Decreased bone resorption in Ezh2 myeloid cell conditional knockout mouse model.

Osteoclasts derived from hematopoietic stem cells control bone resorption. Identifying novel molecules that can epigenetically regulate osteoclastogenesis is important for developing novel treatments for osteoporosis and other disorders associated with bone deterioration and promoting healthy bone formation. The polycomb group (PcG) protein enhancer of zeste homolog 2 (Ezh2), a histone lysine methyltransferase, is associated with epigenetic regulation of numerous cellular processes, but its involvement in bone cell development and homeostasis is not yet clear. Here, LysM-Cre mice were crossed with Ezh2flox/flox mice to delete Ezh2 in myeloid cell lineage mature macrophages. Conditional knockout of Ezh2 (CKO) in myeloid cell line resulted in significant increases in postnatal bone growth in the first 6 months of life for both male and female mice. For female mice, optimal bone mass was seen for mice with Ezh2 deleted in both chromosomes in a pair (f/f Cre+ ; CKO). For male mice, optimal bone mass was found after deletion of Ezh2 from just one chromosome (f/- Cre+ ) with no difference in bone phenotype between f/- Cre+ and CKO male mice. In addition to the gender-specific difference in bone phenotype, Ezh2 CKO mice had significantly less macrophages (CD11b+) present in the bone marrow compared with control mice as well as significantly more mature osteoblasts and bone formation biomarkers present (P1NP, osteocalcin). Inflammatory array for protein lysed from bone tissue revealed deletion of Ezh2 decreased inflammatory milieu in both male and female mice compared with controls. Unexpectedly, myeloid cell deletion of Ezh2 also increased the number of mature osteoblast present in the bone. Deletion of Ezh2 also led to an increase in gene expression of osteoclast-suppressive genes IRF8, MafB, and Arg1 due to a decrease in the presence of the suppressive H3K27me3 epigenetic mark. These findings suggest that manipulation of Ezh2 expression may be a viable strategy to combat bone resorptive disorders such as osteoporosis or arthritis.

Serratia montpellierensis sp. nov., Isolated from Laboratory-Reared Parasitic Wasps Psyttalia lounsburyii Silvestri and Psyttalia ponerophaga Silvestri (Hymenoptera: Braconidae).

Two strains of bacteria, PsyLou2AT and PsyPon4B, were isolated from adult braconid wasps Psyttalia lounsburyii and Psyttalia ponerophaga, respectively. These laboratory-reared wasps were investigated as agents for biological control of the olive fruit fly, Bactrocera oleae. Analysis of 16S rRNA genes of the two isolates demonstrated that they were highly related and belonged to the genus Serratia. Genomic sequencing of these isolates revealed genomes of 5,152,551 bp and 5,154,385 bp for PsyLou2AT and PsyPon4B, respectively, and both genomes had a mol% G+C content of 59.6%. Phylogenetic analyses using BLAST-based average nucleotide identity (ANIb), and digital DNA-DNA hybridization methods indicated that PsyLou2AT was most closely related to Serratia nevei S15T, producing ANIb and dDDH values of 96.11% and 70.2%, respectively. Since these values were literally on the species cutoff threshold, additional S. nevei genome assemblies were analyzed using ANIb and dDDH calculations. This revealed that among assemblies that were clearly identifiable as S. nevei, S. nevei S15T was the most closely related to PsyLou2AT, and that a majority of assemblies produced dDDH values of 68.3-68.7% relative to PsyLou2AT. Additionally, PsyLou2AT differed biochemically from S. nevei S15T in that it produced positive Voges Proskauer tests, produced protease, lacked arginine dihydrolase, and did not utilize D-lactose. Hence, PsyLou2AT represents a novel taxon within the Serratia, for which we propose the name Serratia montpellierensis sp. nov. The type strain is PsyLou2AT (=LMG 32817T =NRRL B-65689T).

MicroRNA-98 reduces nerve growth factor expression in nicotine-induced airway remodeling.

Evolving evidence suggests that nicotine may contribute to impaired asthma control by stimulating expression of nerve growth factor (NGF), a neurotrophin associated with airway remodeling and airway hyperresponsiveness. We explored the hypothesis that nicotine increases NGF by reducing lung fibroblast (LF) microRNA-98 (miR-98) and PPARγ levels, thus promoting airway remodeling. Levels of NGF, miR-98, PPARγ, fibronectin 1 (FN1), endothelin-1 (EDN1, herein referred to as ET-1), and collagen (COL1A1 and COL3A1) were measured in human LFs isolated from smoking donors, in mouse primary LFs exposed to nicotine (50 µg/ml), and in whole lung homogenates from mice chronically exposed to nicotine (100 µg/ml) in the drinking water. In selected studies, these pathways were manipulated in LFs with miR-98 inhibitor (anti-miR-98), miR-98 overexpression (miR-98 mimic), or the PPARγ agonist rosiglitazone. Compared with unexposed controls, nicotine increased NGF, FN1, ET-1, COL1A1, and COL3A1 expression in human and mouse LFs and mouse lung homogenates. In contrast, nicotine reduced miR-98 levels in LFs in vitro and in lung homogenates in vivo Treatment with anti-miR-98 alone was sufficient to recapitulate increases in NGF, FN1, and ET-1, whereas treatment with a miR-98 mimic significantly suppressed luciferase expression in cells transfected with a luciferase reporter linked to the putative seed sequence in the NGF 3'UTR and also abrogated nicotine-induced increases in NGF, FN1, and ET-1 in LFs. Similarly, rosiglitazone increased miR-98 and reversed nicotine-induced increases in NGF, FN1, and ET-1. Taken together, these findings demonstrate that nicotine-induced increases in NGF and other markers of airway remodeling are negatively regulated by miR-98.

On the potential role of globins in brown adipose tissue: a novel conceptual model and studies in myoglobin knockout mice.

Myoglobin (Mb) regulates O2 bioavailability in muscle and heart as the partial pressure of O2 (Po2) drops with increased tissue workload. Globin proteins also modulate cellular NO pools, "scavenging" NO at higher Po2 and converting NO2- to NO as Po2 falls. Myoglobin binding of fatty acids may also signal a role in fat metabolism. Interestingly, Mb is expressed in brown adipose tissue (BAT), but its function is unknown. Herein, we present a new conceptual model that proposes links between BAT thermogenic activation, concurrently reduced Po2, and NO pools regulated by deoxy/oxy-globin toggling and xanthine oxidoreductase (XOR). We describe the effect of Mb knockout (Mb-/-) on BAT phenotype [lipid droplets, mitochondrial markers uncoupling protein 1 (UCP1) and cytochrome C oxidase 4 (Cox4), transcriptomics] in male and female mice fed a high-fat diet (HFD, 45% of energy, ∼13 wk), and examine Mb expression during brown adipocyte differentiation. Interscapular BAT weights did not differ by genotype, but there was a higher prevalence of mid-large sized droplets in Mb-/-. COX4 protein expression was significantly reduced in Mb-/- BAT, and a suite of metabolic/NO/stress/hypoxia transcripts were lower. All of these Mb-/--associated differences were most apparent in females. The new conceptual model, and results derived from Mb-/- mice, suggest a role for Mb in BAT metabolic regulation, in part through sexually dimorphic systems and NO signaling. This possibility requires further validation in light of significant mouse-to-mouse variability of BAT Mb mRNA and protein abundances in wild-type mice and lower expression relative to muscle and heart.NEW & NOTEWORTHY Myoglobin confers the distinct red color to muscle and heart, serving as an oxygen-binding protein in oxidative fibers. Less attention has been paid to brown fat, a thermogenic tissue that also expresses myoglobin. In a mouse knockout model lacking myoglobin, brown fat had larger fat droplets and lower markers of mitochondrial oxidative metabolism, especially in females. Gene expression patterns suggest a role for myoglobin as an oxygen/nitric oxide-sensor that regulates cellular metabolic and signaling pathways.

Metabolic physiology and skeletal muscle phenotypes in male and female myoglobin knockout mice.

Myoglobin (Mb) is a regulator of O2 bioavailability in type I muscle and heart, at least when tissue O2 levels drop. Mb also plays a role in regulating cellular nitric oxide (NO) pools. Robust binding of long-chain fatty acids and long-chain acylcarnitines to Mb, and enhanced glucose metabolism in hearts of Mb knockout (KO) mice, suggest additional roles in muscle intermediary metabolism and fuel selection. To evaluate this hypothesis, we measured energy expenditure (EE), respiratory exchange ratio (RER), body weight gain and adiposity, glucose tolerance, and insulin sensitivity in Mb knockout (Mb-/-) and wild-type (WT) mice challenged with a high-fat diet (HFD, 45% of calories). In males (n = 10/genotype) and females (n = 9/genotype) tested at 5-6, 11-12, and 17-18 wk, there were no genotype effects on RER, EE, or food intake. RER and EE during cold (10°C, 72 h), and glucose and insulin tolerance, were not different compared with within-sex WT controls. At ∼18 and ∼19 wk of age, female Mb-/- adiposity was ∼42%-48% higher versus WT females (P = 0.1). Transcriptomics analyses (whole gastrocnemius, soleus) revealed few consistent changes, with the notable exception of a 20% drop in soleus transferrin receptor (Tfrc) mRNA. Capillarity indices were significantly increased in Mb-/-, specifically in Mb-rich soleus and deep gastrocnemius. The results indicate that Mb loss does not have a major impact on whole body glucose homeostasis, EE, RER, or response to a cold challenge in mice. However, the greater adiposity in female Mb-/- mice indicates a sex-specific effect of Mb KO on fat storage and feed efficiency.NEW & NOTEWORTHY The roles of myoglobin remain to be elaborated. We address sexual dimorphism in terms of outcomes in response to the loss of myoglobin in knockout mice and perform, for the first time, a series of comprehensive metabolic studies under conditions in which fat is mobilized (high-fat diet, cold). The results highlight that myoglobin is not necessary and sufficient for maintaining oxidative metabolism and point to alternative roles for this protein in muscle and heart.

Maternal regulation of SATB2 in osteo-progeniters impairs skeletal development in offspring.

Nutritional status during intrauterine and/or early postnatal life has substantial influence on adult offspring health. Along these lines, there is a growing body of evidence illustrating that high fat diet (HFD)-induced maternal obesity can regulate fetal bone development. Thus, we investigated the effects of maternal obesity on both fetal skeletal development and mechanisms linking maternal obesity to osteoblast differentiation in offspring. Embryonic osteogenic calvarial cells (EOCCs) were isolated from fetuses at gestational day 18.5 (E18.5) of HFD-induced obese rat dams. We observed impaired differentiation of EOCCs to mature osteoblasts from HFD obese dams. ChIP-seq-based genome-wide localization of the repressive histone mark H3K27me3 (mediated via the polycomb histone methyltransferase, enhancer of zeste homologue 2 [Ezh2]) showed that this phenotype was associated with increased enrichment of H3K27me3 on the gene of SATB2, a critical transcription factor required for osteoblast differentiation. Knockdown of Ezh2 in EOCCs and ST2 cells increased SATB2 expression; while Ezh2 overexpression in EOCCs and ST2 cells decreased SATB2 expression. These data were consistent with experimental results showing strong association between H3K27me3, Ezh2, and SATB2 in cells from rats and humans. We have further presented that SATB2 mRNA and protein expression were increased in bones, and increased trabecular bone mass from pre-osteoblast specific Ezh2 deletion (Ezh2flox/flox Osx-Cre+ cko) mice compared with those from control Cre+ mice. These findings indicate that maternal HFD-induced obesity may be associated with decreasing fetal pre-osteoblastic cell differentiation, under epigenetic control of SATB2 expression via Ezh2-dependent mechanisms.

Blockade of equilibrative nucleoside transporter 1/2 protects against Pseudomonas aeruginosa-induced acute lung injury and NLRP3 inflammasome activation.

Pseudomonas aeruginosa infections are increasingly multidrug resistant and cause healthcare-associated pneumonia, a major risk factor for acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Adenosine is a signaling nucleoside with potential opposing effects; adenosine can either protect against acute lung injury via adenosine receptors or cause lung injury via adenosine receptors or equilibrative nucleoside transporter (ENT)-dependent intracellular adenosine uptake. We hypothesized that blockade of intracellular adenosine uptake by inhibition of ENT1/2 would increase adenosine receptor signaling and protect against P. aeruginosa-induced acute lung injury. We observed that P. aeruginosa (strain: PA103) infection induced acute lung injury in C57BL/6 mice in a dose- and time-dependent manner. Using ENT1/2 pharmacological inhibitor, nitrobenzylthioinosine (NBTI), and ENT1-null mice, we demonstrated that ENT blockade elevated lung adenosine levels and significantly attenuated P. aeruginosa-induced acute lung injury, as assessed by lung wet-to-dry weight ratio, BAL protein levels, BAL inflammatory cell counts, pro-inflammatory cytokines, and pulmonary function (total lung volume, static lung compliance, tissue damping, and tissue elastance). Using both agonists and antagonists directed against adenosine receptors A2AR and A2BR, we further demonstrated that ENT1/2 blockade protected against P. aeruginosa -induced acute lung injury via activation of A2AR and A2BR. Additionally, ENT1/2 chemical inhibition and ENT1 knockout prevented P. aeruginosa-induced lung NLRP3 inflammasome activation. Finally, inhibition of inflammasome prevented P. aeruginosa-induced acute lung injury. Our results suggest that targeting ENT1/2 and NLRP3 inflammasome may be novel strategies for prevention and treatment of P. aeruginosa-induced pneumonia and subsequent ARDS.

Novel associations in antibiosis stemming from an insect pupal cell.

The pupal soil cell of the pecan weevil, Curculio caryae (Coleoptera: Curculionidae), was reported previously to exhibit antibiosis to an entomopathogenic fungus, Beauveria bassiana. The objectives of this study were to examine 1) if the antimicrobial effect occurs in other insects that form pupal cells, 2) whether the effect extends to plant pathogenic fungi, and 3) identify the source of antibiosis in pupal soil cells of C. caryae. Antibiosis of pupal cells against B. bassiana was confirmed in-vitro in three additional curculionids, Diaprepes abbreviatus, Conotrachelus nenuphar, and Pissodes nemorensis, all of which had fewer fungal colonies relative to controls. Pupal soil cells were found to suppress phytopathogenic fungi in-vitro, including suppression of Alternaria solani by D. abbreviatus pupal cell, and that of Monilinia fructicola by C. caryae. The detection of antibiosis of soil cells formed by surface-sterilized insects using sterile soil implies the antimicrobial effect stemmed from inside the insect. Further, a novel biotic mechanism was identified: a bacterium related to Serratia nematodiphila was isolated from C. caryae pupal soil cells and was found to be associated with antibiosis. The bacterial cultures with or without autoclave had similar effects but were not as potent as pupal soil cells for suppressing B. bassiana. Also, autoclaved soil cells and autoclaved bacterial culture suppressed M. fructicola but were not as inhibitory as non-autoclaved soil cells. This indicates that antibiosis may be due to bacterial metabolites, although other factors may also be involved. Our findings suggest potential to develop the antibiotic compounds as novel bio-fungicides to control plant diseases.

Transforming growth factor ß1 alters the 3'-UTR of mRNA to promote lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic disease characterized by the pathological remodeling of air sacs as a result of excessive accumulation of extracellular matrix (ECM) proteins, but the mechanism governing the robust protein expression is poorly understood. Our recent findings demonstrate that alternative polyadenylation (APA) caused by NUDT21 reduction is important for the increased expression of fibrotic mediators and ECM proteins in lung fibroblasts by shortening the 3'-untranslated regions (3'-UTRs) of mRNAs and stabilizing their transcripts, therefore activating pathological signaling pathways. Despite the importance of NUDT21 reduction in the regulation of fibrosis, the underlying mechanisms for the depletion are unknown. We demonstrate here that NUDT21 is depleted by TGFß1. We found that miR203, which is increased in IPF, was induced by TGFß1 to target the NUDT21 3'-UTR, thus depleting NUDT21 in human and mouse lung fibroblasts. TGFß1-mediated NUDT21 reduction was attenuated by the miR203 inhibitor antagomiR203 in fibroblasts. TGFß1 transgenic mice revealed that TGFß1 down-regulates NUDT21 in fibroblasts in vivo Furthermore, TGFß1 promoted differential APA of fibrotic genes, including FGF14, RICTOR, TMOD2, and UCP5, in association with increased protein expression. This unique differential APA signature was also observed in IPF fibroblasts. Altogether, our results identified TGFß1 as an APA regulator through NUDT21 depletion amplifying pulmonary fibrosis.

Brown marmorated stink bug, Halyomorpha halys (Stål), genome: putative underpinnings of polyphagy, insecticide resistance potential and biology of a top worldwide pest.

BACKGROUND: Halyomorpha halys (Stål), the brown marmorated stink bug, is a highly invasive insect species due in part to its exceptionally high levels of polyphagy. This species is also a nuisance due to overwintering in human-made structures. It has caused significant agricultural losses in recent years along the Atlantic seaboard of North America and in continental Europe. Genomic resources will assist with determining the molecular basis for this species' feeding and habitat traits, defining potential targets for pest management strategies. RESULTS: Analysis of the 1.15-Gb draft genome assembly has identified a wide variety of genetic elements underpinning the biological characteristics of this formidable pest species, encompassing the roles of sensory functions, digestion, immunity, detoxification and development, all of which likely support H. halys' capacity for invasiveness. Many of the genes identified herein have potential for biomolecular pesticide applications. CONCLUSIONS: Availability of the H. halys genome sequence will be useful for the development of environmentally friendly biomolecular pesticides to be applied in concert with more traditional, synthetic chemical-based controls.

Carnitine palmitoyltransferase 2 knockout potentiates palmitate-induced insulin resistance in C2C12 myotubes.

Saturated fatty acids (SFAs) are implicated in muscle inflammation/cell stress and insulin resistance, but the catalog of factors involved is incomplete. SFA derivatives that accumulate with mismatched FA availability and FA oxidation (FAO) are likely involved, and evidence has emerged that select acylcarnitines should be considered. To understand if excessive long-chain acylcarnitine accumulation and limited FAO associate with lipotoxicity, carnitine palmitoyltransferase 2 knockout C2C12 cells were generated (CPT2 KO). CPT2 KO was confirmed by Western blot, increased palmitoylcarnitine accumulation, and loss of FAO capacity. There was no effect of CPT2 KO on palmitic acid (PA) concentration-dependent increases in media IL-6 or adenylate kinase. PA at 200 and 500 µM did not trigger cell stress responses (phospho-Erk, -JNK, or -p38) above that of vehicle in WT or CPT2 KO cells. In contrast, loss of CPT2 exacerbated PA-induced insulin resistance (acute phospho-Akt; 10 or 100 nM insulin) by as much as ~50-96% compared with WT. Growing cells in carnitine-free media abolished differences between WT and CPT2 KO, but this did not fully rescue PA-induced insulin resistance. The results suggest that PA-induced insulin resistance stems in part from palmitoylcarnitine accumulation, further supporting the hypothesis that select acylcarnitines participate in cell signaling and, when in excess, can compromise cell function. Since carnitine-free conditions could not fully rescue insulin signaling, and CPT2 KO did not alter cell stress responses, the majority of PA-induced "lipotoxicity" in C2C12 myotubes cannot be attributed to palmitoylcarnitine alone.

Implantable cardioverter-defibrillator therapy to reduce sudden cardiac death in adults with congenital heart disease: A registry study.

INTRODUCTION: The adult congenital heart disease (ACHD) population is rapidly expanding. However, a significant proportion of these patients suffer sudden cardiac death. Recommending implantable cardioverter-defibrillator (ICD) insertion requires balancing the need for appropriate therapy in malignant arrhythmia against the consequences of inappropriate therapy and procedural complications. Here we present long-term follow-up data for ICD insertion in patients with ACHD from a large Level 1 congenital cardiac center. METHODS AND RESULTS: All patients with ACHD undergoing ICD insertion over an 18-year period were identified. Data were extracted for baseline characteristics including demographics, initial diagnosis, ventricular function, relevant medication, and indication for ICD insertion. Details regarding device insertion were gathered along with follow-up data including appropriate and inappropriate therapy and complications. A total of 136 ICDs were implanted during this period: 79 for primary and 57 for secondary prevention. The most common congenital cardiac conditions in both groups were tetralogy of Fallot and transposition of the great arteries. Twenty-two individuals in the primary prevention group received appropriate antitachycardia pacing (ATP), 14 underwent appropriate cardioversion, 17 received inappropriate ATP, and 15 received inappropriate cardioversion. In the secondary prevention group, 18 individuals received appropriate ATP, 8 underwent appropriate cardioversion, 8 received inappropriate ATP, and 7 were inappropriately cardioverted. Our data demonstrate low complication rates, particularly with leads without advisories. CONCLUSION: ICD insertion in the ACHD population involves a careful balance of the risks and benefits. Our data show a significant proportion of patients receiving appropriate therapy indicating that ICDs were inserted appropriately.

Chromobacterium paludis sp. nov., a novel bacterium isolated from a Chesapeake Bay marsh.

Two isolates of Gram-reaction-negative, motile, violet-pigmented bacteria were isolated from a small pool in marshland near the mouth of the Nanticoke River in Maryland, USA. The isolates IIBBL 257-1T and IIBBL 257-2 had identical 16S rRNA gene sequences as determined by PCR, and highly similar fatty acid and biochemical profiles. The 16S rRNA gene sequences indicated the isolates belonged to the genus Chromobacterium. Genomic sequencing of IIBBL 257-1T revealed a genome of 4.27 Mb, with a G+C content of 63.6 %. Whole genome comparisons with other members of the Chromobacterium using JSpecies and the genome blast distance phylogeny approach indicated that among described species, IIBBL 257-1T was most closely related to C. amazonense and C. phragmitis. Comparison of the IIBBL 257-1T genome with those of type strains of these species resulted in ANIb and dDDH values of ca. 85 and 30 %, respectively, for both. These results demonstrate that IIBBL 257-1T and IIBBL 257-2 represent a new taxon within the genus Chromobacterium. We propose the name Chromobacterium paludis sp. nov. for this taxon; the type strain is IIBBL 257-1T (=NRRL B-65555T=JCM 33770T).

Muc5b is required for airway defence.

Respiratory surfaces are exposed to billions of particulates and pathogens daily. A protective mucus barrier traps and eliminates them through mucociliary clearance (MCC). However, excessive mucus contributes to transient respiratory infections and to the pathogenesis of numerous respiratory diseases. MUC5AC and MUC5B are evolutionarily conserved genes that encode structurally related mucin glycoproteins, the principal macromolecules in airway mucus. Genetic variants are linked to diverse lung diseases, but specific roles for MUC5AC and MUC5B in MCC, and the lasting effects of their inhibition, are unknown. Here we show that mouse Muc5b (but not Muc5ac) is required for MCC, for controlling infections in the airways and middle ear, and for maintaining immune homeostasis in mouse lungs, whereas Muc5ac is dispensable. Muc5b deficiency caused materials to accumulate in upper and lower airways. This defect led to chronic infection by multiple bacterial species, including Staphylococcus aureus, and to inflammation that failed to resolve normally. Apoptotic macrophages accumulated, phagocytosis was impaired, and interleukin-23 (IL-23) production was reduced in Muc5b(-/-) mice. By contrast, in mice that transgenically overexpress Muc5b, macrophage functions improved. Existing dogma defines mucous phenotypes in asthma and chronic obstructive pulmonary disease (COPD) as driven by increased MUC5AC, with MUC5B levels either unaffected or increased in expectorated sputum. However, in many patients, MUC5B production at airway surfaces decreases by as much as 90%. By distinguishing a specific role for Muc5b in MCC, and by determining its impact on bacterial infections and inflammation in mice, our results provide a refined framework for designing targeted therapies to control mucin secretion and restore MCC.

Interferon regulatory factor 7 (IRF7) represents a link between inflammation and fibrosis in the pathogenesis of systemic sclerosis.

OBJECTIVES: There is considerable evidence that implicates dysregulation of type I interferon signalling (or type I IFN signature) in the pathogenesis of systemic sclerosis (SSc). Interferon regulatory factor 7 (IRF7) has been recognised as a master regulator of type I IFN signalling. The objective of this study was to elucidate the role of IRF7 in dermal fibrosis and SSc pathogenesis. METHODS: SSc and healthy control skin biopsies were investigated to determine IRF7 expression and activation. The role of IRF7 in fibrosis was investigated using IRF7 knockout (KO) mice in the bleomycin-induced and TSK/+mouse models. In vitro experiments with dermal fibroblasts from patients with SSc and healthy controls were performed. RESULTS: IRF7 expression was significantly upregulated and activated in SSc skin tissue and explanted SSc dermal fibroblasts compared with unaffected, matched controls. Moreover, IRF7 expression was stimulated by IFN-α in dermal fibroblasts. Importantly, IRF7 co-immunoprecipitated with Smad3, a key mediator of transforming growth factor (TGF)-ß signalling, and IRF7 knockdown reduced profibrotic factors in SSc fibroblasts. IRF7 KO mice demonstrated attenuated dermal fibrosis and inflammation compared with wild-type mice in response to bleomycin. Specifically, hydroxyproline content, dermal thickness as well as Col1a2, ACTA2 and interleukin-6 mRNA levels were significantly attenuated in IRF7 KO mice skin tissue. Furthermore, IRF7 KO in TSK/+mice attenuated hydroxyproline content, subcutaneous hypodermal thickness, Col1a2 mRNA as well as α-smooth muscle actin and fibronectin expression. CONCLUSIONS: IRF7 is upregulated in SSc skin, interacts with Smad3 and potentiates TGF-ß-mediated fibrosis, and therefore may represent a promising therapeutic target in SSc.

Chromobacterium phragmitis sp. nov., isolated from estuarine marshes.

Thirteen isolates of Gram-stain-negative, motile, violet-pigmented bacteria were isolated from marshes along tidal portions of the Potomac and James rivers in Maryland and Virginia, USA, respectively. 16S rRNA gene sequences and fatty acid analysis revealed a high degree of relatedness among the isolates, and genomic sequencing of two isolates, IIBBL 112-1T and IIBBL 274-1 (from the Potomac and James rivers, respectively), revealed highly similar genomic sequences, with a blast-based average nucleotide identity (ANIb) of ca. 98.7 %. Phylogenetic analysis of 16S rRNA gene sequences suggested that the species most highly related to IIBBL 112-1T were Chromobacterium amazonense, Chromobacterium subtsugae and Chromobacterium sphagni. However, deletion of a 25-nucleotide sequence that may have been horizontally acquired by both IIBBL 112-1T and C. amazonense resulted in a substantially different analysis; in the latter case, the species nearest IIBBL 112-1T were Chromobacterium violaceum, Chromobacterium vaccinii and Chromobacterium piscinae. Whole-genome alignments between either IIBBL 112-1T or IIBBL 274-1 and the type strains of C. vaccinii or C. violaceum resulted in ANIb values in the range of ca. 87 %, while alignment with C. amazonense CBMAI 310T resulted in an ANIb of ca. 83 %. Collectively, these data demonstrate that IIBBL 112-1T and IIBBL 274-1 represent a new taxon within the genus Chromobacterium. We propose the name Chromobacterium phragmitis sp. nov. for this taxon; the type strain is IIBBL 112-1T (=NRRL B-67132T=JCM 31884T).

MicroRNA-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation.

Aberrant proliferation and activation of lung fibroblasts contribute to the initiation and progression of idiopathic pulmonary fibrosis (IPF). However, the mechanisms responsible for the proliferation and activation of fibroblasts are not fully understood. The objective of this study was to investigate the role of miR-101 in the proliferation and activation of lung fibroblasts. miR-101 expression was determined in lung tissues from patients with IPF and mice with bleomycin-induced pulmonary fibrosis. The regulation of miR-101 and cellular signaling was investigated in pulmonary fibroblasts in vitro The role of miR-101 in pulmonary fibrosis in vivo was studied using adenovirus-mediated gene transfer in mice. The expression of miR-101 was down-regulated in fibrotic lungs from patients with IPF and bleomycin-treated mice. The down-regulation of miR-101 occurred via the E26 transformation-specific (ETS) transcription factor. miR-101 suppressed the WNT5a-induced proliferation of lung fibroblasts by inhibiting NFATc2 signaling via targeting Frizzled receptor 4/6 and the TGF-ß-induced activation of lung fibroblasts by inhibition of SMAD2/3 signaling via targeting the TGF-ß receptor 1. Adenovirus-mediated miR-101 gene transfer in the mouse lung attenuated bleomycin-induced lung fibrosis and improved lung function. Our data suggest that miR-101 is an anti-fibrotic microRNA and a potential therapeutic target for pulmonary fibrosis.

Is palmitate truly proinflammatory? Experimental confounders and context-specificity.

Based primarily on cell culture results, saturated fatty acids (SFAs) are proposed to promote inflammation and contribute to metabolic dysfunction through Toll-like receptor activation. Studies are often complicated by a requirement for carriers (e.g., BSA) or solvents (e.g., ethanol) to increase SFA solubility. To ascertain whether these factors influence interpretations of SFA-associated inflammation activity, we measured responses of RAW264.7 monocyte/macrophages and C2C12 myotubes to various BSA, ethanol, and cyclodextrin (alternative FA carrier) conditions. Fatty acid-free, low-endotoxin BSA preparations (0.33% to 2% wt/vol) activated whereas 0.5-1.0% ethanol inhibited RAW264.7 TNFα release. Ethanol modestly increased IL-6 secretion in C2C12 myotubes. Cyclodextrins (0.3-6.0 mM) were tested as alternative carriers of palmitate, but their usefulness was limited due to toxicity and solubility issues. Using a lower-inflammation BSA source and no ethanol, ∼24-h sodium palmitate treatment (≤600 µM) failed to trigger RAW264.7 TNFα release and, in fact, significantly dampened BSA-induced inflammation by >50%. In C2C12 myotubes, only high palmitate concentrations (500-600 µM) elicited IL-6 secretion (>2.5-fold increase). Acute palmitate (200 or 500 µM) treatment did not activate MAP kinase pathways above that of fresh BSA-containing media alone in either cell type. These results highlight the importance of experimental conditions in studies exploring SFA inflammation effects. The limited (or even anti-inflammatory) effects of palmitate that we observed indicate that immunomodulatory effects of SFAs are context-specific. Thus, caution is needed when interpreting the literature related to putative proinflammatory effects of SFA.

Suppression of cleavage factor Im 25 promotes the proliferation of lung cancer cells through alternative polyadenylation.

BACKGROUND: Non-small cell lung cancer (NSCLC) is a life-threatening disease that has a poor prognosis and low survival rate. Cleavage factor Im 25 (CFIm25) is a RNA-binding protein that if down-regulated causes 3'UTR shortening and thus promotes the transcript stability of target genes. It is not clear whether CFIm25 and alternative polyadenylation (APA) play a role during cancer development. The purpose of this study is to explore the role of CFIm25 in lung cancer cell proliferation. METHODS: CFIm25 was knocked down in A549 cells. Western blots were carried out to determine the protein expression of CFIm25, insulin growth factor 1 receptor (IGF1R), CyclinD1 (CCND1) and TP53. Real-time qRT PCR was performed to determine the total transcript levels of CFIm25 targets and the normalized fold changes in their distal PAS (dPAS) usage. Immunofluorescence was carried out to check the expression of CFIm25, IGF1R and CCND1. Cell proliferation over time was determined using the WST-1 reagent. RESULTS: The transcript levels of CCND1 and GSK3ß were significantly increased and the dPAS usage of several oncogenes (IGF1R, CCND1 and GSK3ß) were decreased after CFIm25 knockdown. The protein level of IGF1R was increased, and we detected increased percentage of CCND1 positive cells and cell proliferation over time in CFIm25 knockdown cells. In addition, the mRNA and APA analysis of IGF1R using patient RNA-seq data from the Cancer Genome Atlas indicated that IGF1R is shortened in both lung adenocarcinoma and lung squamous cell carcinoma compared to normal controls. CONCLUSIONS: Our findings suggest that CFIm25 plays an important role in lung cancer cell proliferation through regulating the APA of oncogenes, including IGF1R, and promoting their protein expression.

Dietary factors during early life program bone formation in female rats.

Nutritional status during intrauterine and early postnatal life impacts the risk of chronic diseases; however, evidence for an association between early-life dietary factors and bone health in adults is limited. Soy protein isolate (SPI) may be one such dietary factor that promotes bone accretion during early life with persistent effects into adulthood. In the present study, we fed postnatal day (PND) 24 weanling female rats an SPI diet for 30 d [short-term SPI (ST-SPI)], and on PND 55, we switched SPI diet to control Cas diet until age 6 mo. Rats then underwent either ovariectomy (OVX) or sham surgery and thereafter either continued to be fed an SPI diet or control diet for 1 or 3 wk. We showed significantly increased bone mass in 30-d SPI-fed young rats compared with controls. OVX-induced bone loss was associated with increased osteoblastic cell senescence. On the one hand, both long-term SPI (continuous SPI diet throughout life) and ST-SPI diet only in early life protected against 1 wk post-OVX-associated bone loss. On the other hand, long-term SPI diet diminished the loss of total, trabecular, and cortical bone mineral density, whereas ST-SPI diet only reduced cortical bone mineral density loss 3 wk post-OVX. Persistent and protective effects of SPI diets on OVX-induced bone loss were associated with down-regulation of the caveolin-1/p53-mediated senescence pathway in bone. We recapitulated these results in cell cultures. Reprogramming of cellular senescence signaling by SPI-associated isoflavones in osteoblastic cells may explain the persistent effects of SPI on bone. These results suggest that OVX-induced bone loss, in part, is a result of increased osteoblastic cell senescence, and that ST-SPI diet early in life has modest but persistent programming effects on bone formation to prevent OVX-induced bone loss in adult female rats.-Chen, J.-R., Lazarenko, O. P., Blackburn, M. L., Shankar, K. Dietary factors during early life program bone formation in female rats.