction of a mid-infrared laser pulse and a semiconductor. It is shown that the band gap emission yield is directly connected to the population in the conduction band (CB). By adopting a pump-probe scheme, the time-dependent population in the CB, that is the dynamical Bloch oscillation, can be probed by measuring the band gap emission signal versus pump-probe delay. We also present a model based on accelerated Bloch states to explain the time-resolved measurement of dynamical Bloch oscillation.

Near- and Extended-Edge X-Ray-Absorption Fine-Structure Spectroscopy Using Ultrafast Coherent High-Order Harmonic Supercontinua.

Recent advances in high-order harmonic generation have made it possible to use a tabletop-scale setup to produce spatially and temporally coherent beams of light with bandwidth spanning 12 octaves, from the ultraviolet up to x-ray photon energies >1.6 keV. Here we demonstrate the use of this light for x-ray-absorption spectroscopy at the K- and L-absorption edges of solids at photon energies near 1 keV. We also report x-ray-absorption spectroscopy in the water window spectral region (284-543 eV) using a high flux high-order harmonic generation x-ray supercontinuum with 10^{9} photons/s in 1% bandwidth, 3 orders of magnitude larger than has previously been possible using tabletop sources. Since this x-ray radiation emerges as a single attosecond-to-femtosecond pulse with peak brightness exceeding 10^{26} photons/s/mrad^{2}/mm^{2}/1% bandwidth, these novel coherent x-ray sources are ideal for probing the fastest molecular and materials processes on femtosecond-to-attosecond time scales and picometer length scales.

Environment-induced epigenetic reprogramming in genomic regulatory elements in smoking mothers and their children.

Epigenetic mechanisms have emerged as links between prenatal environmental exposure and disease risk later in life. Here, we studied epigenetic changes associated with maternal smoking at base pair resolution by mapping DNA methylation, histone modifications, and transcription in expectant mothers and their newborn children. We found extensive global differential methylation and carefully evaluated these changes to separate environment associated from genotype-related DNA methylation changes. Differential methylation is enriched in enhancer elements and targets in particular "commuting" enhancers having multiple, regulatory interactions with distal genes. Longitudinal whole-genome bisulfite sequencing revealed that DNA methylation changes associated with maternal smoking persist over years of life. Particularly in children prenatal environmental exposure leads to chromatin transitions into a hyperactive state. Combined DNA methylation, histone modification, and gene expression analyses indicate that differential methylation in enhancer regions is more often functionally translated than methylation changes in promoters or non-regulatory elements. Finally, we show that epigenetic deregulation of a commuting enhancer targeting c-Jun N-terminal kinase 2 (JNK2) is linked to impaired lung function in early childhood.

High-dynamic-range arrival time control for flexible, accurate and precise parametric sub-cycle waveform synthesis.

We introduce a simple all-inline variation of a balanced optical cross-correlator (BOC) that allows to measure the arrival time difference (ATD), over the full Nyquist bandwidth, with increased common-mode rejection and long-term stability. An FPGA-based signal processing unit allows for real-time signal normalization and enables locking to any setpoint with an unprecedented accuracy of 0.07 % within an increased ATD range of more than 400 fs, resulting in attosecond resolution locking. The setup precision is verified with an out-of-loop measurement to be less than 80 as residual jitter paving the way for highly demanding applications such as parametric waveform synthesizers.

Impact of the Electronic Band Structure in High-Harmonic Generation Spectra of Solids.

An accurate analytic model describing the microscopic mechanism of high-harmonic generation (HHG) in solids is derived. Extensive first-principles simulations within a time-dependent density-functional framework corroborate the conclusions of the model. Our results reveal that (i) the emitted HHG spectra are highly anisotropic and laser-polarization dependent even for cubic crystals; (ii) the harmonic emission is enhanced by the inhomogeneity of the electron-nuclei potential; the yield is increased for heavier atoms; and (iii) the cutoff photon energy is driver-wavelength independent. Moreover, we show that it is possible to predict the laser polarization for optimal HHG in bulk crystals solely from the knowledge of their electronic band structure. Our results pave the way to better control and optimize HHG in solids by engineering their band structure.

Identification of Cell Type-Specific Differences in Erythropoietin Receptor Signaling in Primary Erythroid and Lung Cancer Cells.

Lung cancer, with its most prevalent form non-small-cell lung carcinoma (NSCLC), is one of the leading causes of cancer-related deaths worldwide, and is commonly treated with chemotherapeutic drugs such as cisplatin. Lung cancer patients frequently suffer from chemotherapy-induced anemia, which can be treated with erythropoietin (EPO). However, studies have indicated that EPO not only promotes erythropoiesis in hematopoietic cells, but may also enhance survival of NSCLC cells. Here, we verified that the NSCLC cell line H838 expresses functional erythropoietin receptors (EPOR) and that treatment with EPO reduces cisplatin-induced apoptosis. To pinpoint differences in EPO-induced survival signaling in erythroid progenitor cells (CFU-E, colony forming unit-erythroid) and H838 cells, we combined mathematical modeling with a method for feature selection, the L1 regularization. Utilizing an example model and simulated data, we demonstrated that this approach enables the accurate identification and quantification of cell type-specific parameters. We applied our strategy to quantitative time-resolved data of EPO-induced JAK/STAT signaling generated by quantitative immunoblotting, mass spectrometry and quantitative real-time PCR (qRT-PCR) in CFU-E and H838 cells as well as H838 cells overexpressing human EPOR (H838-HA-hEPOR). The established parsimonious mathematical model was able to simultaneously describe the data sets of CFU-E, H838 and H838-HA-hEPOR cells. Seven cell type-specific parameters were identified that included for example parameters for nuclear translocation of STAT5 and target gene induction. Cell type-specific differences in target gene induction were experimentally validated by qRT-PCR experiments. The systematic identification of pathway differences and sensitivities of EPOR signaling in CFU-E and H838 cells revealed potential targets for intervention to selectively inhibit EPO-induced signaling in the tumor cells but leave the responses in erythroid progenitor cells unaffected. Thus, the proposed modeling strategy can be employed as a general procedure to identify cell type-specific parameters and to recommend treatment strategies for the selective targeting of specific cell types.

Jitter analysis of timing-distribution and remote-laser synchronization systems.

We present a powerful jitter analysis method for timing-distribution and remote-laser synchronization systems based on feedback flow between setup elements. We synchronize two different mode-locked lasers in a master-slave configuration locally and remotely over a timing-stabilized fiber link network. Local synchronization reveals the inherent jitter of the slave laser as 2.1 fs RMS (>20 kHz), whereas remote synchronization exhibits an out-of-loop jitter of 8.55 fs RMS integrated for 1 Hz - 1 MHz. Our comprehensive feedback model yields excellent agreement with the experimental results and identifies seven uncorrelated noise sources, out of which the slave laser's jitter dominates with 8.19 fs RMS.

40-µJ passively CEP-stable seed source for ytterbium-based high-energy optical waveform synthesizers.

We demonstrate experimentally for the first time a ~40-µJ two-octave-wide passively carrier-envelope phase (CEP)-stable parametric front-end for seeding an ytterbium (Yb)-pump-based, few-optical-cycle, high-energy optical parametric waveform synthesizer. The system includes a CEP-stable white-light continuum and two-channel optical parametric chirped pulse amplifiers (OPCPAs) in the near- and mid-infrared spectral regions spanning altogether a two-octave-wide spectrum driven by a regenerative amplifier. The output pulses are compressed and fully characterized to demonstrate the well-behaved spectral phase of this seed source.

Cascaded parametric amplification for highly efficient terahertz generation.

A highly efficient, practical approach to high-energy multi-cycle terahertz (THz) generation based on spectrally cascaded optical parametric amplification (THz-COPA) is introduced. Feasible designs are presented that enable the THz wave, initially generated by difference frequency generation between a narrowband optical pump and optical seed (0.1-10% of pump energy), to self-start a cascaded (or repeated) energy downconversion of pump photons in a single pass through a single crystal. In cryogenically cooled, periodically poled lithium niobate, unprecedented energy conversion efficiencies >8% achievable with existing pump laser technology are predicted using realistic simulations. The calculations account for cascading effects, absorption, dispersion, and laser-induced damage. Due to the simultaneous, coupled nonlinear evolution of multiple phase-matched three-wave mixing processes, THz-COPA exhibits physics distinctly different from conventional three-wave mixing parametric amplifiers. This, in turn, governs optimal phase-matching conditions, evolution of optical spectra, and limitations of the nonlinear process. Circumventing these limitations is shown to yield conversion efficiencies ≫10%.

Broadband continuum generation in mode-locked lasers with phase-matched output couplers.

The concept of intracavity phase matching is proposed and demonstrated both theoretically and experimentally with a broadband phase-matched dielectric output coupler for linear-cavity few-cycle Ti:sapphire oscillators. The spectrum in the matched wavelength range is enhanced by >10 dB while maintaining good beam quality via resonantly enhanced continuum generation. The enhanced spectral components can be continuously tuned by varying the intracavity dispersion. Because dielectric coatings offer flexible design capabilities, this approach is applicable to various lasers with different gain media to obtain custom-tailored spectra, which have the potential to benefit several applications, such as shorter pulse generation, seeding of ytterbium lasers for pumping optical parametric amplifiers, and direct f-2f detection of the carrier-envelope phase.

Terahertz generation in lithium niobate driven by Ti:sapphire laser pulses and its limitations.

We experimentally investigate the limits of 800-nm-to-terahertz (THz) energy conversion in lithium niobate at room temperature driven by amplified Ti:sapphire laser pulses with tilted pulse front. The influence of the pump central wavelength, pulse duration, and fluence on THz generation is studied. We achieved a high peak efficiency of 0.12% using transform limited 150 fs pulses and observed saturation of the optical-to-THz conversion efficiency at a fluence of 15 mJ/cm(2) for this pulse duration. We experimentally identify two main limitations for the scaling of optical-to-THz conversion efficiencies: (i) the large spectral broadening of the optical pump spectrum in combination with large angular dispersion of the tilted pulse front and (ii) free-carrier absorption of THz radiation due to multi-photon absorption of the 800 nm radiation.

Predictive value of DNA methylation patterns in AML patients treated with an azacytidine containing induction regimen.

BACKGROUND: Acute myeloid leukemia (AML) is a heterogeneous disease with a poor prognosis. Dysregulation of the epigenetic machinery is a significant contributor to disease development. Some AML patients benefit from treatment with hypomethylating agents (HMAs), but no predictive biomarkers for therapy response exist. Here, we investigated whether unbiased genome-wide assessment of pre-treatment DNA-methylation profiles in AML bone marrow blasts can help to identify patients who will achieve a remission after an azacytidine-containing induction regimen. RESULTS: A total of n = 155 patients with newly diagnosed AML treated in the AMLSG 12-09 trial were randomly assigned to a screening and a refinement and validation cohort. The cohorts were divided according to azacytidine-containing induction regimens and response status. Methylation status was assessed for 664,227 500-bp-regions using methyl-CpG immunoprecipitation-seq, resulting in 1755 differentially methylated regions (DMRs). Top regions were distilled and included genes such as WNT10A and GATA3. 80% of regions identified as a hit were represented on HumanMethlyation 450k Bead Chips. Quantitative methylation analysis confirmed 90% of these regions (36 of 40 DMRs). A classifier was trained using penalized logistic regression and fivefold cross validation containing 17 CpGs. Validation based on mass spectra generated by MALDI-TOF failed (AUC 0.59). However, discriminative ability was maintained by adding neighboring CpGs. A recomposed classifier with 12 CpGs resulted in an AUC of 0.77. When evaluated in the non-azacytidine containing group, the AUC was 0.76. CONCLUSIONS: Our analysis evaluated the value of a whole genome methyl-CpG screening assay for the identification of informative methylation changes. We also compared the informative content and discriminatory power of regions and single CpGs for predicting response to therapy. The relevance of the identified DMRs is supported by their association with key regulatory processes of oncogenic transformation and support the idea of relevant DMRs being enriched at distinct loci rather than evenly distribution across the genome. Predictive response to therapy could be established but lacked specificity for treatment with azacytidine. Our results suggest that a predictive epigenotype carries its methylation information at a complex, genome-wide level, that is confined to regions, rather than to single CpGs. With increasing application of combinatorial regimens, response prediction may become even more complicated.

DNMT and HDAC inhibition induces immunogenic neoantigens from human endogenous retroviral element-derived transcripts.

Immunotherapies targeting cancer-specific neoantigens have revolutionized the treatment of cancer patients. Recent evidence suggests that epigenetic therapies synergize with immunotherapies, mediated by the de-repression of endogenous retroviral element (ERV)-encoded promoters, and the initiation of transcription. Here, we use deep RNA sequencing from cancer cell lines treated with DNA methyltransferase inhibitor (DNMTi) and/or Histone deacetylase inhibitor (HDACi), to assemble a de novo transcriptome and identify several thousand ERV-derived, treatment-induced novel polyadenylated transcripts (TINPATs). Using immunopeptidomics, we demonstrate the human leukocyte antigen (HLA) presentation of 45 spectra-validated treatment-induced neopeptides (t-neopeptides) arising from TINPATs. We illustrate the potential of the identified t-neopeptides to elicit a T-cell response to effectively target cancer cells. We further verify the presence of t-neopeptides in AML patient samples after in vivo treatment with the DNMT inhibitor Decitabine. Our findings highlight the potential of ERV-derived neoantigens in epigenetic and immune therapies.

Dual-chirped optical parametric amplification for generating few hundred mJ infrared pulses.

An ultrafast high-power infrared pulse source employing a dual-chirped optical parametric amplification (DC-OPA) scheme based on a Ti:sapphire pump laser system is theoretically investigated. By chirping both pump and seed pulses in an optimized way, high-energy pump pulses can be utilized for a DC-OPA process without exceeding the damage threshold of BBO crystals, and broadband signal and idler pulses at 1.4 µm and 1.87 µm can be generated with a total conversion efficiency approaching 40%. Furthermore, few-cycle idler pulses with a passively stabilized carrier-envelope phase (CEP) can be generated by the difference frequency generation process in a collinear configuration. DC-OPA, a BBO-OPA scheme pumped by a Ti:sapphire laser, is efficient and scalable in output energy of the infrared pulses, which provides us with the design parameters of an ultrafast infrared laser system with an energy up to a few hundred mJ.