Anionic Pulmonary Surfactant Lipid Treatment Inhibits Rhinovirus A Infection of the Human Airway Epithelium.

Rhinoviruses (RVs) are major instigators of acute exacerbations of asthma, COPD, and other respiratory diseases. RVs are categorized into three species (RV-A, RV-B, and RV-C), which comprise more than 160 serotypes, making it difficult to develop an effective vaccine. Currently, no effective treatment for RV infection is available. Pulmonary surfactant is an extracellular complex of lipids and proteins that plays a central role in regulating innate immunity in the lung. The minor pulmonary surfactant lipids, palmitoyl-oleoyl-phosphatidylglycerol (POPG) and phosphatidylinositol (PI), are potent regulators of inflammatory processes and exert antiviral activity against respiratory syncytial virus (RSV) and influenza A viruses (IAV). In the current study, we examined the potencies of POPG and PI against rhinovirus A16 (RV-A16) in primary human airway epithelial cells (AECs) differentiated at an air-liquid interface (ALI). After AECs were infected with RV-A16, PI reduced the viral RNA copy number by 70% and downregulated (55-75%) the expression of antiviral (MDA5, IRF7, and IFN-lambda) and CXCL11 chemokine genes. In contrast, POPG only slightly decreased MDA5 (24%) and IRF7 (11%) gene expression but did not inhibit IFN-lambda gene expression or RV-A16 replication in AECs. However, both POPG and PI inhibited (50-80%) IL6 gene expression and protein secretion and CXCL11 protein secretion. PI treatment dramatically attenuated global gene expression changes induced by RV-A16 infection alone in AECs. The observed inhibitory effects were indirect and resulted mainly from the inhibition of virus replication. Cell-type enrichment analysis of viral-regulated genes opposed by PI treatment revealed the PI-inhibited viral induction of goblet cell metaplasia and the virus-induced downregulation of ciliated, club, and ionocyte cell types. Notably, the PI treatment also altered the ability of RV-A16 to regulate the expression of some phosphatidylinositol 4-kinase (PI4K); acyl-CoA-binding, domain-containing (ACBD); and low-density lipoprotein receptor (LDLR) genes that play critical roles in the formation and functioning of replication organelles (ROs) required for RV replication in host cells. These data suggest PI can be used as a potent, non-toxic, antiviral agent for RV infection prophylaxis and treatment.

Local simvastatin and inflammation during periodontal mini-flap wound healing: Exploratory results.

BACKGROUND: The objective of this exploratory study was to evaluate inflammatory markers in periodontal maintenance patients from a randomized, double-masked, parallel intervention clinical trial comparing local simvastatin (SIM) to carrier alone following mini-flap access. METHODS: Fifty patients with a 6-9-mm inflamed pocket during periodontal maintenance therapy (PMT) were treated with papilla reflection (PR)/root planing and placement of 2.2-mg simvastatin in methylcellulose (SIM/MCL) or methylcellulose alone (MCL). A small piece of interproximal soft tissue was harvested at baseline and 2 weeks postoperatively, gingival crevicular fluid (GCF) obtained at baseline, 2 weeks and 12 months, and bleeding on probing (BOP) and clinical attachment level (CAL) were measured at baseline and 12 months. Pro-inflammatory interleukin (IL)-6 and anti-inflammatory IL-10 gene activation were determined by reverse transcriptase polymerase chain reaction (rt-PCR). GCF IL-1ß, IL-6, IL-10, and vascular endothelial growth factor (VEGF-A) were measured with multiplex technology. Comparisons between groups and over time used logistic regression and general estimating equations. Associations between inflammatory markers and 12-month outcomes used Wilcoxon rank sum tests or Pearson correlations. RESULTS: Patients in the SIM group had 4.17 greater odds (p = 0.047) of improved BOP at 12 months. Median IL-6 and VEGF were significantly increased for all patients after 2 weeks of healing (p < 0.0001 and p = 0.03, respectively), while median IL-10 gene activation was increased after 2 weeks in SIM/MCL (NS). Overall, elevated GCF IL-10 at 2 weeks was significantly correlated with improved CAL at 12 months (r = -0.32, p = 0.03). CONCLUSIONS: Local SIM/MCL may have anti-inflammatory effects that potentially are associated with improved long-term CAL outcomes.

IL-4-BATF signaling directly modulates IL-9 producing mucosal mast cell (MMC9) function in experimental food allergy.

BACKGROUND: This study group has previously identified IL-9-producing mucosal mast cell (MMC9) as the primary source of IL-9 to drive intestinal mastocytosis and experimental IgE-mediated food allergy. However, the molecular mechanisms that regulate the expansion of MMC9s remain unknown. OBJECTIVES: This study hypothesized that IL-4 regulates MMC9 development and MMC9-dependent experimental IgE-mediated food allergy. METHODS: An epicutaneous sensitization model was used and bone marrow reconstitution experiments were performed to test the requirement of IL-4 receptor α (IL-4Rα) signaling on MMC9s in experimental IgE-mediated food allergy. Flow cytometric, bulk, and single-cell RNA-sequencing analyses on small intestine (SI) MMC9s were performed to illuminate MMC9 transcriptional signature and the effect of IL-4Rα signaling on MMC9 function. A bone marrow-derived MMC9 culture system was used to define IL-4-BATF signaling in MMC9 development. RESULTS: Epicutaneous sensitization- and bone marrow reconstitution-based models of IgE-mediated food allergy revealed an IL-4 signaling-dependent cell-intrinsic effect on SI MMC9 accumulation and food allergy severity. RNA-sequencing analysis of SI-MMC9s identified 410 gene transcripts reciprocally regulated by IL-4 signaling, including Il9 and Batf. Insilico analyses identified a 3491-gene MMC9 transcriptional signature and identified 2 transcriptionally distinct SI MMC9 populations enriched for metabolic or inflammatory programs. Employing an in vitro MMC9-culture model system showed that generation of MMC9-like cells was induced by IL-4 and this was in part dependent on BATF. CONCLUSIONS: IL-4Rα signaling directly modulates MMC9 function and exacerbation of experimental IgE-mediated food allergic reactions. IL-4Rα regulation of MMC9s is in part BATF-dependent and occurs via modulation of metabolic transcriptional programs.

The Clusters of Transcription Factors NFATC2, STAT5, GATA2, AP1, RUNX1 and EGR2 Binding Sites at the Induced Il13 Enhancers Mediate Il13 Gene Transcription in Response to Antigenic Stimulation.

IL-13 plays a critical role in mediating many biological processes responsible for allergic inflammation. Mast cells express Il13 mRNA and produce IL-13 protein in response to antigenic stimulation. Enhancers are essential in promoting gene transcription and are thought to activate transcription by delivering essential accessory cofactors to the promoter to potentiate gene transcription. However, enhancers mediating Il13 have not been identified. Furthermore, which Il13 enhancers detect signals triggered by antigenic stimulation have not yet been defined. In this study, we identified potential mouse Il13 enhancers using histone modification monomethylation at lysine residue 4 on histone 3 (H3K4me1) chromatin immunoprecipitation sequencing and acetylation at lysine residue 27 on histone 3 (H3K27ac) chromatin immunoprecipitation sequencing. We used Omni-assay for transposase-accessible chromatin sequencing to determine which accessible regions within the potential Il13 enhancers that responded to IgE receptor crosslinking. We also demonstrated that the transcription factor cluster consisting of the NFATC2, STAT5, GATA2, AP1, and RUNX1 binding sites at the proximal Il13 enhancer and the transcription factor cluster consisting of the EGR2 binding site at the distal Il13 E+6.5 enhancer are critical in sensing the signals triggered by antigenic stimulation. Those enhancers, which are responsive to antigenic stimulation and are constitutively active, cooperate to generate greater transcriptional outputs. Our study reveals a novel mechanism underlying how antigenic stimulation induces robust Il13 mRNA expression in mouse mast cells.

Engineering Biomimetic Nanofiber Microspheres with Tailored Size, Predesigned Structure, and Desired Composition via Gas Bubble-Mediated Coaxial Electrospray.

Minimally invasive therapies avoiding surgical complexities evoke great interest in developing injectable biomedical devices. Herein, a versatile approach is reported for engineering injectable and biomimetic nanofiber microspheres (NMs) with tunable sizes, predesigned structures, and desired compositions via gas bubble-mediated coaxial electrospraying. The sizes and structures of NMs are controlled by adjusting processing parameters including air flow rate, applied voltage, distance, and spinneret configuration in the coaxial setup. Importantly, unlike the self-assembly method, this technique can be used to fabricate NMs from any material feasible for electrospinning or other nanofiber fabrication techniques. To demonstrate the versatility, open porous NMs are successfully fabricated that consist of various short nanofibers made of poly(ε-caprolactone), poly(lactic-co-glycolic acid), gelatin, methacrylated gelatin, bioglass, and magneto-responsive polymer composites. Open porous NMs support human neural progenitor cell growth in 3D with a larger number and more neurites than nonporous NMs. Additionally, highly open porous NMs show faster cell infiltration and host tissue integration than nonporous NMs after subcutaneous injection to rats. Such a novel class of NMs holds great potential for many biomedical applications such as tissue filling, cell and drug delivery, and minimally invasive tissue regeneration.

Effects of enamel matrix derivative on clinical and inflammatory outcomes in periodontal maintenance patients: Randomized controlled clinical trial.

BACKGROUND: Efficient methods to treat persistent pockets during periodontal maintenance therapy (PMT) require further investigation. The hypothesis of this randomized controlled clinical trial was that local application of enamel matrix derivative (EMD) added to papilla reflection/root preparation (PR/RP) could enhance clinical and inflammatory outcomes, primarily clinical attachment level (CAL). METHODS: Fifty PMT patients with generalized stage III-IV, grade B periodontitis presenting with a 6- to 9-mm interproximal PD were randomly allocated to (PR/RP+EMD; n = 24) and control (PR/RP+saline; n = 26) therapies by sex and smoking status. Roots were treated with reflection of interproximal papillae, root planing assisted with endoscope evaluation, and acid etching, followed by EMD or saline application. Probing depth (PD), CAL, plaque index (PI), and interproximal bone height were evaluated at baseline and 12-months post-therapy. Gingival crevicular fluid, bleeding on probing (BOP), and interleukin-1ß were tested (ELISA) at baseline, 2 weeks, and 6 and 12 months. Groups were compared over time and between groups with Wilcoxon Rank Sum and t-tests. RESULTS: Both PR/RP+ EMD and PR/RP+S resulted in significant improvements in clinical outcomes (PD and CAL, BOP) from baseline to 12 months. No significant differences were found in clinical or inflammatory outcomes between the experimental and control groups. CONCLUSIONS: The addition of EMD to PR/RP does not significantly improve clinical or inflammatory outcomes compared with PR/RP alone during periodontal maintenance therapy.

Dual Delivery of Alendronate and E7-BMP-2 Peptide via Calcium Chelation to Mineralized Nanofiber Fragments for Alveolar Bone Regeneration.

The fixation and stability of dental implants is governed by the quality of the underlying alveolar bone. The current study investigates if the dual delivery of calcium chelating bone therapeutics from mineralized nanofiber fragments can help regenerate alveolar bone in vivo. Alendronate (ALN) or/and bone morphogenetic protein-2-mimicking peptide conjugated to a heptaglutamate moiety (E7-BMP-2) were incorporated onto mineralized nanofiber fragments of polylactide-co-glycolide-collagen-gelatin (PCG in 2:1:1 weight ratios) via calcium coupling/chelation. Two mg of the single-loaded (ALN) and coloaded (ALN + E7-BMP-2) mineralized nanofiber PCG grafts was filled into critical-sized (2 mm diameter × 2 mm depth) alveolar bone defects in rat maxillae and let heal for 4 weeks. X-ray microcomputed tomography analysis of the retrieved maxillae revealed significantly elevated new bone formation parameters for the ALN and ALN + E7-BMP-2 groups compared with the unfilled defect controls. However, no significant differences between the single and coloaded nanofiber grafts were noted. Furthermore, the histopathological analysis of the tissue sections divulged islands of new bone tissue in the ALN and ALN + E7-BMP-2 groups, whereas the control defect was covered with gingival tissue. Together, the presented strategy using mineralized nanofiber fragments in the sustained delivery of dual calcium chelating therapeutics could have potential applications in enhancing bone regeneration.

Tethering peptides onto biomimetic and injectable nanofiber microspheres to direct cellular response.

Biomimetic and injectable nanofiber microspheres (NMs) could be ideal candidate for minimally invasive tissue repair. Herein, we report a facile approach to fabricate peptide-tethered NMs by combining electrospinning, electrospraying, and surface conjugation techniques. The composition and size of NMs can be tuned by varying the processing parameters during the fabrication. Further, bone morphogenic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) mimicking peptides have been successfully tethered onto poly(ε-caprolactone) (PCL):gelatin:(gelatin-methacryloyl) (GelMA)(1:0.5:0.5) NMs through photocrosslinking of the methacrylic group in GelMA and octenyl alanine (OCTAL) in the modified peptides. The BMP-2-OCTAL peptide-tethered NMs significantly promote osteogenic differentiation of bone marrow-derived stem cells (BMSCs). Moreover, human umbilical vein endothelial cells (HUVECs) seeded on VEGF mimicking peptide QK-OCTAL-tethered NMs significantly up-regulated vascular-specific proteins, leading to microvascularization. The strategy developed in this work holds great potential in developing a biomimetic and injectable carrier to efficiently direct cellular response (Osteogenesis and Angiogenesis) for tissue repair.

Mineralized nanofiber segments coupled with calcium-binding BMP-2 peptides for alveolar bone regeneration.

Bone loss around tooth extraction sites can occur, thus making future placement of dental implants difficult. Alveolar bone regeneration can be guided by the application of a nanofibrous bone graft coupled with osteoinductive proteins/peptides, following tooth loss or tooth extraction. In the present study, we demonstrate the potential of mineralized nanofiber segments coupled with calcium-binding bone morphogenetic protein 2 (BMP-2) mimicking peptides for periodontal bone regeneration. Thin electrospun nanofiber membranes of PLGA-collagen-gelatin (2:1:1 wt ratios) were mineralized in 10× modified simulated body fluid (10× mSBF) and cryocut to segments of 20 µm. For predetermined weights of the mineralized nanofiber segments, it was possible to load various amounts of heptaglutamate E7-domain-conjugated BMP-2 peptide. Mineralized short fiber grafts (2 mg), with and without E7-BMP-2 peptides, were implanted into 2 mm × 2 mm (diameter × depth) critical-sized socket defects created in rat maxillae, following extraction of the first molar teeth. A sustained release profile of E7-BMP-2 from the mineralized nanofiber segments was recorded over 4 weeks. X-ray microcomputed tomography (µ-CT) analysis of peptide-loaded nanofiber graft filled defects revealed ∼3 times greater new bone volume and bone mineral density over 4 weeks in comparison to unfilled control defects. Further, histopathology data confirmed the formation of greater new osseous tissue in the BMP2 peptide-loaded, mineralized nanofiber segment group than that of fibrous connective tissue in the unfilled defect group. Altogether, the mineralized nanofiber segments coupled with E7-BMP-2 peptides may be an effective treatment option for alveolar bone loss and defects. STATEMENT OF SIGNIFICANCE: With the high incidence of dental implants/fixtures for missing teeth, the success of the surgical procedures in restorative dentistry is dictated by the quality and quantity of the supporting alveolar bone. To address the problem of alveolar bone loss and defects due to tumor, periodontitis, or even postextraction remodeling, the present study is the first report on the application of mineralized nanofiber fragments coupled with calcium-binding osteoinductive BMP-2 peptides as a synthetic graft material for oral bone regeneration. The ease of fabrication and application of cryocut mineralized nanofiber fragments as maxillofacial bone defect fillers present a promising alternative to the current dental bone graft formulations. Furthermore, the nanofiber segments may also be utilized for several biomedical applications including hemostasis, soft tissue engineering, and wound healing.

The DNMT3A R882H mutant displays altered flanking sequence preferences.

The DNMT3A R882H mutation is frequently observed in acute myeloid leukemia (AML). It is located in the subunit and DNA binding interface of DNMT3A and has been reported to cause a reduction in activity and dominant negative effects. We investigated the mechanistic consequences of the R882H mutation on DNMT3A showing a roughly 40% reduction in overall DNA methylation activity. Biochemical assays demonstrated that R882H does not change DNA binding affinity, protein stability or subnuclear distribution of DNMT3A. Strikingly, DNA methylation experiments revealed pronounced changes in the flanking sequence preference of the DNMT3A-R882H mutant. Based on these results, different DNA substrates with selected flanking sequences were designed to be favored or disfavored by R882H. Kinetic analyses showed that the R882H favored substrate was methylated by R882H with 45% increased rate when compared with wildtype DNMT3A, while methylation of the disfavored substrate was reduced 7-fold. Our data expand the model of the potential carcinogenic effect of the R882H mutation by showing CpG site specific activity changes. This result suggests that R882 is involved in the indirect readout of flanking sequence preferences of DNMT3A and it may explain the particular enrichment of the R882H mutation in cancer patients by revealing mutation specific effects.

Flowering Time Gene Variation in Brassica Species Shows Evolutionary Principles.

Flowering time genes have a strong influence on successful reproduction and life cycle adaptation. However, their regulation is highly complex and only well understood in diploid model systems. For crops with a polyploid background from the genus Brassica, data on flowering time gene variation are scarce, although indispensable for modern breeding techniques like marker-assisted breeding. We have deep-sequenced all paralogs of 35 Arabidopsis thaliana flowering regulators using Sequence Capture followed by Illumina sequencing in two selected accessions of the vegetable species Brassica rapa and Brassica oleracea, respectively. Using these data, we were able to call SNPs, InDels and copy number variations (CNVs) for genes from the total flowering time network including central flowering regulators, but also genes from the vernalisation pathway, the photoperiod pathway, temperature regulation, the circadian clock and the downstream effectors. Comparing the results to a complementary data set from the allotetraploid species Brassica napus, we detected rearrangements in B. napus which probably occurred early after the allopolyploidisation event. Those data are both a valuable resource for flowering time research in those vegetable species, as well as a contribution to speciation genetics.

Loss-of-function but not dominant-negative intragenic IKZF1 deletions are associated with an adverse prognosis in adult BCR-ABL-negative acute lymphoblastic leukemia.

Genetic alterations of the transcription factor IKZF1 ("IKAROS") are detected in around 15-30% of cases of BCR-ABL-negative B-cell precursor acute lymphoblastic leukemia. Different types of intragenic deletions have been observed, resulting in a functionally inactivated allele ("loss-of-function") or in "dominant-negative" isoforms. The prognostic impact of these alterations especially in adult acute lymphoblastic leukemia is not well defined. We analyzed 482 well-characterized cases of adult BCR-ABL-negative B-precursor acute lymphoblastic leukemia uniformly treated in the framework of the GMALL studies and detected IKZF1 alterations in 128 cases (27%). In 20%, the IKZF1 alteration was present in a large fraction of leukemic cells ("high deletion load") while in 7% it was detected only in small subclones ("low deletion load"). Some patients showed more than one IKZF1 alteration (8%). Patients exhibiting a loss-of-function isoform with high deletion load had a shorter overall survival (OS at 5 years 28% vs. 59%; P<0.0001), also significant in a subgroup analysis of standard risk patients according to GMALL classification (OS at 5 years 37% vs. 68%; P=0.0002). Low deletion load or dominant-negative IKZF1 alterations had no prognostic impact. The results thus suggest that there is a clear distinction between loss-of-function and dominant-negative IKZF1 deletions. Affected patients should thus be monitored for minimal residual disease carefully to detect incipient relapses at an early stage and they are potential candidates for alternative or intensified treatment regimes. (clinicaltrials.gov identifiers: 00199056 and 00198991).

Three Novel Species with Peptidoglycan Cell Walls form the New Genus Lacunisphaera gen. nov. in the Family Opitutaceae of the Verrucomicrobial Subdivision 4.

The cell wall of free-living bacteria consists of peptidoglycan (PG) and is critical for maintenance of shape as dissolved solutes cause osmotic pressure and challenge cell integrity. Surprisingly, the subdivision 4 of the phylum Verrucomicrobia appears to be exceptional in this respect. Organisms of this subdivision are described to be devoid of muramic or diaminopimelic acid (DAP), usually found as components of PG in bacterial cell walls. Here we describe three novel bacterial strains from a freshwater lake, IG15T, IG16bT, and IG31T, belonging to a new genus in the subdivision 4 of Verrucomicrobia which we found to possess PG as part of their cell walls. Biochemical analysis revealed the presence of DAP not only in these novel strains, but also in Opitutus terrae PB90-1T, the closest described relative of strains IG15T, IG16bT, and IG31T. Furthermore, we found that nearly all genes necessary for peptidoglycan synthesis are present in genomes of subdivision 4 members, as well as in the complete genome sequence of strain IG16bT. In addition, we isolated and visualized PG-sacculi for strain IG16bT. Thus, our results challenge the concept of peptidoglycan-less free-living bacteria. Our polyphasic taxonomy approach places the novel strains in a new genus within the family Opitutaceae, for which the name Lacunisphaera gen. nov. is proposed. Strain designations for IG15T, IG16bT and IG31T are Lacunisphaera parvula sp. nov. (=DSM 26814 = LMG 29468), L. limnophila sp. nov. (=DSM 26815 = LMG 29469) and L. anatis sp. nov. (=DSM 103142 = LMG 29578) respectively, with L. limnophila IG16bT being the type species of the genus.

Targeted deep sequencing of flowering regulators in Brassica napus reveals extensive copy number variation.

Gene copy number variation (CNV) is increasingly implicated in control of complex trait networks, particularly in polyploid plants like rapeseed (Brassica napus L.) with an evolutionary history of genome restructuring. Here we performed sequence capture to assay nucleotide variation and CNV in a panel of central flowering time regulatory genes across a species-wide diversity set of 280 B. napus accessions. The genes were chosen based on prior knowledge from Arabidopsis thaliana and related Brassica species. Target enrichment was performed using the Agilent SureSelect technology, followed by Illumina sequencing. A bait (probe) pool was developed based on results of a preliminary experiment with representatives from different B. napus morphotypes. A very high mean target coverage of ~670x allowed reliable calling of CNV, single nucleotide polymorphisms (SNPs) and insertion-deletion (InDel) polymorphisms. No accession exhibited no CNV, and at least one homolog of every gene we investigated showed CNV in some accessions. Some CNV appear more often in specific morphotypes, indicating a role in diversification.

Post-polyploidisation morphotype diversification associates with gene copy number variation.

Genetic models for polyploid crop adaptation provide important information relevant for future breeding prospects. A well-suited model is Brassica napus, a recent allopolyploid closely related to Arabidopsis thaliana. Flowering time is a major adaptation trait determining life cycle synchronization with the environment. Here we unravel natural genetic variation in B. napus flowering time regulators and investigate associations with evolutionary diversification into different life cycle morphotypes. Deep sequencing of 35 flowering regulators was performed in 280 diverse B. napus genotypes. High sequencing depth enabled high-quality calling of single-nucleotide polymorphisms (SNPs), insertion-deletions (InDels) and copy number variants (CNVs). By combining these data with genotyping data from the Brassica 60 K Illumina® Infinium SNP array, we performed a genome-wide marker distribution analysis across the 4 ecogeographical morphotypes. Twelve haplotypes, including Bna.FLC.A10, Bna.VIN3.A02 and the Bna.FT promoter on C02_random, were diagnostic for the diversification of winter and spring types. The subspecies split between oilseed/kale (B. napus ssp. napus) and swedes/rutabagas (B. napus ssp. napobrassica) was defined by 13 haplotypes, including genomic rearrangements encompassing copies of Bna.FLC, Bna.PHYA and Bna.GA3ox1. De novo variation in copies of important flowering-time genes in B. napus arose during allopolyploidisation, enabling sub-functionalisation that allowed different morphotypes to appropriately fine-tune their lifecycle.

Efficient targeted DNA methylation with chimeric dCas9-Dnmt3a-Dnmt3L methyltransferase.

DNA methylation plays a critical role in the regulation and maintenance of cell-type specific transcriptional programs. Targeted epigenome editing is an emerging technology to specifically regulate cellular gene expression in order to modulate cell phenotypes or dissect the epigenetic mechanisms involved in their control. In this work, we employed a DNA methyltransferase Dnmt3a-Dnmt3L construct fused to the nuclease-inactivated dCas9 programmable targeting domain to introduce DNA methylation into the human genome specifically at the EpCAM, CXCR4 and TFRC gene promoters. We show that targeting of these loci with single gRNAs leads to efficient and widespread methylation of the promoters. Multiplexing of several guide RNAs does not increase the efficiency of methylation. Peaks of targeted methylation were observed around 25 bp upstream and 40 bp downstream of the PAM site, while 20-30 bp of the binding site itself are protected against methylation. Potent methylation is dependent on the multimerization of Dnmt3a/Dnmt3L complexes on the DNA. Furthermore, the introduced methylation causes transcriptional repression of the targeted genes. These new programmable epigenetic editors allow unprecedented control of the DNA methylation status in cells and will lead to further advances in the understanding of epigenetic signaling.

Staphylococcus aureus Exploits a Non-ribosomal Cyclic Dipeptide to Modulate Survival within Epithelial Cells and Phagocytes.

Community-acquired (CA) Staphylococcus aureus cause various diseases even in healthy individuals. Enhanced virulence of CA-strains is partly attributed to increased production of toxins such as phenol-soluble modulins (PSM). The pathogen is internalized efficiently by mammalian host cells and intracellular S. aureus has recently been shown to contribute to disease. Upon internalization, cytotoxic S. aureus strains can disrupt phagosomal membranes and kill host cells in a PSM-dependent manner. However, PSM are not sufficient for these processes. Here we screened for factors required for intracellular S. aureus virulence. We infected escape reporter host cells with strains from an established transposon mutant library and detected phagosomal escape rates using automated microscopy. We thereby, among other factors, identified a non-ribosomal peptide synthetase (NRPS) to be required for efficient phagosomal escape and intracellular survival of S. aureus as well as induction of host cell death. By genetic complementation as well as supplementation with the synthetic NRPS product, the cyclic dipeptide phevalin, wild-type phenotypes were restored. We further demonstrate that the NRPS is contributing to virulence in a mouse pneumonia model. Together, our data illustrate a hitherto unrecognized function of the S. aureus NRPS and its dipeptide product during S. aureus infection.

cDNA Library Enrichment of Full Length Transcripts for SMRT Long Read Sequencing.

The utility of genome assemblies does not only rely on the quality of the assembled genome sequence, but also on the quality of the gene annotations. The Pacific Biosciences Iso-Seq technology is a powerful support for accurate eukaryotic gene model annotation as it allows for direct readout of full-length cDNA sequences without the need for noisy short read-based transcript assembly. We propose the implementation of the TeloPrime Full Length cDNA Amplification kit to the Pacific Biosciences Iso-Seq technology in order to enrich for genuine full-length transcripts in the cDNA libraries. We provide evidence that TeloPrime outperforms the commonly used SMARTer PCR cDNA Synthesis Kit in identifying transcription start and end sites in Arabidopsis thaliana. Furthermore, we show that TeloPrime-based Pacific Biosciences Iso-Seq can be successfully applied to the polyploid genome of bread wheat (Triticum aestivum) not only to efficiently annotate gene models, but also to identify novel transcription sites, gene homeologs, splicing isoforms and previously unidentified gene loci.

miRNA expression profiling of Epstein-Barr virus-associated NKTL cell lines by Illumina deep sequencing.

The aim of this work was to establish the microRNA profile of SNK6 and SNT16, two Epstein-Barr virus (EBV)-infected cell lines derived from nasal NK/T-cell lymphoma (NKTL). The oncogenic EBV is strongly associated with the pathogenesis of nasal and extranodal NK/T-cell lymphoma and expresses 44 mature microRNAs and two noncoding EBV-encoded RNAs (EBERs). miRNAs are 19-25nt noncoding RNAs that affect host and viral gene expression post-transcriptionally. Deregulated miRNA patterns are frequently linked to a variety of human cancers including lymphomas. miRNA profiling of the two NK/T cell lines vs. primary cells revealed 10 and 4 up-regulated and 10 and 12 down-regulated miRNAs in SNK6 and SNT16 cells respectively. The results were validated by qRT-PCR for selected miRNAs. Target gene analyses confirmed cullin 5 (CUL5) and sphingosin-1-phosphate receptor 1 (S1PR1) as targets for the down-regulated hsa-miR-148a and viral ebv-miR-BART16 respectively. As recently demonstrated for the regulation of IL1-alpha by miR-142-3p, coexpression of the EBERs selectively exerted corepression of S1PR1 by BART16 but not of CUL5 by miR-148a, indicating selective corepression by the EBERs.

Methylation Markers for the Identification of Body Fluids and Tissues from Forensic Trace Evidence.

The identification of body fluids is an essential tool for clarifying the course of events at a criminal site. The analytical problem is the fact that the biological material has been very often exposed to detrimental exogenous influences. Thereby, the molecular substrates used for the identification of the traces may become degraded. So far, most protocols utilize cell specific proteins or RNAs. Instead of measuring these more sensitive compounds this paper describes the application of the differential DNA-methylation. As a result of two genome wide screenings with the Illumina HumanMethylation BeadChips 27 and 450k we identified 150 candidate loci revealing differential methylation with regard to the body fluids venous blood, menstrual blood, vaginal fluid, saliva and sperm. Among them we selected 9 loci as the most promising markers. For the final determination of the methylation degree we applied the SNuPE-method. Because the degree of methylation might be modified by various endogenous and exogenous factors, we tested each marker with approximately 100 samples of each target fluid in a validation study. The stability of the detection procedure is proved in various simulated forensic surroundings according to standardized conditions. We studied the potential influence of 12 relatively common tumors on the methylation of the 9 markers. For this purpose the target fluids of 34 patients have been analysed. Only the cervix carcinoma might have an remarkable effect because impairing the signal of both vaginal markers. Using the Illumina MiSeq device we tested the potential influence of cis acting sequence variants on the methylation degree of the 9 markers in the specific body fluid DNA of 50 individuals. For 4 marker loci we observed such an influence either by sole SNPs or haplotypes. The identification of each target fluid is possible in arbitrary mixtures with the remaining four body fluids. The sensitivity of the individual body fluid tests is in the same range as for the forensic STR-analysis. It is the first forensic body fluid protocol which considers the exogenic and endogenic parameters potentially interfering with the true results.