CHIP facilitates ubiquitination of inducible nitric oxide synthase and promotes its proteasomal degradation

Inducible nitric oxide synthase (iNOS) is responsible for nitric oxide (NO) synthesis from l-arginine in response to inflammatory mediators. It is reported that iNOS is degraded mainly by the ubiquitin-proteasome pathway in RAW264.7 cells and human embryonic kidney (HEK) 293 cells. In this study, we showed that iNOS was ubiquitinated and degraded dependent on CHIP (COOH terminus of heat shock protein 70-interacting protein), a chaperone-dependent ubiquitin ligase. The results from overexpression and RNAi experiments demonstrated that CHIP decreased the protein level of iNOS, shortened the half-life of iNOS and attenuated the production of NO. Furthermore, CHIP promoted ubiquitination and proteasomal degradation of iNOS by associating with iNOS. These results suggest that CHIP plays an important role in regulation iNOS activity.     

microRNA-338-3p promotes ox-LDL-induced endothelial cell injury through targeting BAMBI and activating TGF-β/Smad pathway

microRNAs (miRNAs) have been revealed to participate in the pathological process of atherosclerosis (AS). However, the exact role of miR-338-3p, a target miRNA of BMP and activin membrane-bound inhibitor (BAMBI), and its possible molecular mechanism in AS remain unidentified. In this study, we found that BAMBI was significantly decreased, whereas miR-338-3p increased in patients with AS and oxidized low-density lipoprotein (ox-LDL)-induced HUVEC cells. Furthermore, overexpression of miR-338-3p significantly decreased cell viability and elevated cell apoptosis, whereas its inhibition significantly promoted cell viability and inhibited cell apoptosis in ox-LDL-induced HUVEC cells. Moreover, miR-338-3p overexpression increased TGF-β/Smad pathway activation in ox-LDL-induced HUVEC cells. A dual-luciferase reporter assay confirmed the direct interaction between miR-338-3p and the 3′-untranslated region of BAMBI messenger RNA. Furthermore, the suppression of BAMBI ameliorated the effect of miR-338-3p inhibition against ox-LDL-induced HUVEC cell injury. In conclusion, our study thus suggests that miR-338-3p promoted ox-LDL-induced HUVEC cell injury by targeting BAMBI and activating the TGF-β/Smad pathway, which may provide a novel and promising therapeutic target for AS.     

海南红厚壳花香气成分采集和TCT-GC-MS分析

用鲜花循环式动态顶空技术采集红厚壳鲜花的香气成分,TCT-GC-MS联用技术分析鉴定。4-羟基-2-丁酮,双烯酮,1、2-环氧-2-甲基丁烷,1、2-环氧-3-甲基丁烷,2-乙基戊烷,3-己醇,甲基环戊烷,2-丁醇等24个成分被检测出,占总离子流出峰面积的90.28%。并对结果进行了分析讨论。     

Systems Mapping for Hematopoietic Progenitor Cell Heterogeneity

Cells with the same genotype growing under the same conditions can show different phenotypes, which is known as “population heterogeneity”. The heterogeneity of hematopoietic progenitor cells has an effect on their differentiation potential and lineage choices. However, the genetic mechanisms governing population heterogeneity remain unclear. Here, we present a statistical model for mapping the quantitative trait locus (QTL) that affects hematopoietic cell heterogeneity. This strategy, termed systems mapping, integrates a system of differential equations into the framework for systems mapping, allowing hypotheses regarding the interplay between genetic actions and cell heterogeneity to be tested. A simulation approach based on cell heterogeneity dynamics has been designed to test the statistical properties of the model. This model not only considers the traditional QTLs, but also indicates the methylated QTLs that can illustrate non-genetic individual differences. It has significant implications for probing the molecular, genetic and epigenetic mechanisms of hematopoietic progenitor cell heterogeneity.     

Partial Protective Effect of Intranasal Immunization with Recombinant Toxoplasma gondii Rhoptry Protein 17 against Toxoplasmosis in Mice

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite that infects a variety of mammals, including humans. An effective vaccine for this parasite is therefore needed. In this study, RH strain T. gondii rhoptry protein 17 was expressed in bacteria as a fusion with glutathione S-transferase (GST) and the recombinant proteins (rTgROP17) were purified via GST-affinity chromatography. BALB/c mice were nasally immunised with rTgROP17, and induction of immune responses and protection against chronic and lethal T. gondii infections were investigated. The results revealed that mice immunised with rTgROP17 produced high levels of specific anti-rTgROP17 IgGs and a mixed IgG1/IgG2a response of IgG2a predominance. The systemic immune response was associated with increased production of Th1 (IFN-γand IL-2) and Th2 (IL-4) cytokines, and enhanced lymphoproliferation (stimulation index, SI) in the mice immunised with rTgROP17. Strong mucosal immune responses with increased secretion of TgROP17-specific secretory IgA (SIgA) in nasal, vaginal and intestinal washes were also observed in these mice. The vaccinated mice displayed apparent protection against chronic RH strain infection as evidenced by their lower liver and brain parasite burdens (59.17% and 49.08%, respectively) than those of the controls. The vaccinated mice also exhibited significant protection against lethal infection of the virulent RH strain (survival increased by 50%) compared to the controls. Our data demonstrate that rTgROP17 can trigger strong systemic and mucosal immune responses against T. gondii and that ROP17 is a promising candidate vaccine for toxoplasmosis.     

Redox State of Pentraxin 3 as a Novel Biomarker for Resolution of Inflammation and Survival in Sepsis

In an endotoxaemic mouse model of sepsis, a tissue-based proteomics approach for biomarker discovery identified long pentraxin 3 (PTX3) as the lead candidate for inflamed myocardium. When the redox-sensitive oligomerization state of PTX3 was further investigated, PTX3 accumulated as an octamer as a result of disulfide-bond formation in heart, kidney, and lung—common organ dysfunctions seen in patients with sepsis. Oligomeric moieties of PTX3 were also detectable in circulation. The oligomerization state of PTX3 was quantified over the first 11 days in critically ill adult patients with sepsis. On admission day, there was no difference in the oligomerization state of PTX3 between survivors and non-survivors. From day 2 onward, the conversion of octameric to monomeric PTX3 was consistently associated with a greater survival after 28 days of follow-up. For example, by day 2 post-admission, octameric PTX3 was barely detectable in survivors, but it still constituted more than half of the total PTX3 in non-survivors (p < 0.001). Monomeric PTX3 was inversely associated with cardiac damage markers NT-proBNP and high-sensitivity troponin I and T. Relative to the conventional measurements of total PTX3 or NT-proBNP, the oligomerization of PTX3 was a superior predictor of disease outcome.Severe sepsis is a common acute illness in intensive care units (ICUs)¹ and is associated with high mortality rates and chronic morbidity. When it is associated with hypotension (termed septic shock), the mortality rate is very high (50% to 80%). Cardiovascular dysfunction during sepsis is multifactorial and often associated with minimal loss of myocardial tissue, but with the release of myocardial-specific markers such as troponins. A key unmet clinical need is the availability of a biomarker that predicts myocardial dysfunction early, monitors response to treatment, and thus identifies a cohort of patients at higher risk of septic shock to aid in targeted interventions and improve outcome (1).In the present study, we used proteomics for biomarker discovery. Over the past decade, the field of proteomics has made impressive progress. Plasma and serum, however, are the most complex proteomes of the human body (2), and less abundant proteins tend to be missed in untargeted proteomics analyses of body fluids (3). Thus, we pursued an alternative strategy: the application of proteomics to diseased tissue (4), in which the potential biomarkers are less dilute and have a less uncertain cellular origin (5–7). We employed a solubility-based protein-subfractionation methodology to analyze inflammatory proteins that are retained with sepsis tissue. This innovative proteomics approach shall reveal inflammatory molecules that reside and persist within inflamed tissue. We hypothesized that proteins that accumulate in the susceptible tissues are more likely to be biomarker candidates for organ dysfunction than proteins that just circulate in plasma or serum. We then validated our proteomics findings in the preclinical model using samples from sepsis patients admitted to ICUs.     

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each year. While several animal models exist for this procedure and mice are the species that is most commonly used. The reasons for using mice are the relative cost of using this species, the existence of many genetically defined strains that allow for the study of immune responses, and the existence of an extensive array of reagents that can be used to further define responses in this species. This model has been used to define factors in the cornea that are responsible for the relative immune privilege status of this tissue that enables corneal allografts to survive acute rejection in the absence of immunosuppressive therapy. It has also been used to define those factors that are most important in rejection of such allografts. Consequently, much of what we know concerning mechanisms of both corneal allograft acceptance and rejection are due to studies using a murine model of corneal transplantation. In addition to describing a model for acute corneal allograft rejection, we also present for the first time a model of late-term corneal allograft rejection.     

TRPV1 activation impedes foam cell formation by inducing autophagy in oxLDL-treated vascular smooth muscle cells

Vascular smooth muscle cells (VSMCs) are an important origin of foam cells besides macrophages. The mechanisms underlying VSMC foam cell formation are relatively little known. Activation of transient receptor potential vanilloid subfamily 1 (TRPV1) and autophagy have a potential role in regulating foam cell formation. Our study demonstrated that autophagy protected against foam cell formation in oxidized low-density lipoprotein (oxLDL)-treated VSMCs; activation of TRPV1 by capsaicin rescued the autophagy impaired by oxLDL and activated autophagy–lysosome pathway in VSMCs; activation of TRPV1 by capsaicin impeded foam cell formation of VSMCs through autophagy induction; activation of TRPV1 by capsaicin induced autophagy through AMP-activated protein kinase (AMPK) signaling pathway. This study provides evidence that autophagy plays an important role in VSMC foam cell formation and highlights TRPV1 as a promising therapeutic target in atherosclerosis.