Data from successful attenuated lentiviral vaccine studies indicate that fully mature Env-specific antibodies characterized by high titer, high avidity, and the predominant recognition of conformational epitopes are associated with protective efficacy. Although vaccination with a DNA prime/recombinant vaccinia-vectored vaccine boost strategy has been found to be effective in some trials with non-human primate/simian/human immunodeficiency virus (SHIV) models, it remains unclear whether this vaccination strategy could elicit mature equine infectious anemia virus (EIAV) Env-specific antibodies, thus protecting vaccinated horses against EIAV infection. Therefore, in this pilot study we vaccinated horses using a strategy based on DNA prime/recombinant Tiantan vaccinia (rTTV)-vectored vaccines encoding EIAV env and gag genes, and observed the development of Env-specific antibodies, neutralizing antibodies, and p26-specific antibodies. Vaccination with DNA induced low titer, low avidity, and the predominant recognition of linear epitopes by Env-specific antibodies, which was enhanced by boosting vaccinations with rTTV vaccines. However, the maturation levels of Env-specific antibodies induced by the DNA/rTTV vaccines were significantly lower than those induced by the attenuated vaccine EIAVFDDV. Additionally, DNA/rTTV vaccines did not elicit broadly neutralizing antibodies. After challenge with a virulent EIAV strain, all of the vaccinees and control horses died from EIAV disease. These data indicate that the regimen of DNA prime/rTTV vaccine boost did not induce mature Env-specific antibodies, which might have contributed to immune protection failure.
T cells must undergo two critical differentiation processes before they become competent effectors that can mediate actual immune responses. Progenitor T cells undergo defined stages of differentiation in the thymus, which include positive and negative selection, to generate a repertoire of T cells that will respond to foreign but not self antigens. When these immunocompetent T cells first migrate out of thymus into peripheral lymphoid tissues, they are naïve and are unable to mediate immune responses. However, upon antigen encounter, peripheral CD4+ naïve T cells undergo another differentiation process to become armed effector T cells including Th1, Th2, Th17 or regulatory T cells, all of which are capable of regulating immune responses. A canonical Wnt/β-catenin/T cell factor (TCF) pathway has been shown to regulate T cell differentiation in both the thymus and in peripheral lymphoid tissues. Dysfunction of this pathway at any stage of T cell differentiation could lead to severe autoimmunity including experimental autoimmune encephalomyelitis or immune deficiency. Understanding the role played by β-catenin/TCF-1 in T cell differentiation will facilitate our understanding of the mechanisms that regulate T cell function and assist in identifying novel therapy targets for treating both autoimmune and immune diseases. Therefore, in this review, we will focus on the function of β-catenin/TCF-1 pathway in the regulation of thymic and peripheral T cell differentiation processes.
HIF-1 activates various genes in cancer progression and metastasis. HIF-1α 1772 C/T and 1790 G/A polymorphisms are reportedly associated with cancer risk; however, the results are inconclusive.
A meta-analysis of 34 studies that involved 7522 cases and 9847 controls for 1772 C/T and 24 studies that involved 4884 cases and 8154 controls for 1790 G/A was conducted to identify the association of C/T and G/A polymorphisms with cancer risk. Odds ratio (OR) and 95% confidence intervals (95% CI) were used to assess the strength of association.
HIF-1α 1772 C/T and 1790 G/A polymorphisms were associated with higher cancer risk in homozygote comparison (1772C/T: TT vs. CC: OR = 2.45, 95% CI: 1.52, 3.96; Pheterogeneity = 0.028; 1790G/A: AA vs. GG: OR=4.74, 95% CI: 1.78, 12.6; Pheterogeneity < 0.01), dominant model (1772C/T: TT/CT vs. CC: OR = 1.27, 95% CI: 1.04, 1.55; Pheterogeneity < 0.01, 1790G/A: AA/GA vs. GG: OR = 1.65, 95% CI: 1.05, 2.60; Pheterogeneity < 0.01), T allele versus C allele (T vs. C: OR = 1.42, 95% CI: 1.18, 1.70; Pheterogeneity < 0.01), and A allele versus G allele (A vs. G: OR = 1.83, 95% CI: 1.13, 2.96; Pheterogeneity < 0.01). On a subgroup analysis, the 1772 C/T polymorphism was significantly linked to higher risks for breast cancer, lung cancer, prostate cancer, and cervical cancer, whereas the 1790 G/A polymorphism was significantly linked to higher risks for lung cancer and prostate cancer. A significantly increased cancer risk was found in both Asians and Caucasians for 1772C/T polymorphism, whereas a significantly increased cancer risk was found in Caucasians in the heterozygote comparison and recessive model for 1790G/A polymorphism.
HIF-1α 1772 C/T and 1790 G/A polymorphisms are significantly associated with higher cancer risk.
Structural changes of chromosomes are a primary mechanism of genome rearrangement over the course of evolution and detailed knowledge of such changes in a given species and its close relatives should increase the efficiency and precision of chromosome engineering in crop improvement. We have identified sequences bordering each of the main translocation and inversion breakpoints on chromosomes 4A, 5A and 7B of the modern bread wheat genome. The locations of these breakpoints allow, for the first time, a detailed description of the evolutionary origins of these chromosomes at the gene level. Results from this study also demonstrate that, although the strategy of exploiting sorted chromosome arms has dramatically simplified the efforts of wheat genome sequencing, simultaneous analysis of sequences from homoeologous and non-homoeologous chromosomes is essential in understanding the origins of DNA sequences in polyploid species.
Dysregulation of Wnt/β-catenin signaling contributes to the development of diabetic retinopathy by inducing retinal inflammation, vascular leakage, and neovascularization. Here, we evaluated the inhibitory effect of a monoclonal antibody (Mab) specific for the E1E2 domain of Wnt coreceptor low-density lipoprotein receptor–related protein 6, Mab2F1, on canonical Wnt signaling and its therapeutic potential for diabetic retinopathy. Mab2F1 displayed robust inhibition on Wnt signaling with a half-maximal inhibitory concentration (IC50) of 20 μg/mL in retinal pigment epithelial cells. In addition, Mab2F1 also attenuated the accumulation of β-catenin and overexpression of vascular endothelial growth factor, intercellular adhesion molecule-1, and tumor necrosis factor-α induced by high-glucose medium in retinal endothelial cells. In vivo, an intravitreal injection of Mab2F1 significantly reduced retinal vascular leakage and decreased preretinal vascular cells in oxygen-induced retinopathy (OIR) rats, demonstrating its inhibitory effects on ischemia-induced retinal neovascularization. Moreover, Mab2F1 blocked the overexpression of the inflammatory/angiogenic factors, attenuated leukostasis, and reduced retinal vascular leakage in both early and late stages of streptozotocin-induced diabetes. In conclusion, Mab2F1 inhibits canonical Wnt signaling, vascular leakage, and inflammation in the retina of diabetic retinopathy models, suggesting its potential to be used as a therapeutic agent in combination with other antiangiogenic compounds.
Acid soluble collagen (ASC) from scales of croceine croaker (ASC-C) was successfully isolated with the yield of 0.37% ± 0.08% (dry weight basis), and characterized as type I collagen on the basis of amino acid analysis and electrophoretic pattern. The antioxidant hydrolysate of ASC-C (ACH) was prepared through a two-stage in vitro digestion (4-h trypsin followed by 4-h pepsin), and three antioxidant peptides (ACH-P1, ACH-P2, and ACH-P3) were further isolated from ACH using ultrafiltration, gel chromatography, and RP-HPLC, and their amino acid sequences were identified as GFRGTIGLVG (ACH-P1), GPAGPAG (ACH-P2), and GFPSG (ACH-P3). ACH-P1, ACH-P2, and ACH-P3 showed good scavenging activities on hydroxyl radical (IC50 0.293, 0.240, and 0.107 mg/mL, respectively), DPPH radical (IC50 1.271, 0.675, and 0.283 mg/mL, respectively), superoxide radical (IC50 0.463, 0.099, and 0.151 mg/mL, respectively), and ABTS radical (IC50 0.421, 0.309, and 0.210 mg/mL, respectively). ACH-P3 was also effectively against lipid peroxidation in the model system. The antioxidant activities of three collagen peptides were due to the presence of hydrophobic amino acid residues within the peptide sequences. The collagen peptides might be used as antioxidant for the therapy of diseases associated with oxidative stress, or reducing oxidative changes during storage.
croceine croaker (Pseudosciaena crocea); scale; acid soluble collagen (ASC); peptide; antioxidant activity
To assess the safety and efficacy of balloon kyphoplasty (KP) compared with percutaneous vertebroplasty (VP) and provide recommendations for using these procedures to treat osteoporotic vertebral compression fractures (OVCF).
A systematic search of all studies published through March 2012 was conducted using the MEDLINE, EMBASE, OVID, ScienceDirect and Cochrane CENTRAL databases. The randomized controlled trials (RCTs) and non-randomized controlled trials that compared KP to VP and provided data on safety and clinical effects were identified. Demographic characteristics, adverse events and clinical outcomes were manually extracted from all of the selected studies. The evidence quality levels and recommendations were assessed using the GRADE system.
Twelve studies encompassing 1,081 patients met the inclusion criteria. Subgroup meta-analyses were performed according to the study design. In the RCT subgroup, there were significant differences between the two procedures in short-term visual analog scale (VAS), long-term kyphosis angles, operative times and anterior vertebrae heights. In the cohort study subgroup, there were significant differences between the two procedures in short- and long-term VAS, short- and long-term Oswestry Disability Index (ODI), cement leakage rates, short- and long-term kyphosis angles, operative times and anterior vertebrae heights. However, there were no significant differences in long-term VAS or adjacent vertebral fracture rates in the RCT subgroup. There were no significant differences in short- or long-term VAS, short- or long-term ODI, cement leakage rates, adjacent vertebral fracture rates, short- or long-term kyphosis angles or anterior vertebrae heights in the CCT subgroup, and the adjacent vertebral fracture rates did not differ significantly in the cohort study subgroup. The overall GRADE system evidence quality was very low, which lowers our confidence in their recommendations.
KP and VP are both safe and effective surgical procedures for treating OVCF. KP may be superior to VP in patients with large kyphosis angles, vertebral fissures, fractures in the posterior edge of the vertebral body or significant height loss in the fractured vertebrae. Due to the poor quality of the evidence currently available, high-quality RCTs are required.
Osteoporosis; Kyphoplasty; Vertebroplasty; Vertebral compression fracture; Systematic review
MicroRNAs (miRNAs) are known to participate in post-transcriptional regulation of gene expression and are involved in multiple pathogenic processes. Here, we identified miRNA expression changes in the retinas of Akita mice, a genetic model of type 1 diabetes, and investigated the potential role of miRNA in diabetic retinopathy.
Visual function of Akita and control mice was evaluated by electroretinography. MiRNA expression changes in the retinas of Akita mice were identified by miRNA-specific microarray and confirmed by quantitative RT-PCR (qRT-PCR). The potential downstream targets of identified miRNAs were predicted by bioinformatic analysis using web-based applications and confirmed by dual luciferase assay. The mRNA and protein changes of identified downstream targets were examined by qRT-PCR and Western blot analysis.
MiRNA-specific microarray and qRT-PCR showed that miR-200b was upregulated significantly in the Akita mouse retina. Sequence analysis and luciferase assay identified oxidation resistance 1 (Oxr1) as a downstream target gene regulated by miR-200b. In a human Müller cell line, MIO-M1, transfection of a miR-200b mimic downregulated Oxr1 expression. Conversely, transfection of MIO-M1 with a miR-200b inhibitor resulted in upregulated Oxr1. Furthermore, overexpression of recombinant Oxr1 attenuated oxidative stress marker, nitration of cellular proteins, and ameliorated apoptosis induced by 4-hydroxynonenal (4-HNE), an oxidative stressor. Similarly, transfection of a miR-200b inhibitor decreased, whereas transfection of miR-200b mimic increased the number of apoptotic cells following 4-HNE treatment.
These results suggested that miR-200b–regulated Oxr1 potentially has a protective role in diabetic retinopathy.
This study demonstrated that miR-200b was upregulated in the Akita retina, and miR-200b downregulates the expression of Oxr1, which potentially has a protective role in diabetic retinopathy.
Artificial selection played an important role in the origin of modern Glycine max cultivars from the wild soybean Glycine soja. To elucidate the consequences of artificial selection accompanying the domestication and modern improvement of soybean, 25 new and 30 published whole-genome re-sequencing accessions, which represent wild, domesticated landrace, and Chinese elite soybean populations were analyzed.
A total of 5,102,244 single nucleotide polymorphisms (SNPs) and 707,969 insertion/deletions were identified. Among the SNPs detected, 25.5% were not described previously. We found that artificial selection during domestication led to more pronounced reduction in the genetic diversity of soybean than the switch from landraces to elite cultivars. Only a small proportion (2.99%) of the whole genomic regions appear to be affected by artificial selection for preferred agricultural traits. The selection regions were not distributed randomly or uniformly throughout the genome. Instead, clusters of selection hotspots in certain genomic regions were observed. Moreover, a set of candidate genes (4.38% of the total annotated genes) significantly affected by selection underlying soybean domestication and genetic improvement were identified.
Given the uniqueness of the soybean germplasm sequenced, this study drew a clear picture of human-mediated evolution of the soybean genomes. The genomic resources and information provided by this study would also facilitate the discovery of genes/loci underlying agronomically important traits.
Artificial selection; Evolution; Genetic diversity; Population genomics; Soybean
Manganese (Mn) is an essential micronutrient for plants, but is toxic when present in excess. The rice plant (Oryza sativa L.) accumulates high concentrations of Mn in the aerial parts; however, the molecular basis for Mn tolerance is poorly understood. In the present study, genes encoding Mn tolerance were screened for by expressing cDNAs of genes from rice shoots in Saccharomyces cerevisiae. A gene encoding a cation diffusion facilitator (CDF) family member, OsMTP8.1, was isolated, and its expression was found to enhance Mn accumulation and tolerance in S. cerevisiae. In plants, OsMTP8.1 and its transcript were mainly detected in shoots. High or low supply of Mn moderately induced an increase or decrease in the accumulation of OsMTP8.1, respectively. OsMTP8.1 was detected in all cells of leaf blades through immunohistochemistry. OsMTP8.1 fused to green fluorescent protein was localized to the tonoplast. Disruption of OsMTP8.1 resulted in decreased chlorophyll levels, growth inhibition in the presence of high concentrations of Mn, and decreased accumulation of Mn in shoots and roots. However, there was no difference in the accumulation of other metals, including Zn, Cu, Fe, Mg, Ca, and K. These results suggest that OsMTP8.1 is an Mn-specific transporter that sequesters Mn into vacuoles in rice and is required for Mn tolerance in shoots.
CDF family; manganese tolerance; OsMTP8.1; rice; transporter; vacuole
Reduced sensitivity of prostate cancer (PC) cells to radiation therapy poses a significant challenge in the clinic. Activation of epidermal growth factor receptor (EGFR), type 1 insulin-like growth factor receptor (IGF1R), and crosstalk between these two signaling pathways have been implicated in the development of radiation resistance in PC. This study assessed the effects of targeting both receptors on the regulation of radio-sensitivity in PC cells. Specific inhibitors of EGFR and IGF1R, Erlotinib and AG1024, as well as siRNA targeting EGFR and IGF1R, were used to radio-sensitize PC cells. Our results showed that co-inhibiting both receptors significantly dampened cellular growth and DNA damage repair, and increased radio-sensitivity in PC cells. These effects were carried out through synergistic inhibition of homologous recombination-directed DNA repair (HRR), but not via inhibition of non-homologous end joining (NHEJ). Furthermore, the compromised HRR capacity was caused by reduced phosphorylation of insulin receptor substrate 1 (IRS1) and its subsequent interaction with Rad51. The synergistic effect of the EGFR and IGF1R inhibitors was also confirmed in nude mouse xenograft assay. This is the first study testing co-inhibiting EGFR and IGF1R signaling in the context of radio-sensitivity in PC and it may provide a promising adjuvant therapeutic approach to improve the outcome of PC patients to radiation treatment.
Diaquacobinamide (H2O)2Cbi2+ or its conjugate base hydroxyaquacobinamide (OH(H2O)Cbi+)) can bind up to two cyanide ions, making dicyanocobinamide. This transition is accompanied by a significant change in color, previously exploited for cyanide determination. The reagent OH(H2O)Cbi+ is used in excess; when trace amounts of cyanide are added, CN(H2O)Cbi+ should be formed. But the spectral absorption of CN(H2O)Cbi+ is virtually the same as that of OH(H2O)Cbi+. It has been inexplicable how trace amounts of cyanide are sensitively measured by this reaction. It is shown here that even with excess OH(H2O)Cbi+, (CN)2Cbi is formed first due to kinetic reasons; this only slowly forms CN(H2O)Cbi+. This understanding implies that CN(H2O)Cbi+ will itself be a better reagent.
We describe a single valve merging zone flow analyzer that allows both sample and reagent economy. With a 50 cm liquid core waveguide (LCW) flow cell and an inexpensive fiber optic - charge coupled device array spectrometer, a S/N=3 limit of detection of 8 nM, a linear dynamic range to 6 μM, and excellent precision (RSD 0.49% and 1.07% at 50 and 100 nM, respectively, n=5 each) are formed. At 1% carryover, sample throughput is 40 h−1. The setup is readily used to measure thiocyanate with different reagents. We demonstrate applicability to real samples by analyzing human saliva samples and hydrolyzed extracts of apple seeds, peach pits, and almonds.
Cyanide; Cobinamide; Merging-Zone; Liquid Core waveguide
A total of 19 novel (3a–3s) N′-benzoyl-3-(4-bromophenyl)-1H-pyrazole-5-carbohydrazide analogs were designed, synthesized, and evaluated for biological activities as potential DNA gyrase inhibitors. The results showed that compound 3k can strongly inhibit Staphylococcus aureus DNA gyrase and Bacillus subtilis DNA gyrase (with IC50 of 0.15 µg/mL and 0.25 µg/mL, respectively). Structure-activity relationships were also discussed base on the biological and docking simulation results.
To gain a better understanding of North American population history, complete mitochondrial genomes (mitogenomes) were generated from four ancient and three living individuals of the northern Northwest Coast of North America, specifically the north coast of British Columbia, Canada, current home to the indigenous Tsimshian, Haida, and Nisga’a. The mitogenomes of all individuals were previously unknown and assigned to new sub-haplogroup designations D4h3a7, A2ag and A2ah. The analysis of mitogenomes allows for more detailed analyses of presumed ancestor–descendant relationships than sequencing only the HVSI region of the mitochondrial genome, a more traditional approach in local population studies. The results of this study provide contrasting examples of the evolution of Native American mitogenomes. Those belonging to sub-haplogroups A2ag and A2ah exhibit temporal continuity in this region for 5000 years up until the present day. Of possible associative significance is that archaeologically identified house structures in this region maintain similar characteristics for this same period of time, demonstrating cultural continuity in residence patterns. The individual dated to 6000 years before present (BP) exhibited a mitogenome belonging to sub-haplogroup D4h3a. This sub-haplogroup was earlier identified in the same general area at 10300 years BP on Prince of Wales Island, Alaska, and may have gone extinct, as it has not been observed in any living individuals of the Northwest Coast. The presented case studies demonstrate the different evolutionary paths of mitogenomes over time on the Northwest Coast.
Choroidal neovascularization (CNV) is a severe complication of AMD. The Wnt signaling pathway has been shown to mediate angiogenesis. The purpose of this study was to investigate the pathogenic role of the Wnt pathway in CNV and explore the therapeutic potential of a novel Wnt signaling inhibitor in CNV.
Adult rats and mice were photocoagulated using diode laser to induce CNV. On the same day, the animals were intravitreally injected with a monoclonal antibody (Mab2F1) blocking LRP6 or nonspecific mouse IgG. The Wnt signaling activation and target gene expression in the eyecup were determined by Western blot analysis. Fundus angiography was used to examine leakage from the laser lesion. CNV areas were measured on choroidal flatmount using FITC-dextran.
Levels of Wnt pathway components and Wnt target gene expression were elevated in both laser-induced CNV rat and mouse eyecups, suggesting activation of the Wnt pathway. Significant suppression of Wnt signaling was observed in the Mab2F1 treatment group. Mab2F1 decreased vascular leakage from CNV lesions and reduced the neovascular area in laser-induced CNV rats. Mab2F1 inhibited the hypoxia-induced activation of Wnt signaling in cultured RPE cells. Mab2F1 also ameliorated retinal inflammation and vascular leakage in the eyecups of very low-density lipoprotein receptor knockout mice, a model of subretinal neovascularization.
The Wnt pathway is activated in the laser-induced CNV models and plays a pathogenic role in CNV. Blockade of Wnt signaling using an anti-LRP6 antibody has therapeutic potential in CNV.
The present study demonstrated that Wnt pathway plays a key pathogenic role in CNV.
Excessive accumulation of lipofuscin is associated with pathogenesis of atrophic age-related macular degeneration (AMD) and Stargardt disease. Pharmacologic inhibition of the retinol-induced interaction of retinol-binding protein 4 (RBP4) with transthyretin (TTR) in the serum may decrease the uptake of serum retinol to the retina and reduce formation of lipofuscin bisretinoids. We evaluated in vitro and in vivo properties of the new nonretinoid RBP4 antagonist, A1120.
RBP4 binding potency, ability to antagonize RBP4-TTR interaction, and compound specificity were analyzed for A1120 and for the prototypic RBP4 antagonist fenretinide. A1120 ability to inhibit RPE65-mediated isomerohydrolase activity was assessed in the RPE microsomes. The in vivo effect of A1120 administration on serum RBP4, visual cycle retinoids, lipofuscin bisretinoids, and retinal visual function was evaluated using a combination of biochemical and electrophysiologic techniques.
In comparison to fenretinide, A1120 did not act as a RARα agonist, while exhibiting superior in vitro potency in RBP4 binding and RBP4-TTR interaction assays. A1120 did not inhibit isomerohydrolase activity in the RPE microsomes. A1120 dosing in mice induced 75% reduction in serum RBP4, which correlated with reduction in visual cycle retinoids and ocular levels of lipofuscin fluorophores. A1120 dosing did not induce changes in kinetics of dark adaptation.
A1120 significantly reduces accumulation of lipofuscin bisretinoids in the Abca4−/− animal model. This activity correlates with reduction in serum RBP4 and visual cycle retinoids confirming the mechanism of action for A1120. In contrast to fenretinide, A1120 does not act as a RARα agonist indicating a more favorable safety profile for this nonretinoid compound.
A1120, a nonretinoid RBP4 antagonist, reduces bisretinoids in Abca4−/− mice, which correlates with reduction in serum RBP4 and ocular retinyl palmitate confirming the mechanism of action. In contrast to fenretinide, A1120 lacks RARa agonistic activity, which indicates a more favorable safety profile.
Tea is the most popular non-alcoholic health beverage in the world. The tea plant (Camellia sinensis (L.) O. Kuntze) needs to undergo a cold acclimation process to enhance its freezing tolerance in winter. Changes that occur at the molecular level in response to low temperatures are poorly understood in tea plants. To elucidate the molecular mechanisms of cold acclimation, we employed RNA-Seq and digital gene expression (DGE) technologies to the study of genome-wide expression profiles during cold acclimation in tea plants.
Using the Illumina sequencing platform, we obtained approximately 57.35 million RNA-Seq reads. These reads were assembled into 216,831 transcripts, with an average length of 356 bp and an N50 of 529 bp. In total, 1,770 differentially expressed transcripts were identified, of which 1,168 were up-regulated and 602 down-regulated. These include a group of cold sensor or signal transduction genes, cold-responsive transcription factor genes, plasma membrane stabilization related genes, osmosensing-responsive genes, and detoxification enzyme genes. DGE and quantitative RT-PCR analysis further confirmed the results from RNA-Seq analysis. Pathway analysis indicated that the “carbohydrate metabolism pathway” and the “calcium signaling pathway” might play a vital role in tea plants’ responses to cold stress.
Our study presents a global survey of transcriptome profiles of tea plants in response to low, non-freezing temperatures and yields insights into the molecular mechanisms of tea plants during the cold acclimation process. It could also serve as a valuable resource for relevant research on cold-tolerance and help to explore the cold-related genes in improving the understanding of low-temperature tolerance and plant-environment interactions.
Camellia Sinensis; Cold Acclimation; RNA-Seq; DGE; Genome-wide Expression Profiles; Tea Plants
Although protein kinase C-θ (PKC-θ)-deficient mice are resistant to the induction of Th17-dependent experimental autoimmune encephalomyelitis, the function of PKC-θ in Th17 differentiation remains unknown. Here we show that purified, naive CD4 PKC-θ−/− T cells were defective in Th17 differentiation, whereas Th1 and Th2 differentiation appeared normal. Activation of PKC-θ with PMA promoted Th17 differentiation in wild type (WT) but not PKC-θ−/− T cells. Furthermore, PKC-θ−/− T cells had notably lower levels of Stat3, a transcription factor required for Th17 differentiation, and PMA markedly stimulated the expression of Stat3 in WT but not PKC-θ−/− T cells. In contrast, activation of Stat4 and Stat6, which are critical for Th1 and Th2 differentiation, was normal in PKC-θ−/− T cells. Forced expression of Stat3 significantly increased Th17 differentiation in PKC-θ−/− T cells, suggesting that reduced Stat3 levels were responsible for impaired Th17 differentiation and that Stat3 lies downstream of PKC-θ. Constitutively active PKC-θ or WT PKC-θ activated by either PMA or TCR cross-linking, stimulated expression of a luciferase reporter gene driven by the Stat3 promoter. PKC-θ-mediated activation of the Stat3 promoter was inhibited by dominant negative AP-1 and IκB kinase-β, but stimulated by WT AP-1 and IκB kinase-β, suggesting that PKC-θ stimulates Stat3 transcription via the AP-1 and NF-κB pathways. Lastly, conditions favoring Th17 differentiation induced the highest activation level of PKC-θ. Altogether the data indicate that PKC-θ integrates the signals from TCR signaling and Th17 priming cytokines to up-regulate Stat3 via NF-κB and AP-1, resulting in the stimulation of Th17 differentiation.
We report here the genomic sequence of Legionella pneumophila strain LPE509 from the water distribution system of a hospital in Shanghai, China. This is the first complete genome sequence of an environmental L. pneumophila isolate. Genomic analyses identified approximately 600 genes unique to LPE509 compared to those of the 7 available L. pneumophila genomes.
Protein Kinase C-θ (PKC-θ) has been shown to be a critical T cell receptor (TCR) signaling molecule that promotes the activation and differentiation of naïve T cells into inflammatory effector T cells. We demonstrate here that PKC-θ-mediated signals inhibit iTreg differentiation via an AKT-Forkhead Box O1/3a (FoxO1/3A) pathway. Transforming growth factor β-induced iTreg differentiation was enhanced in PKC-θ−/− T cells or WT cells treated with a specific PKC-θ inhibitor, but was inhibited by the PKC-θ activator PMA, or by CD28 crosslinking which enhances PKC-θ activation. PKC-θ−/− T cells had reduced activity of the AKT kinase, and the expression of a constitutively active form of AKT in PKC-θ−/− T cells restored ability to inhibit iTreg differentiation. Furthermore, knockdown or over expression of the AKT downstream targets FoxO1 and FoxO3a was found to inhibit or promote iTreg differentiation in PKC-θ−/− T cells accordingly, indicating that the AKT-FoxO1/3A pathway is responsible for the inhibition of iTreg differentiation of iTreg downstream of PKC-θ. We conclude that PKC-θ is able to control T cell-mediated immune responses by shifting the balance between the differentiation of effector T cells and inhibitory Tregs.
Serum retinol-binding protein 4 (RBP4) is the sole specific vitamin A (retinol) transporter in blood. Elevation of serum RBP4 in patients has been linked to cardiovascular disease and diabetic retinopathy. However, the significance of RBP4 elevation in the pathogenesis of these vascular diseases is unknown. Here we show that RBP4 induces inflammation in primary human retinal capillary endothelial cells (HRCEC) and human umbilical vein endothelial cells (HUVEC) by stimulating expression of proinflammatory molecules involved in leukocyte recruitment and adherence to endothelium, including vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), E-selectin, monocyte chemoattractant protein 1 (MCP-1), and interleukin-6 (IL-6). We demonstrate that these novel effects of RBP4 are independent of retinol and the RBP4 membrane receptor STRA6 and occur in part via activation of NADPH oxidase and NF-κB. Importantly, retinol-free RBP4 (apo-RBP4) was as potent as retinol-bound RBP4 (holo-RBP4) in inducing proinflammatory molecules in both HRCEC and HUVEC. These studies reveal that RBP4 elevation can directly contribute to endothelial inflammation and therefore may play a causative role in the development or progression of vascular inflammation during cardiovascular disease and microvascular complications of diabetes.
The efficient recycling of the chromophore of visual pigments, 11-cis retinal, through the retinoid visual cycle is an essential process for maintaining normal vision. RPE65 is the isomerohydrolase in retinal pigment epithelium and generates predominantly 11-cis retinol (11cROL) and a minor amount of 13-cis retinol (13cROL), from all-trans retinyl ester (atRE). We recently identified and characterized novel homologs of RPE65, RPE65c and 13-cis isomerohydrolase (13cIMH), which are expressed in the zebrafish inner retina and brain, respectively. Although these two homologs share 97% amino acid sequence identity, they exhibit distinct product specificities. Under the same assay conditions, RPE65c generated predominantly 11cROL, similar to RPE65, while 13cIMH generated exclusively 13cROL from atRE substrate. To study the impacts of the key residues determining isomerization product specificity of RPE65, we replaced candidate residues by site-directed mutagenesis in RPE65c and 13cIMH. Point mutations at residues Tyr58, Phe103 and Leu133 in RPE65c resulted in significantly altered isomerization product specificities. Particularly, our results showed that residue 58 is a primary determinant of isomerization specificity, since the Y58N mutation in RPE65c and its reciprocal N58Y mutation in 13cIMH completely reversed the respective enzyme isomerization product specificities. These findings will contribute to the elucidation of molecular mechanisms underlying the isomerization reaction catalyzed by RPE65.
isomerohydrolase; product specificity; visual cycle; 11-cis retinoid; 13-cis retinoid; site-directed mutagenesis
AIM: To compare synchronous laparoscopic cholecystectomy (LC) combined with endoscopic sphincterotomy (EST) and sequential LC combined with EST for treating cholecystocholedocholithiasis.
METHODS: A total of 150 patients were included and retrospectively studied. Among these, 70 were selected for the synchronous operation, in which the scheme was endoscopic retrograde cholangiopancreatography combined with EST during LC. The other 80 patients were selected for the sequential operation, in which the scheme involved first cutting the papillary muscle under endoscopy and then performing LC. The indexes in the two groups, including the operation time, the success rate, the incidence of complications, and the length of the hospital stay, were observed.
RESULTS: There were no significant differences between the groups in terms of the numbers of patients, sex distribution, age, American Society of Anesthesiologists score, serum bilirubin, γ-glutamyl transpeptidase, mean diameter of common bile duct stones, and previous medical and surgical history (P = 0.54, P = 0.18, P = 0.52, P = 0.22, P = 0.32, P = 0.42, P = 0.68, P = 0.70, P = 0.47 and P = 0.57). There was no significant difference in the surgical operation time between the two groups (112.1 ± 30.8 min vs 104.9 ± 18.2 min). Compared with the sequential operation group, the incidence of pancreatitis was lower (1.4% vs 6.3%), the incidence of hyperamylasemia (1.4% vs 10.0%, P < 0.05) was significantly reduced, and the length of the hospital stay was significantly shortened in the synchronous operation group (3 d vs 4.5 d, P < 0.001).
CONCLUSION: For treatment of cholecystocholedocholithiasis, synchronous LC combined with EST reduces incidence of complications, decreases length of hospital stay, simplifies the surgical procedure, and reduces operation time.
Laparoscopic cholecystectomy; Endoscopic sphincterotomy; Endoscopic retrograde cholangiopancreatography; Cholecystolithiasis; Choledocholithiasis
Cell-in-cell structures refer to a unique phenomenon that one living cell enters into another living cell intactly, occurring between homotypic tumor cells or tumor (or other tissue cells) and immune cells (named as heterotypic cell-in-cell structure). In the present study, through a large scale of survey we observed that heterotypic cell-in-cell structure formation occurred commonly in vitro with host cells derived from different human carcinomas as well as xenotypic mouse tumor cell lines. Most of the lineages of human immune cells, including T, B, NK cells, monocytes as well as in vitro activated LAK cells, were able to invade tumor cell lines. Poorly differentiated stem cells were capable of internalizing immune cells as well. More significantly, heterotypic tumor/immune cell-in-cell structures were observed in a higher frequency in tumor-derived tissues than those in adjacent tissues. In mouse hepatitis models, heterotypic immune cell/hepatocyte cell-in-cell structures were also formed in a higher frequency than in normal controls. After in vitro culture, different forms of internalized immune cells in heterotypic cell-in-cell structures were observed, with one or multiple immune cells inside host cells undergoing resting, degradation or mitosis. More strikingly, some internalized immune cells penetrated directly into the nucleus of target cells. Multinuclear cells with aneuploid nucleus were formed in target tumor cells after internalizing immune cells as well as in situ tumor regions. Therefore, with the prevalence of heterotypic cell-in-cell structures observed, we suggest that shielding of immune cells inside tumor or inflammatory tissue cells implies the formation of aneuploidy with the increased multinucleation as well as fine-tuning of microenvironment under pathological status, which may define distinct mechanisms to influence the etiology and progress of tumors.