Glioblastoma epigenomics discloses a complex biology and potential therapeutic targets.

Background and purpose:

Glioblastoma (GBM), a highly aggressive form of brain tumors, has been extensively studied using OMICS methods, and the most characteristic molecular determinants have been incorporated into the histopathological diagnosis. Research data, nevertheless, only partially have been adopted in clinical practice. Here we aimed to present results of our epige­no­mic GBM profiling to better understand early and late determinants of these tumors, and to share main elements of our findings with practicing professionals.

GBM specimens were surgically obtained after first diagnosis (GBM1) and at recurrence (GBM2). DNA was extracted from 24 sequential pairs of formalin-fixed, paraffin-embedded tumor tissues. The Reduced Representation Bisulfite Sequencing kit was used for library preparation. Pooled libraries were sequenced on an Illumina NextSeq 550 instrument. Methylation controls (MC) were obtained from a publicly available database. Bioinformatic analyses were performed to identify differentially methylated pathways and their elements in cohorts of MC, GBM1 and GBM2.

Several differentially methylated pathways involved in basic intracellular and brain tissue developmental processes were identified in the GBM1 vs. MC and GBM2 vs. MC comparisons. Among differentially me­thylated pathways, those involved in immune regulation, neurotransmitter (particularly dopaminergic, noradrenergic and glutaminergic) responses and regulation of stem cell differentiation and proliferation stood out in the GBM2 vs. GBM1 comparisons.

Our study revealed biological complexity of early and late gliomagenesis encompassing mechanisms from basic intracellular through distorted neurodevelopmental processes to more specific immune and highjacked neurotransmitter pathways in the tumor microenvironment. These findings may offer considerations for therapeutic approaches.

Missing Heritability in Albinism: Deep Characterization of a Hungarian Albinism Cohort Raises the Possibility of the Digenic Genetic Background of the Disease.

Albinism is characterized by a variable degree of hypopigmentation affecting the skin and the hair, and causing ophthalmologic abnormalities. Its oculocutaneous, ocular and syndromic forms follow an autosomal or X-linked recessive mode of inheritance, and 22 disease-causing genes are implicated in their development. Our aim was to clarify the genetic background of a Hungarian albinism cohort. Using a 22-gene albinism panel, the genetic background of 11 of the 17 Hungarian patients was elucidated. In patients with unidentified genetic backgrounds (n = 6), whole exome sequencing was performed. Our investigations revealed a novel, previously unreported rare variant (N687S) of the two-pore channel two gene (TPCN2). The N687S variant of the encoded TPC2 protein is carried by a 15-year-old Hungarian male albinism patient and his clinically unaffected mother. Our segregational analysis and in vitro functional experiments suggest that the detected novel rare TPCN2 variant alone is not a disease-causing variant in albinism. Deep genetic analyses of the family revealed that the patient also carries a phenotype-modifying R305W variant of the OCA2 protein, and he is the only family member harboring this genotype. Our results raise the possibility that this digenic combination might contribute to the observed differences between the patient and the mother, and found the genetic background of the disease in his case.

Biofilm formation initiating rotifer-specific biopolymer and its predicted components.

The rotifer-specific biopolymer, namely Rotimer, is a recently discovered group of the biomolecule family. Rotimer has an active role in the biofilm formation initiated by rotifers (e.g., Euchlanis dilatata or Adineta vaga) or in the female-male sexual interaction of monogononts. To understand the Ca2+- and polarity-dependent formation of this multifunctional viscoelastic material, it is essential to explore its molecular composition. The investigation of the rotifer-enhanced biofilm and Rotimer-inductor conglomerate (RIC) formation yielded several protein candidates to predict the Rotimer-specific main components. The exudate of E. dilatata males was primarily applied from different biopolimer-containing samples (biofilm or RIC). The advantage of males over females lies in their degenerated digestive system and simple anatomy. Thus, their exudate is less contaminated with food and endosymbiont elements. The sequenced and annotated genome and transcriptome of this species opened the way for identifying Rotimer proteins by mass spectrometry. The predicted rotifer-biopolymer forming components are SCO-spondins and 14-3-3 protein. The characteristics of Rotimer are similar to Reissner's fiber, which is found in the central nervous system of vertebrates and is mainly formed from SCO-spondins. This molecular information serves as a starting point for its interdisciplinary investigation and application in biotechnology, biomedicine, or neurodegeneration-related drug development.

Serum Insights: Leveraging the Power of miRNA Profiling as an Early Diagnostic Tool for Non-Small Cell Lung Cancer.

Non-small cell lung cancer is the predominant form of lung cancer and is associated with a poor prognosis. MiRNAs implicated in cancer initiation and progression can be easily detected in liquid biopsy samples and have the potential to serve as non-invasive biomarkers. In this study, we employed next-generation sequencing to globally profile miRNAs in serum samples from 71 early-stage NSCLC patients and 47 non-cancerous pulmonary condition patients. Preliminary analysis of differentially expressed miRNAs revealed 28 upregulated miRNAs in NSCLC compared to the control group. Functional enrichment analyses unveiled their involvement in NSCLC signaling pathways. Subsequently, we developed a gradient-boosting decision tree classifier based on 2588 miRNAs, which demonstrated high accuracy (0.837), sensitivity (0.806), and specificity (0.859) in effectively distinguishing NSCLC from non-cancerous individuals. Shapley Additive exPlanations analysis improved the model metrics by identifying the top 15 miRNAs with the strongest discriminatory value, yielding an AUC of 0.96 ± 0.04, accuracy of 0.896, sensitivity of 0.884, and specificity of 0.903. Our study establishes the potential utility of a non-invasive serum miRNA signature as a supportive tool for early detection of NSCLC while also shedding light on dysregulated miRNAs in NSCLC biology. For enhanced credibility and understanding, further validation in an independent cohort of patients is warranted.

miRNA-Seq Tissue Diagnostic Signature: A Novel Model for NSCLC Subtyping.

Non-small cell lung cancer (NSCLC) encompasses distinct histopathological subtypes, namely adenocarcinoma (AC) and squamous cell lung carcinoma (SCC), which require precise differentiation for effective treatment strategies. In this study, we present a novel molecular diagnostic model that integrates tissue-specific expression profiles of microRNAs (miRNAs) obtained through next-generation sequencing (NGS) to discriminate between AC and SCC subtypes of NSCLC. This approach offers a more comprehensive and precise molecular characterization compared to conventional methods such as histopathology or immunohistochemistry. Firstly, we identified 31 miRNAs with significant differential expression between AC and SCC cases. Subsequently, we constructed a 17-miRNA signature through rigorous multistep analyses, including LASSO/elastic net regression. The signature includes both upregulated miRNAs (hsa-miR-326, hsa-miR-450a-5p, hsa-miR-1287-5p, hsa-miR-556-5p, hsa-miR-542-3p, hsa-miR-30b-5p, hsa-miR-4728-3p, hsa-miR-450a-1-3p, hsa-miR-375, hsa-miR-147b, hsa-miR-7705, and hsa-miR-653-3p) and downregulated miRNAs (hsa-miR-944, hsa-miR-205-5p, hsa-miR-205-3p, hsa-miR-149-5p, and hsa-miR-6510-3p). To assess the discriminative capability of the 17-miRNA signature, we performed receiver operating characteristic (ROC) curve analysis, which demonstrated an impressive area under the curve (AUC) value of 0.994. Our findings highlight the exceptional diagnostic performance of the miRNA signature as a stratifying biomarker for distinguishing between AC and SCC subtypes in lung cancer. The developed molecular diagnostic model holds promise for providing a more accurate and comprehensive molecular characterization of NSCLC, thereby guiding personalized treatment decisions and improving clinical management and prognosis for patients.

Characterization of antibiotic resistomes by reprogrammed bacteriophage-enabled functional metagenomics in clinical strains.

Functional metagenomics is a powerful experimental tool to identify antibiotic resistance genes (ARGs) in the environment, but the range of suitable host bacterial species is limited. This limitation affects both the scope of the identified ARGs and the interpretation of their clinical relevance. Here we present a functional metagenomics pipeline called Reprogrammed Bacteriophage Particle Assisted Multi-species Functional Metagenomics (DEEPMINE). This approach combines and improves the use of T7 bacteriophage with exchanged tail fibres and targeted mutagenesis to expand phage host-specificity and efficiency for functional metagenomics. These modified phage particles were used to introduce large metagenomic plasmid libraries into clinically relevant bacterial pathogens. By screening for ARGs in soil and gut microbiomes and clinical genomes against 13 antibiotics, we demonstrate that this approach substantially expands the list of identified ARGs. Many ARGs have species-specific effects on resistance; they provide a high level of resistance in one bacterial species but yield very limited resistance in a related species. Finally, we identified mobile ARGs against antibiotics that are currently under clinical development or have recently been approved. Overall, DEEPMINE expands the functional metagenomics toolbox for studying microbial communities.

Spectrum and Density of Gamma and X-ray Induced Mutations in a Non-Model Rice Cultivar.

Physical mutagens are a powerful tool used for genetic research and breeding for over eight decades. Yet, when compared to chemical mutagens, data sets on the effect of different mutagens and dosages on the spectrum and density of induced mutations remain lacking. To address this, we investigated the landscape of mutations induced by gamma and X-ray radiation in the most widely cultivated crop species: rice. A mutant population of a tropical upland rice, Oryza sativa L., was generated and propagated via self-fertilization for seven generations. Five dosages ranging from 75 Gy to 600 Gy in both X-ray and gamma-irradiated material were applied. In the process of a forward genetic screens, 11 unique rice mutant lines showing phenotypic variation were selected for mutation analysis via whole-genome sequencing. Thousands of candidate mutations were recovered in each mutant with single base substitutions being the most common, followed by small indels and structural variants. Higher dosages resulted in a higher accumulation of mutations in gamma-irradiated material, but not in X-ray-treated plants. The in vivo role of all annotated rice genes is yet to be directly investigated. The ability to induce a high density of single nucleotide and structural variants through mutagenesis will likely remain an important approach for functional genomics and breeding.

Epigenetic Suppression of the IL-7 Pathway in Progressive Glioblastoma.

BACKGROUND: Immune evasion in glioblastoma (GBM) shields cancer cells from cytotoxic immune response. METHODS: We investigated CpG methylation in promoters, genes, and pathways in 22 pairs of formalin-fixed paraffin-embedded sequential (FFPE) GBM using restricted resolution bisulfite sequencing (RRBS) and bioinformatic analyses. RESULTS: Gene ontology revealed hypermethylation in elements of the innate and adaptive immune system when recurrent GBM samples (GBMrec) were compared to control (CG) and primary GBM samples (GBMprim). Higher methylation levels of the IL-7 signaling pathway and response to IL-7 were found in GBMrec suggesting a progressive blockade of the IL-7 driven T cell response in sequential GBM. Analyses of the Cancer Genome Atlas array-based data confirmed hypermethylation of the IL-7 pathway in recurrent compared with primary GBM. We also quantified DNA CpG methylation in promoter and gene regions of the IL-7 ligand and IL-7 α-receptor subunit in individual samples of a large RRBS-based sequential cohort of GBM in a Viennese database and found significantly higher methylation levels in the IL-7 receptor α-subunit in GBMrec compared with GBMprim. CONCLUSIONS: This study revealed the progressive suppression of the IL-7 receptor-mediated pathway as a means of immune evasion by GBM and thereby highlighted it as a new treatment target.

Molecular archaeology of human cognitive traits.

The brains and minds of our human ancestors remain inaccessible for experimental exploration. Therefore, we reconstructed human cognitive evolution by projecting nonsynonymous/synonymous rate ratios (ω values) in mammalian phylogeny onto the anatomically modern human (AMH) brain. This atlas retraces human neurogenetic selection and allows imputation of ancestral evolution in task-related functional networks (FNs). Adaptive evolution (high ω values) is associated with excitatory neurons and synaptic function. It shifted from FNs for motor control in anthropoid ancestry (60-41 mya) to attention in ancient hominoids (26-19 mya) and hominids (19-7.4 mya). Selection in FNs for language emerged with an early hominin ancestor (7.4-1.7 mya) and was later accompanied by adaptive evolution in FNs for strategic thinking during recent (0.8 mya-present) speciation of AMHs. This pattern mirrors increasingly complex cognitive demands and suggests that co-selection for language alongside strategic thinking may have separated AMHs from their archaic Denisovan and Neanderthal relatives.

Particle-dependent reproduction and exogenic biopolymer secretion of protozoa co-cultured rotifers.

The rotifer-specific exogenic biopolymer, named Rotimer and its related molecular processes are affected by physical and chemical factors (e.g., temperature, pH or metal ions); however, the study of biological influences (e.g., the presence protozoa) concerning the particle-dependent reproduction (egg laying) and 'biopolymer producing capacity' (BPC) of rotifers is the objective of the present work. Non-planktonic rotifer species (Philodina acuticornis, Adineta vaga, Euchlanis dilatata, and Lecane bulla) were studied in paired micrometazoa-protozoa co-cultures involving Paramecium, Diplonema, and Amoeba. These protozoa can be beneficial food sources, enhancing reproduction, or even toxic factors for the above-mentioned animals, but can also function as particle-like mechanical stimulators. Furthermore, current studies reveal that bdelloids, similarly to monogonants, produce filamentous exudate; moreover, the body of bdelloids is covered by their exudate, unlike that of monogonants, especially in the case of A. vaga. A mathematical formula was developed as an improved version of a previously published viability marker to characterize the BPC and the relative amount of produced exudate in different conditions. Rotifer species secreting biopolymers appear to be a general trait indicating a common evolutionary background (e.g., calcium- and particle dependency) of such molecules; therefore, the BPC becomes an experiential sublethal influencing marker to these micrometazoans.

TUBB4B gene mutation in Leber phenotype of congenital amaurosis syndrome associated with early-onset deafness.

Beta-tubulin 4B isotype is one of the subunits of microtubules encoded by TUBB4B gene on chromosome 9, which is responsible for the maintenance of microtubule stability. In humans, mutations in microtubule-encoding genes have been associated with several tubulinopathies with very heterogeneous symptoms. So far, only two missense mutations in TUBB4B gene have been found to have pathological implications in this disorder. Here we report a Hungarian family with three affected members, mother and her 12- and 14-year-old children, who suffer from ophthalmologic and hearing impairments probably due to c.1171C > T missense variant in the TUBB4B gene. The presented case is the second report, and unique in the literature because of three affected family members carrying the same mutation and the family provides evidence for a quite similar but not identical phenotype of LCAEOD in subjects carrying this mutation.

The interacting rotifer-biopolymers are anti- and disaggregating agents for human-type beta-amyloid in vitro.

Neurodegeneration-related human-type beta-amyloid 1-42 aggregates (H-Aß) are one of the biochemical markers and executive molecules in Alzheimer's disease. The exogenic rotifer-specific biopolymer, namely Rotimer, has a protective effect against H-Aß toxicity on Euchlanis dilatata and Lecane bulla monogonant rotifers. Due to the external particle-dependent secreting activity of these animals, this natural exudate exists in a bound form on the surface of epoxy-metal beads, named as Rotimer Inductor Conglomerate (RIC). In this current work the experiential in vitro molecular interactions between Rotimer and Aßs are presented. The RIC form was uniformly used against H-Aß aggregation processes in stagogram- and fluorescent-based experiments. These well-known cell-toxic aggregates stably and quickly (only taking a few minutes) bind to RIC. The epoxy beads (as carriers) alone or the scrambled version of H-Aß (with random amino acid sequence) were the ineffective and inactive negative controls of this experimental system. The RIC has significant interacting, anti-aggregating and disaggregating effects on H-Aß. To detect these experiments, Bis-ANS and Thioflavin T were applied during amyloid binding, two aggregation-specific functional fluorescent dyes with different molecular characteristics. This newly described empirical interaction of Rotimer with H-Aß is a potential starting point and source of innovation concerning targeted human- and pharmaceutical applications.

External modulation of Rotimer exudate secretion in monogonant rotifers.

The Rotimer, a rotifer-specific biopolymer, is an exogenic bioactive exudate secreted by different monogonant species (e.g. Euchlanis dilatata or Lecane bulla). The production of this viscoelastic biomolecule is induced by different micro-particles, thereby forming a special Rotimer-Inductor Conglomerate (RIC) in a web format. In this case, the water insoluble Carmine crystals, filtered to size (max. diameter was 50 µm), functioned as an inductor. The RIC production is an adequate empirical indicator to follow up this filamentous biopolymer secretion experientially; moreover, this procedure is very sensitive to the environmental factors (temperature, pH, metals and possible natural pollutant agents). The above mentioned species show completely different reactions to these factors, except to the presence of calcium and to the modulating effects of different drugs. One of the novelties of this work is that the Rotimer secretion and consequently, the RIC-formation is a mutually obligatory and evolutionary calcium-dependent process in the concerned monogonants. This in vivo procedure needs calcium, both for the physiology of animals and for fiber formation, particularly in the latter case. The conglomerate covered area (%) and the detection of the longest filament (mm) of the given RIC were the generally and simultaneously applied methods in the current modulating experiments. Exploring the regulatory (e.g. calcium-dependency) and stimulating (e.g. Lucidril effect) possibilities of biopolymer secretion are the basis for optimizing the RIC-production capacities of these micro-metazoans.

DNA methylation and protein expression of Wnt pathway markers in progressive glioblastoma.

BACKGROUND: Wnt signaling plays important roles in tumorigenesis, invasiveness and therapeutic resistance of glioblastoma (GBM). METHODS: We simultaneously investigated six Wnt pathway markers (Wnt5a, Fzd-2, beta-catenin, Wnt3a, Wnt7b, Fzd-10) at epigenetic and protein levels in 21 sequential formalin-fixed paraffin-embedded GBM pairs and controls. RESULTS: Expression levels of Wnt5a, beta-catenin and Wnt3a proteins either moderately or significantly increased, while those of Fzd-2, Wnt7b and Fzd-10 decreased in the primary (GBM-P) and recurrent (GBM-R) tumors compared to the controls. Methylation levels within promoters and genes showed corresponding decreases for Wnt5a, beta-catenin and Wnt3a in tumors vs. controls, while that of Fzd-10 was uniformly high. Comparing the GBM-P and GBM-R pairs, proteins of Fzd-2, beta-catenin and Wnt3a were either moderately or significantly up-, while that of Wnt7b was downregulated in GBM-R, but these patterns were not accompanied by inverse methylation patterns in the corresponding promoters and genes over time. No methylation differences were noted within promoters and genes of the same markers in 112 pairs of primary and recurrent GBMs in a database, suggesting that the observed changes in protein expression levels may not be explained by CpG methylation status alone. The promoter and gene methylation rate was the highest for Fzd-10 in the database cohort too, supporting the noted low Fzd-10 protein expression. DISCUSSION: These analyses underscore the relevance of Wnt pathway molecules in the context of their methylation profiles in the development and evolution of GBM, and suggest that Wnt pathway regulation as a potential treatment target merits further studies.

Involvement of the Catecholamine Pathway in Glioblastoma Development.

Glioblastoma (GBM) is the most aggressive tumor of the central nervous system (CNS). The standard of care improves the overall survival of patients only by a few months. Explorations of new therapeutic targets related to molecular properties of the tumor are under way. Even though neurotransmitters and their receptors normally function as mediators of interneuronal communication, growing data suggest that these molecules are also involved in modulating the development and growth of GBM by acting on neuronal and glioblastoma stem cells. In our previous DNA CpG methylation studies, gene ontology analyses revealed the involvement of the monoamine pathway in sequential GBM. In this follow-up study, we quantitated the expression levels of four selected catecholamine pathway markers (alpha 1D adrenergic receptor-ADRA1D; adrenergic beta receptor kinase 1 or G protein-coupled receptor kinase 2-ADRBK1/GRK2; dopamine receptor D2-DRD2; and synaptic vesicle monoamine transporter-SLC18A2) by immunohistochemistry, and compared the histological scores with the methylation levels within the promoters + genes of these markers in 21 pairs of sequential GBM and in controls. Subsequently, we also determined the promoter and gene methylation levels of the same markers in an independent database cohort of sequential GBM pairs. These analyses revealed partial inverse correlations between the catecholamine protein expression and promoter + gene methylation levels, when the tumor and control samples were compared. However, we found no differences in the promoter + gene methylation levels of these markers in either our own or in the database primary-recurrent GBM pairs, despite the higher protein expression of all markers in the primary samples. This observation suggests that regulation of catecholamine expression is only partially related to CpG methylation within the promoter + gene regions, and additional mechanisms may also influence the expression of these markers in progressive GBM. These analyses underscore the involvement of certain catecholamine pathway markers in GBM development and suggest that these molecules mediating or modulating tumor growth merit further exploration.

NGS-Based Application for Routine Non-Invasive Pre-Implantation Genetic Assessment in IVF.

Although non-invasive pre-implantation genetic testing for aneuploidy (NIPGT-A) is potentially appropriate to assess chromosomal ploidy of the embryo, practical application of it in a routine IVF centre have not been started in the absence of a recommendation. Our objective in this study was to provide a comprehensive workflow for a clinically applicable strategy for NIPGT-A based on next-generation sequencing (NGS) technology with the corresponding bioinformatic pipeline. In a retrospective study, we performed NGS on spent blastocyst culture media of Day 3 embryos fertilised with intracytoplasmic sperm injection (ICSI) with quality score on morphology assessment using the blank culture media as background control. Chromosomal abnormalities were identified by an optimised bioinformatics pipeline applying copy number variation (CNV) detecting algorithm. In this study, we demonstrate a comprehensive workflow covering both wet- and dry-lab procedures supporting a clinically applicable strategy for NIPGT-A that can be carried out within 48 h, which is critical for the same-cycle blastocyst transfer. The described integrated approach of non-invasive evaluation of embryonic DNA content of the culture media can potentially supplement existing pre-implantation genetic screening methods.

Neurodegeneration-related beta-amyloid as autocatabolism-attenuator in a micro-in vivo system.

Investigation of human neurodegeneration-related aggregates of beta-amyloid 1-42 (Aß42) on bdelloid rotifers is a novel interdisciplinary approach in life sciences. We reapplied an organ size-based in vivo monitoring system, exploring the autocatabolism-related alterations evoked by Aß42, in a glucose-supplemented starvation model. The experientially easy-to-follow size reduction of the bilateral reproductive organ (germovitellaria) in fasted rotifers was rescued by Aß42, serving as a nutrient source- and peptide sequence-specific attenuator of the organ shrinkage phase and enhancer of the regenerative one including egg reproduction. Recovery of the germovitellaria was significant in comparison with the greatly shrunken form. In contrast to the well-known neurotoxic Aß42 (except the bdelloids) with specific regulatory roles, the artificially designed scrambled version (random order of amino acids) was inefficient in autocatabolism attenuation, behaving as negative control. This native Aß42-related modulation of the 'functionally reversible organ shrinkage' can be a potential experiential and supramolecular marker of autocatabolism in vivo.

DNA CpG methylation in sequential glioblastoma specimens.

PURPOSE: Glioblastoma is the most aggressive form of brain tumors. A better understanding of the molecular mechanisms leading to its evolution is essential for the development of treatments more effective than the available modalities. Here, we aim to identify molecular drivers of glioblastoma development and recurrence by analyzing DNA CpG methylation patterns in sequential samples. METHODS: DNA was isolated from 22 pairs of primary and recurrent formalin-fixed, paraffin-embedded glioblastoma specimens, and subjected to reduced representation bisulfite sequencing. Bioinformatic analyses were conducted to identify differentially methylated sites and pathways, and biostatistics was used to test correlations among clinical and pathological parameters. RESULTS: Differentially methylated pathways likely involved in primary tumor development included those of neuronal differentiation, myelination, metabolic processes, synapse organization and endothelial cell proliferation, while pathways differentially active during glioblastoma recurrence involved those associated with cell processes and differentiation, immune response, Wnt regulation and catecholamine secretion and transport. CONCLUSION: DNA CpG methylation analyses in sequential clinical specimens revealed hypomethylation in certain pathways such as neuronal tissue development and angiogenesis likely involved in early tumor development and growth, while suggested altered regulation in catecholamine secretion and transport, Wnt expression and immune response contributing to glioblastoma recurrence. These pathways merit further investigations and may represent novel therapeutic targets.

Chromatin Organization in Early Land Plants Reveals an Ancestral Association between H3K27me3, Transposons, and Constitutive Heterochromatin.

Genome packaging by nucleosomes is a hallmark of eukaryotes. Histones and the pathways that deposit, remove, and read histone modifications are deeply conserved. Yet, we lack information regarding chromatin landscapes in extant representatives of ancestors of the main groups of eukaryotes, and our knowledge of the evolution of chromatin-related processes is limited. We used the bryophyte Marchantia polymorpha, which diverged from vascular plants circa 400 mya, to obtain a whole chromosome genome assembly and explore the chromatin landscape and three-dimensional genome organization in an early diverging land plant lineage. Based on genomic profiles of ten chromatin marks, we conclude that the relationship between active marks and gene expression is conserved across land plants. In contrast, we observed distinctive features of transposons and other repetitive sequences in Marchantia compared with flowering plants. Silenced transposons and repeats did not accumulate around centromeres. Although a large fraction of constitutive heterochromatin was marked by H3K9 methylation as in flowering plants, a significant proportion of transposons were marked by H3K27me3, which is otherwise dedicated to the transcriptional repression of protein-coding genes in flowering plants. Chromatin compartmentalization analyses of Hi-C data revealed that repressed B compartments were densely decorated with H3K27me3 but not H3K9 or DNA methylation as reported in flowering plants. We conclude that, in early plants, H3K27me3 played an essential role in heterochromatin function, suggesting an ancestral role of this mark in transposon silencing.

Dissection of subclonal evolution by temporal mutation profiling in chronic lymphocytic leukemia patients treated with ibrutinib.

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib is inducing durable responses in chronic lymphocytic leukemia (CLL) patients with refractory/relapsed disease or with TP53 defect, with BTK and phospholipase C gamma 2 (PLCG2) mutations representing the predominant mechanisms conferring secondary ibrutinib resistance. To understand the landscape of genomic changes and the dynamics of subclonal architecture associated with ibrutinib treatment, an ultra-deep next-generation sequencing analysis of 30 recurrently mutated genes was performed on sequential samples of 20 patients, collected before and during single-agent ibrutinib treatment. Mutations in the SF3B1, MGAand BIRC3 genes were enriched during ibrutinib treatment, while aberrations in the BTK, PLCG2, RIPK1, NFKBIE and XPO1 genes were exclusively detected in posttreatment samples. Besides the canonical mutations, four novel BTK mutations and three previously unreported PLCG2 variants were identified. BTK and PLCG2 mutations were backtracked in five patients using digital droplet PCR and were detectable on average 10.5 months before clinical relapse. With a median follow-up time of 36.5 months, 7/9 patients harboring BTK mutations showed disease progression based on clinical and/or laboratory features. In conclusion, subclonal heterogeneity, dynamic clonal selection and various patterns of clonal variegation were identified with novel resistance-associated BTK mutations in individual patients treated with ibrutinib.