Brahma-related gene 1 has time-specific roles during brain and eye development.

During development, gene expression is tightly controlled to facilitate the generation of the diverse cell types that form the central nervous system. Brahma-related gene 1 (Brg1, also known as Smarca4) is the catalytic subunit of the SWItch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex that regulates transcription. We investigated the role of Brg1 between embryonic day 6.5 (E6.5) and E14.5 in Sox2-positive neural stem cells (NSCs). Being without major consequences at E6.5 and E14.5, loss of Brg1 between E7.5 and E12.5 resulted in the formation of rosette-like structures in the subventricular zone, as well as morphological alterations and enlargement of neural retina (NR). Additionally, Brg1-deficient cells showed decreased survival in vitro and in vivo. Furthermore, we uncovered distinct changes in gene expression upon Brg1 loss, pointing towards impaired neuron functions, especially those involving synaptic communication and altered composition of the extracellular matrix. Comparison with mice deficient for integrase interactor 1 (Ini1, also known as Smarcb1) revealed that the enlarged NR was Brg1 specific and was not caused by a general dysfunction of the SWI/SNF complex. These results suggest a crucial role for Brg1 in NSCs during brain and eye development.

Integration of Sequencing and Epidemiologic Data for Surveillance of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infections in a Tertiary-Care Hospital.

BACKGROUND: The ongoing coronavirus disease 2019 pandemic significantly burdens hospitals and other healthcare facilities. Therefore, understanding the entry and transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for effective prevention and preparedness measures. We performed surveillance and analysis of testing and transmission of SARS-CoV-2 infections in a tertiary-care hospital in Germany during the second and third pandemic waves in fall/winter 2020. METHODS: Between calendar week 41 in 2020 and calendar week 1 in 2021, 40%, of all positive patient and staff samples (284 total) were subjected to full-length viral genome sequencing. Clusters were defined based on similar genotypes indicating common sources of infection. We integrated phylogenetic, spatial, and temporal metadata to detect nosocomial infections and outbreaks, uncover transmission chains, and evaluate containment measures' effectiveness. RESULTS: Epidemiologic data and contact tracing readily recognize most healthcare-associated (HA) patient infections. However, sequencing data reveal that temporally preceding index cases and transmission routes can be missed using epidemiologic methods, resulting in delayed interventions and serially linked outbreaks being counted as independent events. While hospital-associated transmissions were significantly elevated at a moderate rate of community transmission during the second wave, systematic testing and high vaccination rates among staff have led to a substantial decrease in HA infections at the end of the second/beginning of the third wave despite high community transmissions. CONCLUSIONS: While epidemiologic analysis is critical for immediate containment of HA SARS-CoV-2 outbreaks, integration of genomic surveillance revealed weaknesses in identifying staff contacts. Our study underscores the importance of high testing frequency and genomic surveillance to detect, contain and prevent SARS-CoV-2-associated infections in healthcare settings.

The CCR6/CCL20 axis expands RORγt+ Tregs to protect from glomerulonephritis.

Previous studies have identified a unique Treg population, which expresses the Th17 characteristic transcription factor RORγt. These RORγt+ Tregs possess enhanced immunosuppressive capacity, which endows them with great therapeutic potential. However, as a caveat, they are also capable of secreting pro-inflammatory IL-17A. Since the sum function of RORγt+ Tregs in glomerulonephritis (GN) remains unknown, we studied the effects of their absence. Purified CD4+ T cell populations, containing or lacking RORγt+ Tregs, were transferred into immunocompromised RAG1 knockout mice and the nephrotoxic nephritis model of GN was induced. Absence of RORγt+ Tregs significantly aggravated kidney injury, demonstrating overall kidney-protective properties. Analyses of immune responses showed that RORγt+ Tregs were broadly immunosuppressive with no preference for a particular type of T cell response. Further characterization revealed a distinct functional and transcriptional profile, including enhanced production of IL-10. Expression of the chemokine receptor CCR6 marked a particularly potent subset, whose absence significantly worsened GN. As an underlying mechanism, we found that chemokine CCL20 acting through receptor CCR6 signaling mediated expansion and activation of RORγt+ Tregs. Finally, we also detected an increase of CCR6+ Tregs in kidney biopsies, as well as enhanced secretion of chemokine CCL20 in 21 patients with anti-neutrophil cytoplasmic antibody associated GN compared to that of 31 healthy living donors, indicating clinical relevance. Thus, our data characterize RORγt+ Tregs as anti-inflammatory mediators of GN and identify them as promising target for Treg directed therapies.

Novel marine metalloprotease-new approaches for inhibition of biofilm formation of Stenotrophomonas maltophilia.

Many marine organisms produce bioactive molecules with unique characteristics to survive in their ecological niches. These enzymes can be applied in biotechnological processes and in the medical sector to replace aggressive chemicals that are harmful to the environment. Especially in the human health sector, there is a need for new approaches to fight against pathogens like Stenotrophomonas maltophilia which forms thick biofilms on artificial joints or catheters and causes serious diseases. Our approach was to use enrichment cultures of five marine resources that underwent sequence-based screenings in combination with deep omics analyses in order to identify enzymes with antibiofilm characteristics. Especially the supernatant of the enrichment culture of a stony coral caused a 40% reduction of S. maltophilia biofilm formation. In the presence of the supernatant, our transcriptome dataset showed a clear stress response (upregulation of transcripts for metal resistance, antitoxins, transporter, and iron acquisition) to the treatment. Further investigation of the enrichment culture metagenome and proteome indicated a series of potential antimicrobial enzymes. We found an impressive group of metalloproteases in the proteome of the supernatant that is responsible for the detected anti-biofilm effect against S. maltophilia. KEY POINTS: • Omics-based discovery of novel marine-derived antimicrobials for human health management by inhibition of S. maltophilia • Up to 40% reduction of S. maltophilia biofilm formation by the use of marine-derived samples • Metalloprotease candidates prevent biofilm formation of S. maltophilia K279a by up to 20.

High-resolution analysis of Merkel Cell Polyomavirus in Merkel Cell Carcinoma reveals distinct integration patterns and suggests NHEJ and MMBIR as underlying mechanisms.

Merkel Cell Polyomavirus (MCPyV) is the etiological agent of the majority of Merkel Cell Carcinomas (MCC). MCPyV positive MCCs harbor integrated, defective viral genomes that constitutively express viral oncogenes. Which molecular mechanisms promote viral integration, if distinct integration patterns exist, and if integration occurs preferentially at loci with specific chromatin states is unknown. We here combined short and long-read (nanopore) next-generation sequencing and present the first high-resolution analysis of integration site structure in MCC cell lines as well as primary tumor material. We find two main types of integration site structure: Linear patterns with chromosomal breakpoints that map closely together, and complex integration loci that exhibit local amplification of genomic sequences flanking the viral DNA. Sequence analysis suggests that linear patterns are produced during viral replication by integration of defective/linear genomes into host DNA double strand breaks via non-homologous end joining, NHEJ. In contrast, our data strongly suggest that complex integration patterns are mediated by microhomology-mediated break-induced replication, MMBIR. Furthermore, we show by ChIP-Seq and RNA-Seq analysis that MCPyV preferably integrates in open chromatin and provide evidence that viral oncogene expression is driven by the viral promoter region, rather than transcription from juxtaposed host promoters. Taken together, our data explain the characteristics of MCPyV integration and may also provide a model for integration of other oncogenic DNA viruses such as papillomaviruses.

Atypical teratoid/rhabdoid tumors (ATRTs) with SMARCA4 mutation are molecularly distinct from SMARCB1-deficient cases.

Atypical teratoid/rhabdoid tumors (ATRTs) are very aggressive childhood malignancies of the central nervous system. The underlying genetic cause are inactivating bi-allelic mutations in SMARCB1 or (rarely) in SMARCA4. ATRT-SMARCA4 have been associated with a higher frequency of germline mutations, younger age, and an inferior prognosis in comparison to SMARCB1 mutated cases. Based on their DNA methylation profiles and transcriptomics, SMARCB1 mutated ATRTs have been divided into three distinct molecular subgroups: ATRT-TYR, ATRT-SHH, and ATRT-MYC. These subgroups differ in terms of age at diagnosis, tumor location, type of SMARCB1 alterations, and overall survival. ATRT-SMARCA4 are, however, less well understood, and it remains unknown, whether they belong to one of the described ATRT subgroups. Here, we examined 14 ATRT-SMARCA4 by global DNA methylation analyses. We show that they form a separate group segregating from SMARCB1 mutated ATRTs and from other SMARCA4-deficient tumors like small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) or SMARCA4 mutated extra-cranial malignant rhabdoid tumors. In contrast, medulloblastoma (MB) samples with heterozygous SMARCA4 mutations do not group separately, but with established MB subgroups. RNA sequencing of ATRT-SMARCA4 confirmed the clustering results based on DNA methylation profiling and displayed an absence of typical signature genes upregulated in SMARCB1 deleted ATRT. In summary, our results suggest that, in line with previous clinical observations, ATRT-SMARCA4 should be regarded as a distinct molecular subgroup.

TDP-43 enhances translation of specific mRNAs linked to neurodegenerative disease.

The RNA-binding protein TDP-43 is heavily implicated in neurodegenerative disease. Numerous patient mutations in TARDBP, the gene encoding TDP-43, combined with data from animal and cell-based models, imply that altered RNA regulation by TDP-43 causes Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. However, underlying mechanisms remain unresolved. Increased cytoplasmic TDP-43 levels in diseased neurons suggest a possible role in this cellular compartment. Here, we examined the impact on translation of overexpressing human TDP-43 and the TDP-43A315T patient mutant protein in motor neuron-like cells and primary cultures of cortical neurons. In motor-neuron like cells, TDP-43 associates with ribosomes without significantly affecting global translation. However, ribosome profiling and additional assays revealed enhanced translation and direct binding of Camta1, Mig12, and Dennd4a mRNAs. Overexpressing either wild-type TDP-43 or TDP-43A315T stimulated translation of Camta1 and Mig12 mRNAs via their 5'UTRs and increased CAMTA1 and MIG12 protein levels. In contrast, translational enhancement of Dennd4a mRNA required a specific 3'UTR region and was specifically observed with the TDP-43A315T patient mutant allele. Our data reveal that TDP-43 can function as an mRNA-specific translational enhancer. Moreover, since CAMTA1 and DENND4A are linked to neurodegeneration, they suggest that this function could contribute to disease.

The basic helix-loop-helix transcription factor TCF4 impacts brain architecture as well as neuronal morphology and differentiation.

Germline mutations in the basic helix-loop-helix transcription factor 4 (TCF4) cause the Pitt-Hopkins syndrome (PTHS), a developmental disorder with severe intellectual disability. Here, we report findings from a new mouse model with a central nervous system-specific truncation of Tcf4 leading to severe phenotypic abnormalities. Furthermore, it allows the study of a complete TCF4 knockout in adult mice, circumventing early postnatal lethality of previously published mouse models. Our data suggest that a TCF4 truncation results in an impaired hippocampal architecture affecting both the dentate gyrus as well as the cornu ammonis. In the cerebral cortex, loss of TCF4 generates a severe differentiation delay of neural precursors. Furthermore, neuronal morphology was critically affected with shortened apical dendrites and significantly increased branching of dendrites. Our data provide novel information about the role of Tcf4 in brain development and may help to understand the mechanisms leading to intellectual deficits observed in patients suffering from PTHS.

Expression of a Structural Protein of the Mycovirus FgV-ch9 Negatively Affects the Transcript Level of a Novel Symptom Alleviation Factor and Causes Virus Infection-Like Symptoms in Fusarium graminearum.

Infections of fungi by mycoviruses are often symptomless but sometimes also fatal, as they perturb sporulation, growth, and, if applicable, virulence of the fungal host. Hypovirulence-inducing mycoviruses, therefore, represent a powerful means to defeat fungal epidemics on crop plants. Infection with Fusarium graminearum virus China 9 (FgV-ch9), a double-stranded RNA (dsRNA) chrysovirus-like mycovirus, debilitates Fusarium graminearum, the causal agent of fusarium head blight. In search for potential symptom alleviation or aggravation factors in F. graminearum, we consecutively infected a custom-made F. graminearum mutant collection with FgV-ch9 and found a mutant with constantly elevated expression of a gene coding for a putative mRNA-binding protein that did not show any disease symptoms despite harboring large amounts of virus. Deletion of this gene, named virus response 1 (vr1), resulted in phenotypes identical to those observed in the virus-infected wild type with respect to growth, reproduction, and virulence. Similarly, the viral structural protein coded on segment 3 (P3) caused virus infection-like symptoms when expressed in the wild type but not in the vr1 overexpression mutant. Gene expression analysis revealed a drastic downregulation of vr1 in the presence of virus and in mutants expressing P3. We conclude that symptom development and severity correlate with gene expression levels of vr1 This was confirmed by comparative transcriptome analysis, showing a large transcriptional overlap between the virus-infected wild type, the vr1 deletion mutant, and the P3-expressing mutant. Hence, vr1 represents a fundamental host factor for the expression of virus-related symptoms and helps us understand the underlying mechanism of hypovirulence.IMPORTANCE Virus infections of phytopathogenic fungi occasionally impair growth, reproduction, and virulence, a phenomenon referred to as hypovirulence. Hypovirulence-inducing mycoviruses, therefore, represent a powerful means to defeat fungal epidemics on crop plants. However, the poor understanding of the molecular basis of hypovirulence induction limits their application. Using the devastating fungal pathogen on cereal crops, Fusarium graminearum, we identified an mRNA binding protein (named virus response 1, vr1) which is involved in symptom expression. Downregulation of vr1 in the virus-infected fungus and vr1 deletion evoke virus infection-like symptoms, while constitutive expression overrules the cytopathic effects of the virus infection. Intriguingly, the presence of a specific viral structural protein is sufficient to trigger the fungal response, i.e., vr1 downregulation, and symptom development similar to virus infection. The advancements in understanding fungal infection and response may aid biological pest control approaches using mycoviruses or viral proteins to prevent future Fusarium epidemics.

BRD4 promotes p63 and GRHL3 expression downstream of FOXO in mammary epithelial cells.

Bromodomain-containing protein 4 (BRD4) is a member of the bromo- and extraterminal (BET) domain-containing family of epigenetic readers which is under intensive investigation as a target for anti-tumor therapy. BRD4 plays a central role in promoting the expression of select subsets of genes including many driven by oncogenic transcription factors and signaling pathways. However, the role of BRD4 and the effects of BET inhibitors in non-transformed cells remain mostly unclear. We demonstrate that BRD4 is required for the maintenance of a basal epithelial phenotype by regulating the expression of epithelial-specific genes including TP63 and Grainy Head-like transcription factor-3 (GRHL3) in non-transformed basal-like mammary epithelial cells. Moreover, BRD4 occupancy correlates with enhancer activity and enhancer RNA (eRNA) transcription. Motif analyses of cell context-specific BRD4-enriched regions predicted the involvement of FOXO transcription factors. Consistently, activation of FOXO1 function via inhibition of EGFR-AKT signaling promoted the expression of TP63 and GRHL3. Moreover, activation of Src kinase signaling and FOXO1 inhibition decreased the expression of FOXO/BRD4 target genes. Together, our findings support a function for BRD4 in promoting basal mammary cell epithelial differentiation, at least in part, by regulating FOXO factor function on enhancers to activate TP63 and GRHL3 expression.

Runx1 downregulates stem cell and megakaryocytic transcription programs that support niche interactions.

Disrupting mutations of the RUNX1 gene are found in 10% of patients with myelodysplasia (MDS) and 30% of patients with acute myeloid leukemia (AML). Previous studies have revealed an increase in hematopoietic stem cells (HSCs) and multipotent progenitor (MPP) cells in conditional Runx1-knockout (KO) mice, but the molecular mechanism is unresolved. We investigated the myeloid progenitor (MP) compartment in KO mice, arguing that disruptions at the HSC/MPP level may be amplified in downstream cells. We demonstrate that the MP compartment is increased by more than fivefold in Runx1 KO mice, with a prominent skewing toward megakaryocyte (Meg) progenitors. Runx1-deficient granulocyte-macrophage progenitors are characterized by increased cloning capacity, impaired development into mature cells, and HSC and Meg transcription signatures. An HSC/MPP subpopulation expressing Meg markers was also increased in Runx1-deficient mice. Rescue experiments coupled with transcriptome analysis and Runx1 DNA-binding assays demonstrated that granulocytic/monocytic (G/M) commitment is marked by Runx1 suppression of genes encoding adherence and motility proteins (Tek, Jam3, Plxnc1, Pcdh7, and Selp) that support HSC-Meg interactions with the BM niche. In vitro assays confirmed that enforced Tek expression in HSCs/MPPs increases Meg output. Interestingly, besides this key repressor function of Runx1 to control lineage decisions and cell numbers in progenitors, our study also revealed a critical activating function in erythroblast differentiation, in addition to its known importance in Meg and G/M maturation. Thus both repressor and activator functions of Runx1 at multiple hematopoietic stages and lineages likely contribute to the tumor suppressor activity in MDS and AML.

Essential control of early B-cell development by Mef2 transcription factors.

The sequential activation of distinct developmental gene networks governs the ultimate identity of a cell, but the mechanisms involved in initiating downstream programs are incompletely understood. The pre-B-cell receptor (pre-BCR) is an important checkpoint of B-cell development and is essential for a pre-B cell to traverse into an immature B cell. Here, we show that activation of myocyte enhancer factor 2 (Mef2) transcription factors (TFs) by the pre-BCR is necessary for initiating the subsequent genetic network. We demonstrate that B-cell development is blocked at the pre-B-cell stage in mice deficient for Mef2c and Mef2d TFs and that pre-BCR signaling enhances the transcriptional activity of Mef2c/d through phosphorylation by the Erk5 mitogen-activating kinase. This activation is instrumental in inducing Krüppel-like factor 2 and several immediate early genes of the AP1 and Egr family. Finally, we show that Mef2 proteins cooperate with the products of their target genes (Irf4 and Egr2) to induce secondary waves of transcriptional regulation. Our findings uncover a novel role for Mef2c/d in coordinating the transcriptional network that promotes early B-cell development.

A transplant "immunome" screening platform defines a targetable epitope fingerprint of multiple myeloma.

Multiple myeloma (MM) is a hematological cancer for which immune-based treatments are currently in development. Many of these rely on the identification of highly disease-specific, strongly and stably expressed antigens. Here, we profiled the myeloma B-cell immunome both to explore its predictive role in the context of autologous and allogeneic hematopoietic stem cell transplantation (HSCT) and to identify novel immunotherapeutic targets. We used random peptide phage display, reverse immunization, and next-generation sequencing-assisted antibody phage display to establish a highly myeloma-specific epitope fingerprint targeted by B-cell responses of 18 patients in clinical remission. We found that allogeneic HSCT more efficiently allowed production of myeloma-specific antibodies compared with autologous HSCT and that a highly reactive epitope recognition signature correlated with superior response to treatment. Next, we performed myeloma cell surface screenings of phage-displayed patient transplant immunomes. Although some of the screenings yielded clear-cut surface binders, the majority of screenings did not, suggesting that many of the targeted antigens may in fact not be accessible to the B-cell immune system in untreated myeloma cells. This fit well with the identification of heat-shock proteins as a class of antigens that showed overall the broadest reactivity with myeloma patient sera after allogeneic HSCT and that may be significantly translocated to the cell surface upon treatment as a result of immunogenic cell death. Our data reveal a disease-specific epitope signature of MM that is predictive for response to treatment. Mining of transplant immunomes for strong myeloma surface binders may open up avenues for myeloma immunotherapy.

Histone Chaperone SSRP1 is Essential for Wnt Signaling Pathway Activity During Osteoblast Differentiation.

Cellular differentiation is accompanied by dramatic changes in chromatin structure which direct the activation of lineage-specific transcriptional programs. Structure-specific recognition protein-1 (SSRP1) is a histone chaperone which is important for chromatin-associated processes such as transcription, DNA replication and repair. Since the function of SSRP1 during cell differentiation remains unclear, we investigated its potential role in controlling lineage determination. Depletion of SSRP1 in human mesenchymal stem cells elicited lineage-specific effects by increasing expression of adipocyte-specific genes and decreasing the expression of osteoblast-specific genes. Consistent with a role in controlling lineage specification, transcriptome-wide RNA-sequencing following SSRP1 depletion and the induction of osteoblast differentiation revealed a specific decrease in the expression of genes involved in biological processes related to osteoblast differentiation. Importantly, we observed a specific downregulation of target genes of the canonical Wnt signaling pathway, which was accompanied by decreased nuclear localization of active ß-catenin. Together our data uncover a previously unknown role for SSRP1 in promoting the activation of the Wnt signaling pathway activity during cellular differentiation. Stem Cells 2016;34:1369-1376.

Pulmonary Targeting of Adeno-associated Viral Vectors by Next-generation Sequencing-guided Screening of Random Capsid Displayed Peptide Libraries.

Vectors mediating strong, durable, and tissue-specific transgene expression are mandatory for safe and effective gene therapy. In settings requiring systemic vector administration, the availability of suited vectors is extremely limited. Here, we present a strategy to select vectors with true specificity for a target tissue from random peptide libraries displayed on adeno-associated virus (AAV) by screening the library under circulation conditions in a murine model. Guiding the in vivo screening by next-generation sequencing, we were able to monitor the selection kinetics and to determine the right time point to discontinue the screening process. The establishment of different rating scores enabled us to identify the most specifically enriched AAV capsid candidates. As proof of concept, a capsid variant was selected that specifically and very efficiently delivers genes to the endothelium of the pulmonary vasculature after intravenous administration. This technical approach of selecting target-specific vectors in vivo is applicable to any given tissue of interest and therefore has broad implications in translational research and medicine.

Identification of a Novel Hepacivirus in Domestic Cattle from Germany.

UNLABELLED: Hepatitis C virus (HCV) continues to represent one of the most significant threats to human health. In recent years, HCV-related sequences have been found in bats, rodents, horses, and dogs, indicating a widespread distribution of hepaciviruses among animals. By applying unbiased high-throughput sequencing, a novel virus of the genus Hepacivirus was discovered in a bovine serum sample. De novo assembly yielded a nearly full-length genome coding for a polyprotein of 2,779 amino acids. Phylogenetic analysis confirmed that the virus represents a novel species within the genus Hepacivirus. Viral RNA screening determined that 1.6% (n = 5) of 320 individual animals and 3.2% (n = 5) of 158 investigated cattle herds in Germany were positive for bovine hepacivirus. Repeated reverse transcription-PCR (RT-PCR) analyses of animals from one dairy herd proved that a substantial percentage of cows were infected, with some of them being viremic for over 6 months. Clinical and postmortem examination revealed no signs of disease, including liver damage. Interestingly, quantitative RT-PCR from different organs and tissues, together with the presence of an miR-122 binding site in the viral genome, strongly suggests a liver tropism for bovine hepacivirus, making this novel virus a promising animal model for HCV infections in humans. IMPORTANCE: Livestock animals act as important sources for emerging pathogens. In particular, their large herd size and the existence of multiple ways of direct and food-borne infection routes emphasize their role as virus reservoirs. Apart from the search for novel viruses, detailed characterization of these pathogens is indispensable in the context of risk analysis. Here, we describe the identification of a novel HCV-like virus in cattle. In addition, determination of the prevalence and of the course of infection in cattle herds provides valuable insights into the biology of this novel virus. The results presented here form a basis for future studies targeting viral pathogenesis of bovine hepaciviruses and their potential to establish zoonotic infections.

DNA methylation in an engineered heart tissue model of cardiac hypertrophy: common signatures and effects of DNA methylation inhibitors.

DNA methylation affects transcriptional regulation and constitutes a drug target in cancer biology. In cardiac hypertrophy, DNA methylation may control the fetal gene program. We therefore investigated DNA methylation signatures and their dynamics in an in vitro model of cardiac hypertrophy based on engineered heart tissue (EHT). We exposed EHTs from neonatal rat cardiomyocytes to a 12-fold increased afterload (AE) or to phenylephrine (PE 20 µM) and compared DNA methylation signatures to control EHT by pull-down assay and DNA methylation microarray. A 7-day intervention sufficed to induce contractile dysfunction and significantly decrease promoter methylation of hypertrophy-associated upregulated genes such as Nppa (encoding ANP) and Acta1 (α-skeletal actin) in both intervention groups. To evaluate whether pathological consequences of AE are affected by inhibiting de novo DNA methylation we applied AE in the absence and presence of DNA methyltransferase (DNMT) inhibitors: 5-aza-2'-deoxycytidine (aza, 100 µM, nucleosidic inhibitor), RG108 (60 µM, non-nucleosidic) or methylene disalicylic acid (MDSA, 25 µM, non-nucleosidic). Aza had no effect on EHT function, but RG108 and MDSA partially prevented the detrimental consequences of AE on force, contraction and relaxation velocity. RG108 reduced AE-induced Atp2a2 (SERCA2a) promoter methylation. The results provide evidence for dynamic DNA methylation in cardiac hypertrophy and warrant further investigation of the potential of DNA methylation in the treatment of cardiac hypertrophy.

Next-generation sequencing of peripheral B-lineage cells pinpoints the circulating clonotypic cell pool in multiple myeloma.

The identity of the proliferative compartment of myeloma progenitor cells remains a matter of debate. Polymerase chain reaction-based studies suggested pre-switch "clonotypic" B cells sharing the immunoglobulin (Ig) rearrangement of the malignant plasma cell (M-PC), to circulate in the blood and possess stem cell-like properties. Here, we disprove this hypothesis. We screened peripheral blood IgM, IgG, and IgA repertoires of myeloma patients for the clonotypic rearrangement by next-generation sequencing. None of 12 cases showed pre-switch clonotypic transcripts. In the post-switch IgG/IgA repertoires, however, the clonotypic rearrangement was detected at high frequency in 6 of 8 patients with active disease, whereas it was undetectable after treatment, correlating with flow cytometric presence or absence of circulating M-PCs. Minor subclones with alternative post-switch isotypes suggested ongoing switch events and clonal evolution at the M-PC level. Our findings consistently show an absence of pre-switch clonotypic B cells, while M-PCs circulate in the peripheral blood and may contribute to spreading of the disease.

Rapid Capture Next-Generation Sequencing in Clinical Diagnostics of Kinase Pathway Aberrations in B-Cell Precursor ALL.

Comprehensive next-generation sequencing (NGS) applications have recently identified various recurrent kinase and cytokine receptor rearrangements in Ph-like B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) amenable to tyrosin kinase inhibitor treatment. For rapid diagnostics of kinase pathway aberrations in minimal residual disease (MRD) high-risk BCP-ALL, we developed a PCR-independent NGS custom enrichment capture panel targeting recurrent genomic alterations, which allows for the identification of unknown 5' fusion partner genes and precise mapping of variable genomic breakpoints. Using a standardized bioinformatics algorithm, we identified kinase and cytokine receptor rearrangements in the majority of ALL patients with high burden of postinduction MRD and enrichment of IKZF1 mutation or deletion (IKZF1(del) ).

Deciphering the microRNA signature of pathological cardiac hypertrophy by engineered heart tissue- and sequencing-technology.

Pathological cardiac hypertrophy and fibrosis are modulated by a set of microRNAs, most of which have been detected in biologically complex animal models of hypertrophy by arrays with moderate sensitivity and disregard of passenger strand (previously "star") microRNAs. Here, we aimed at precisely analyzing the microRNA signature of cardiac hypertrophy and fibrosis by RNA sequencing in a standardized in vitro hypertrophy model based on engineered heart tissue (EHT). Spontaneously beating, force-generating fibrin EHTs from neonatal rat heart cells were subjected to afterload enhancement for 7days (AE-EHT), and EHTs without intervention served as controls. AE resulted in reduced contractile force and relaxation velocity, fibrotic changes and reactivation of the fetal gene program. Small RNAs were extracted from control and AE-EHTs and sequencing yielded almost 750 different mature microRNAs, many of which have never been described before in rats. The detection of both arms of the precursor stem-loop (pre-miRNA), namely -3p and -5p miRs, was frequent. 22 abundantly sequenced microRNAs were >1.3× upregulated and 15 abundantly sequenced microRNAs downregulated to <0.77×. Among the upregulated microRNAs were 3 pairs of guide and passenger strand microRNAs (miR-21-5p/-3p, miR-322-5p/-3p, miR-210-3p/-5p) and one single passenger strand microRNA (miR-140-3p). Among downregulated microRNAs were 3 pairs (miR-133a-3p/-5p, miR-30e-5p/3p, miR-30c-5p/-3p). Preincubating EHTs with anti-miR-21-5p markedly attenuated the AE-induced contractile failure, cardiomyocyte hypertrophy and fibrotic response, recapitulating prior results in whole animals. Taken together, AE-induced pathological hypertrophy in EHTs is associated with 37 differentially regulated microRNAs, including many passenger strands. Antagonizing miR-21-5p ameliorates dysfunction in this model.