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Periodontitis (PD) is a severe inflammatory gum pathology that damages the periodontal soft tissue and bone. It is highly prevalent in the US, affecting more than 47% of adults. Besides routine scaling and root planing, there are few effective treatments for PD. Developed as an effective treatment for hyperlipidemia, simvastatin (SIM) is also known for its well-established anti-inflammatory and osteogenic properties, suggesting its potential utility in treating PD. Its clinical translation, however, has been impeded by its poor water-solubility, lack of osteotropicity, and side effects (e.g., hepatoxicity) associated with systemic exposure. To address these challenges, an N -(2-hydroxypropyl) methacrylamide (HPMA) copolymer-based thermoresponsive polymeric prodrug of SIM (ProGel-SIM) was developed as a local therapy for PD. Its aqueous solution is free-flowing at 4 °C and transitions into a hydrogel at ∼30 °C, allowing for easy local application and retention. After a thorough characterization of its physicochemical properties, ProGel-SIM was administered weekly into the periodontal pocket of an experimental rat model of PD. At 3 weeks post initiation of the treatment, the animals were euthanized with palate isolated for μ-CT and histological analyses. When compared to dose equivalent simvastatin acid (SMA, active form of SIM) treatment, the rats in the ProGel-SIM treated group showed significantly higher periodontal bone volume (0.34 mm 3 vs 0.20 mm 3 , P = 0.0161) and less neutrophil (PMN) infiltration ( P < 0.0001) and IL-1β secretion ( P = 0.0036). No measurable side effect was observed. Collectively, these results suggest that ProGel-SIM may be developed as a promising drug candidate for the effective clinical treatment of PD.

Background: The purpose of this 6-week, single-blinded, randomized clinical trial was to determine if the use of an interproximal brush, with or without a tracking device, is more effective than an oral irrigator in improving interproximal probing depth (PD), clinical attachment level (CAL), plaque index (PI), gingival index (GI), bleeding on probing (BOP), and inflammatory markers., Methods: Seventy-six patients with Stages III-IV, Grade B periodontitis and a 5-7 mm posterior interproximal PD with BOP were randomized: (1) interproximal brush alone (IB; n = 26), (2) interproximal brush with tracking device (TD; n = 23), (3) oral irrigator (OI; n = 27). Participants used devices once daily for 6 weeks. Clinical measurements (PD, CAL, PI, BOP, GI) and gingival crevicular fluid (GCF) samples were collected at baseline and 6 weeks., Results: All groups showed a significant reduction in PD and CAL (≥1.1 mm, p < 0.0001) and improvement in BOP (≥56%, p < 0.0001) and GI (≥82%, p < 0.001) at the experimental site with no differences among groups. The IB and IB+TD groups showed a significant reduction in PI (≥0.9, p ≤ 0.01). Interleukin (IL)-1β was reduced in all groups (p = 0.006), but IB+TB more than OI (p ≤ 0.05). IL-10 was reduced among all groups (p = 0.01), while interferon-gamma significantly increased (p = 0.01) in all groups., Conclusions: IB and OI improved clinical parameters of PD and CAL and reduced inflammatory markers (BOP, GI, GCF IL-1β). IB had better interproximal plaque reduction. Tracking did not significantly improve clinical parameters compared with the IB and OI groups, suggesting future modifications are needed., (© 2023 The Authors. Journal of Periodontology published by Wiley Periodicals LLC on behalf of American Academy of Periodontology.)

Differences in transcriptomes, transcription factor usage, and function have identified T follicular helper 2 (Tfh2) cells and T helper 2 (Th2) cells as distinct clusters of differentiation 4+",(CD4) T-cell subsets in settings of type-2 inflammation. Although the transcriptional programs driving Th2 cell differentiation and cytokine production are well defined, dependence on these classical Th2 programs by Tfh2 cells is less clear. Using cytokine reporter mice in combination with transcription factor inference analysis, the b-Zip transcription factor c-Maf and its targets were identified as an important regulon in both Th2 and Tfh2 cells. Conditional deletion of c-Maf in T cells confirmed its importance in type-2 cytokine expression by Th2 and Tfh2 cells. However, while c-Maf was not required for Th2-driven helminth clearance or lung eosinophilia, it was required for Tfh2-driven Immunoglobulin E production and germinal center formation. This differential regulation of cell-mediated and humoral immunity by c-Maf was a result of redundant pathways in Th2 cells that were absent in Tfh2 cells, and c-Maf-specific mechanisms in Tfh2 cells that were absent in Th2 cells. Thus, despite shared expression by Tfh2 and Th2 cells, c-Maf serves as a unique regulator of Tfh2-driven humoral hallmarks during type-2 immunity., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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-1β, 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., (© 2022 The Authors. Journal of Periodontology published by Wiley Periodicals LLC on behalf of American Academy of Periodontology.)

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.

Immune system molecules are expressed by neurons, yet their functions are often unknown. We have identified IL-13 and its receptor IL-13Ra1 as neuronal, synaptic proteins in mouse, rat, and human brains, whose engagement upregulates the phosphorylation of NMDAR and AMPAR subunits and, in turn, increases synaptic activity and CREB-mediated transcription. We demonstrate that increased IL-13 is a hallmark of traumatic brain injury (TBI) in male mice as well as in two distinct cohorts of human patients. We also provide evidence that IL-13 upregulation protects neurons from excitotoxic death. We show IL-13 upregulation occurring in several cohorts of human brain samples and in cerebrospinal fluid (CSF). Thus, IL-13 is a physiological modulator of synaptic physiology of neuronal origin, with implications for the establishment of synaptic plasticity and the survival of neurons under injury conditions. Furthermore, we suggest that the neuroprotection afforded through the upregulation of IL-13 represents an entry point for interventions in the pathophysiology of TBI., (© 2023. The Author(s).)

Background: The purpose of this double-masked, randomized, controlled trial was to determine if the local application of simvastatin (SIM), combined with minimally invasive papilla reflection and root planing (PR/RP), is effective in improving clinical attachment level (CAL), probing depth (PD) reduction, and increasing interproximal bone height (IBH) in persistent 6-9 mm periodontal pockets in patients receiving periodontal maintenance therapy (PMT)., Methods: Fifty patients with Stage III, Grade B periodontitis presenting with a 6-9 mm interproximal PD with a history of bleeding on probing (BOP) were included in the study. Experimental [PR/RP+SIM/methylcellulose (MCL); n = 27] and control (PR/RP+MCL; n = 23) therapies were randomly assigned. Root surfaces were accessed via reflection of interproximal papillae, followed by RP assisted with endoscope evaluation, acid etching, and SIM/MCL or MCL application. CAL, PD, BOP, plaque presence, and IBH (using standardized vertical bitewing radiographs) were evaluated at baseline and 12 months. Measurements were compared by group and time using Chi-square, Wilcoxon rank-sum, and t-tests., Results: Both PR/RP+SIM/MCL and PR/RP+MCL, respectively, resulted in improvements in clinical outcomes (CAL: -1.9 ± 0.3 mm, p < 0.0001; -1.0 ± 0.3 mm, p < 0.003; PD: -2.3 mm ± 0.3, p < 0.0001; -1.3 mm ± 0.3, p < 0.0001; BOP: -58.7%; -41.7%, p < 0.05) and stable IBH (-0.2 ± 0.12, -0.4 ± 0.2, p = 0.22) from baseline to 12 months post-therapy. PR/RP+SIM/MCL had more improvement in CAL (p = 0.03), PD (p = 0.007), and BOP (p = 0.047)., Conclusions: The addition of SIM/MCL to PR/RP improved CAL, PD, and BOP compared with PR/RP alone in periodontal maintenance patients., (© 2022 The Authors. Journal of Periodontology published by Wiley Periodicals LLC on behalf of American Academy of Periodontology.)

Large quantities of organic matter are continuously deposited, and (a)biotic gradients intersect in the soil-rhizosphere, where biodegradation contributes to the global cycles of elements. The betaproteobacterial genus Aromatoleum comprises cosmopolitan, facultative denitrifying degradation specialists. Aromatoleum aromaticum. pCyN1 stands out for anaerobically decomposing plant-derived monoterpenes in addition to monoaromatic hydrocarbons, polar aromatics and aliphatics. The catabolic network's structure and flexibility in A. aromaticum pCyN1 were studied across 34 growth conditions by superimposing proteome profiles onto the manually annotated 4.37 Mbp genome. Strain pCyN1 employs three fundamentally different enzymes for C-H-bond cleavage at the methyl groups of p-cymene/4-ethyltoluene, toluene and p-cresol respectively. Regulation of degradation modules displayed substrate specificities ranging from narrow (toluene and cyclohexane carboxylate) via medium-wide (one module shared by p-cymene, 4-ethyltoluene, α-phellandrene, α-terpinene, γ-terpinene and limonene) to broad (central benzoyl-CoA pathway serving 16 aromatic substrates). Remarkably, three variants of ATP-dependent (class I) benzoyl-CoA reductase and four different β-oxidation routes establish a degradation hub that accommodates the substrate diversity. The respiratory system displayed several conspicuous profiles, e.g. the presence of nitrous oxide reductase under oxic and of low-affinity oxidase under anoxic conditions. Overall, nutritional versatility in conjunction with network regulation endow A. aromaticum pCyN1 with broad adaptability., (© 2022 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Copepods encompass numerous ecological roles including parasites, detrivores and phytoplankton grazers. Nonetheless, copepod genome assemblies remain scarce. Lepeophtheirus salmonis is an economically and ecologically important ectoparasitic copepod found on salmonid fish. We present the 695.4 Mbp L. salmonis genome assembly containing ≈60% repetitive regions and 13,081 annotated protein-coding genes. The genome comprises 14 autosomes and a ZZ-ZW sex chromosome system. Assembly assessment identified 92.4% of the expected arthropod genes. Transcriptomics supported annotation and indicated a marked shift in gene expression after host attachment, including apparent downregulation of genes related to circadian rhythm coinciding with abandoning diurnal migration. The genome shows evolutionary signatures including loss of genes needed for peroxisome biogenesis, presence of numerous FNII domains, and an incomplete heme homeostasis pathway suggesting heme proteins to be obtained from the host. Despite repeated development of resistance against chemical treatments L. salmonis exhibits low numbers of many genes involved in detoxification., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: To track plaque scores on a subset of teeth in general dental practice patients to determine if plaque scores could improve along with periodontal, restorative and extraction outcomes., Methods: Percentage of surfaces with subgingival plaque were recorded and graphed on five teeth (#3, 8, 14, 19, 30) at each appointment, followed by focused oral hygiene instructions, in 343 patients over a 5-10-year period. Patient age, gender, prophylaxes/year, and experimental teeth periodontitis stage, % 4-5 and ≥ 6 mm pockets, % bleeding on probing, % surfaces restored and patients with extractions were recorded. Relationships among average plaque scores and the longitudinal periodontal, restorative and extraction changes were analyzed using Chi-Square, Kruskal-Wallis, and Wilcoxon Rank Sum tests., Results: Plaque scores improved from median 40% to 25% (P< 0.0001) over the 5-10 years. Plaque scores and periodontitis stages were associated (P= 0.03) with few periodontally healthy patients (9%) having poor plaque scores (> 50% plaque surfaces). Furthermore, good plaque scores (≤ 25%) and periodontal health (Stage I) were linked to the need for few restorations (P< 0.0001), while prophylaxes/year had no significant relationship. Extractions were related more with Stage III/IV (advanced) periodontitis (P< 0.0001) than with plaque score (NS)., Clinical Significance: In a general dental practice, tracking plaque scores at each appointment on a subset of representative teeth can be time-efficient, and is associated with improved oral hygiene, stable periodontal status and reduced restorative needs., Competing Interests: The authors declared no conflict of interest., (Copyright©American Journal of Dentistry.)

RNA helicases play crucial functions in RNA biology. In plants, RNA helicases are encoded by large gene families, performing roles in abiotic stress responses, development, the post-transcriptional regulation of gene expression as well as house-keeping functions. Several of these RNA helicases are targeted to the organelles, mitochondria and chloroplasts. Cyanobacteria are the direct evolutionary ancestors of plant chloroplasts. The cyanobacterium Synechocystis 6803 encodes a single DEAD-box RNA helicase, CrhR, that is induced by a range of abiotic stresses, including low temperature. Though the ΔcrhR mutant exhibits a severe cold-sensitive phenotype, the physiological function(s) performed by CrhR have not been described. To identify transcripts interacting with CrhR, we performed RNA co-immunoprecipitation with extracts from a Synechocystis crhR deletion mutant expressing the FLAG-tagged native CrhR or a K57A mutated version with an anticipated enhanced RNA binding. The composition of the interactome was strikingly biased towards photosynthesis-associated and redox-controlled transcripts. A transcript highly enriched in all experiments was the crhR mRNA, suggesting an auto-regulatory molecular mechanism. The identified interactome explains the described physiological role of CrhR in response to the redox poise of the photosynthetic electron transport chain and characterizes CrhR as an enzyme with a diverse range of transcripts as molecular targets., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Except for routine scaling and root planing, there are few effective nonsurgical therapeutic interventions for periodontitis and associated alveolar bone loss. Simvastatin (SIM), one of the 3-hydroxy-3-methylglutaryl-cosenzyme A reductase inhibitors, which is known for its capacity as a lipid-lowering medication, has been proven to be an effective anti-inflammatory and bone anabolic agent that has shown promising benefits in mitigating periodontal bone loss. The local delivery of SIM into the periodontal pocket, however, has been challenging due to SIM's poor water solubility and its lack of osteotropicity. To overcome these issues, we report a novel SIM formulation of a thermoresponsive, osteotropic, injectable hydrogel (PF127) based on pyrophosphorolated pluronic F127 (F127-PPi). After mixing F127-PPi with F127 at a 1:1 ratio, the resulting PF127 was used to dissolve free SIM to generate the SIM-loaded formulation. The thermoresponsive hydrogel's rheologic behavior, erosion and SIM release kinetics, osteotropic property, and biocompatibility were evaluated in vitro . The therapeutic efficacy of SIM-loaded PF127 hydrogel on periodontal bone preservation and inflammation resolution was validated in a ligature-induced periodontitis rat model. Given that SIM is already an approved medication for hyperlipidemia, the data presented here support the translational potential of the SIM-loaded PF127 hydrogel for better clinical management of periodontitis and associated pathologies.

Several studies have attempted to understand the origin and evolution of single-exon genes (SEGs) in eukaryotic organisms, including fishes, but few have examined the functional and evolutionary relationships between SEGs and multiple-exon gene (MEG) paralogs, in particular the conservation of promoter regions. Given that SEGs originate via the reverse transcription of mRNA from a "parental" MEGs, such comparisons may enable identifying evolutionarily-related SEG/MEG paralogs, which might fulfill equivalent physiological functions. Here, the relationship of SEG proportion with MEG count, gene density, intron count, and chromosome size was assessed for the genome of the European sea bass, Dicentrarchus labrax . Then, SEGs with an MEG parent were identified, and promoter sequences of SEG/MEG paralogs were compared, to identify highly conserved functional motifs. The results revealed a total count of 1,585 (8.3% of total genes) SEGs in the European sea bass genome, which was correlated with MEG count but not with gene density. The significant correlation of SEG content with the number of MEGs suggests that SEGs were continuously and independently generated over evolutionary time following species divergence through retrotranscription events, followed by tandem duplications. Functional annotation showed that the majority of SEGs are functional, as is evident from their expression in RNA-seq data used to support homology-based genome annotation. Differences in 5'UTR and 3'UTR lengths between SEG/MEG paralogs observed in this study may contribute to gene expression divergence between them and therefore lead to the emergence of new SEG functions. The comparison of nonsynonymous to synonymous changes (Ka/Ks) between SEG/MEG parents showed that 74 of them are under positive selection (Ka/Ks > 1; p  = .0447). An additional fifteen SEGs with an MEG parent have a common promoter, which implies that they are under the influence of common regulatory networks., Competing Interests: Eventual conflicts of interest (including personal communications or additional permissions, related manuscripts), sources of financial support, corporate involvement, and patent holdings are disclosed., (© 2021 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

The genus Desulfonema belongs to the deltaproteobacterial family Desulfobacteraceae and comprises marine, sulfate-reducing bacteria that form filaments and move by gliding. This study reports on the complete, manually annotated genomes of Dn. limicola 5ac10T (6.91 Mbp; 6,207 CDS) and Dn. magnum 4be13T (8.03 Mbp; 9,970 CDS), integrated with substrate-specific proteome profiles (8 vs. 11). The richness in mobile genetic elements is shared with other Desulfobacteraceae members, corroborating horizontal gene transfer as major driver in shaping the genomes of this family. The catabolic networks of Dn. limicola and Dn. magnum have the following general characteristics: 98 versus 145 genes assigned (having genomic shares of 1.7 vs. 2.2%), 92.5 versus 89.7% proteomic coverage, and scattered gene clusters for substrate degradation and energy metabolism. The Dn. magnum typifying capacity for aromatic compound degradation (e.g., p-cresol, 3-phenylpropionate) requires 48 genes organized in operon-like structures (87.7% proteomic coverage; no homologs in Dn. limicola). The protein complements for aliphatic compound degradation, central pathways, and energy metabolism are highly similar between both genomes and were identified to a large extent (69-96%). The differential protein profiles revealed a high degree of substrate-specificity for peripheral reaction sequences (forming central intermediates), agreeing with the high number of sensory/regulatory proteins predicted for both strains. By contrast, central pathways and modules of the energy metabolism were constitutively formed under the tested substrate conditions. In accord with their natural habitats that are subject to fluctuating changes of physicochemical parameters, both Desulfonema strains are well equipped to cope with various stress conditions. Next to superoxide dismutase and catalase also desulfoferredoxin and rubredoxin oxidoreductase are formed to counter exposure to molecular oxygen. A variety of proteases and chaperones were detected that function in maintaining cellular homeostasis upon heat or cold shock. Furthermore, glycine betaine/proline betaine transport systems can respond to hyperosmotic stress. Gliding movement probably relies on twitching motility via type-IV pili or adventurous motility. Taken together, this proteogenomic study demonstrates the adaptability of Dn. limicola and Dn. magnum to its dynamic habitats by means of flexible catabolism and extensive stress response capacities., (© 2021 The Author(s) Published by S. Karger AG, Basel.)

The betaproteobacterial genus Aromatoleum comprises facultative denitrifiers specialized in the anaerobic degradation of recalcitrant organic compounds (aromatic and terpenoid). This study reports on the complete and manually annotated genomes of Ar. petrolei ToN1T (5.41 Mbp) and Ar. bremense PbN1T (4.38 Mbp), which cover the phylogenetic breadth of the genus Aromatoleum together with previously genome sequenced Ar. aromaticum EbN1T [Rabus et al., Arch Microbiol. 2005 Jan;183(1):27-36]. The gene clusters for the anaerobic degradation of aromatic and terpenoid (strain ToN1T only) compounds are scattered across the genomes of strains ToN1T and PbN1T. The richness in mobile genetic elements is shared with other Aromatoleum spp., substantiating that horizontal gene transfer should have been a major driver in shaping the genomes of this genus. The composite catabolic network of strains ToN1T and PbN1T comprises 88 proteins, the coding genes of which occupy 86.1 and 76.4 kbp (1.59 and 1.75%) of the respective genome. The strain-specific gene clusters for anaerobic degradation of ethyl-/propylbenzene (strain PbN1T) and toluene/monoterpenes (strain ToN1T) share high similarity with their counterparts in Ar. aromaticum strains EbN1T and pCyN1, respectively. Glucose is degraded via the ED-pathway in strain ToN1T, while gluconeogenesis proceeds via the reverse EMP-pathway in strains ToN1T, PbN1T, and EbN1T. The diazotrophic, endophytic lifestyle of closest related genus Azoarcus is known to be associated with nitrogenase and type-6 secretion system (T6SS). By contrast, strains ToN1T, PbN1T, and EbN1T lack nif genes for nitrogenase (including cofactor synthesis and enzyme maturation). Moreover, strains PbN1T and EbN1T do not possess tss genes for T6SS, while strain ToN1T does and facultative endophytic "Aromatoleum" sp. CIB is known to even have both. These findings underpin the functional heterogeneity among Aromatoleum members, correlating with the high plasticity of their genomes., (© 2021 The Author(s) Published by S. Karger AG, Basel.)

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., (Copyright © 2020 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Periodontitis is a chronic inflammatory disease caused by complex interactions between the host immune system and pathogens that affect the integrity of periodontium. To prevent disease progression and thus preserve alveolar bone structure, simultaneous anti-inflammatory and osteogenic intervention are essential. Hence, a glycogen synthase kinase 3 beta inhibitor (BIO) was selected as a potent inflammation modulator and osteogenic agent to achieve this treatment objective. BIO's lack of osteotropicity, poor water solubility, and potential long-term systemic side effects, however, have hampered its clinical applications. To address these limitations, pyrophosphorylated Pluronic F127 (F127-PPi) was synthesized and mixed with regular F127 to prepare an injectable and thermoresponsive hydrogel formulation (PF127) of BIO, which could adhere to hard tissue and gradually release BIO to exert its therapeutic effects locally. Comparing to F127 hydrogel, PF127 hydrogels exhibited stronger binding to hydroxyapatite (HA). Additionally, BIO's solubility in PF127 solution was dramatically improved over F127 solution and the improvement was proportional to the polymer concentration. When evaluated on a rat model of periodontitis, PF127-BIO hydrogel treatment was found to be very effective in preserving alveolar bone and ligament, and preventing periodontal inflammation, as shown by the micro-CT and histological data, respectively. Altogether, these findings suggested that the thermoresponsive PF127 hydrogel is an effective local drug delivery system for better clinical management of periodontitis and associated pathologies., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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-1β 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., (© 2020 American Academy of Periodontology.)

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., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The sRNA Yfr1 and members of the Yfr2 sRNA family are almost universally present within cyanobacteria. The conserved motifs of these sRNAs are nearly complementary to each other, suggesting their ability to participate in crosstalk. The conserved motif of Yfr1 is shared by members of the Yfr10 sRNA family, members of which are otherwise less conserved in sequence, structure, and synteny compared to Yfr1. The different structural properties enable the discrimination of unique targets of Yfr1 and Yfr10. Unlike most studied regulatory sRNAs, Yfr1 gene expression only slightly changes under the tested stress conditions and is present at high levels at all times. In contrast, cellular levels of Yfr10 increase during the course of acclimation to darkness, and levels of Yfr2 increase when cells are shifted to high light or nitrogen limitation conditions. In this study, we investigated the targetomes of Yfr2, Yfr1, and Yfr10 in Prochlorococcus MED4, establishing CRAFD-Seq as a new method for identifying direct targets of these sRNAs that is applicable to all bacteria, including those that are not amenable to genetic modification. The results suggest that these sRNAs are integrated within a regulatory network of unprecedented complexity in the adjustment of carbon and nitrogen-related primary metabolism.

Clearance of intracellular pathogens, such as Leishmania ( L .) major , depends on an immune response with well-regulated cytokine signaling. Here we describe a pathogen-mediated mechanism of evading CXCL10, a chemokine with diverse antimicrobial functions, including T cell recruitment. Infection with L. major in a human monocyte cell line induced robust CXCL10 transcription without increasing extracellular CXCL10 protein concentrations. We found that this transcriptionally independent suppression of CXCL10 is mediated by the virulence factor and protease, glycoprotein-63 ( gp63) . Specifically, GP63 cleaves CXCL10 after amino acid A81 at the base of a C-terminal alpha-helix. Cytokine cleavage by GP63 demonstrated specificity, as GP63 cleaved CXCL10 and its homologs, which all bind the CXCR3 receptor, but not distantly related chemokines, such as CXCL8 and CCL22. Further characterization demonstrated that CXCL10 cleavage activity by GP63 was produced by both extracellular promastigotes and intracellular amastigotes. Crucially, CXCL10 cleavage impaired T cell chemotaxis in vitro , indicating that cleaved CXCL10 cannot signal through CXCR3. Ultimately, we propose CXCL10 suppression is a convergent mechanism of immune evasion, as Salmonella enterica and Chlamydia trachomatis also suppress CXCL10. This commonality suggests that counteracting CXCL10 suppression may provide a generalizable therapeutic strategy against intracellular pathogens., Importance: Leishmaniasis, an infectious disease that annually affects over one million people, is caused by intracellular parasites that have evolved to evade the host's attempts to eliminate the parasite. Cutaneous leishmaniasis results in disfiguring skin lesions if the host immune system does not appropriately respond to infection. A family of molecules called chemokines coordinate recruitment of the immune cells required to eliminate infection. Here, we demonstrate a novel mechanism that Leishmania (L.) spp. employ to suppress host chemokines: a Leishmania- encoded protease cleaves chemokines known to recruit T cells that fight off infection. We observe that other common human intracellular pathogens, including Chlamydia trachomatis and Salmonella enterica , reduce levels of the same chemokines, suggesting a strong selective pressure to avoid this component of the immune response. Our study provides new insights into how intracellular pathogens interact with the host immune response to enhance pathogen survival.

Size and composition of γδ T cell populations change dramatically with tissue location, during development, and in disease. Given the functional differentiation of γδ T cell subsets, such shifts might alter the impact of γδ T cells on the immune system. To test this concept, and to determine if γδ T cells can affect other immune cells prior to an immune response, we examined non-immunized mice derived from strains with different genetically induced deficiencies in γδ T cells, for secondary changes in their immune system. We previously saw extensive changes in pre-immune antibodies and B cell populations. Here, we report effects on αβ T cells. Similarly to the B cells, αβ T cells evidently experience the influence of γδ T cells at late stages of their pre-immune differentiation, as single-positive heat stable antigen-low thymocytes. Changes in these and in mature αβ T cells were most prominent with memory-phenotype cells, including both CD8+ and CD4+ populations. As previously observed with B cells, most of the effects on αβ T cells were dependent on IL-4. Unexpectedly, IL-4 seemed to be produced mainly by αβ T cells in the non-immunized mice, albeit strongly regulated by γδ T cells. Similarly to our findings with B cells, changes of αβ T cells were less pronounced in mice lacking all γδ T cells than in mice lacking only some, suggesting that the composition of the γδ T cell population determines the nature of the γδ-influence on the other pre-immune lymphocytes., Competing Interests: The authors have declared that no competing interests exist.

In addition to the BAC-based reference sequence of the accession Columbia-0 from the year 2000, several short read assemblies of THE plant model organism Arabidopsis thaliana were published during the last years. Also, a SMRT-based assembly of Landsberg erecta has been generated that identified translocation and inversion polymorphisms between two genotypes of the species. Here we provide a chromosome-arm level assembly of the A. thaliana accession Niederzenz-1 (AthNd-1&#95;v2c) based on SMRT sequencing data. The best assembly comprises 69 nucleome sequences and displays a contig length of up to 16 Mbp. Compared to an earlier Illumina short read-based NGS assembly (AthNd-1&#95;v1), a 75 fold increase in contiguity was observed for AthNd-1&#95;v2c. To assign contig locations independent from the Col-0 gold standard reference sequence, we used genetic anchoring to generate a de novo assembly. In addition, we assembled the chondrome and plastome sequences. Detailed analyses of AthNd-1&#95;v2c allowed reliable identification of large genomic rearrangements between A. thaliana accessions contributing to differences in the gene sets that distinguish the genotypes. One of the differences detected identified a gene that is lacking from the Col-0 gold standard sequence. This de novo assembly extends the known proportion of the A. thaliana pan-genome., Competing Interests: The authors have declared that no competing interests exist.

Throughout the temperate zones, plants face combined drought and heat spells in increasing frequency and intensity. Here, we compared periodic (intermittent, i.e., high-frequency) versus chronic (continuous, i.e., high-intensity) drought-heat stress scenarios in gray poplar ( Populus × canescens ) plants for phenotypic and transcriptomic effects during stress and after recovery. Photosynthetic productivity after stress recovery exceeded the performance of poplar trees without stress experience. We analyzed the molecular basis of this stress-related memory phenotype and investigated gene expression responses across five major tree compartments including organs and wood tissues. For each of these tissue samples, transcriptomic changes induced by the two stress scenarios were highly similar during the stress phase but strikingly divergent after recovery. Characteristic molecular response patterns were found across tissues but involved different genes in each tissue. Only a small fraction of genes showed similar stress and recovery expression profiles across all tissues, including type 2C protein phosphatases, the LATE EMBRYOGENESIS ABUNDANT PROTEIN4-5 genes, and homologs of the Arabidopsis ( Arabidopsis thaliana ) transcription factor HOMEOBOX7. Analysis of the predicted transcription factor regulatory networks for these genes suggested that a complex interplay of common and tissue-specific components contributes to the coordination of post-recovery responses to stress in woody plants., (© 2019 American Society of Plant Biologists. All rights reserved.)

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., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

DNA methylation in bacteria is important for defense against foreign DNA, but is also involved in DNA repair, replication, chromosome partitioning, and regulatory processes. Thus, characterization of the underlying DNA methyltransferases in genetically tractable bacteria is of paramount importance. Here, we characterized the methylome and orphan methyltransferases in the model cyanobacterium Synechocystis sp. PCC 6803. Single molecule real-time (SMRT) sequencing revealed four DNA methylation recognition sequences in addition to the previously known motif m5CGATCG, which is recognized by M.Ssp6803I. For three of the new recognition sequences, we identified the responsible methyltransferases. M.Ssp6803II, encoded by the sll0729 gene, modifies GGm4CC, M.Ssp6803III, encoded by slr1803, represents the cyanobacterial dam-like methyltransferase modifying Gm6ATC, and M.Ssp6803V, encoded by slr6095 on plasmid pSYSX, transfers methyl groups to the bipartite motif GGm6AN7TTGG/CCAm6AN7TCC. The remaining methylation recognition sequence GAm6AGGC is probably recognized by methyltransferase M.Ssp6803IV encoded by slr6050. M.Ssp6803III and M.Ssp6803IV were essential for the viability of Synechocystis, while the strains lacking M.Ssp6803I and M.Ssp6803V showed growth similar to the wild type. In contrast, growth was strongly diminished of the Δsll0729 mutant lacking M.Ssp6803II. These data provide the basis for systematic studies on the molecular mechanisms impacted by these methyltransferases.

Purpose: The purpose of this study was to evaluate the effects of repeated scaling and root planing (SRP), with or without locally-delivered minocycline microspheres (MM) on residual pockets in patients undergoing periodontal maintenance (PMT). Methods: Patients on PMT were randomized into two groups for treatment of one posterior interproximal inflamed pocket (≥5 mm) with a history of bleeding on probing every 6 months: SRP plus MM (n=30) or exclusively SRP (n=30). Baseline and 24-month measurements included radiographic interproximal alveolar bone height, probing depths (PD), clinical attachment level (CAL), bleeding on probing (BOP), gingival crevicular fluid (GCF), and salivary interleukin (IL) - 1β, (24 month only). Results were analyzed for baseline data or change in measurements after 24 months of treatment between different treatment groups, as well as whether significant changes occurred after 24 months of treatment for each treatment group individually. Results: Alveolar bone height and GCF IL-1β remained stable over the 24 months. The SRP + MM and SRP groups each demonstrated reduced PD (0.8 ± 0.9 mm and 1.1 ±0.6 mm, respectively, p < 0.001 each), CAL (0.8 ± 0.9 mm and 1.0 ± 0.6 mm, respectively, p < 0.001 each) and BOP (55% and 48%, respectively, p = 0.001 each). However, there were no differences between groups over the 24-month study period. Conclusion: Scaling and root planning alone, of moderately inflamed periodontal pockets at 6-month intervals, produced stable interproximal alveolar bone height as well as sustained improvements in probing depths, clinical attachment level, bleeding on probing over 24 months; minocycline microspheres were not shown to enhance these results., (Copyright © 2018 The American Dental Hygienists’ Association.)

Purpose: Simvastatin (SIM), a HMG-CoA reductase inhibitor widely prescribed for hypercholesterolemia, has been reported to ameliorate inflammation and promote osteogenesis. Its clinical applications on these potential secondary indications, however, have been hampered by its lack of osteotropicity and poor water solubility. To address this challenge, we propose to design and evaluate the therapeutic efficacy of a novel simvastatin prodrug with better water solubility and bone affinity., Method: The prodrug (SIM-PPi) was synthesized by directly conjugating a SIM trimer to a pyrophosphate (PPi). It was characterized and evaluated in vitro for its water solubility, osteotropicity, toxicity, anti-inflammatory and osteoinductive properties. It was then tested for anti-inflammatory and osteoinductive properties in vivo by three weekly injections into gingiva of a ligature-induced experimental periodontitis rat model., Results: In vitro studies showed that SIM-PPi has greatly improved water-solubility of SIM and shows strong binding to hydroxyapatite (HA). In macrophage culture, SIM-PPi inhibited LPS-induced pro-inflammatory cytokines (IL-1β, IL-6). In osteoblast culture, it was found to significantly increase alkaline phosphatase (ALP) activity with accelerated mineral deposition, confirming the osteogenic potential of SIM-PPi. When tested in vivo on an experimental periodontal bone-loss model, SIM-PPi exhibited a superior prophylactic effect compared to dose equivalent SIM in reducing inflammatory cells and in preserving alveolar bone structure, as shown in the histological and micro-CT data., Conclusion: SIM-PPi may have the potential to be further developed for better clinical management of bone loss associated with periodontitis.

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.

Interactions between co-existing microorganisms deeply affect the physiology of the involved organisms and, ultimately, the function of the ecosystem as a whole. Copiotrophic Alteromonas are marine gammaproteobacteria that thrive during the late stages of phytoplankton blooms in the marine environment and in laboratory co-cultures with cyanobacteria such as Trichodesmium. The response of this heterotroph to the sometimes rapid and transient changes in nutrient supply when the phototroph crashes is not well understood. Here, we isolated and sequenced the strain Alteromonas macleodii str. Te101 from a laboratory culture of Trichodesmium erythraeum IMS101, yielding a chromosome of 4.63 Mb and a single plasmid of 237 kb. Increasing salinities to ≥43 ppt inhibited the growth of Trichodesmium but stimulated growth of the associated Alteromonas. We characterized the transcriptomic responses of both microorganisms and identified the complement of active transcriptional start sites in Alteromonas at single-nucleotide resolution. In replicate cultures, a similar set of genes became activated in Alteromonas when growth rates of Trichodesmium declined and mortality was high. The parallel activation of fliA, rpoS and of flagellar assembly and growth-related genes indicated that Alteromonas might have increased cell motility, growth, and multiple biosynthetic activities. Genes with the highest expression in the data set were three small RNAs (Aln1a-c) that were identified as analogs of the small RNAs CsrB-C in E. coli or RsmX-Z in pathogenic bacteria. Together with the carbon storage protein A (CsrA) homolog Te101&#95;05290, these RNAs likely control the expression of numerous genes in responding to changes in the environment.

Recently, the Mucin-1 (MUC1) gene has been identified as a causal gene of autosomal dominant tubulointerstitial kidney disease (ADTKD). Most causative mutations are buried within a GC-rich 60 basepair variable number of tandem repeat (VNTR), which escapes identification by massive parallel sequencing methods due to the complexity of the VNTR. We established long read single molecule real time sequencing (SMRT) targeted to the MUC1-VNTR as an alternative strategy to the snapshot assay. Our approach allows complete VNTR assembly, thereby enabling the detection of all variants residing within the VNTR and simultaneous determination of VNTR length. We present high resolution data on the VNTR architecture for a cohort of snapshot positive (n = 9) and negative (n = 7) ADTKD families. By SMRT sequencing we could confirm the diagnosis in all previously tested cases, reconstruct both VNTR alleles and determine the exact position of the causative variant in eight of nine families. This study demonstrates that precise positioning of the causative mutation(s) and identification of other coding and noncoding sequence variants in ADTKD-MUC1 is feasible. SMRT sequencing could provide a powerful tool to uncover potential factors encoded within the VNTR that associate with intra- and interfamilial phenotype variability of MUC1 related kidney disease.

SETDB1 is an essential H3K9 methyltransferase involved in silencing of retroviruses and gene regulation. We show here that its triple Tudor domain (3TD) specifically binds to doubly modified histone H3 containing K14 acetylation and K9 methylation. Crystal structures of 3TD in complex with H3K14ac/K9me peptides reveal that peptide binding and K14ac recognition occurs at the interface between Tudor domains (TD) TD2 and TD3. Structural and biochemical data demonstrate a pocket switch mechanism in histone code reading, because K9me1 or K9me2 is preferentially recognized by the aromatic cage of TD3, while K9me3 selectively binds to TD2. Mutations in the K14ac/K9me binding sites change the sub-nuclear localization of 3TD. ChIP-seq analyses show that SETDB1 is enriched at H3K9me3 regions and K9me3/K14ac is enriched at SETDB1 binding sites overlapping with LINE elements, suggesting that recruitment of the SETDB1 complex to K14ac/K9me regions has a role in silencing of active genomic regions.

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.

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 )., (Copyright© 2017 Ferrata Storti Foundation.)

Background: Histone post-translational modifications (PTMs) play central roles in chromatin-templated processes. Combinations of two or more histone PTMs form unique interfaces for readout and recruitment of chromatin interacting complexes, but the genome-wide mapping of coexisting histone PTMs remains an experimentally difficult task., Results: We introduce here a novel type of affinity reagents consisting of two fused recombinant histone modification interacting domains (HiMIDs) for direct detection of doubly modified chromatin. To develop the method, we fused the MPP8 chromodomain and DNMT3A PWWP domain which have a binding specificity for H3K9me3 and H3K36me2/3, respectively. We validate the novel reagent biochemically and in ChIP applications and show its specific interaction with H3K9me3-H3K36me2/3 doubly modified chromatin. Modification specificity was confirmed using mutant double-HiMIDs with inactivated methyllysine binding pockets. Using this novel tool, we mapped coexisting H3K9me3-H3K36me2/3 marks in human cells by chromatin interacting domain precipitation (CIDOP). CIDOP-seq data were validated by qPCR, sequential CIDOP/ChIP and by comparison with CIDOP- and ChIP-seq data obtained with single modification readers and antibodies. The genome-wide distribution of H3K9me3-H3K36me2/3 indicates that it represents a novel bivalent chromatin state, which is enriched in weakly transcribed chromatin segments and at ZNF274 and SetDB1 binding sites., Conclusions: The application of double-HiMIDs allows the single-step study of co-occurrence and distribution of combinatorial chromatin marks. Our discovery of a novel H3K9me3-H3K36me2/3 bivalent chromatin state illustrates the power of this approach, and it will stimulate numerous follow-up studies on its biological functions.

Background: Loss of alveolar ridge width and height after tooth extraction is well documented, but models to evaluate ridge preservation are neither standardized nor cost-effective. This rat model characterizes the pattern of bone turnover and inflammation after extraction and bone grafting with or without local simvastatin (SIM)., Methods: Fifty retired-breeder rats underwent extraction of the maxillary right first molar and standard surgical defect creation under inhalation/local anesthesia. The left side of each animal served as unmanipulated control. Untreated groups (n = 8 to 9 per group) were compared (analysis of variance, t test) at days 0, 7, 14, and 28 for alveolar ridge height and width and for markers of inflammation and bone turnover by microcomputed tomography, histology, and enzyme-linked immunosorbent assay. Seventeen additional specimens had defects grafted with either bone mineralized matrix (BMM) or a BMM+SIM conjugate., Results: Extraction-induced bone loss (BL) was noted on buccal, palatal, and interproximal height (P <0.05) and ridge width (P <0.01). Week 1 inflammation positively correlated with ridge height; thereafter, a more intense inflammatory reaction corresponded to reduction in alveolar bone height and density (r = 0.74; P <0.05; Spearman). BMM+SIM preserved the most interproximal bone height (P <0.01), increased ridge width and bone density (P <0.01), enhanced 7-day prostaglandin E2 (P <0.01), and reduced 28-day inflammation density (P <0.05)., Conclusions: The standard defect used in the current study paralleled human postextraction alveolar BL. Defect grafting, especially BMM+SIM, reduced inflammation and preserved bone.

Background: Fully isogenic lines in fish can be developed using "mitotic" gynogenesis (suppression of first zygotic mitosis following inactivation of the sperm genome). However, genome-wide verification of the steps in this process has seldom been applied. We used ddRADseq to generate SNP markers in a meiotic gynogenetic family of European seabass (Dicentrarchus labrax): (i) to verify the lack of paternal contribution in a meiotic gynogenetic family; (ii) to generate a gene-centromere map from this family; (iii) to identify telomeric markers that could distinguish mitotic gynogenetics from meiotic gynogenetics, which sometimes arise spontaneously in mitotic gynogenetic families., Results: From a single meiotic gynogenetic family consisting of 79 progeny, 42 million sequencing reads (Illumina, trimmed to 148 bases) resolved 6866 unique RAD-tags. The 340 male-informative SNP markers that were identified confirmed the lack of paternal contribution. A gene-centromere map was constructed based on 804 female-informative SNPs in 24 linkage groups (2n = 48) with a total length of 1251.02 cM (initial LG assignment was based on the seabass genome assembly, dicLab v1). Chromosome arm structure could be clearly discerned from the pattern of heterozygosity in each linkage group in 18 out of 24 LGs: the other six showed anomalies that appeared to be related to issues in the genome assembly., Conclusion: Genome-wide screening enabled substantive verification of the production of the gynogenetic family used in this study. The large number of telomeric and subtelomeric markers with high heterozygosity values in the meiotic gynogenetic family indicate that such markers could be used to clearly distinguish between meiotic and mitotic gynogenetics.

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.

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., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Circular RNAs (circRNAs) belong to a recently re-discovered species of RNA that emerge during RNA maturation through a process called back-splicing. A downstream 5' splice site is linked to an upstream 3' splice site to form a circular transcript instead of a canonical linear transcript. Recent advances in next-generation sequencing (NGS) have brought circRNAs back into the focus of many scientists. Since then, several studies reported that circRNAs are differentially expressed across tissue types and developmental stages, implying that they are actively regulated and not merely a by-product of splicing. Though functional studies have shown that some circRNAs could act as miRNA-sponges, the function of most circRNAs remains unknown. To expand our understanding of possible roles of circular RNAs, we propose a new pipeline that could fully characterizes candidate circRNA structure from RNAseq data-FUCHS: FU ll CH aracterization of circular RNA using RNA- S equencing. Currently, most computational prediction pipelines use back-spliced reads to identify circular RNAs. FUCHS extends this concept by considering all RNA-seq information from long reads (typically >150 bp) to learn more about the exon coverage, the number of double break point fragments, the different circular isoforms arising from one host-gene, and the alternatively spliced exons within the same circRNA boundaries. This new knowledge will enable the user to carry out differential motif enrichment and miRNA seed analysis to determine potential regulators during circRNA biogenesis. FUCHS is an easy-to-use Python based pipeline that contributes a new aspect to the circRNA research., Competing Interests: The authors declare there are have no competing interests.

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, IG15 T , IG16b T , and IG31 T , 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 PB90-1 T , the closest described relative of strains IG15 T , IG16b T , and IG31 T . 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 IG16b T . In addition, we isolated and visualized PG-sacculi for strain IG16b T . 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 IG15 T , IG16b T and IG31 T 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 IG16b T being the type species of the genus.

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&#95;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., Competing Interests: The authors declare no competing financial interests.

Understanding RNA processing and turnover requires knowledge of cleavages by major endoribonucleases within a living cell. We have employed TIER-seq (transiently inactivating an endoribonuclease followed by RNA-seq) to profile cleavage products of the essential endoribonuclease RNase E in Salmonella enterica. A dominating cleavage signature is the location of a uridine two nucleotides downstream in a single-stranded segment, which we rationalize structurally as a key recognition determinant that may favor RNase E catalysis. Our results suggest a prominent biogenesis pathway for bacterial regulatory small RNAs whereby RNase E acts together with the RNA chaperone Hfq to liberate stable 3' fragments from various precursor RNAs. Recapitulating this process in vitro, Hfq guides RNase E cleavage of a representative small-RNA precursor for interaction with a mRNA target. In vivo, the processing is required for target regulation. Our findings reveal a general maturation mechanism for a major class of post-transcriptional regulators., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Sulfate-reducing bacteria (SRB) are key players of the carbon- and sulfur-cycles in the sediments of the world's oceans. Habitat relevant SRBs are often members of the Desulfosarcina-Desulfococcus clade belonging to the deltaproteobacterial family of Desulfobacteraceae. Despite this environmental recognition, their molecular (genome-based) physiology and their potential to contribute to organic carbon mineralization as well as to adapt to changing environmental conditions have been scarcely investigated. A metabolically versatile representative of this family is Desulfococcus multivorans that is able to completely oxidize (to CO 2 ) a variety of organic acids, including fatty acids up to C 14, as well as aromatic compounds., Results: In this study the complete 4.46 Mbp and manually annotated genome of metabolically versatile Desulfococcus multivorans DSM 2059 is presented with particular emphasis on a proteomics-driven metabolic reconstruction. Proteomic profiling covered 17 substrate adaptation conditions (6 aromatic and 11 aliphatic compounds) and comprised 2D DIGE, shotgun proteomics and analysis of the membrane protein-enriched fractions. This comprehensive proteogenomic dataset allowed for reconstructing a metabolic network of degradation pathways and energy metabolism that consists of 170 proteins (154 detected; ~91 % coverage). Peripheral degradation routes feed via central benzoyl-CoA, (modified) β-oxidation or methylmalonyl-CoA pathways into the Wood-Ljungdahl pathway for complete oxidation of acetyl-CoA to CO 2 . Dissimilatory sulfate reduction is fueled by a complex electron transfer network composed of cytoplasmic components (e.g., electron transfer flavoproteins) and diverse membrane redox complexes (Dsr, Qmo, Hmc, Tmc, Qrc, Nuo and Rnf). Overall, a high degree of substrate-specific formation of catabolic enzymes was observed, while most complexes involved in electron transfer appeared to be constitutively formed., Conclusions: A highly dynamic genome structure in combination with substrate-specifically formed catabolic subproteomes and a constitutive subproteome for energy metabolism and electron transfer appears to be a common trait of Desulfobacteraceae members.

The functional annotation of transcriptomes and identification of noncoding RNA (ncRNA) classes has been greatly facilitated by the advent of next-generation RNA sequencing which, by reading the nucleotide order of transcripts, theoretically allows the rapid profiling of all transcripts in a cell. However, primary sequence per se is a poor predictor of function, as ncRNAs dramatically vary in length and structure and often lack identifiable motifs. Therefore, to visualize an informative RNA landscape of organisms with potentially new RNA biology that are emerging from microbiome and environmental studies requires the use of more functionally relevant criteria. One such criterion is the association of RNAs with functionally important cognate RNA-binding proteins. Here we analyze the full ensemble of cellular RNAs using gradient profiling by sequencing (Grad-seq) in the bacterial pathogen Salmonella enterica, partitioning its coding and noncoding transcripts based on their network of RNA-protein interactions. In addition to capturing established RNA classes based on their biochemical profiles, the Grad-seq approach enabled the discovery of an overlooked large collective of structured small RNAs that form stable complexes with the conserved protein ProQ. We show that ProQ is an abundant RNA-binding protein with a wide range of ligands and a global influence on Salmonella gene expression. Given its generic ability to chart a functional RNA landscape irrespective of transcript length and sequence diversity, Grad-seq promises to define functional RNA classes and major RNA-binding proteins in both model species and genetically intractable organisms., Competing Interests: The authors declare no conflict of interest.

Background: Minocycline microspheres (MMs) are being used to treat residual inflamed periodontal pockets during periodontal maintenance therapy (PMT), but evidence for efficacy from randomized clinical trials is lacking. The purpose of this study is to evaluate the effect of MMs plus scaling and root planing (SRP) on these sites., Methods: Sixty patients with chronic periodontitis on 6-month PMT intervals to be followed for 1 year were randomized (51 completed the study) into two statistically similar groups, SRP + MM (aged 66.8 years) and SRP alone (aged 67 years), to treat a ≥5 mm posterior interproximal pocket during PMT with a history of bleeding on probing (BOP). Group treatments were applied to the site at baseline and 6 months. Clinical attachment levels (CALs; primary outcome), probing depths (PDs), plaque, and BOP also were recorded at baseline and 6 and 12 months. In addition, gingival crevicular fluid was analyzed for an inflammation index ratio of interleukin (IL)-1β/IL-1 receptor antagonist (ra) using enzyme-linked immunosorbent assays., Results: All clinical parameters improved significantly (P <0.005) from baseline in both groups with no differences between groups at any time point. CAL decreased 17% (0.9 ± 0.8 mm) and 13% (0.7 ± 0.9 mm) in SRP + MM and 11% (0.7 ± 1.1 mm) and 21% (1.2 ± 0.9 mm) in SRP at 6 and 12 months, respectively. The odds of having BOP decreased 90% (down to 38% of patients) and 95% (26%) in SRP + MM and 82% (42%) and 82% (41%) in SRP at 6 and 12 months, respectively. IL-1β/IL-1ra decreased a significant 61% (P = 0.009) only in SRP + MM at 6 months., Conclusions: SRP of inflamed moderate pockets during 6-month PMT, with or without MMs, improves CALs, along with PDs and BOP over a 1-year period. The use of MMs did not result in an additional benefit over SRP alone.

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., Competing Interests: The authors have declared that no competing interests exist.