Description: Background— Two endophenotypes of arterial calcification, calcification on arterial wall and calcification in atherosclerotic plaques, are associated with different types of cardiovascular events. Mgp -deficient mice showed matrix Gla protein (MGP) is strongly associated with calcification on arterial wall without atherosclerotic plaques, and MGP variants were not significantly associated with myocardial infarction. MGP may play different roles in the 2 endophenotypes. Methods and Results— We analyzed the associations of MGP variants rs4236, rs1800801, and rs1800802 with the 2 endophenotypes determined by multidetector computed tomography angiography. A total of 585 with calcification on coronary artery wall, 675 with calcification in coronary atherosclerotic plaques, 454 with calcification on aortic wall, and 725 controls were enrolled. After Bonferroni correction, rs4236 and rs1800801 were still associated with calcification on arterial wall, the odds ratios were 0.708 (95% confidence interval, 0.540–0.928) for rs4236 and 0.652 (95% confidence interval, 0.479–0.888) for rs1800801 in coronary artery wall calcification, and 0.699 (95% confidence interval, 0.525–0.931) for rs4236 and 0.650 (95% confidence interval, 0.467–0.905) for rs1800801 in aortic wall calcification, respectively. The variants were correlated with calcification severity by ln(CAC Agatston score+1) in coronary artery wall calcification but not in atherosclerotic plaque calcification. In accordance with their associations with calcification on arterial wall, rs4236C and rs1800801A were associated with higher MGP plasma levels, whereas rs1800802C was associated with lower MGP levels in normal controls. Because of the role of calcification in plaque vulnerability, their associations with acute myocardial infarction were also determined in 771 controls and 752 patients, no association was found. Conclusions— MGP genetic variants showed association with calcification on arterial wall but not with calcification in atherosclerotic plaques.

Description: Long intergenic noncoding RNAs (lincRNAs) are long noncoding transcripts (〉200 nt) from the intergenic regions of annotated protein-coding genes. We report here that the lincRNA gene lincRNA-Tnfaip3 , located at mouse chromosome 10 proximal to the tumor necrosis factor α-induced protein 3 ( Tnfaip3 ) gene, is an early-primary response gene controlled by nuclear factor-B (NF-B) signaling in murine macrophages. Functionally, lincRNA- Tnfaip3 appears to mediate both the activation and repression of distinct classes of inflammatory genes in macrophages. Specifically, induction of lincRNA-Tnfaip3 is required for the transactivation of NF-B-regulated inflammatory genes in response to bacterial LPSs stimulation. LincRNA-Tnfaip3 physically interacts with the high-mobility group box 1 (Hmgb1), assembling a NF-B/Hmgb1/lincRNA-Tnfaip3 complex in macrophages after LPS stimulation. This resultant NF-B/Hmgb1/lincRNA-Tnfaip3 complex can modulate Hmgb1-associated histone modifications and, ultimately, transactivation of inflammatory genes in mouse macrophages in response to microbial challenge. Therefore, our data indicate a new regulatory role of NF-B-induced lincRNA-Tnfaip3 to act as a coactivator of NF-B for the transcription of inflammatory genes in innate immune cells through modulation of epigenetic chromatin remodeling.—Ma, S., Ming, Z., Gong, A.-Y., Wang, Y., Chen, X., Hu, G., Zhou, R., Shibata, A., Swanson, P. C., Chen, X.-M. A long noncoding RNA, LincRNA-Tnfaip3, acts as a coregulator of NF-B to modulate inflammatory gene transcription in mouse macrophages.

Description: Erwinia amylovora causes a devastating disease called fire blight in rosaceous plants. The type III secretion system (T3SS) is one of the important virulence factors utilized by E. amylovora in order to successfully infect its hosts. By using a green fluorescent protein (GFP) reporter construct combined with a high-throughput flow cytometry assay, a library of phenolic compounds and their derivatives was studied for their ability to alter the expression of the T3SS. Based on the effectiveness of the compounds on the expression of the T3SS pilus, the T3SS inhibitors 4-methoxy-cinnamic acid (TMCA) and benzoic acid (BA) and one T3SS inducer, trans -2-(4-hydroxyphenyl)-ethenylsulfonate (EHPES), were chosen for further study. Both the T3SS inhibitors (TMCA and BA) and the T3SS inducer (EHPES) were found to alter the expression of T3SS through the HrpS-HrpL pathway. Additionally, TMCA altered T3SS expression through the rsmB Ea -RsmA Ea system. Finally, we found that TMCA and BA weakened the hypersensitive response (HR) in tobacco by suppressing the T3SS of E. amylovora . In our study, we identified phenolic compounds that specifically targeted the T3SS. The T3SS inhibitor may offer an alternative approach to antimicrobial therapy by targeting virulence factors of bacterial pathogens.

Description: Background and Purpose— A novel quantitative susceptibility mapping (QSM) processing technology has been developed to map tissue susceptibility property without blooming artifacts. We hypothesize that hematoma volume measurement on QSM is independent of imaging parameters, eliminating its echo time dependence on gradient echo MRI. Methods— Gradient echo MRI of 16 patients with intracerebral hemorrhage was processed with susceptibility-weighted imaging, R 2 * (=1/T2*) mapping, and QSM at various echo times. Hematoma volumes were measured from these images. Results— Linear regression of hematoma volume versus echo time showed substantial slopes for gradient echo magnitude (0.45±0.31 L/s), susceptibility-weighted imaging (0.52±0.46), and R 2 * (0.39±0.30) but nearly zero slope for QSM (0.01±0.05). At echo time=20 ms, hematoma volume on QSM was 0.80 x that on gradient echo magnitude image ( R 2 =0.99). Conclusions— QSM can provide reliable measurement of hematoma volume, which can be performed rapidly and accurately using a semiautomated segmentation tool.

Description: Clostridium difficile is a spore-forming bacillus that produces toxin-mediated enteric disease. C. difficile expresses two major virulence factors, toxin A (TcdA) and toxin B (TcdB). Human and animal studies demonstrate a clear association between humoral immunity to these toxins and protection against C. difficile infection (CDI). The receptor binding-domains (RBDs) of TcdA and TcdB are known to be immunogenic. Here, we tested the immunoadjuvant properties of Salmonella enterica serovar Typhimurium flagellin (FliC) subunit D1 as an innate immune agonist expressed as a recombinant fusion vaccine targeting the RBDs of TcdA and TcdB in mice. Intraperitoneally immunized mice developed prominent anti-TcdA and anti-TcdB immunoglobulin G in serum. The protective efficacy of the recombinant vaccines, with or without an adjuvant, was tested in a mouse model of CDI that closely represents the human disease. Following intraperitoneal immunization equivalent to two doses of toxoid A and toxoid B vaccine adjuvanted with alum and oral challenge with C. difficile VPI 10463, C57BL/6 mice were able to mount a protective immune response that prevented diarrhea and death compared to mice immunzed with alum alone. These results are significantly different from those for control mice ( P 〈 0.001). These results provide evidence that a recombinant protein-based vaccine targeting the RBDs of the C. difficile toxins adjuvanted with S . Typhimurium flagellin can induce rapid, high-level protection in a mouse model of CDI when challenged with the homologous strain from which the vaccine antigens were derived and warrant further preclinical testing against clinically relevant C. difficile strains in the mouse and hamster models of CDI.

Description: 18 F-FPPRGD2, which was approved for clinical study recently, has favorable properties for integrin targeting and showed potential for antiangiogenic therapy and early response monitoring. However, the time-consuming multiple-step synthesis may limit its widespread applications in the clinic. In this study, we developed a simple lyophilized kit for labeling PRGD2 peptide ( 18 F-AlF-NOTA-PRGD2, denoted as 18 F-alfatide) using a fluoride–aluminum complex that significantly simplified the labeling procedure. Methods: Nine patients with a primary diagnosis of lung cancer were examined by both static and dynamic PET imaging with 18 F-alfatide, and 1 tuberculosis patient was investigated using both 18 F-alfatide and 18 F-FDG imaging. Standardized uptake values were measured in tumors and other main organs at 30 min and 1 h after injection. Kinetic parameters were calculated by Logan graphical analysis. Immunohistochemistry and staining intensity quantification were performed to confirm the expression of integrin α v β 3 . Results: Under the optimal conditions, the whole radiosynthesis including purification was accomplished within 20 min with a decay-corrected yield of 42.1% ± 2.0% and radiochemical purity of more than 95%. 18 F-alfatide PET imaging identified all tumors, with mean standardized uptake values of 2.90 ± 0.10. Tumor-to-muscle and tumor-to-blood ratios were 5.87 ± 2.02 and 2.71 ± 0.92, respectively. Conclusion: 18 F-alfatide can be produced with excellent radiochemical yield and purity via a simple, 1-step, lyophilized kit. PET scanning with 18 F-alfatide allows specific imaging of α v β 3 expression with good contrast in lung cancer patients. This technique might be used for the assessment of angiogenesis and for planning and response evaluation of cancer therapies that would affect angiogenesis status and integrin expression levels.

Description: Nebivolol is a selective β1-blocker with nitric oxide–enhancing effects. MicroRNAs are small noncoding RNA molecules that downregulate gene expression. We compared the effects of nebivolol and atenolol, a first generation β1-selective blocker, on left ventricular hypertrophy, fibrosis, and function and microRNA expression in a rodent model of hypertension. Dahl salt-sensitive rats received either low-salt chow (control) or AIN-76A high-salt (8% NaCl) diet and randomized to vehicle (high-salt), nebivolol (20 mg/kg per day), or atenolol (50 mg/kg per day) for 8 weeks. High-salt induced left ventricular hypertrophy and fibrosis and decreased the expression of miR-27a, -29a, and -133a. Nebovolol attenuated deterioration of left ventricular systolic function, remodeling, and fibrosis more than atenolol, despite similar effects on heart rate and blood pressure. Nebivolol, but not atenolol, prevented the decrease in miR-27a and -29a induced by high-salt. Nebivolol and atenolol equally attenuated the decrease in miR-133a. In vitro overexpression of miR-27a,-29a, and -133a inhibited cardiomyocyte hypertrophy and reduced collagen expression. Both miR-27a and -29a target Sp1, and miR-133a targets Cdc42. Pharmacological inhibition of Sp1 and Cdc42 decreased myocardial fibrosis and hypertrophy. Our data support a differential microRNAs expression profile in salt-induced hypertension. Nebivolol substantially attenuated cardiac remodeling, hypertrophy, and fibrosis more than atenolol. These effects are related to attenuation of the hypertension-induced decrease in miR-27a and -29a (with a subsequent decrease in Sp1 expression) and miR-133a (with a subsequent decrease in Cdc42).

Description: Clostridium difficile infection (CDI) is a common and debilitating nosocomial infection with high morbidity and mortality. C. difficile mediates diarrhea and colitis by releasing two toxins, toxin A and toxin B. Since both toxins stimulate proinflammatory signaling pathways in human colonocytes and both are involved in the pathophysiology of CDI, neutralization of toxin A and B activities may represent an important therapeutic approach against CDI. Recent studies indicated that human monoclonal antibodies (MAbs) against toxins A and B reduce their cytotoxic and secretory activities and prevent CDI in hamsters. Moreover, anti-toxin A and anti-toxin B MAbs together with antibiotics also effectively reduced recurrent CDI in humans. However, whether these MAbs neutralize toxin A- and toxin B-associated immune responses in human colonic mucosa or human peripheral blood monocyte cells (PBMCs) has never been examined. We used fresh human colonic biopsy specimens and peripheral blood monocytes to evaluate the effects of these antibodies against toxin A- and B-associated cytokine release, proinflammatory signaling, and histologic damage. Incubation of anti-toxin A (MK3415) or anti-toxin B (MK6072) MAbs with human PBMCs significantly inhibited toxin A- and toxin B-mediated tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) expression. MK3415 and MK6072 also diminished toxin A- and toxin B-mediated NF-B p65 phosphorylation in human monocytes, respectively, and significantly reduced toxin A- and B-induced TNF-α and IL-1β expression as well as histologic damage in human colonic explants. Our results underline the effectiveness of MK3415 and MK6072 in blocking C. difficile toxin A- and toxin B-mediated inflammatory responses and histologic damage.

Description: Rationale: Transplantation of stem cells into damaged hearts has had modest success as a treatment for ischemic heart disease. One of the limitations is the poor stem cell survival in the diseased microenvironment. Prolyl hydroxylase domain protein 2 (PHD2) is a cellular oxygen sensor that regulates 2 key transcription factors involved in cell survival and inflammation: hypoxia-inducible factor and nuclear factor-B. Objective: We studied whether and how PHD2 silencing in human adipose-derived stem cells (ADSCs) enhances their cardioprotective effects after transplantation into infarcted hearts. Methods and Results: ADSCs were transduced with lentiviral short hairpin RNA against prolyl hydroxylase domain protein 2 (shPHD2) to silence PHD2. ADSCs, with or without shPHD2, were transplanted after myocardial infarction in mice. ADSCs reduced cardiomyocyte apoptosis, fibrosis, and infarct size and improved cardiac function. shPHD2-ADSCs exerted significantly more protection. PHD2 silencing induced greater ADSC survival, which was abolished by short hairpin RNA against hypoxia-inducible factor-1α. Conditioned medium from shPHD2-ADSCs decreased cardiomyocyte apoptosis. Insulin-like growth factor-1 (IGF-1) levels were significantly higher in the conditioned medium of shPHD2-ADSCs versus ADSCs, and depletion of IGF-1 attenuated the cardioprotective effects of shPHD2-ADSC–conditioned medium. Nuclear factor-B activation was induced by shPHD2 to induce IGF-1 secretion via binding to IGF-1 gene promoter. Conclusions: PHD2 silencing promotes ADSCs survival in infarcted hearts and enhances their paracrine function to protect cardiomyocytes. The prosurvival effect of shPHD2 on ADSCs is hypoxia-inducible factor-1α dependent, and the enhanced paracrine function of shPHD2-ADSCs is associated with nuclear factor-B–mediated IGF-1 upregulation. PHD2 silencing in stem cells may be a novel strategy for enhancing the effectiveness of stem cell therapy after myocardial infarction.

Description: This study aimed to test the hypothesis that the brain of Protopterus annectens expressed l -gulono--lactone oxidase ( gulo /Gulo), the enzyme catalyzing the last step of ascorbate biosynthesis, and could maintain high concentrations of ascorbate during estivation. We cloned and sequenced gulo from the kidney of P. annectens and performed quantitative PCR to determine its mRNA expression in kidney and brain. Gulo activity was assayed and its protein abundance was determined by Western blot using custom-made anti-Gulo antibody. Effects of estivation on concentrations of ascorbate and dehydroascorbate in the kidney and brain were also determined. Both brain and kidney, but not liver, of P. annectens expressed gulo /Gulo. Desiccation induced P. annectens to estivate, and 6 mo of estivation led to drastic decreases in gulo /Gulo expression and ascorbate concentration in the kidney. However, high concentrations of ascorbate and ascorbate + dehydroascorbate were maintained in the brain during estivation, probably resulting from in situ ascorbate synthesis. Control fish were placed in freshwater, where they were fully active in a favorable environment unlike estivation on land. The ability to synthesize ascorbate to ameliorate oxidative stress directly in the brain might contribute to the ability of P. annectens to undergo prolonged estivation on land.—Ching, B., Ong, J. L. Y., Chng, Y. R., Chen, X. L., Wong, W. P., Chew, S. F., Ip, Y. K. l -gulono--lactone oxidase expression and vitamin C synthesis in the brain and kidney of the African lungfish, Protopterus annectens.