Cystatin M/E ameliorates bone resorption through increasing osteoclastic cell estrogen influx.

In multiple myeloma (MM), increased osteoclast differentiation leads to the formation of osteolytic lesions in most MM patients. Bisphosphonates, such as zoledronic acid (ZA), are used to ameliorate bone resorption, but due to risk of serious side effects as well as the lack of repair of existing lesions, novel anti-bone resorption agents are required. Previously, the absence of osteolytic lesions in MM was strongly associated with elevated levels of cystatin M/E (CST6), a cysteine protease inhibitor, secreted by MM cells. In this study, both MM- and ovariectomy (OVX)-induced osteoporotic mouse models were used to compare the effects of recombinant mouse CST6 (rmCst6) and ZA on preventing bone loss. µCT showed that rmCst6 and ZA had similar effects on improving percent bone volume, and inhibited differentiation of non-adherent bone marrow cells into mature osteoclasts. Single-cell RNA sequencing showed that rmCst6 and not ZA treatment reduced bone marrow macrophage percentage in the MM mouse model compared to controls. Protein and mRNA arrays showed that both rmCst6 and ZA significantly inhibit OVX-induced expression of inflammatory cytokines. For OVX mice, ERα protein expression in bone was brought to sham surgery level by only rmCst6 treatments. rmCst6 significantly increased mRNA and protein levels of ERα and significantly increased total intracellular estrogen concentrations for ex vivo osteoclast precursor cell cultures. Based on these results, we conclude that CST6 improves MM or OVX bone loss models by increasing the expression of estrogen receptors as well as the intracellular estrogen concentration in osteoclast precursors, inhibiting their maturation.

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).

Phenolic acids prevent sex-steroid deficiency-induced bone loss and bone marrow adipogenesis in mice.

Phenolic acids, such as hippuric acid (HA) and 3-(3-hydroxyphenyl) propionic acid (3-3-PPA), can be produced from microbiome digestion of polyphenols. Previously it was found that HA and 3-3-PPA facilitate bone formation and suppress bone resorption. However, the mechanism of action by which HA and 3-3-PPA protect bone from degeneration is currently unknown. In this report, we present that HA and 3-3-PPA suppression of bone resorption is able to ameliorate bone loss in an ovariectomy (OVX) osteopenic mouse model though not to the extent of Zoledronic acid (ZA). HA and 3-3-PPA treatments were shown to significantly decrease bone marrow adipocyte-like cell formation and inhibited gene expression of key adipogenesis regulator peroxisome proliferator activated receptor gamma (PPARγ) and lipoprotein lipase (Lpl) in bone from OVX mice. In addition, ChIP experiments showed that the association between PPARγ and Lpl promoter region in preadipocyte-like cells was significantly suppressed following HA or 3-3-PPA treatment. Contrasting HA and 3-3-PPA, ZA significantly increased TRAP activity in the area close to growth plate and significantly suppressed bone cell proliferation. These data suggest that phenolics acids such as HA or 3-3-PPA may prevent bone degeneration after OVX through suppression of inflammatory milieu in the bone.

Genomic sequencing of fourteen bacillus thuringiensis isolates: insights into geographic variation and phylogenetic implications.

OBJECTIVE: This work was performed in support of a separate study investigating the activity of pesticidal proteins produced by Bacillus thuringiensis against the Asian citrus psyllid, Diaphorina citri. The fourteen Bacillus isolates chosen were selected from a large, geographically diverse collection that was characterized only by biochemical phenotype and morphology of the parasporal crystal, hence, for each isolate it was desired to determine the specific pesticidal proteins produced, assign each to a Bacillus cereus multilocus sequence type (ST), and predict their placement within the classical Bt serotyping system. In addition, phylogenetic distances between the isolates and Bacillus thuringiensis serovar type strains were determined by calculating digital DNA-DNA hybridization (dDDH) values among the isolates. RESULTS: Based on the assembled sequence data, the isolates were found to be likely representatives of the Bt serovars kurstaki (ST 8), pakistani (ST 550), toumanoffi (ST 240), israelensis (ST 16), thuringiensis (ST 10), entomocidus (ST 239), and finitimus (ST 171). In cases where multiple isolates occurred within a predicted serovar, pesticidal protein profiles were found to be identical, despite the geographic diversity of the isolates. As expected, the dDDH values calculated for pairwise comparisons of the isolates and their apparent corresponding Bt serovar type strains were quite high (> 98%), however dDDH comparisons of the isolates with other serovar type strains were often surprisingly low (< 70%) and suggest unrecognized taxa within Bt and the Bacillus cereus sensu lato.

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.

Immunocapture LC-MS(/MS) assays for biotherapeutic and biomarker proteins: the European Bioanalysis Forum continuing discussions on scientific and regulatory challenges.

The use of LC-MS(/MS) assays to quantify (biotherapeutic or biomarker) proteins is commonplace and well accepted across industry. There is a good understanding on the added value over conventional analytical technologies (i.e., ligand-binding assays). In fact, the impact of combining small- and large-molecule technologies for large-molecule analysis has played a significant part in bringing the bioanalytical communities closer together and building a mutual respect and understanding between scientists. This paper from the European Bioanalysis Forum presents a history of the journey and future perspectives for hybrid assays, with focus on the unanswered scientific questions, including regulatory discussions to be had. Hybrid assays are essentially a combination of ligand-binding assays and MS, and the ICH M10 guideline does not address this approach directly. Decision-based acceptance criteria are still being discussed, and the industry should continue to do so.

Maternal high-fat diet modifies epigenetic marks H3K27me3 and H3K27ac in bone to regulate offspring osteoblastogenesis in mice.

Studies from both humans and animal models indicated that maternal chronic poor-quality diet, especially a high fat diet (HFD), is significantly associated with reduced bone density and childhood fractures in offspring. When previously studied in a rat model, our data suggested that maternal HFD changes epigenetic marks such as DNA methylation and histone modifications to control osteoblast metabolism. In mouse embryonic and postnatal offspring bone samples, a ChIP-sequencing (ChIP-Seq)-based genome-wide method was used to locate the repressive histone mark H3K27me3 (mediated via the polycomb histone methyltransferase, Ezh2) and expressive histone mark H3K27ac (p300/CBP mediated) throughout the genome. Using isolated mouse embryonic cells from foetal calvaria (osteoblast-like cells), H3K27me3 ChIP-Seq showed that 147 gene bodies and 26 gene promoters in HFD embryotic samples had a greater than twofold increase in H3K27me peaks compared to controls. Among the HFD samples, Pthlh and Col2a1 that are important genes playing roles during chondro- and osteogenesis had significantly enriched levels of H3K27me3. Their decreased mRNA expression was confirmed by real-time PCR and standard ChIP analysis, indicating a strong association with Ezh2 mediated H3K27me3 epigenetic changes. Using embryonic calvaria osteoblastic cells and offspring bone samples, H3K27ac ChIP-Seq analysis showed that osteoblast inhibitor genes Tnfaip3 and Twist1 had significantly enriched peaks of H3K27ac in HFD samples compared to controls. Their increased gene expression and association with H3K27ac were also confirmed by real-time PCR and standard ChIP analysis. These findings indicate that chronic maternal HFD changes histone trimethylation and acetylation epigenetic marks to regulate expression of genes controlling osteoblastogenesis.

Nox4 expression in osteo-progenitors controls bone development in mice during early life.

Tightly regulated and cell-specific NADPH-oxidases (Nox) represent one of the major sources of reactive oxygen species (ROS) signaling molecules that are involved in tissue development and stem cell self-renewal. We have characterized the role of Nox4 in osteo-progenitors during postnatal bone development. Nox4 expression in bone and ROS generation were increased during early osteoblast differentiation and bone development. Stromal osteoblastic cell self-renewal, proliferation and ROS production were significantly lower in samples from whole-body Nox4 knockout mice (Nox4-/-) and conditional knockout (CKO) mice with depletion of Nox4 in the limb bud mesenchyme compared with those from control mice (Nox4fl/fl), but they were reversed after 9 passages. In both sexes, bone volume, trabecular number and bone mineral density were significantly lower in 3-week old CKO and Nox4-/- mice compared with Nox4fl/fl controls. This was reflected in serum levels of bone formation markers alkaline phosphatase (ALP) and procollagen 1 intact N-terminal propeptide (P1NP). However, under-developed bone formation in 3-week old CKO and Nox4-/- mice quickly caught up to levels of control mice by 6-week of age, remained no different at 13-week of age, and was reversed in 32-week old male mice. Osteoclastogenesis showed no differences among groups, however, CTX1 reflecting osteoclast activity was significantly higher in 3-week old male CKO and Nox4-/- mice compared with control mice, and significantly lower in 32-week old Nox4-/- mice compared with control mice. These data suggest that Nox4 expression and ROS signaling in bone and osteoblastic cells coordinately play an important role in osteoblast differentiation, proliferation and maturation.

GPR109A gene deletion ameliorates gonadectomy-induced bone loss in mice.

Sex steroid deficiency plays critical roles in the pathophysiology of bone as the result of uncertain bone remodeling, i.e., increased bone resorption with equivocal bone formation. We have previously shown that GPR109A, a G protein coupled receptor, controls osteoclastogenesis and bone resorption, where global GPR109A deletion decreased osteoclast bone resorption and increased bone mass. Here, we used global GPR109A gene deletion, ovariectomized (OVX) and orchidectomized (ORX) mouse models to probe the role of GPR109A in gonadectomy-induced bone loss in female and male mice. Six months old GPR109A-/- mice and their wild type littermates were allocated to Sham or gonadectomized groups for six weeks. Using densitometric micro-CT confirmed by peripheral quantitative CT (pQCT) scans on tibia and spine, and three-point bending test on femur ex vivo, we found the bone volume, trabecular number, as well as bone mineral density and content in both trabecular and cortical sites were significantly decreased in wild type OVX and ORX compared with respective Sham groups. While bone mass in both male and female GPR109A-/- Sham groups were significantly higher compared with their respective wild type Sham groups, global GPR109A gene deletion ameliorated gonadectomy-induced bone loss. In GPR109A-/- females, most of bone mass and strength parameters measured by micro-CT, pQCT and three-point bending test were not different between Sham and OVX groups. In wild type but not in GPR109-/- mice, bone remodeling marker measurements indicated that both bone resorption (Cathepsin K) and bone formation (osteocalcin) markers were increased in gonadectomized mice compared to sham, with the exception of bone specific ALP, which was decreased in gonadectomized mice. Expression of bone resorption markers (Cathepsin K) were significantly lower, but ß-catenin expression was higher in GPR109A-/- mice compared with their wild type littermates. Collectively, these data indicate that global GPR109A deletion ameliorates gonadectomy-induced bone loss through suppression of bone resorption.

Soy Formula Is Not Estrogenic and Does Not Result in Reproductive Toxicity in Male Piglets: Results from a Controlled Feeding Study.

Soy infant formula which is fed to over half a million infants per year contains isoflavones such as genistein, which have been shown to be estrogenic at high concentrations. The developing testis is sensitive to estrogens, raising concern that the use of soy formulas may result in male reproductive toxicity. In the current study, male White-Dutch Landrace piglets received either sow milk (Sow), or were provided milk formula (Milk), soy formula (Soy), milk formula supplemented with 17-beta-estradiol (2 mg/kg/d) (M + E2) or supplemented with genistein (84 mg/L of diet; (M + G) from postnatal day 2 until day 21. E2 treatment reduced testis weight (p < 0.05) as percentage of body weight, significantly suppressed serum androgen concentrations, increased tubule area, Germ cell and Sertoli cell numbers (p < 0.05) relative to those of Sow or Milk groups. Soy formula had no such effects relative to Sow or Milk groups. mRNAseq revealed 103 differentially expressed genes in the M + E2 group compared to the Milk group related to endocrine/metabolic disorders. However, little overlap was observed between the other treatment groups. These data suggest soy formula is not estrogenic in the male neonatal piglet and that soy formula does not significantly alter male reproductive development.

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.

Short-Term Increased Physical Activity During Early Life Affects High-Fat Diet-Induced Bone Loss in Young Adult Mice.

Mechanical stresses associated with physical activity (PA) have beneficial effects on increasing BMD and improving bone quality. However, a high-fat diet (HFD) and obesity tend to have negative effects on bone, by increasing bone marrow adiposity leading to increased excretion of proinflammatory cytokines, which activate RANKL-induced bone resorption. In the current study, whether short-term increased PA via access to voluntary wheel running during early life has persistent and protective effects on HFD-induced bone resorption was investigated. Sixty 4-week-old male C57BL6/J mice were divided into two groups postweaning: without or with PA (access to voluntary running wheel 7-8 km/day) for 4 weeks. After 4 weeks with or without PA, mice were further subdivided into control diet or HFD groups for 8 weeks, and then all animals were switched back to control diet for an additional 4 weeks. Mice from the HFD groups were significantly heavier and obese; however, after 4 weeks of additional control diet their body weights returned to levels of mice on continuous control diet. Using µ-CT and confirmed by pQCT of tibias and spines ex vivo, it was determined that bone volume and trabecular BMD were significantly increased with PA in control diet animals compared with sedentary animals without access to wheels, and such anabolic effects of PA on bone were sustained after ceasing PA in adult mice. Eight weeks of a HFD deteriorated bone development in mice. Unexpectedly, early-life PA did not prevent persistent effects of HFD on deteriorating bone quality; in fact, it exacerbated a HFD-induced inflammation, osteoclastogenesis, and trabecular bone loss in adult mice. In accordance with these data, signal transduction studies revealed that a HFD-induced Ezh2, DNA methyltransferase 3a, and nuclear factor of activated T-cells 1 expression were amplified in nonadherent hematopoietic cells. In conclusion, short-term increased PA in early life is capable of increasing bone mass; however, it alters the HFD-induced bone marrow hematopoietic cell-differentiation program to exacerbate increased bone resorption if PA is halted. © 2021 Arkansas Children's Nutrition Center. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

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.

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.

Enhancing Extracellular Adenosine Levels Restores Barrier Function in Acute Lung Injury Through Expression of Focal Adhesion Proteins.

Background: Acute respiratory distress syndrome (ARDS) is a clinical presentation of acute lung injury (ALI) with often fatal lung complication. Adenosine, a nucleoside generated following cellular stress provides protective effects in acute injury. The levels of extracellular adenosine can be depleted by equilibrative nucleoside transporters (ENTs). ENT inhibition by pharmaceutical agent dipyridamole promotes extracellular adenosine accumulation and is protective in ARDS. However, the therapeutic potential of dipyridamole in acute lung injury has not yet been evaluated. Methods: Adenosine acts on three adenosine receptors, the adenosine A1 (Adora1), A2a (Adora2a), the A2b (Adora2b) or the adenosine A3 (Adora 3) receptor. Accumulation of adenosine is usually required to stimulate the low-affinity Adora2b receptor. In order to investigate the effect of adenosine accumulation and the contribution of epithelial-specific ENT2 or adora2b expression in experimental ALI, dipyridamole, and epithelial specific ENT2 or Adora2b deficient mice were utilized. MLE12 cells were used to probe downstream Adora2b signaling. Adenosine receptors, transporters, and targets were determined in ARDS lungs. Results: ENT2 is mainly expressed in alveolar epithelial cells and is negatively regulated by hypoxia following tissue injury. Enhancing adenosine levels with ENT1/ENT2 inhibitor dipyridamole at a time when bleomycin-induced ALI was present, reduced further injury. Mice pretreated with the ADORA2B agonist BAY 60-6583 were protected from bleomycin-induced ALI by reducing vascular leakage (558.6 ± 50.4 vs. 379.9 ± 70.4, p < 0.05), total bronchoalveolar lavage fluid cell numbers (17.9 ± 1.8 to 13.4 ± 1.4 e4, p < 0.05), and neutrophil infiltration (6.42 ± 0.25 vs. 3.94 ± 0.29, p < 0.05). While mice lacking Adora2b in AECs were no longer protected by dipyridamole. We also identified occludin and focal adhesion kinase as downstream targets of ADORA2B, thus providing a novel mechanism for adenosine-mediated barrier protection. Similarly, we also observed similar enhanced ADORA2B (3.33 ± 0.67 to 16.12 ± 5.89, p < 0.05) and decreased occludin (81.2 ± 0.3 to 13.3 ± 0.4, p < 0.05) levels in human Acute respiratory distress syndrome lungs. Conclusion: We have highlighted a role of dipyridamole and adenosine signaling in preventing or treating ALI and identified Ent2 and Adora2b as key mediators in important for the resolution of ALI.

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.

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).

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.

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.

Neonatal diet impacts liver mitochondrial bioenergetics in piglets fed formula or human milk.

BACKGROUND: Neonatal diet impacts many physiological systems and can modify risk for developing metabolic disease and obesity later in life. Less well studied is the effect of postnatal diet (e.g., comparing human milk (HM) or milk formula (MF) feeding) on mitochondrial bioenergetics. Such effects may be most profound in splanchnic tissues that would have early exposure to diet-associated or gut microbe-derived factors. METHODS: To address this question, we measured ileal and liver mitochondrial bioenergetics phenotypes in male piglets fed with HM or MF from day 2 to day 21 age. Ileal and liver tissue were processed for mitochondrial respiration (substrate only [pyruvate, malate, glutamate], substrate + ADP, and proton "leak" post-oligomycin; measured by Oroboros methods), mitochondrial DNA (mtDNA) and metabolically-relevant gene expression analyses. RESULTS: No differences between the diet groups were observed in mitochondrial bioenergetics indices in ileal tissue. In contrast, ADP-dependent liver Complex I-linked OXPHOS capacity and Complex I + II-linked OXPHOS capacity were significantly higher in MF animals relative to HM fed piglets. Interestingly, p53, Trap1, and Pparß transcript abundances were higher in MF-fed relative to HM-fed piglets in the liver. Mitochondrial DNA copy numbers (normalized to nuclear DNA) were similar within-tissue regardless of postnatal diet, and were ~ 2-3 times higher in liver vs. ileal tissue. CONCLUSION: While mechanisms remain to be identified, the data indicate that neonatal diet can significantly impact liver mitochondrial bioenergetics phenotypes, even in the absence of a change in mtDNA abundance. Since permeabilized liver mitochondrial respiration was increased in MF piglets only in the presence of ADP, it suggests that formula feeding led to a higher ATP turnover. Specific mechanisms and signals involved with neonatal diet-associated differences in liver bioenergetics remain to be elucidated.