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Due to the worldwide epidemic of allergic disease and a cure nowhere in sight, there is a crucial need to explore its pathophysiological mechanisms. As allergic disease has been associated with gut dysbiosis, we searched for a possible mechanism from the perspective of the molecular interface between host and microbiota with concurrent metabolomics and microbiome composition analysis. Sprague-Dawley rats were injected with Artemisia pollen extract to stimulate a hyper reaction to pollen. This hyper reaction decreased the circulation of valine, isoleucine, aspartate, glutamate, glutamine, indole-propionate (IPA), and myo-inositol, and reduced short-chain fatty acids (SCFAs) in feces. Several beneficial genera belonging to Ruminococcaceae, Lachnospiraceae, and Clostridiales declined in the model group, whereas Helicobacter and Akkermansia were only expressed in the model group. Furthermore, the expression of intestinal claudin-3 and liver fatty acid binding protein was downregulated in the model group and associated with metabolic changes and bacteria. Our results suggest that alterations in amino acids as well as their derivatives (especially valine, and IPA which is the reductive product of tryptophan) , SCFAs, and the gut microbiome (specifically Akkermansia and Helicobacter) may disrupt the intestinal barrier function by inhibiting the expression of claudin proteins and affecting the mucus layer, which further results in hay fever.

Two new premyrsinane-type diterpenes (2 and 3) as diastereomers were synthesized from lathyrane-type diterpene euphorbia factor L3 (1) for the first time via an efficient Fe(acac)3-catalyzed skeleton conversion process. This conversion features a biogenetically inspired strategy that relies on a concise reductive olefin coupling involving intramolecular Michael addition with free radicals. The structures of 2 and 3 were elucidated by a combination of the interpretation of their spectroscopic data and single-crystal X-ray diffraction analysis. The premyrsinane diterpenes 2 and 3 exhibited cytotoxic activity against the 4T1 breast cancer cell line, while the parent compound euphorbia factor L3 (1) was inactive. The current results not only confirmed the biogenetic relationship between lathyranes and premyrsinanes for the first time but also suggested a novel method for the preparation of naturally rare premyrsinane diterpenes with high bioactivity from the more abundant natural lathyrane diterpenes.

Objective Rap2c is a member of the Ras superfamily that has been implicated in various types of cancers. However, its role in glioma remains elusive. This study aimed to elucidate the role of Rap2c in glioma and its specific molecular mechanism. Methods We determined the expression of Rap2c in glioma tissues by Western blotting and immunohistochemistry (IHC) assays. The proliferation and apoptosis of cells were explored using CCK-8 and flow cytometry assay, whereas the migration and invasion of glioma cells were determined using transwell assay. The potential mechanism of Rap2c in the migration of glioma cell lines was investigated through Western blotting analysis and transwell assay. BALB/c nude mice were used to establish tumor models to test the effect of Rap2c on cancer metastasis in vivo. Results Our data showed that the protein expression of Rap2c was significantly up-regulated in glioma tissues compared with normal brain tissues, and Rap2c overexpression negatively correlated with 5-year overall survival rate. However, there was no correlation between Rap2c expression and clinicopathological parameters of glioma patients. Overexpression of Rap2c promoted the migration and invasion abilities of glioma cells but had no significant effect on the proliferation of glioma cells. Western blotting analysis revealed that Rap2c overexpression increased the phosphorylation level of extracellular signal-related kinase1/2 (ERK1/2), and this effect was abolished with U0126, a selective MEK inhibitor. Furthermore, overexpression of Rap2c induced lung metastasis of glioma cells in xenograft models. Conclusion These findings indicate that high Rap2c expression predicts poor prognosis in glioma. Rap2c-mediated ERK1/2 phosphorylation initiates EMT cascade and promotes migration and invasion of glioma cells. Thus, targeting Rap2c and ERK signaling pathway could be a novel treatment modality for glioma.

BACKGROUND Artemisia annua is an important autumnal pollen allergen for seasonal allergic rhinitis (SAR) in northern China. To date, no study has investigated allergen immunotherapy with A annua. We aimed to investigate the efficacy and mechanisms underlying A annua-sublingual immunotherapy (SLIT). METHODS This was a randomized, double-blind, placebo-controlled phase III clinical trial involving 71 SAR patients, randomized to SLIT with A annua extract (n = 47) or placebo (n = 24) for 32 weeks. Total nasal symptom score (TNSS; primary clinical end point) was evaluated at baseline (peak pollen phase (PPP) in the previous year), initiation of A annua-SLIT, 1st PPP during SLIT, end of SLIT and 2nd PPP during follow-up. Blood samples and nasal secretions were collected at beginning and after SLIT for assessment of T cells and inflammatory mediators. Safety was assessed according to adverse events (AEs) reported. RESULTS Artemisia annua-SLIT significantly reduced TNSS to a greater level from baseline (from 9.45 ± 1.68 to 6.16 ± 2.27) than placebo (from 9.29 ± 2.09 to 9.05 ± 2.40) at the 1st PPP (P

Aim Magnesium (Mg) deficiency (known as grass tetany) is a serious metabolic disorder that affects grazing ruminants. We tested whether Mg-fertiliser can increase Mg concentration of Italian ryegrasses (Lolium multiflorum L.) including a cultivar (cv. Bb2067; ‘Magnet’), bred to accumulate larger concentrations of Mg. Methods Under controlled environment (CE) conditions, three cultivars (cv. Bb2067, cv. Bb2068, cv. RvP) were grown in low-nutrient compost at six fertiliser rates (0–1500 μM MgCl2.6H2O). Under field conditions, the three cultivars in the CE condition and cv. Alamo were grown at two sites, and four rates of MgSO4 fertiliser application rates (0–200 kg ha−1 MgO). Multiple grass cuts were taken over two-years. Results Grass Mg concentration increased with increasing Mg-fertiliser application rates in all cultivars and conditions. Under field conditions, cv. Bb2067 had 11–73% greater grass Mg concentration and smaller forage tetany index (FTI) than other cultivars across the Mg-fertiliser application rates, sites and cuts. Grass dry matter (DM) yield of cv. Bb2067 was significantly (p p ≥ 0.05). Conclusions Biofortification of grass with Mg through breeding and agronomy can improve the forage Mg concentration for grazing ruminants, even in high-growth spring grass conditions when hypomagnesaemia is most prevalent. Response to agronomic biofortification varied with cultivar, Mg-fertiliser rate, site and weather. The cost:benefit of these approaches and farmer acceptability, and the impact on cattle and sheep grazing on grasses biofortified with Mg requires further investigation.

To evaluate the relationship between the positions of cytoplasmic granulation and the oocytes developmental potential in human, we detected the developmental potentials of oocytes with centrally located cytoplasmic granulation (CLCG). The patients’ age, body mass index (BMI), Infertility duration, follicle stimulation hormone (FSH) levels, average stimulate ovulation days, gonadotropin (GN) total dose, fertilization rate, cleavage rate, high quality embryo rate, embryo utilization rate and pregnancy rate were analyzed. The results showed that there were no significant difference on patients’ age, BMI, infertility duration, FSH levels, average stimulate ovulation days, GN total dose, pregnancy rate and birth rate between CLCG group and control group in patients with BMI P > 0.05). However, there was no significant difference in fertilization rate, cleavage rate, and high quality embryo rate in patients with BMI P > 0.05). The pregnancy rate was low in both groups, but 35 and 15 healthy fetuses were born in each group. We also found that the central granulated area size did not affect fertilization rate, cleavage rate, embryo utilization rate, and high quality embryo rate (P > 0.05). These results suggested CLCG might be a normal morphology of oocyte. The oocytes from patients with or without CLCG had no significant difference in their developmental potentials. The patients who transferred CLCG embryos had successful delivery. The developmental potentials of oocytes with different CLCG grades had no obvious differences.

The PhosPhAt 4.0 database contains information on Arabidopsis phosphorylation sites identified by mass spectrometry in large-scale experiments from different research groups. So far PhosPhAt 4.0 has been one of the most significant large-scale data resources for plant phosphorylation studies. Functionalities of the web application, besides display of phosphorylation sites, include phosphorylation site prediction and kinase-target relationships retrieval. Here, we present an overview and user instructions for the PhosPhAt 4.0 database, with strong emphasis on recent renewals regarding protein annotation by SUBA4.0 and Mapman4, and additional phosphorylation site information imported from other databases, such as UniProt. Here, we provide a user guide for the retrieval of phosphorylation motifs from the kinase-target database and how to visualize these results. The improvements incorporated into the PhosPhAt 4.0 database have produced much more functionality and user flexibility for phosphoproteomic analysis.

The transmembrane receptor kinase family is the largest protein kinase family in Arabidopsis. Many members of this family play critical roles in plant signaling pathways. However, many of these kinases have yet uncharacterized functions and very little is known about the direct substrates of these kinases. We have developed the "ShortPhos" method, an efficient and simple mass spectrometry (MS)-based phosphoproteomics protocol to perform comparative phosphopeptide profiling of knockout mutants of receptor-like kinases. Through this method, we are able to better understand the functional roles of plant kinases in the context of their signaling networks.

Protein phosphorylation is an important cellular regulatory mechanism affecting the activity, localization, conformation, and interaction of proteins. Protein phosphorylation is catalyzed by kinases, and thus kinases are the enzymes regulating cellular signaling cascades. In the model plant Arabidopsis, 940 genes encode for kinases. The substrate proteins of kinases are phosphorylated at defined sites, which consist of common patterns around the phosphorylation site, known as phosphorylation motifs. The discovery of kinase specificity with a preference of phosphorylation of certain motifs and application of such motifs in deducing signaling cascades helped to reveal underlying regulation mechanisms, and facilitated the prediction of kinase-target pairs. In this mini-review, we took advantage of retrieved data as examples to present the functions of kinase families along with their commonly found phosphorylation motifs from their substrates.

Protective immunity to the obligate intracellular bacterium Chlamydia is thought to rely on CD4 T cell-dependent IFNγ production. Nevertheless, whether IFNγ is produced by other cellular source during Chlamydia infection and how CD4 T cell-dependent and -independent IFNγ contribute differently to host resistance has not been carefully evaluated. In this study, we dissect the requirements of IFNγ produced by innate immune cells and CD4 T cells for resolution of Chlamydia muridarum female reproductive tract (FRT) infection. After C. muridarum intravaginal inoculation, IFNγ-deficient and T cell-deficient mice exhibited opposite phenotypes for survival and bacterial shedding at the FRT mucosa, demonstrating the distinct requirements for IFNγ and CD4 T cells in host defense against Chlamydia. In Rag-deficient mice, IFNγ produced by innate lymphocytes (ILCs) accounted for early bacterial containment and prolonged survival in the absence of adaptive immunity. Although group I ILCs are potent IFNγ producers, we found that mature NK cells and ILC1 were not the sole source for innate IFNγ in response to Chlamydia. T cell adoptive transfer experiments revealed that WT and IFNγ-deficient CD4 T cells were equally capable of mediating effective bacterial killing in the FRT during the early stage of Chlamydia infection. Together, our results revealed that innate IFNγ is essential for preventing systemic Chlamydia dissemination, whereas IFNγ produced by CD4 T cells is largely dispensable at the FRT mucosa.

The application of tetracycline (TET) is very common in medical treatment, fisheries, and animal husbandry, resulting in its frequent detection with abundant concentrations in the aquatic environment. Though the effects of TET on zebrafish (Danio rerio) at embryonic and larval stages have been reported, there is very limited information on the possible long-term effect on aquatic fishes at the juvenile stage, especially at environmentally relevant levels. In this study, we have exposed juvenile zebrafish to two levels of TET at 1 and 100 µg/L for one month until their adulthood. The result showed that both levels of TET can significantly increase the body weight of the zebrafish, while there is no change in the body length. TET exposure also affected the liver microstructure by lipid vacuoles generation and global lipidomics analysis revealed a significant upregulation in hepatic triglyceride (TAG) levels. The metabolomics analysis showed great dysregulations in hepatic metabolic pathways including linoleic acid metabolism, tyrosine metabolism, and methionine metabolism, which are known to be linked with increased body weight gain through hepatic lipid accumulation. The hepatic gene expression involved in lipid transport (e.g., apoa4 and fabp11) and lipogenic factors (e.g., ppar) have been significantly upregulated in the livers of TET exposed zebrafish. Interestingly, the 16 rRNA gene sequence-based zebrafish gut microbial community analysis revealed an enhanced community diversity and altered microbial community composition upon TET exposure. To our knowledge, this is the first study showing that TET exposure can increase the body weight in juvenile zebrafish and the study on the ecotoxicity of antibiotic occurrences in the aquatic system can be further warranted.

Background The incidence of insulin resistance (IR) has rapidly increased worldwide over the last 20 years, no perfect solution has yet been identified. Finding new therapeutic drugs will help improve this situation. As a traditional Chinese medicine, PPM (processed Polygonum multiflorum) has widely been used in the clinic. Recently, other clinical functions of PPM have been widely analyzed. Results Administration of the water extract from PPM decreased the level of FBG, TC, and TG, and increased the level of FGC, thereby reducing the IR index and improving IR. Furthermore, Western blot analysis revealed that PPM significantly increased GPR43 and AMPK expression when compared with the MOD group, and GPR43, AMPK were known as glucose metabolism-related proteins. In addition, treatment with PPM can restore the balance of gut microbiota by adjusting the relative abundance of bacteria both at the phylum and genus level, and these changes have been reported to be related to IR. Methods Sprague Dawley (SD) rats were fed a high-fat diet and were gavaged daily with either normal saline solution or PPM for 12 weeks. Major biochemical indexes, such as fasting blood glucose (FBG), fasting glucagon (FGC), total cholesterol (TC), and triglyceride (TG) were measured. Then the protein expression of adenosine 5′-monophosphate -activated protein kinase (AMPK) and G protein-coupled receptor 43 (GPR43) was evaluated by using Western blot analysis. Moreover, the composition of gut microbiota was assessed by analyzing 16S rRNA sequences. Conclusions Our findings showed that PPM reversed the increasing of FBG and the decreasing of IRI, PPM accelerated the expression of glucose metabolism-related proteins and regulated the intestinal microecological balance. Therefore, we hold the opinion that PPM may be an effective option for treating IR.

Broilers stocked in high densities may be prone to oxidative and inflammatory insults, resulting in impaired health status, growth performance, and meat quality. This study was to determine if 30% extra supplemental dl-methionine alleviated or prevented those adverse effects of a higher stocking density in broiler chickens. A total of 560 male Cornish Cross cockerels (day old) were divided into four groups: two stocking densities (9 and 12 birds/m2) and two supplementations of methionine (grower: 2.90 or 3.77 g/kg and finisher: 2.60 or 3.38 g/kg). Growth performance was recorded weekly. Blood and tissues were sampled at the end of each period. High stocking density decreased (P < 0.05) body weight and growth performance of growers and (or) finishers. Those differences were partially attenuated by the extra methionine supplementation. The high methionine elevated (P < 0.05) glutathione (GSH) concentration in the thigh at both ages (> 24%). The high stocking density elevated (>28%, P < 0.05) glutathione concentration in the plasma, breast, and thigh of growers, but decreased (P < 0.05) it in the liver of growers and thigh of finishers. Interaction effects (P < 0.05) between dietary methionine and stocking density were found on activities of the antioxidant enzyme glutathione S-transferase in the liver of growers and breast, thigh, and adipose tissue of finishers. The interaction effect was also found on activities of glutathione peroxidase and superoxide dismutase in the thigh of growers. The extra methionine decreased (P < 0.05) hepatic gene expression of heat shock protein 90 (18%) and thigh and breast malondialdehyde concentrations of the finishers (35%). In conclusion, the 30% extra dl-methionine supplementation was able to partially mitigate adverse effects caused by the higher stocking density and to improve the redox status of the broilers.

Pteris cretica var. nervosa is a dominant fern species found in antimony (Sb) mining areas, capable of forming symbiosis with arbuscular mycorrhizal fungi (AMF), especially with those members of the Glomus genus. Despite this fern's relevance and the potential contribution of mycorrhizal symbiosis to phytoremediation, the AMF's impact on P. var. nervosa phytoremediation of Sb remains unknown. Here, we exposed P. var. nervosa to different concentrations of Sb for 6 months. Our results showed that Sb reduced shoot biomass, enlarged the root/shoot ratio, and disrupted the fronds' intracellular structure. AMF inoculation, however, was able to moderate these phenotypic changes and increased the accumulation level of Sb in plants. From a proteomics analysis of this plant's fronds, a total of 283 proteins were identified. Notably, those proteins with catalytic function, carbon fixing and ATP metabolic function were highly enriched. K-means clustering demonstrated protein-changing patterns involved in multiple metabolic pathways during exposure to Sb. Further, these patterns can be moderated by AMF inoculation. Pearson correlations were used to assess the plant biomarkers-soil Sb relationships; This revealed a strong correlation between ribosome alteration and the root/shoot ratio when inoculated with AMF, and a positive correlation between photosynthesis proteins and chlorophyll (SPAD value). Our results indicate AMF could moderate the fronds impairment by maintaining the sufficient protein levels for ribosomal functioning, photosynthesis activity and to counter ROS production. We demonstrate the effective use of AMF associated with P. cretica var. nervosa for Sb phytoremediation and the potential of applying proteomics to better understand the mechanism behind this symbiotic plant physiological response.

We find that the DREB subfamily transcription factor, CmERF053, has a novel function to regulate the development of shoot branching and lateral root in addition to affecting abiotic stress. Dehydration-responsive element binding proteins (DREBs) are important plant transcription factors that regulate various abiotic stresses. Here, we isolated an APETALA2/ethylene-responsive factor (AP2/ERF) transcription factor from chrysanthemum (Chrysanthemum morifolium 'Jinba'), CmERF053, the expression of which was rapidly up-regulated by main stem decapitation. Phylogenetic analysis indicated that it belongs to the A-6 group of the DREB subfamily, and the subcellular localization assay confirmed that CmERF053 was a nuclear protein. Overexpression of CmERF053 in Arabidopsis exhibited positive effects of plant lateral organs, which had more shoot branching and lateral roots than did the wild type. We also found that the expression of CmERF053 in axillary buds was induced by exogenous cytokinins. These results suggested that CmERF053 may be involved in cytokinins-related shoot branching pathway. In this study, an altered auxin distribution was observed during root elongation in the seedlings of the overexpression plants. Furthermore, overexpress CmERF053 gene could enhance drought tolerance. Together, these findings indicated that CmERF053 plays crucial roles in regulating shoot branching, lateral root, and drought stress in plant. Moreover, our study provides potential application value for improving plant productivity, ornamental traits, and drought tolerance.

OBJECTIVES: Maternal and infant undernutrition is highly prevalent in developing countries, leading to serious fetus/infant mortality, intrauterine growth restriction, stunting, and severe wasting. However, the effects of maternal undernutrition have generally focused on the reduced maternal nutrient supply to the fetus. The potential impairment of fetal metabolic pathways has not been well studied. METHODS: Pregnant gilts (Landrace x Yorkshire x Duroc) received the NRC gestation diet with (n = 4) or without (n = 4) 50% intake restriction at insemination day and 70% for the following gestation period. Full term fetuses were obtained via C-section, two piglets were selected from each gilt in both groups and subject to hepatic tissue collection. MicroRNA and mRNA deep sequencing analysis was performed using the Illumina GAIIx system. The mRNA-miRNA correlation and associated signaling pathways were analyzed via CLC workbench, Ingenuity Pathway Analysis Software. RESULTS: A total of 42 differentially expressed miRNAs were identified between intake-restriction and full-nutrition group. Among of these, mir-206, mir-133b, mir-1246, mir-1843 and mir-7139 are the most downregulated and mir-10b, mir-708 and mir-222 are the most upregulated miRNAs. A total of 1215 mRNAs were identified to differentially expressed between two groups. Two metabolic pathways: retinol biosynthesis and oxidative phosphorylation were significantly modified, and the modification was associated with the miRNA changes induced by the maternal feed restriction. Briefly, the retinol biosynthesis pathway was upregulated (p

Immune mechanisms responsible for pathogen clearance from the female reproductive tract (FRT) are incompletely defined; in particular, the contribution of lymphocyte trafficking to this process is unclear. CCR7-deficient mice have profoundly altered lymphocyte recirculation and display ectopic formation of lymphocyte aggregates within mucosal nonlymphoid tissues, including the FRT. In this study, we investigated how altered lymphocyte distribution in CCR7-deficient mice would affect host responses to Chlamydia muridarum within the reproductive tract. As expected, CCR7-deficient mice exhibited reduced lymphocyte trafficking to lymph nodes and a corresponding increase in T cell populations within the FRT. After intravaginal infection with Chlamydia, CCR7-deficient mice displayed markedly reduced Ag-specific CD4 T cell responses within the local draining iliac lymph nodes, yet robust Th1 and Th17 responses were prominent in the FRT. In addition, Chlamydia-specific Ab responses were dysregulated in CCR7-deficient mice, displaying an unexpected increase in the systemic IgA responses. Importantly, prominent mucosal immune responses in CCR7-deficient mice increased the efficiency of bacteria clearance from the FRT while reducing tissue-associated inflammation and pathology. Thus, increased numbers of lymphocytes within the FRT result in pathogen clearance with reduced immune-mediated pathology.

There are few diseases in modern biomedicine that have garnered as much scientific interest and public concern as Alzheimer's disease (AD). The amyloid hypothesis has become the dominant model of AD pathogenesis; however, the details of the hypothesis are changing over time. Recently, given the increasing recognition, subtle effects of amyloid β protein (Aβ) on synaptic efficacy may be critical to AD progression. Synaptic plasticity is the important neurochemical foundation of learning and memory. Recent studies have identified that soluble Aβ oligomers combine with certain receptors to impair synaptic plasticity in AD, which advanced the amyloid hypothesis. The aim of the present review was to summarize the role of Aβ-relevant receptors in regulating synaptic plasticity and their downstream signaling cascades, which may provide novel insights into the understanding of the pathogenesis of AD and the development of therapeutic strategies to slow down the progression of AD-associated memory decline in the early stages.

With a view to improving the establishment of Aphidius gifuensis Ashmead, a potential natural enemy for suppression of aphids on vegetables, the effects of host deprivation and honey feeding before release on the parasitoid’s egg load and reproduction were studied under laboratory conditions. Host deprivation significantly affected mummy production and survival rate of female parasitoids. A. gifuensis produced more mummies when exposed to a short period (1 and 2 days) of host deprivation, but when host deprivation lasted for 3 days, their mummy production sharply decreased. Offspring produced by parasitoids that had been deprived of hosts for 2 days were much heavier than those produced by parasitoids deprived of hosts for 1 and 3 days. However, host deprivation did not affect emergence rate or offspring sex ratio; the emergence rate was always above 80%, and the offspring sex ratio was always female biased irrespective of whether the host-deprivation period was 1, 2, or 3 days. Honey feeding greatly increased egg load, longevity and decelerated oosorption of A. gifuensis when hosts were absent, and significantly increased parasitoid mummy production in late age. We discuss the results in the context of augmentative biological control, with the view to understanding how to increase parasitoid performance after they are released.

The obligate intracellular bacterium Chlamydia trachomatis causes the most prevalent bacterial sexually transmitted infection worldwide. CD4 T cells play a central role in the protective immunity against Chlamydia female reproductive tract (FRT) infection, while B cells are thought to be dispensable for resolution of primary Chlamydia infection in mouse models. We recently reported an unexpected requirement of B cells in local Chlamydia-specific CD4 T-cell priming and bacterial containment within the FRT. Here, we sought to tackle the precise effector function of B cells during Chlamydia primary infection. Using mixed bone marrow chimeras that lack B-cell-dependent Ag presentation (MHCIIB - / - ) or devoid of circulating antibodies (AID-/- × μS-/- ), we show that Chlamydia-specific CD4 T-cell expansion does not rely on Ag presentation by B cells. Importantly, we demonstrate that antibody, but not B-cell-dependent Ag presentation, is required for preventing systemic bacterial dissemination following Chlamydia FRT infection.

Receptor kinases (RK) constitute the largest protein kinase family in plants. In particular, members of the leucine-rich repeat-receptor kinases (LRR-RKs) are involved in the perception of various signals at the plasma membrane. Experimental evidence over the past years revealed a conserved activation mechanism through ligand-inducible heterodimer formation: a ligand is recognized by a receptor kinase with a large extracellular domain (ECD). This ligand binding receptor directly interacts with a so-called co-receptor with a small ECD for ligand fixation and kinase activation. A large proportion of LRR-RKs is functionally still uncharacterized and the dynamic complexity of the plasma membrane makes it difficult to precisely define receptor kinase heterodimer pairs and their functions. In this review, we give an overview of the current knowledge of LRR receptor and co-receptor functions. We use ECD lengths to classify the LRR receptor kinase family and describe different interaction properties of ligand-binding receptors and their respective co-receptor from a network perspective.

Gastric cancer is the fourth most common type of cancer and the second cause of cancer‑related mortalities worldwide despite the use of multimodal therapy. Cadherins are transmembrane glycoproteins that are involved in tumorigenesis. CDH17 has been found to be over‑expressed in gastric cancer and its overexpression was associated with lymph node metastasis and tumor‑node‑metastasis stage of the patients, yet the exact role and molecular mechanism of CDH17 in gastric cancer have not been determined. Using a lentiviral system as a delivery mediator of RNA interference, we found that inhibition of CDH17 can lead to reduce proliferation and increase apoptosis of gastric cancer cell line MKN28 in vitro and significantly diminish their tumorigenicity in vivo. Our results of the present study suggest that CDH17 may be a promising candidate for the therapeutic targeting of gastric cancer.

The Nogo-66 receptor (NgR1), a receptor for Nogo-A, contributes to the inhibition of axonal regeneration in the adult central nervous system after traumatic injuries. Thus, NgR1 has been considered a critical target in axon regeneration therapy. Here, we identified a specific NgR1 antagonist peptide (HIYTALV, named NAP2) which promotes neurite regeneration in vitro from a phage display heptapeptide library. NAP2 was co-localized with NgR1 on the surface of PC12 cells and cerebellar granule cells (CGCs) by immunofluorescence assay. Horseradish peroxidase (HRP)-streptavidin-biotin assay further showed that NAP2 binds to NgR1 and the dissociation constant (Kd) was 0.45 μM Functional analyses indicated that NAP2 could reduce the inhibitory effects of Nogo-66 on neurite outgrowth in differentiated PC12 cells and CGCs by blocking the Nogo-66-induced activation of Rho-associated coiled coil-containing protein kinase (ROCK), collapsin response mediator protein 2 (CRMP2) and myosin light chain (MLC). Taken together, the small molecule NgR1 antagonist peptide NAP2 (MW: 815.98Da) has a potential ability in crossing blood brain barrier and will be a promising therapeutic agent for the treatment of spinal cord injury and neurodegenerative diseases.

The aim of this study was to investigate the toxic effects of aflatoxins and evaluate the effectiveness of Bacillus subtilis ANSB060 in detoxifying aflatoxicosis in broilers. A total of 360 one-week-old male broilers (Ross 308) were assigned to six dietary treatments for five weeks. The treatment diets were: C0 (basal diet), C1.0 (C0 + 1.0 g B. subtilis ANSB060/kg diet), M0 (basal diet formulated with moldy peanut meal), M0.5, M1.0 and M2.0 (M0 + 0.5, 1.0 and 2.0 g B. subtilis ANSB060/kg diet, respectively). The contents of aflatoxin B1, B2, G1 and G2 in the diets formulated with moldy peanut meal were 70.7 ± 1.3, 11.0 ± 1.5, 6.5 ± 0.8 and 2.0 ± 0.3 µg/kg, respectively. The results showed that aflatoxins increased (p &lt, 0.05) serum aspartate transaminase activity, decreased (p &lt, 0.05) serum glutathione peroxidase activity, and enhanced (p &lt, 0.05) malondialdehyde contents in both the serum and liver. Aflatoxins also caused gross and histological changes in liver tissues, such as bile duct epithelium hyperplasia, vacuolar degeneration and lymphocyte infiltration. The supplementation of ANSB060 reduced aflatoxin levels in the duodenum and counteracted the negative effects of aflatoxins, leading to the conclusion that ANSB060 has a protective effect against aflatoxicosis and this protection is dose-related.

GhNPR1 shares similar functions as Arabidopsis NPR1 . Silencing of GhNPR1 in Gladiolus results in an enhanced susceptibility to Curvularia gladioli. We propose that GhNPR1 plays important roles in plant immunity. Gladiolus plants and corms are susceptible to various types of pathogens including fungi, bacteria and viruses. Understanding the innate defense mechanism in Gladiolus is a prerequisite for the development of new protection strategies. The non-expressor of pathogenesis-related gene 1 (NPR1) and bzip transcription factor TGA2 play a key role in regulating salicylic acid (SA)-mediated systemic acquired resistance (SAR). In this study, the homologous genes, GhNPR1 and GhTGA2, were isolated from Gladiolus and functionally characterized. Expression of GhNPR1 exhibited a 3.8-fold increase in Gladiolus leaves following salicylic acid treatment. A 1332 bp fragment of the GhNPR1 promoter from Gladiolus hybridus was identified. Inducibility of the GhNPR1 promoter by SA was demonstrated using transient expression assays in the leaves of Nicotiana benthamiana. The GhNPR1 protein is located in the nucleus and cytomembrane. GhNPR1 interacts with GhTGA2, as observed using the bimolecular fluorescence complementation system. Overexpression of GhNPR1 in an Arabidopsis npr1 mutant can restore its basal resistance to Pseudomonas syringae pv. tomato DC3000. Silencing of GhNPR1, using a tobacco rattle virus-based silencing vector, resulted in an enhanced susceptibility to Curvularia gladioli. In conclusion, these results suggest that GhNPR1 plays a pivotal role in the SA-dependent systemic acquired resistance in Gladiolus.

We investigated the toxicity of Karenia mikimotoi toward three model grazers, the cladoceran Moina mongolica, the copepod Pseudodiaptomus annandalei, and the crustacean Artemia salina, and explored its chemical response upon zooplankton grazing. An induction experiment, where K. mikimotoi was exposed to grazers or waterborne cues from the mixed cultures revealed that K. mikimotoi might be toxic or nutritionally inadequate toward the three grazers. In general, direct exposure to the three grazers induced the production of hemolytic toxins and the synthesis of eicosapentaenoic acid (EPA). Both EPA and the hemolytic toxins from K. mikimotoi decreased the survival rate of the three grazers. In addition, the survival rates of M. mongolica, P. annandalei, and A. salina in the presence of induced K. mikimotoi that had previously been exposed to a certain grazer were lower than their counterparts caused by fresh K. mikimotoi, suggesting that exposure to some grazers might increase the toxicity of K. mikimotoi. The chemical response and associated increased resistance to further grazing suggested that K. mikimotoi could produce deterrents to protect against grazing by zooplankton and that the substances responsible might be hemolytic toxins and EPA.

Toll-like receptors (TLRs) can recognize microbial patterns and utilize adaptor molecules, such as-MyD88 or (TRIF TIR-domain-containing adapter-inducing interferon-β), to initiate downstream signaling that ultimately affects the initiation of adaptive immunity. In addition to this inflammatory role, TLR5 expression on dendritic cells can favor antigen presentation of flagellin peptides and thus increase the sensitivity of flagellin-specific T-cell responses in vitro and in vivo. Here, we examined the role of alternative signaling pathways that might regulate flagellin antigen presentation in addition to MyD88. These studies suggest a requirement for spleen tyrosine kinase, a noncanonical TLR-signaling adaptor molecule, and its downstream molecule CARD9 in regulating the sensitivity of flagellin-specific CD4(+) T-cell responses in vitro and in vivo. Thus, a previously unappreciated signaling pathway plays an important role in regulating the dominance of flagellin-specific T-cell responses.

Mass spectrometry (MS)-based large scale phosphoproteomics has facilitated the investigation of plant phosphorylation dynamics on a system-wide scale. However, generating large scale data sets for membrane phosphoproteins usually requires fractionation of samples and extended hands-on laboratory time. To overcome these limitations, we developed “ShortPhos,” an efficient and simple phosphoproteomics protocol optimized for research on plant membrane proteins. The optimized workflow allows fast and efficient identification and quantification of phosphopeptides, even from small amounts of starting plant materials. “ShortPhos” can produce label-free datasets with a high quantitative reproducibility. In addition, the “ShortPhos” protocol recovered more phosphorylation sites from membrane proteins, especially plasma membrane and vacuolar proteins, when compared to our previous workflow and other membrane-based data in the PhosPhAt 4.0 database. We applied “ShortPhos” to study kinase-substrate relationships within a nitrate-induction experiment on Arabidopsis roots. The “ShortPhos” identified significantly more known kinase-substrate relationships compared to previous phosphoproteomics workflows, producing new insights into nitrate-induced signaling pathways.

There is no effective drug for the therapy of acute carbon monoxide (CO) poisoning. The purpose of the present study was to investigate the potential preventive and therapeutic effects of hemin on an animal model of acute CO poisoning and to provide a potential therapeutic candidate drug. A total of 80 Kunming mice were randomly divided into four groups, namely the air control, acute CO poisoning, hemin-treatment + CO and hemin-pretreatment + CO groups (n=20 each). Furthermore, the mortality rate of mice, blood carboxyhaemoglobin (HbCO) concentration and serum malondialdehyde (MDA) concentration were measured, and pathological changes of the hippocampal area were determined using histochemical staining. The mice with acute CO poisoning had a 50% mortality rate at 1 h, with an increase in blood HbCO, serum MDA levels and pathological impairments of the hippocampus. Furthermore, the mortality rate, blood HbCO and serum MDA levels of mice with pretreatment and treatment of hemin were decreased. Additionally, the pathological changes of the hippocampal area were improved in the hemin-treatment and hemin-pretreatment groups compared with the mice treated with CO. These results suggest that hemin is a novel effective chemical for the prevention and treatment of acute CO poisoning in mice. Therefore, the present study provides a novel method and experimental basis for the application of hemin in treating patients with acute CO poisoning.

Genome-wide association studies (GWASs) have been performed extensively in diverse populations to identify single nucleotide polymorphisms (SNPs) associated with complex diseases or traits. However, to date, the SNPs identified fail to explain a large proportion of the variance of the traits/diseases. GWASs on type 2 diabetes (T2D) and obesity are generally focused on individual traits independently, and genetic intercommunity (common genetic contributions or the product of over correlated phenotypic world) between them are largely unknown, despite extensive data showing that these two phenotypes share both genetic and environmental risk factors. Here, we applied a recently developed genetic pleiotropic conditional false discovery rate (cFDR) approach to discover novel loci associated with BMI and T2D by incorporating the summary statistics from existing GWASs of these two traits. Conditional Q-Q and fold enrichment plots were used to visually demonstrate the strength of pleiotropic enrichment. Adopting a cFDR nominal significance level of 0.05, 287 loci were identified for BMI and 75 loci for T2D, 23 of which for both traits. By incorporating related traits into a conditional analysis framework, we observed significant pleiotropic enrichment between obesity and T2D. These findings may provide novel insights into the etiology of obesity and T2D, individually and jointly.

The US swine industry experiences severe production and profit losses from respiratory infections despite the wide spread use of vaccines and antibiotics. Long-chain PUFAs can modulate immune cell ...

Chlamydia trachomatis genital infection is the most common sexually transmitted bacterial disease, causing a significant burden to females due to reproductive dysfunction. Intensive screening and antibiotic treatment are unable to completely prevent female reproductive dysfunction, thus, efforts have become focused on developing a vaccine. A major impediment is identifying a safe and effective adjuvant which induces cluster of differentiation 4 (CD4) cells with attributes capable of halting genital infection and inflammation. Previously, we described a natural nanocapsule called the vault which was engineered to contain major outer membrane protein (MOMP) and was an effective vaccine which significantly reduced early infection and favored development of a cellular immune response in a mouse model. In the current study, we used another chlamydial antigen, a polymorphic membrane protein G-1 (PmpG) peptide, to track antigen-specific cells and evaluate, in depth, the vault vaccine for its protective capacity in the absence of an added adjuvant. We found PmpG-vault immunized mice significantly reduced the genital bacterial burden and histopathologic parameters of inflammation following a C. muridarum challenge. Immunization boosted antigen-specific CD4 cells with a multiple cytokine secretion pattern and reduced the number of inflammatory cells in the genital tract making the vault vaccine platform safe and effective for chlamydial genital infection. We conclude that vaccination with a Chlamydia-vault vaccine boosts antigen-specific immunities that are effective at eradicating infection and preventing reproductive tract inflammation.

Fungal laccases are typically unstable at high temperatures and alkaline conditions, thereby limiting their practical application. In this study, the novel laccase-producing Bacillus pumilus strain W3 was isolated from raw gallnut honey samples. The CotA–laccase gene was cloned from W3 and efficiently expressed by recombinant Escherichia coli in its biologically active form. The purified recombinant laccase had an extensive pH range for substrate catalysis. The enzyme was highly stable in alkaline pH and high temperatures, with considerable tolerance to NaCl and organic solvents. Laccase activity remained constant after 10 d of incubation at pH 9.0, whereas approximately 45% of the initial activity was detected after 10 h incubation at 80 °C. Two azo dyes and two anthraquinonic dyes could be efficiently decolorized by purified laccase in the presence of a mediator under alkaline condition. More than 90% decolorization was observed at pH 9.0 after incubation for 5 h. These unusual properties indicated a high potential of the novel CotA–laccase for industrial application on decolorisation of textile dyeing effluent.

A protocorm-like bodies (PLBs) regeneration system has been established for several Rosa spp. Greater understanding of the PLB formation process, in particular how PLBs are triggered from the rhizoid meristem, would contribute to the establishment of PLB protocols for cut roses that could be applied for rapid propagation and molecular breeding. The present study investigated the morphogenesis of Rosa canina PLBs. Callus rhizoids of R. canina were cultured on thidiazuron medium to induce PLBs, and the developmental process was examined by stereoscope, scanning electron microscopy, and transmission electron microscopy via paraffin sections and ultrathin sections. The results showed that 5 days after callus rhizoids were transferred to thidiazuron medium (DAT), vascular tissue comprising phloem and xylem was formed from the leaf callus to the rhizoid meristem, where a group of cells dividing periclinally emerged at 9 DAT. In the rhizoid tip, the division and enlargement of those dividing cells induced the formation of starch-rich parenchyma cells, and the totipotent cells adjacent to the epidermis differentiated into meristematic centers at about 20 DAT. PLBs were formed from meristematic centers through globular embryos at approximately 35 DAT, and then germinated precociously without going through the heart, torpedo, and cotyledon stages. This research suggests that R. canina PLBs were derived from parenchyma cells adjacent to the epidermis of the rhizoid tip. Vascular tissue of callus rhizoid was essential for the formation of parenchyma cells in the rhizoid meristem.

T cell effector functions can be elicited by non-cognate stimuli, but the mechanism and contribution of this pathway to the resolution of intracellular-macrophage infections has not been defined. Here we have shown that CD4+ T helper-1 (Th1) cells can be rapidly stimulated by microbe-associated molecular patterns (MAMPs) during active infection with Salmonella or Chlamydia. Further, maximal stimulation of Th1 cells by lipopolysaccharide (LPS) did not require T cell-intrinsic expression of toll-like receptor-4 (TLR4), interleukin-1 receptor (IL-1R), or interferon-γ receptor (IFN–γR), but instead required the adaptor protein Myd88, IL-18R, and IL-33R. Innate stimulation of Th1 cells also required host expression of TLR4 and inflammasome components that together increased serum concentrations of IL-18. Finally, the elimination of non-cognate Th1 cell stimulation hindered the resolution of primary Salmonella infection. Thus, the in vivo bactericidal capacity of Th1 cells is regulated by the response to non-cognate stimuli elicited by multiple innate immune receptors.

To determine whether caveolae and caveolin-1 affect the distribution of calcitonin receptor-like receptors (CLR) in vascular smooth muscle cell (VSMC) membranes, we have used VSMCs cell line A10. We found that calcitonin gene-related peptide (CGRP) reduced CLR protein in the VSMC membrane in a time-dependent manner, which was dramatically decreased after 4 h CGRP treatment, and remained at a low level after 16 h. CGRP8-37 or β-cyclodextrin (β-CD) blocked this effect, without changing the total levels of CLR protein and mRNA in the cells. Co-immunoprecipitation experiments showed that CLR bound to caveolin-1 in cell membrane fractions. Confocal laser microscopic studies confirmed this co-localization relationship at the cell plasma membrane. Thus, our data indicate that the structural integrity of caveolae plays an important role in regulating subcellular distribution of CLR.

The objective of this study was to examine the mechanism by which conjugated linoleic acid (CLA) reduces body fat. Young male mice were fed three combinations of fatty acids at three doses (0.06%, 0.2%, and 0.6%, w/w) incorporated into AIN76 diets for 7 weeks. The types of fatty acids were linoleic acid (control), an equal mixture of trans-10, cis-12 (10,12) CLA plus linoleic acid, and an equal isomer mixture of 10,12 plus cis-9, trans-11 (9,11) CLA. Mice receiving the 0.2% and 0.6% dose of 10,12 CLA plus linoleic acid or the CLA isomer mixture had decreased white adipose tissue (WAT) and brown adipose tissue (BAT) mass and increased incorporation of CLA isomers in epididymal WAT and liver. Notably, in mice receiving 0.2% of both CLA treatments, the mRNA levels of genes associated with browning, including uncoupling protein 1 (UCP1), UCP1 protein levels, and cytochrome c oxidase activity, were increased in epididymal WAT. CLA-induced browning in WAT was accompanied by increases in mRNA levels of markers of inflammation. Muscle cytochrome c oxidase activity and BAT UCP1 protein levels were not affected by CLA treatment. These data suggest a linkage between decreased adiposity, browning in WAT, and low-grade inflammation due to consumption of 10,12 CLA.

Salmonella infection profoundly affects host erythroid development, but the mechanisms responsible for this effect remain poorly understood. We monitored the impact of Salmonella infection on erythroid development and found that systemic infection induced anemia, splenomegaly, elevated erythropoietin (EPO) levels, and extramedullary erythropoiesis in a process independent of Salmonella pathogenicity island 2 (SPI2) or flagellin. The circulating EPO level was also constitutively higher in mice lacking the expression of signal-regulatory protein α (SIRPα). The expression level of EPO mRNA was elevated in the kidney and liver but not increased in the spleens of infected mice despite the presence of extramedullary erythropoiesis in this tissue. In contrast to data from a previous report, mice lacking EPO receptor (EPOR) expression on nonerythroid cells (EPOR rescued) had bacterial loads similar to those of wild-type mice following Salmonella infection. Indeed, treatment to reduce splenic erythroblasts and mature red blood cells correlated with elevated bacterial burdens, implying that extramedullary erythropoiesis benefits the host. Together, these findings emphasize the profound effect of Salmonella infection on erythroid development and suggest that the modulation of erythroid development has both positive and negative consequences for host immunity.

Ajudes rebudes: Marie Curie Career Integration Grant; Dexeus Foundation for Women's Health Research; i Contratos Ramón y Cajal CD11c is an α integrin classically employed to define myeloid dendritic cells. Although there is little information about CD11c expression on human T cells, mouse models have shown an association of CD11c expression with functionally relevant T cell subsets. In the context of genital tract infection, we have previously observed increased expression of CD11c in circulating T cells from mice and women. Microarray analyses of activated effector T cells expressing CD11c derived from naïve mice demonstrated enrichment for natural killer (NK) associated genes. Here we find that murine CD11c+ T cells analyzed by flow cytometry display markers associated with non-conventional T cell subsets, including γδ T cells and invariant natural killer T (iNKT) cells. However, in women, only γδ T cells and CD8+ T cells were enriched within the CD11c fraction of blood and cervical tissue. These CD11c+ cells were highly activated and had greater interferon (IFN)-γ secretory capacity than CD11c- T cells. Furthermore, circulating CD11c+ T cells were associated with the expression of multiple adhesion molecules in women, suggesting that these cells have high tissue homing potential. These data suggest that CD11c expression distinguishes a population of circulating T cells during bacterial infection with innate capacity and mucosal homing potential.

Recent studies have shed light on the connection between elevated erythropoetin production/spleen erythropoiesis and increased susceptibility to Salmonella infection. In this article, we provide another mouse model, the SIRPα-deficient (Sirpα−/−) mouse, that manifests increased erythropoiesis as well as heightened susceptibility to Salmonella infection. Sirpα−/− mice succumbed to systemic infection with attenuated Salmonella, possessing significantly higher bacterial loads in both the spleen and the liver. Moreover, Salmonella-specific Ab production and Ag-specific CD4 T cells were reduced in Sirpα−/− mice compared with wild-type controls. To further characterize the potential mechanism underlying SIRPα-dependent Ag-specific CD4 T cell priming, we demonstrate that lack of SIRPα expression on dendritic cells results in less efficient Ag processing and presentation in vitro. Collectively, these findings demonstrate an indispensable role of SIRPα for protective immunity to Salmonella infection.

Altres ajuts: Marie Curie Career Integration Grant i una beca Fundació Dexeus Salut de la Dona Efforts to develop vaccines that can elicit mucosal immune responses in the female genital tract against sexually transmitted infections have been hampered by an inability to measure immune responses in these tissues. The differential expression of adhesion molecules is known to confer site-dependent homing of circulating effector T cells to mucosal tissues. Specific homing molecules have been defined that can be measured in blood as surrogate markers of local immunity (e.g. α4β7 for gut). Here we analyzed the expression pattern of adhesion molecules by circulating effector T cells following mucosal infection of the female genital tract in mice and during a symptomatic episode of vaginosis in women. While CCR2, CCR5, CXCR6 and CD11c were preferentially expressed in a mouse model of Chlamydia infection, only CCR5 and CD11c were clearly expressed by effector T cells during bacterial vaginosis in women. Other homing molecules previously suggested as required for homing to the genital mucosa such as α4β1 and α4β7 were also differentially expressed in these patients. However, CD11c expression, an integrin chain rarely analyzed in the context of T cell immunity, was the most consistently elevated in all activated effector CD8+ T cell subsets analyzed. This molecule was also induced after systemic infection in mice, suggesting that CD11c is not exclusive of genital tract infection. Still, its increase in response to genital tract disorders may represent a novel surrogate marker of mucosal immunity in women, and warrants further exploration for diagnostic and therapeutic purposes.

In the present study, two experiments were conducted to investigate the effect of Mn source on Mn transport and the expression of a Mn transporter, divalent metal transporter 1 (DMT1), in the small intestine of broilers. In Expt 1, in situ ligated duodenal loops from Mn-deficient chicks (29-d-old) were perfused with solutions containing 0–8·74 mmol Mn/l from either MnSO4, or one of two organic chelates of Mn and amino acids with moderate (OM) or strong (OS) chelation strength (Qf) up to 30 min. In Expt 2, Mn-deficient intact broilers (14-d-old) were fed a control diet (12·45 mg Mn/kg) or the control diet supplemented with 100 mg Mn/kg as one of all Mn sources for 14 d. The uptake kinetics of Mn from different Mn sources in the ligated duodenal loops followed a saturable process as determined by regression analysis of concentration-dependent uptake rates. The maximum transport rate (Jmax) and Km values, and DMT1 mRNA levels in the ligated duodenal loops were higher (P 4. DMT1 mRNA levels were much higher (P P 4 Qf showed higher Mn transport and absorption, and DMT1 might be involved in the regulation of organic Mn transport in the proximal small intestine of broilers.

Enteric pathogens can cause relapsing infections in a proportion of treated patients, but greater understanding of this phenomenon is hindered by the lack of appropriate animal models. We report here a robust animal model of relapsing primary typhoid that initiates after apparently successful antibiotic treatment of susceptible mice. Four days of enrofloxacin treatment were sufficient to reduce bacterial loads below detectable levels in all major organs, and mice appeared otherwise healthy. However, any interruption of further antibiotic therapy allowed renewed fecal shedding and renewed bacterial growth in systemic tissues to occur, and mice eventually succumbed to relapsing infection. In vivo imaging of luminescent Salmonella identified the mesenteric lymph nodes (MLNs) as a major reservoir of relapsing infection. A magnetic-bead enrichment strategy isolated MLN-resident CD11b + Gr-1 − monocytes associated with low numbers of persistent Salmonella . However, the removal of MLNs increased the severity of typhoid relapse, demonstrating that this organ serves as a protective filter to restrain the dissemination of bacteria during antibiotic therapy. Together, these data describe a robust animal model of typhoid relapse and identify an important intestinal phagocyte subset involved in protection against the systemic spread of enteric infection.

The antimicrobial resistance and the character of integrons were determined in 58 Shigella flexneri strains isolated from China. All isolates were multi-drug resistant and found to carry integrons of class 1 (94.8%), class 2 (100%), or both (94.8%). No intI3 was detected. The typical class 1 integrons were found in conjugative plasmids and could be transferred to the recipient E. coli DH5α. The gene cassettes of typical class 1 integrons dfrA17-aadA5 and dfrA12-orfF-aadA2 were detected in 54 strains (93.1%) and 1 strain, respectively. Atypical class 1 integrons located on the chromosome with gene cassettes bla (oxa-30)-aadA1 were detected in 55 isolates (94.8%). All the intI2 positive isolates carried gene cassettes dfrA1-sat1-aadA1. To our knowledge, this is the first report that atypical and typical class 1 integrons coexisted with class 2 integron in multi-drug resistant S. flexneri strains.

To explore the mechanisms involved in ox-LDL transcytosis across endothelial cells and the role of caveolae in this process. An in vitro model was established to investigate the passage of oxidized low density lipoprotein (ox-LDL) through a tight monolayer of human umbilical vein endothelial cells (HUVEC) cultured on a collagen-coated filter. Passage of DiI-labeled ox-LDL through the monolayer was measured using a fluorescence spectrophotometer. The uptake and efflux of ox-LDL by HUVEC were determined using fluorescence microscopy and HPLC. Caveolae inhibitors – carrageenan (250 μg/mL), filipin (5 μg/mL), and nocodazole (33 μmol/L)–decreased the transport of ox-LDL across the monolayer by 48.9%, 72.4%, and 79.8% as compared to the control group. In addition, they effectively decreased ox-LDL uptake and inhibited the efflux of ox-LDL. Caveolin-1 and LOX-1 were up-regulated by ox-LDL in a time-dependent manner and decreased gradually after depletion of ox-LDL (P

Regulatory T cell (Treg) development proceeds via a two-step process in which naive CD4+ thymocytes are first converted into CD4+CD25+CD122+GITR+Foxp3− Treg progenitors, followed by a second step in which IL-2 converts these Treg progenitors into CD4+Foxp3+ Tregs. The costimulatory molecule CD28 is required for efficient Treg development. However, the stage at which CD28 affects Treg development remains undefined. In this article, we demonstrate that Cd28−/− mice lack Treg progenitors. Furthermore, the P187YAP motif in the cytoplasmic tail of CD28, which links CD28 to Lck activation, is required for this process. In contrast, the Y170MNM motif, which links CD28 to PI3K activation, is not required for Treg progenitor development. Finally, the CD28/Lck pathway was shown to activate the NF-κB family of transcription factors. We demonstrate that c-Rel, but not NF-κB1, promotes the development of Treg progenitors. Thus, a CD28/c-Rel–dependent pathway is involved in initiating Treg development.