Phocarctos hookeri) is a Threatened marine mammal with a restricted distribution and a small, declining, population size. The species is susceptible to bacterial pathogens, having suffered three mass mortality events since 1998. Understanding the genetic factors linked to this susceptibility is important in mitigating population decline. The gene solute carrier family 11 member a1 (Slc11a1) plays an important role in mammalian resistance or susceptibility to a wide range of bacterial pathogens. At present, Slc11a1 has not been characterised in many taxa, and despite its known roles in mediating the effects of infectious disease agents, has not been examined as a candidate gene in susceptibility or resistance in any wild population of conservation concern. Here we examine components of Slc11a1 in NZSLs and identify: i) a polymorphic nucleotide in the promoter region; ii) putative shared transcription factor binding motifs between canids and NZSLs; and iii) a conserved polymorphic microsatellite in the first intron of Slc11a1, which together suggest conservation of Slc11a1 gene structure in otariids. At the promoter polymorphism, we demonstrate a shift away from normal allele frequency distributions and an increased likelihood of death from infectious causes with one allelic variant. While this increased likelihood is not statistically significant, lack of significance is potentially due to the complexity of genetic susceptibility to disease in wild populations. Our preliminary data highlight the potential significance of this gene in disease resistance in wild populations; further exploration of Slc11a1 will aid the understanding of susceptibility to infection in mammalian species of conservation significance.The New Zealand sea lion (NZSL,  Phocarctos hookeri, NZSL) appears to be highly susceptible to infection by bacterial pathogens. Episodic disease events have been a frequent occurrence in the recent history of the NZSL population; three outbreaks have been documented since 1998, resulting in high levels of mortality of pups especially, but also of adults in one event [The regularity with which marine mammals are subject to epizootic episodes is well documented  and massne event , 10, 11.ne event . Resourcne event –15, but ne event . Thus, uIt is suggested that novel disease episodes within a species are not usually caused by new infectious organisms, rather they are caused by ‘host shifts’, where known agents infect new hosts . TherefoSlc11a1, previously known as natural resistance associated macrophage protein 1 Nramp1), which is involved in resistance to a broad array of bacterial pathogens [Slc11a1 plays an important role in innate immunity, preventing bacterial growth in the early stages of infection [Slc11a1 is expressed exclusively in macrophages and polymorphonuclear leukocytes [Sllc11a1 homologs are widely identified and highly conserved from yeast to humans [As a step towards addressing the absence of data on the elements of innate immunity, here we investigate the gene solute carrier family 11 member a1 . Dead pups had been assigned a known cause of death at autopsy, and here we include those that died from bacterial infection (n = 23), hookworm-related enteritis (n = 32) and ‘other’ (n = 37) where ‘other’ includes pups that died from causes other than pathogenic .The NZSL is a protected species under the Marine Mammal Protection Act (1978), administered by the New Zealand Department of Conservation (DOC). Samples presented in this paper were collected with funding from the NZ Department of Conservation, and approvals for sample collection were obtained from the DOC Animal Ethics Committee .et al. 2013 [Slc11a1 (377bp) was amplified using oligonucleotides previously designed for application to the dog genome [ca. 50ng template DNA, 20mM Tris-HCl, 50mM KCl, 5nmol each dATP, dTTT, dGTP and dCTP, 1pmol each primer, 2mM MgCl2, 6% DMSO and 0.1 unit Taq-Ti DNA polymerase . Touch-down thermal cycling parameters [°C for 5 minutes, followed by 10 cycles of 94°C for 20 seconds, 65°C for 20 seconds, and 72°C for 30 seconds. Next, 40 cycles of 94°C for 20 seconds, annealing temperature (Tx) for 20 seconds and 72C for 30 seconds were performed, where Tx decreased by 0.5°C per cycle, beginning at 65°C. This was finalised by an extension step of 72°C for 5 minutes. PCR products were sequenced using an ABI 3730xl DNA Analyser  using the Genetic Analysis Service at the University of Otago and analysed with the program Geneious [Slc11a1 were designed with the program Primer3 [DNA was extracted as described in Osborne al. 2013 . Part ofg genome : NRAMP1-rameters  were as Geneious . NZSL-sp Primer3 : NZSL_F TGTAAAACGACGGCCAGTGAGTCTGCTTGAGATTCTCTC—3’, NRMICRI1-R 5’—TATCACCTCCACCCTTCAAAC—3’. The forward primer was fluorescently labelled with a 5’ M13 tag (underlined above) for subsequent genotyping [ca. 50ng template DNA, 20mM Tris-HCl, 50mM KCl, 5nmol each dATP, dTTT, dGTP and dCTP, 1pmol M13 primer, 1pmol reverse primer, 0.25pmol forward primer, 2mM MgCl2, 6% DMSO, 75mM TMAC and 0.1 unit Taq-Ti DNA polymerase . Thermal cycling parameters were as follows: initial denaturation 94°C for 5mins; 35 cycles of: 94°C for 30 seconds, 45°C for 30 seconds, 72°C for 45 seconds; 8 cycles of: 94°C for 30 seconds, 53°C for 30 seconds, 72°C for 45 seconds; final extension 72°C for 10 mins. PCR products were genotyped using an ABI 3730xl DNA Analyser and analysed with the program GeneMapper . For microsatellite analyses only, the number of individuals analysed were: n = 18 (bacteria), 30 (enteritis), 34 (other). These differences are due to differing PCR success between the Slc11a1 partial promoter sequence and the microsatellite.The intron 1 microsatellite was amplified using primers designed to the dog genome : NRMICRInotyping , for whiSlc11a1 sequence upstream of the repeat region, and the reverse strand aligned (in reverse complement) downstream of the repeat region, indicating that the correct region of NZSL DNA was being amplified.The products amplified from oligonucleotide primers for the intron 1 microsatellite were sequenced in dead NZSL pups by Sanger sequencing methods  to ensurThe program MatInspector  was usedBos taurus, AY438096), sheep , pig  human  and dog  Slc11a1 sequences. Mammalian sequences were aligned to the NZSL promoter region using the default alignment method in Geneious [http://www.ncbi.nlm.nih.gov/SNP/), with search terms limited to exclude ‘homo’ (Homo sapiens) sequence submissions. Retrieved sequences were aligned using MAFFT .The integrity of the region of the NZSL promoter amplified here was investigated in other mammals through multiple sequence alignment of cattle . A single polymorphic site was detected in this region, a guanine (G) to adenine (A) substitution at residue number 317 of the 377bp amplified here  designed to NZSL DNA from those individuals that were successfully amplified and sequenced with the canine primers resulted in consistent amplification of 377bp of promoter sequence after trimming . This seied here . Both heied here  and geno5-6GCCAA-3’) and seven putative IFN-γ  sites  sites . These t1a1 gene . A putatOther predicted binding sites that occur in the NZSL promoter sequence are: AP1 , AP2 , GM-CSF .SLC11A1 identified as highly variable and associated with disease susceptibility [Slc11a1 , Canis familiaris , Mus musculus , Ovis aries , Pan troglodytes , Pongo abelii  and Rattus norvegicus . Based on MAFFT alignment, intraspecific polymorphism was detected at the NZSL polymorphic site in all species above, but for those species where only one sequence was retrieved (cow and sheep).A region of canine tibility , 57 was [Slc11a1 . Two not[Slc11a1 . A databIn the first instance, genotypes at the promoter polymorphism were identified through Sanger sequencing methods , and eacNo differences in genotype frequency distribution were observed between status groups, as determined by Fisher’s Exact Tests, Genotype frequencies for animals dying of bacteria and enteritis appear to be inconsistent with neutrality when displayed on a bar plot , and disData from animals dying of infection  were combined and compared with those that died of other causes . Expected heterozygosity at this locus for all dead pups is 0.28 versus an observed heterozygosity of 0.22 indicating that the microsatellite, while polymorphic, shows little variation. We find no evidence for linkage disequilibrium between the intron 1 microsatellite and the promoter polymorphism for any status group, or across the population .Amplified alleles were assigned numerical identifiers based on their observed fragment size. Of the alleles amplified, two (165bp and 167bp) were common; allele 165 was more frequent than allele 167 . Data from infected pups were grouped (where ‘infected’ is defined as those pups dying of both bacteria and enteritis) and compared to data from pups dying from ‘other’ causes. Odds ratios as calculated by Fisher’s Exact Test showed that animals possessing haplotype H3 were twice as likely to be ‘infected’ than animals possessing haplotype H2 . However, because both haplotypes contain the same microsatellite allele, the likelihood is that this pattern is driven by the promoter polymorphism frequency differences. We note here that while the odds ratios are of interest, they were not statistically significant.As a consequence of low frequencies for some of the haplotypes, only haplotypes H2, H3, H4 and H5 are considered in further analyses. A bar plot of haplotype frequencies, grouped by cause of death, shows that animals dying of ‘other’ causes have a haplotype distribution of the most common haplotypes, H2 and H3, that is more reflective of neutrality than those dying of bacteria or enteritis ; animalsSlc11a1 gene. The polymorphic site lies in a putative transcription factor binding site, which was identified using canid transcription factor recognition sequences. A greater number of pups dying from infectious causes possess the G variant of this polymorphism, and while this pattern is not statistically significant, we have shown that pups with the G variant are twice as likely to be dead from infectious causes, rather than from ‘other’ causes . We provide evidence of intraspecific polymorphism in other mammalian species using the data currently available. We demonstrate conservation of a polymorphic microsatellite between canids and otariids; however there was no significant difference in frequency of the two most common microsatellite alleles between causes of death, and the locus was determined to be in Hardy Weinberg equilibrium.Here we have demonstrated conservation of gene structural elements between canids and otariids, implying potential conserved function across mammalian taxa. We have identified one polymorphism in 377bp of sequence obtained from the promoter region of the New Zealand sea lion et al. (2005), these genomic features do not appear to be interacting to produce susceptible or resistant haplotypes in the NZSL. We note that the G variant leads to a two-fold increase in likelihood that an animal is infected. This increased likelihood suggests that the polymorphism may have a role in susceptibility to infectious agents; however post-hoc power analysis of this comparison suggests that the study lacks sufficient power to test this association rigorously. A larger study including more animals with known causes of death would be required to effectively test for an association in this species.Previous work with dogs has shown that haplotypes constructed of combinations of polymorphic loci analysed together (e.g. the intron 1 microsatellite and polymorphisms in G-rich regions on the promoter ), show aSlc11a1 is expressed in phagocytic cells [Slc11a1 promoter region, consistent with the observation that IFN-γ increases the induction of Slc11a1 gene expression in mice [Slc11a1 promoter region has many putative regulatory motifs which are conserved between the canid and NZSL genomes [Slc11a1 in macrophage activation in response to IFN-γ stimulation [Transcription factor binding sites identified in promoter regions, by bioinformatic analysis, can provide some clues to the function of the genes downstream of those promoters. ic cells , 37 and ic cells . Putativ in mice . Indeed, genomes , consistmulation .Slc11a1 gene expression. However, it is difficult to predict whether this SNP is affecting transcription factor binding, and confirming this would require direct biochemical analysis. We have previously shown that a high degree of homology exists between the canid and NZSL genomes [Slc11a1 expression in the NZSL [The binding motif for nuclear factor I (NF-I), which is involved in the differentiation between erythrocytes and granulocytes , brain d genomes , and it  genomes . Howeverthe NZSL , 79. Thuthe NZSL .Slc11a1 promoter could have an effect on its expression [Slc11a1 promoter region could be altering promoter activity and therefore also gene function. There is evidence of intraspecific polymorphism in published Slc11a1 nucleotide sequences, which could mean that the NZSL polymorphic nucleotide is a commonly variable one. While the majority of the information on intraspecific polymorphism is limited to rodents, we find evidence that the polymorphic site is commonly variable in dogs as well as NZSLs . The detection of intraspecific polymorphism in two related species that share a large amount of homology suggests a functional role for the nucleotide, however further work, such as in vitro reporter gene assays, is needed to investigate this fully.The NZSL displays a polymorphic site in a region of guanine nucleotide repeats in the promoter region, and is reflective of an identified variable region in dogs, where polymorphisms in G-rich regions have been associated with disease susceptibility , 57. Polpression  and humapression . It is pSLC11A1 [Microsatellites within genes often affect their function , 84 and SLC11A1 . While sSLC11A1 , normallSLC11A1 . The demWhile distinct allele frequency differences at the promoter polymorphism were observed in relation to NZSL pup mortality and infection status, and were accompanied by a strong likelihood of infection with the G variant, these differences were not statistically significant, leading one to question the potential cause of non-significance. The NZSL is an observed population only, meaning that any variable factors can not be controlled. This is in contrast to domesticated agricultural populations where there is more control over environmental variables, and higher degrees of genetic homogeneity with populations. Higher homogeneity can lessen the variability in the phenotypes observed within the population and may strengthen the ability to detect associations between genotype and phenotype within and between populations. Environmental variability within the NZSL population is much greater than for most agricultural, laboratory and captive populations; therefore a much larger number of individuals is likely to be required to explore genetic associations with the polygenic traits likely to underlie fitness. It is likely that the factors above account for the higher p-values in detected here, despite strong odds ratios.Slc11a1 affects disease resistance in this species, our preliminary data, encompassing the conservation of Slc11a1 cross-species, and the likelihoods identified here, illustrate the promising nature of this gene as a candidate for disease resistance and/or susceptibility in this and other mammalian species. We suggest that its investigation should be broadened to include wild populations and those of conservation significance.Thus, while a larger sample of individuals with assigned cause of death would be required to investigate more rigorously whether S1 Fig377bp of NZSL SLC11A1 promoter region sequence. The polymorphic site is underlined. (DOCX)Click here for additional data file.S2 FigPutative transcription factor binding site motifs  for NZSL SLC11A1 promoter sequence, with SNP (A—G) indicated in gold. Figure output from Geneious with annotations manually added.(DOCX)Click here for additional data file.S3 FigNZSL sequence for SLC11A1 partial promoter region aligned against canine SLC11A1 promoter region. Green bar represents sequence similarity with white gaps showing divergence. Green arrows indicate where NZSL primers bind, with missing NZSL sequence at these sites due to trimming of the sequence. Red arrow indicates the transcription start site in canine sequence. Pale arrow shows the G-rich stretch in dogs associated with susceptibility to infection. This region is interrupted in the NZSL.(DOCX)Click here for additional data file.S4 FigMultiple sequence alignment of SLC11A1 sequences from cattle, sheep, pig, human, dog and NZSL. Variable region in canine sequence is shown shaded in blue and promoter SNP is shaded in gold in all species.(DOCX)Click here for additional data file.S5 FigHaplotype frequencies (%) of combined promoter polymorphism and microsatellite variant, grouped by cause of death. Haplotype (H) numbers refer to haplotype numbers indicated in (DOCX)Click here for additional data file.


Arabidopsis  from diverse plant species. Apoplastic interactions are integral to plant signaling, growth, defense, physiology, and reproduction. In addition, the plant apoplast serves as a hub of pathogen effectors and a great deal of pathogenic proteins and small molecules are cataloged in literature. In spite of its importance in plant biology, the knowledge about apoplastic lipids and their carriers have been limited to few seminal studies and sporadic efforts. Here, the attempt is to comprehend the gained knowledge and remaining gaps in plant apoplastic lipidomes and address means as to how efforts can unveil the black box of plant apoplastic lipidome. The apoplast is defined as the extracellular matrix, the plant cell wall and the intercellular spaces where the apoplastic fluid circulates  and lysophospholipids such as lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), and lysophosphatidic acid (LPA) are produced from structural phospholipids by specific enzymatic pathways and are minor constituents of the cell membranes —the most abundant class of storage lipids, wax esters, sterols, sterol esters, acylated sterol glycosides, phytoglycolipids, ceramides, glucosylceramides. For instance only phospholipids consists of major classes of phospholipids such as phosphatidylcholines (PC), phosphatidylglycerols (PG), phosphatidylethanolamines (PE), phosphatidylserines (PS), and phosphatidylinositols (PI) are structural phospholipids that have distinct and specific distributions in the cellular membranes, contributing to their identity  and cuticles which is consisted of lipid and hydrocarbon polymers impregnated with wax among others. Cell walls include macromolecular polymeric structures such as lignins, pectins, cellulose among others. Suberin is an apoplastic biopolymer that contributes to the control of diffusion of water and solutes across internal root tissues and in periderms  mutant exhibited wild-type local resistance to avirulent and virulent Pseudomonas syringae, failed to develop systemic acquired resistance (SAR) to virulent pathogens such as Pseudomonas or Peronospora parasitica  are secreted into the apoplast, that contain a hydrophobic pocket which binds long-chain fatty acids (FA) to transfer lipids via an unknown mechanism  function as a apoplastic carrier when lipid components from the senescent cells of the endosperm , a cytochrome P450 monooxygenase catalyzing fatty acid oxidation was reported in the apoplast  leaves infected with a cloned isolate of the apple scab Venturia inaequalis  hydrolyzes phospholipids at the sn-2 position to yield lysophospholipids and free fatty acids. With leaf maturity and upon pathogenic challenge the paralog PLA2α is translocated from ER/Golgi locations to apoplastic space  the apoplastic (exoplasmic) leaflet, as well as in rafts, phospholipids did not include digalactosyldiacylglycerol (DGDG), but showed the presence of acylated sterol glycosides  seeds imbibed for 2 h contained diverse phospholipids, i.e., PA and PI being the major phospholipids  leaf cells, the characteristic oxylipins for oxidation were not detected in apoplastic fluid over the period of the apoplastic burst, although linolenic and linoleic acids were detected in apoplastic fluids before elicitation (Bolwell et al., Suberin is a heteropolymer with polymeric aliphatic and associated aromatic materials. Long-chain oxygenated fatty acids are the core constituents of the suberin polyester. Arabidopsis (Degenkolbe et al., in situ lipidomic visualization/ imaging (Horn and Chapman, We have just started exploring and understanding the apoplastic lipidomes at the levels of singular carrier proteins only, i.e., more specifically the LTPs and their functionalities in pathogenesis and stress conditions, mostly. Only a handful of lipids have been localized to the apoplast or roughly assigned any function in the apoplast. Thus, we have only touched the tip of the ice berg of the entire plant lipidome which must be dynamic and diverse. However, with expanding lipidomic studies in model systems such as The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Magnetic resonance imaging (3.0-T) was performed at baseline, 6th hour and 1 week after procedure. Cardiomyocyte apoptosis was detected by cardiac-TUNEL staining, and caspase-3 and caspase-8 were detected by RT-PCR and immunohistochemistry. Furthermore, serum TNF-α, IL-6 and troponin T were analysed, while myocardial expressions of TNF-α and IL-6 were detected. Both TNF-α expression  and average number of apoptotic cardiomyocyte nuclei were significantly increased in CME group compared with the sham-operation group. Six hours after CME, left ventricular end-systolic volume (LVESV) was increased and the left ventricular ejection fraction (LVEF) was decreased in CME group. Pre-treatment with adalimumab not only significantly improved LVEF after CME , but also suppressed cardiomyocyte apoptosis and the expression of caspase-3 and caspase-8. Meanwhile, the average number of apoptotic cardiomyocytes nuclei was inversely correlated with LVEF . TNF-α-induced cardiomyocyte apoptosis is likely involved in cardiac dysfunction after CME. TNF-α antibody therapy suppresses cardiomyocyte apoptosis and improves early cardiac function after CME.This experimental study was designed to clarify the relationship between cardiomyocyte apoptosis and tumour necrosis factor-alpha (TNF-α) expression, and confirm the effect of TNF-α on cardiac dysfunction after coronary microembolization (CME) in mini-pigs. Nineteen mini-pigs were divided into three groups: sham-operation group ( This allows for the initiation of apoptosis in vascular endothelial cells and cardiomyocytes Our objective of this study focused on the occurrence of cardiac apoptosis after CME, and tried to explore direct relation between the effect of TNF-α and cardiac apoptosis. We also wanted to clarify whether reduction in cardiac apoptosis was involved in the cardiac protection of TNF-α antibody in CME.n = 5); Group two: CME group (n = 7) and Group three: TNF-α antibody pre-treatment group (n = 7)—intracoronary received 2 mg/kg adalimumab  30 min. before CME. Animals were initially sedated via intramuscular injection of combination ketamine (20 mg/kg) and diazepam (2 mg/kg). Anaesthesia was maintained with intravenous 3% pentobarbital sodium. Vascular sheaths (one 7F and one 6F) were placed in the right femoral artery and vein respectively.Nineteen mini-pigs (22–26 kg) [SCXK (Shanghai) 2007-0013] of either sex were divided into three groups. Group one: Sham-operation group , and the protocols were approved by the Animal Care and Use Committee of Fudan University, China (A5732-01).Detailed experimental steps to generate the mini-pig CME model have been previously described One week after the procedures, hearts were excised and sectioned into five slices from apex to base in a plane parallel to the atrioventricular groove. We selected the second and third slices approached to apex for histological detection. Approximately, 200–300 mg of transmural myocardium, from the left ventricle anterior walls, was immediately frozen in liquid nitrogen and stored at −70°C for RT-PCR and Western blot. The formaldehyde-fixed specimens were embedded in paraffin and sectioned into slices of 5 μm thickness for haematoxylin and eosin staining, immunohistochemical analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) staining. Other myocardium was immersed in a 0.2 mol/l sodium phosphate buffer (pH 7.4) containing 1.0% nitroblue tetrazolium chloride  for 20 min. at 37°C to demonstrate the presence of myocardial necrosis after operation.Haematoxylin and eosin staining was also applied to demonstrate the presence of myocardial microinfarcts. The area of necrosis was calculated from 10 random fields (×200) of each slice using Leica DFC 320 digital software , and the percentage of each slice was calculated and averaged. The observers who performed histological analyses were blinded to the results of study grouping.Magnetic resonance imaging (MRI) was performed at baseline, 6th hour and 1 week after operation using 3.0-T Siemens scanner  with a maximum slew rate of 200 mT/m/msec. and maximum gradient strength of 40 mT/m. Transverse, two-chamber and four-chamber LV long-axis scout images were obtained to determine the final short-axis image plane. Cine MRI was acquired in contiguous short-axis planes from the apex to the base of the heart to measure LV function. After the cine was obtained, mini-pigs received an intravenous bolus of 0.05 mmol/kg gadolinium DTPA  at a rate of 4 ml/sec. by means of an infusion pump followed by a 10 ml flush of saline at a rate of 4 ml/sec. Electrocardiograph was recorded simultaneously, and MRI detection included the evaluation of left ventricular ejection fraction (LVEF), left ventricular end-systolic volume (LVESV) and left ventricular end-diastolic volume (LVEDV). Two observers performed MRI analysis were blinded to the results of grouping.Serum samples were obtained at baseline, 2nd hour, 6th hour and 1 week after CME or sham-operation, and stored at −70°C for detection. Serum concentrations of TNF-α and IL-6 were determined by ELISA assay . Serum level troponin T was analysed by immunoturbidimetry .Myocardial apoptosis was detected by TUNEL staining using a commercially available kit , which was supplemented with a cation that specifically enhanced the labelling of apoptotic cardiac cells. In each specimen, paraffin slices were incubated with TUNEL labelling mixture and counterstained with nuclear fast red. TUNEL-positive cardiomyocyte nuclei, which were blue in colour, were counted in 10 random high-power fields , and the average number of TUNEL-positive cell nuclei in each HPF was calculated by a Leica DFC 320 digital camera (Leica Microsystems Imaging Solutions Ltd).Total RNA was extracted from the anterior wall of myocardium using trizol , and dissolved in distilled water. RNA concentration was determined by measurement of optical density at 260 nm. Reverse transcription was performed with 1 μg of total RNA and oligo(dT) primers in a 20-μl reaction according to the manufacturer's protocol . The sequences of the 5′-sense primers and the 3′-anti-sense primers used in this study were shown in TableAll of 19 mini-pigs' heart were subjected to immunohistochemistry and Western blot analyses. Paraffin-embedded heart specimens were cut into 5-μm sections, deparaffinized in xylene, and dehydrated in graded alcohol. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide. After rinsing with PBS, slices of each myocardium were incubated overnight at 4°C with mouse monoclonal anti-porcine TNF-α antibody , rabbit polyclonal anti-porcine IL-6 antibody , rabbit polyclonal anti-porcine caspase-3 antibody  and goat polyclonal anti-porcine caspase-8 antibody  for immunohistochemistry. According to the protocol of Superpicture Polymer Detection Kit , HRP-polymer conjugate was added and incubated for 10 min. After a wash step, the DAB chromogen was added. Slices were counterstained with haematoxylin and examined by light microscopy. Positive-staining area per cent was calculated in 10 random high-power fields  by a Leica DFC 320 digital camera (Leica Microsystems Imaging Solutions Ltd).Detail experimental steps of the Western blot procedure have been previously described The extent of fibrosis was detected by Masson Trichrome stain. It was performed to distinguish areas of collagen, which was shown in blue colour. The percentage of blue staining was examined by light microscopy, and analysed by a Leica DFC 320 digital camera (Leica Microsystems Imaging Solutions Ltd). Collagen volume fraction (CVF) was calculated by averaging the percentage area of stained tissue within 10 fields (×200) randomly selected from the infarct area on each slice.anova analysis, while one-way anova was performed for analysis of apoptosis (average apoptosis nuclei), RT-PCR , Western blot (average band density), positive-staining area per cent in immunohistochemistry and CVF among three groups. Student's t-test was performed for necrosis-area analysis between CME and antibody pre-treatment group. Correlation analysis (Spearman test) was performed to evaluate the correlations among cardiac volume, LVEF and average number of apoptosis nuclei. Statistical analyses were performed with SPSS software (Version 18.0). All P-values were two-sided, and P < 0.05 was considered statistically significant.Data were expressed as mean ± SD. Data from MRI  and serum levels of TNF-α, IL-6 and troponin T at each time-point were estimated by two-way There were no significant changes in heart rate, blood pressure or coronary flow evaluated by Thrombolysis in Myocardial Infarction before and after the procedure Table.NBT and haematoxylin and eosin staining were performed. We detected obvious microinfarcts and polystyrene microspheres in the anterior wall of the CME group (microembolization-associated myocardium), which were not observed in sham-operation group. Leucocyte infiltration in the embolized myocardium was also increased in the CME group. Adalimumab (TNF-α antibody) not only greatly prevented leucocyte infiltration, but also decreased total areas of necrosis and microinfarcts in treatment group Fig..versus 50.4 ± 3.9%, P = 0.036; LVEF-1 week: 56.7 ± 4.2% versus 52.7 ± 2.9%, P = 0.041).MRI using 3.0-T Siemens scanner was performed at baseline, 6th hour and 1 week after operation. Changes in cardiac volume and function at different time-points were shown in TableP < 0.01); this effect was maintained up to 6 hrs after CME . One week later, the TNF-α level was decreased to basal level . In the TNF-α antibody pre-treatment group, serum levels of TNF-α were slightly decreased. However, the decrease was not statistically significant , the serum level of TNF-α in the CME group was increased significantly at 2nd hour after CME . Pre-treatment with TNF-α antibody significantly decreased apoptosis  and negatively correlated with LVEF . There was no significant correlation with LVEDV . TNF-α antibody treatment greatly reduced CVF after CME  was significantly higher than that in sham-operation group  or method of administration  or compensatory mechanism. Adalimumab (Humira), which was intracoronary injection in our study, was one of recombinant human monoclonal anti-TNF-α antibodies. It could combine with TNF-α specially, and block the interaction between TNF-α and its receptors (p55 and p75). Its pharmacokinetics half-time was 12 to 14 days. We found that TNF-α antibody improved cardiac dysfunction after CME Table, which c mg/kg orCardiomyocyte apoptosis has been documented as a critical form of cell death in ischaemia and reperfusion damage. A key component to apoptotic signalling pathways is the activation of caspases, a group of cysteinyl-aspartate-directed proteases. Caspases are typically activated by proteolytic cleavage in vitro research is required to clarify the detailed signal transduction involved cardiomyocyte apoptosis. Finally, microspheres are chemically inert and lack any chemoattractant properties; thus, they are quite different from microemboli identified in patients at autopsy or retrieved during PCI. In conclusion, further research should be performed taking these limitations into account.We should note some of our study's limitations. First, there were only a small number of mini-pigs investigated. Second, a prolonged experimental period should be warranted to see the long-term effects of TNF-α on cardiac apoptosis after CME. Third, multiple doses of TNF-α antibody should be taken into account. In addition, more TNF-α-induced cardiomyocyte apoptosis is likely involved in cardiac dysfunction after CME. TNF-α antibody therapy suppresses cardiomyocyte apoptosis and improves early cardiac function after CME.


Sturnus vulgaris). Although waves of agitation in starling flocks have been suggested to reflect density waves, exact escape manoeuvres cannot be distinguished because flocks are spatially too far away. Therefore, waves may also reflect orientation waves (due to escape by rolling). In the present study, we investigate this issue in a computational model, StarDisplay. We use this model because its flocks have been shown to resemble starling flocks in many traits. In the model, we show that agitation waves result from changes in orientation rather than in density. They resemble empirical data both qualitatively in visual appearance and quantitatively in wave speed. In the model, local interactions with only two to seven closest neighbours suffice to generate empirical wave speed. Wave speed increases with the number of neighbours mimicked or repeated from and the distance to them. It decreases with reaction time and with time to identify the escape manoeuvre of others and is not affected by flock size. Our findings can be used as predictions for empirical studies.Fast transfer of information in groups can have survival value. An example is the so-called wave of agitation observed in groups of animals of several taxa under attack. It has been shown to reduce predator success. It usually involves the repetition of a manoeuvre throughout the group, transmitting the information of the attack quickly, faster than the group moves itself. The specific manoeuvre underlying a wave is typically known, but not so in starlings (The online version of this article (doi:10.1007/s00265-015-1891-3) contains supplementary material, which is available to authorized users. Fast transfer of information in groups can have survival value  has shown that the dark band moves continuously in a line from one end of the flock to the other end always away from the predator , reaction time, cue identification time  and flock size. We compare wave speeds to those reported for empirical data , its attraction to the roost (site for sleeping), the simplified aerodynamics of flight which includes banking while turning, and reaction time  induced many turns of the flock  . The location of the body of each individual relative to the origin is indicated by a vector aw) Fig. . The indrear Eq.  and the rear Eq. .1\documeirear, escape by one of the three escape manoeuvres, sidewards and back, namely zigzag and zig, or accelerate forward . Because the social coordination , back and sideward to the other side again Fig. .4\documementclass2pt{minimTzside seconds and Tzback seconds back again , especially of starlings, see supplementary material Table σu of 10 ms  by calculating for each escaping individual its spatial distance to the first bird escaping and the time interval between the escape of the first individual and itself. The wave speed is the average of all these measurements. We took the average rather than the median because the variation in reaction time has been drawn from a normal distribution.For each parameter value of NND, range of repetition, reaction time, cue identification time and flock size, we have run 30 replicas.In empirical studies of flocks of starlings, an agitation wave has been described as a dark band moving away from the attack by the predator to the other side of the flock Fig. .Our model did not produce an agitation wave, if the flock members merely adjusted their movement but did not repeat an escape manoeuvre Fig.  movie S1The zig movement generated an agitation wave merely because temporarily rolling laterally changed the orientation of the birds towards the observer which implies a difference in the visibility of the wing area  for potential manoeuvres of danger, we expected the speed of the wave to increase. Indeed, extending the range of repetition from two till seven neighbours increased the speed of the wave in the model Fig. .Fig. 4SpIf a flock was less dense, meaning that on average individuals were further apart, wave speed was expected to be higher too because the reaction time remains the same . Empirically, flock density (measured as the average distance to the nearest neighbours) varies at least between 0.68 and 1.51 m  sufficed to generate the same range of speeds of waves as recorded in the empirical data for ranges of repeating escape manoeuvres from two till seven neighbours and for densities of the flocks between 0.71 and 1.93 m Table . UnfortuWe show that only if individuals repeat a ‘cue of danger’ an agitation wave results. It results not in the form of a density wave as has been assumed by us formerly . Thus, when we see the flock from the side, the projection of the wing surface visible to us increases when birds have rolled sideward, and it decreases again when they fly level. As a consequence of the repetition of the escape manoeuvre by rolling, we see a dark band (of birds that are temporarily rolled sideward) moving away from the predator. There are no visible changes in density in the flock during an agitation wave (since no wave is visible if birds are represented as spheres).The reasons why speeding up forward does not lead to a visible density wave may be twofold: individuals may not be coming sufficiently close to others (because of collision avoidance) and may be moving too slow to create visible changes in density (since rolling is faster) Warrick .Agitation waves in flocks of dunlins and in schools of anchovies are due to changes of orientation also, but in contrast to our model in dunlins and anchovies, changes in orientation are associated with changes in colour Radakov . BesidesAptenodytes forsteri). In a comparison between a mathematical model and empirical data, it was shown that a single step forward from a single penguin located in a densely packed huddle could trigger a complete wave , cue identification time (data available on request) and flock size.Calidris pusilla, when they depart Movie S1The escape manoeuvre of rolling sideward, not repeated by others. This movie shows that this escape manoeuvre without being repeated by other (others merely adjust their movement direction) does not result in an agitation wave. (MP4 503 kb)Movie S2Repetition of the escape manoeuvre of speeding-up-forward repeated by neighbours. This movie shows that from repetition of this escape manoeuvre an agitation wave is not observed. (MP4 745 kb)Movie S3Repetition of the zigzag-escape manoeuvre of rolling to one side, back, rolling to the other side and back again Movie S5A spherical body shape and repetition of the zig-escape manoeuvre of sideward rolling and back. This movie shows that the repetition of the zig-escape manoeuvre does not generate an observable agitation wave when the body shape is circular. (MP4 154 kb)Below is the link to the electronic supplementary material.


The BioVis 2015 symposium is affiliated with ISMB, the Intelligent Systems for Molecular Biology conference, as a Special Interest Group (SIG). BioVis 2015 took place in Dublin, Ireland, July 10-11 2015.The expanding experimental methodology and resulting data of biological research create significant challenges for computational visualization techniques. The goal of BioVis 2015 - BMC Proceedings Section Editor together selected those that would be published in BMC Proceedings.All papers were reviewed by reviewers from both the bioinformatics and visualization fields and were evaluated for improvements over state-of-the-art and for scientific soundness. The review process was organized in two review cycles. In the first review cycle, each paper was reviewed by three to four reviewers. In the second review cycle, the primary reviewers checked whether the required revisions for conditionally accepted papers were successfully included. Based on the reviewers' scores, reviews, and recommendations, the BioVis 2015 Paper and Publication Chairs and BMC Bioinformatics supplement and as "Proceedings of the 5th Symposium on Biological Data Visualization: Part 2" in these BMC Proceedings. The highest quality papers were selected for the BMC Bioinformatics supplement. Those papers that feature high quality work but were not accepted for the BMC Bioinformatics supplement are published in BMC Proceedings. From the 21 papers submitted to BioVis 2015, 9 papers are published in the BMC Bioinformatics supplement and 5 papers are published in these BMC Proceedings.The papers from BioVis 2015 appear in two different proceedings: As "Proceedings of the 5th Symposium on Biological Data Visualization: Part 1" in a The articles in these Proceedings cover an interesting spectrum of challenging problems in biological data visualization and their solutions. First, several papers address the issue of pathway visualization. Dang et al. make two contributions, presenting both a glyph/matrix-based visualization to investigate one-to-one relationships between proteins , and a mWe hope that the collection of papers in these Proceedings will educate, inspire, and engage visualization researchers in problems in biological data visualization, as well as bioinformatics and biology researchers in state-of-the-art visualization research.The authors declare that they have no competing interests.


Niphargus is a speciose amphipod genus found in groundwater habitats across Europe. Three Niphargus species living in the sulphidic Frasassi caves in Italy harbour sulphur-oxidizing Thiothrix bacterial ectosymbionts. These three species are distantly related, implying that the ability to form ectosymbioses with Thiothrix may be common among Niphargus. Therefore, Niphargus–Thiothrix associations may also be found in sulphidic aquifers other than Frasassi. In this study, we examined this possibility by analysing niphargids of the genera Niphargus and Pontoniphargus collected from the partly sulphidic aquifers of the Southern Dobrogea region of Romania, which are accessible through springs, wells and Movile Cave. Molecular and morphological analyses revealed seven niphargid species in this region. Five of these species occurred occasionally or exclusively in sulphidic locations, whereas the remaining two were restricted to nonsulphidic areas. Thiothrix were detected by PCR on all seven Dobrogean niphargid species and observed using microscopy to be predominantly attached to their hosts' appendages. 16S rRNA gene sequences of the Thiothrix epibionts fell into two main clades, one of which (herein named T4) occurred solely on niphargids collected in sulphidic locations. The other Thiothrix clade was present on niphargids from both sulphidic and nonsulphidic areas and indistinguishable from the T3 ectosymbiont clade previously identified on Frasassi-dwelling Niphargus. Although niphargids from Frasassi and Southern Dobrogea are not closely related, the patterns of their association with Thiothrix are remarkably alike. The finding of similar Niphargus–Thiothrix associations in aquifers located 1200 km apart suggests that they may be widespread in European groundwater ecosystems. Thiothrix bacteria and three species of the groundwater amphipod genus Niphargus were reported from Frasassi , which are predominantly attached to hairs (setae) and spines of their legs and antennae . The three ectosymbiont clades do not form a monophyletic group  as well as in anchihaline caves in Croatia, where sulphide is present but has not yet been quantified  provides the ideal locality to start examining this question, as it has a sulphidic aquifer that can be accessed through artificial wells, springs and Movile Cave. Discovered in 1986, Movile Cave was the first terrestrial chemoautotrophic ecosystem described  and is oin situ hybridization (FISH), and in 2.5% glutaraldehyde prepared in phosphate-buffered saline (PBS) for scanning electron microscopy (SEM). A microbial mat sample was collected in April 2011 from Airbell 2 of Movile Cave (see Thiothrix found in Movile Cave.Amphipods were collected between April 2011 and September 2012 in the Southern Dobrogea region of Romania (see TablesDNA extractions for niphargid sequencing and PCR screenings (see below) were performed as described in Synurella sp. and Gammarus sp.). PCR amplifications and sequencing of a fragment of the mitochondrial cytochrome c oxidase (COI) gene, of the complete nuclear internal transcribed spacer (ITS) and of a fragment of the nuclear 28S rRNA gene were performed as reported previously (http://www.mnhn.fr/jfflot/champuru), and the haplotypes of other heterozygotes were determined using Clark's method , comprising 69 niphargids and two outgroups . Sample preparation and analysis were done as described previously  and its competitor probe G123T-C .Three other individuals  were investigated using FISH. The ommended  using thThiothrix-specific forward primer THIO714F  was designed as described above and used in combination with THIO1492R for direct sequencing of PCR products.The m MgCl2, 0.3 mm dNTP mix (Bioline), 25–30 ng of DNA , 1.25 units of BioTaq DNA polymerase (Bioline) and 25 pm each of the primers THIO714F and THIO1492R. PCRs were performed in a Labcycler , with an initial denaturation at 94 °C for 5 min, followed by 35 cycles of 94 °C for 1 min, 56 °C for 25 s, 72 °C for 2.5 min and a final extension at 72 °C for 5 min. PCR products were checked on a 1% agarose gel. Bands of the expected size (∼800 bp) were excised and extracted using the QIAquick Gel Extraction Kit . PCR products were cloned and sequenced as described previously . PCR mixtures (50 μL) contained 1× ammonium buffer , 2 mThiothrix DNA by direct PCR screenings. PCR mixtures (10 μL) contained the same ingredients as described for Thiothrix clone library construction. Cycling conditions were as follows: initial denaturation at 94 °C for 3 min, followed by 50 cycles of 94 °C for 45 s, 56 °C for 30 s and 72 °C for 1.5 min. PCR products were checked on a 1% agarose gel, and samples revealing bands of the expected size (∼800 bp) were sequenced directly using the primers THIO718Fseq and THIO1492R. In cases where mixtures of two overlapping Thiothrix sequences were obtained from the same PCR product, the individual sequences were resolved by comparison with Thiothrix epibiont sequences from the clone libraries. All sequences were assembled and screened for chimeras as described above.DNA extracts obtained from the 71 amphipod specimens  were examined for Thiothrix species and to sequences previously obtained from Niphargus species and microbial mats in Frasassi. 81 of these 84 sequences (leaving out three redundant Thiothrix sequences from samples AH_10.4 and SS_10.1 that were obtained in both PCR screenings and clone libraries) were used for phylogenetic analyses, together with 65 closely related Thiothrix sequences downloaded from GenBank. All sequences were aligned using the MAFFT version 6 multiple sequence alignment tool , whereas sequencing of the ITS marker failed for one specimen and the COI sequences of four of them were of bacterial origin. Both the nuclear ITS and the mitochondrial COI markers were congruent in delineating seven species among our samples  or identical (for ITS and the 28S rRNA gene) to the ones from Dobrogea, despite a geographical distance of over 1000 km. We were not able to obtain material from the type locality of N. gallicus in France to verify whether it is really the same species . The bacteria were found attached predominantly to hairs and spines on legs and antennae of the niphargids .SEM revealed accumulations of filamentous bacteria on two gids Fig.. The ThiThiothrix-related partial 16S rRNA gene sequences were obtained from 21 of the 71 amphipod DNA extracts using PCR screenings with Thiothrix-specific primers . From three of these 21 DNA extracts , mixtures of two overlapping Thiothrix sequences were obtained, which were resolved by comparison with Thiothrix epibiont sequences in clone libraries obtained from P. ruffoi (AH_10.4) and N. cf. stygius (SS_10.1). From 11 of the remaining 50 amphipod samples, bands of the expected size (∼800 bp) were obtained on the agarose gel, but the top BLAST hits of the corresponding sequences belonged to bacteria other than Thiothrix . The clone library constructed from the Movile microbial mat DNA yielded 52 Thiothrix sequences, which were used for comparison with Thiothrix sequences obtained from the niphargids.Thiothrix epibiont sequences obtained in this study fell into two clades  and from Frasassi microbial mats (The majority (86%) of ades Fig.. One of ons Table. Four reial mats ., hydrothermal vent crabs of the genus Kiwa . On the basis of the present data, it is not possible to say whether these additional bacteria represent yet unknown symbionts or free-living bacteria that were loosely attached to the niphargid individuals at the time of collection.Direct PCR screenings revealed that individuals of all seven niphargid species harboured Niphargus–Thiothrix epibioses found in Southern Dobrogea and Frasassi. First, the Thiothrix filaments are attached to the base of hairs on the amphipod appendages in both cases . Third, clade T3 is present in both sulphidic and nonsulphidic waters, whereas the other clades (T4 in the case of Southern Dobrogea and T1-T2 in the case of Frasassi) are only abundant in sulphidic locations , and Thiothrix filaments were missing on 5 of 10 niphargid specimens examined by SEM . It seems unlikely that all these apparently Thiothrix-lacking individuals had lost their epibionts due to moulting. Instead, our results suggest that the Thiothrix ectosymbionts may not be obligate for their hosts. Whether the Thiothrix bacteria benefit from associating with amphipods remains to be investigated.Epibiotic ediments . Just asThiothrix ectosymbionts of clades T3 and/or T4, which are attached to their hosts in a manner similar to that previously observed in the Frasassi caves in Italy. Thus, Niphargus–Thiothrix associations are not restricted to Frasassi, where they were first discovered, but may be widespread in European sulphidic and nonsulphidic aquifers.Six of the seven niphargid species identified in the Southern Dobrogea region of Romania harbour putative


Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are characterized by acute lung inflammation. Intercellular adhesion molecule-1 (ICAM-1) and interleukin-8 (IL-8) play an important role in the development of these diseases. Mitogen-activated protein kinase (MAPK) p38/activated protein kinase 2 (MK2) regulates the expression of ICAM-1 and IL-8 in human lung microvascular endothelial cells (HPMECs) stimulated by tumor necrosis factor-α (TNF-α); however, the underlying molecular mechanism remains unclear. Here, we show that human antigen R (HuR), an RNA binding protein which binds preferentially to AU-rich elements (AREs) and stabilizes mRNAs, regulates TNF-α-induced ICAM-1 expression in the MK2/HuR signaling pathway.MK2 and HuR were silenced respectively in HPMECs and then HPMECs were stimulatied with TNF-α. Nucleo-cytoplasmic shuttling of HuR was detected by subcellular fractionation and confocal microscopy in MK2 knockdown HPMECs. In HuR silencing cells, protein and mRNA levels of ICAM-1 and IL-8 were measured by western blot analysis, ELISA and real-time PCR; mRNA stabilization were measured by real-time PCR after actinomycin D (ActD) blocking transcription. Furthermore, we performed neutrophil adhesion assay to assess the adhering capacity after HuR silencing.MK2 were subjected to a knockdown by interfering RNA, the mRNA and protein levels of HuR in human pulmonary microvascular endothelial cells (HPMECs) were not affected. However, after the stimulation of TNF-α, silencing MK2 inhibited HuR accumulation to cytoplasm from nucleus in HPMECs. Consequently, knockdown of HuR by RNA interference in HPMECs, there was reduction in the stability of ICAM-1 mRNA and ICAM-1 protein level. This event was accompanied by a decrease in the adhesion of neutrophils towards HPMECs. Nevertheless, HuR silencing had no effect on the mRNA and protein levels of IL-8.These results indicate that MK2 post-transcriptionally regulates TNF-α-induced ICAM-1 expression by altering the cytoplasmic localization of HuR in HPMECs. Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are two acute inflammatory diseases. In the lungs, neutrophil recruitment and activation play a pivotal role in ALI/ARDS , 2. TumoPrevious studies have shown that the stability of mRNA containing AU-rich elements (ARE)(AU is the abbreviated form of adenylate uridylate) is regulated through an MK2-dependent and ARE-targeted mechanism –11. MoreActinomycin D (ActD) was purchased from Sigma-Aldrich . Lipofectamine 2000 (11668–027) and Opti-MEM I reduced serum medium were obtained from Invitrogen . MK2 siRNA, HuR siRNA and negative control siRNA were received from GenePharma . TNF-α (300-01A) was purchased from PeproTech . Phosphatase Inhibitor Cocktail (04906845001) was from Roche . An enhanced chemiluminescent (ECL) kit were obtained from Pierce . The protein assay kit, nuclear and cytoplasmic protein extraction kit (P0027) and the RIPA Lysis Buffer (P0013C) were purchased from Beyotime Institute of Biotechnology . Antibodies raised against MK2 (#3042), phospho-MK2 , ICAM-1 (#4915) was obtained from Cell Signaling ; a monoclonal antibody against HuR (ab14371) was received from Abcam ; antibodies against β-actin (P30002) and histone (P30266) were from Abmart ;IL-8 ELISA Kit (BMS204/3) was received from eBioscience . RevertAid First Strand cDNA Synthesis Kit (#K1622) was purchased from Fermentas UAB . FastStart Universal SYBR Green Master (Rox) (04913914001) was purchased from Roche . Endothelial cell medium and HPMECs were purchased from ScienCell .HPMECs were grown in ECM medium supplemented with 5 % fetal bovine serum.MK2 siRNA and HuR siRNA were respectively reverse transfected at a concentration of 40 nm with Lipofectamine 2000 diluted in Opti-MEM. After 24 h, to allow for gene silencing, cells were stimulated with TNF-α. The final concentration of TNF-αwas 10 ng/ml. The sequences of siRNAs are as follows: MK2 siRNA 1: sense (5’-GAC CAG GCAUUC ACA GAA ATT-3’) and antisense (5’-UUU CUG UGA AUG CCU GGU CTT-3’); MK2 siRNA 2: sense (5’-CAC CCU UGG AUC AUG CAA UTT-3’) and antisense (5’-AUU GCA UGA UCC AAG GGU GTT-3’); MK2 siRNA 3: sense (5’- GUU AUA CAC CGU ACU AUG UUU-3’) and antisense (5’-ACA UAG UAC GGU GUA UAA CUU-3’); HuR siRNA: sense (5’-GGA UGA GUU ACG AAG CCU GTT-3’) and antisense (5’-CAG GCU UCG UAA CUC AUC CTG-3’). Controls were treated with negative control siRNA anLipofectamine 2000.http://rsb.info.nih.gov/ij/, National Institutes of Health, USA), and the data were normalized to β-actin or histone.The total cellular lysates were prepared as previously described . Cells wThe Nuclear and Cytoplasmic Protein Extraction Kit (P0027) were purchased from Beyotime Institute of Biotechnology . Nuclear and cytoplasmic protein fractions of HPMEC were obtained according to manufacturer’s directions. In brief, extraction reagent A for cytoplasmic protein was added with 1 mM phenylmethylsulfonyl fluoride (PMSF) and incubation on ice for 15 min. Then extraction reagent B for cytoplasmic protein was added. The cytosolic fractions were prepared by high-speed centrifugation (16000 g for 5 min at 4 °C) and the supernatant was collected. For preparing nuclear fractions, extraction reagent for nuclear protein with PMSF in supplemented was added to nuclear sediment, centrifuged  after 30 min, and the supernatant was saved.HPMECs seeded on coverslips were transfected with siRNA or negative control siRNA as described above. After treatment with TNF-α, cells were washed using with phosphate buffered saline (PBS) and fixed with 4 % paraformaldehyde for 15 min at room temperature. The fixed HPMECs were permeabilized with 0.5 % Triton X-100 in PBS for 10 min at room temperature. Subsequently, they were blocked with 1 % BSA in PBST for 30 min at room temperature. Finally, they were washed with PBS and incubated overnight with primary HuR antibody (100-fold diluted with 1 % BSA in PBST) at 4 °C, followed by 3 × 5 min washes with PBS. The HPMECs were then incubated with fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG (green) (200-fold diluted with 1 % BSA in PBST) for 1 h in the dark. Following 3 × 5 min washes with PBS, the 4’-6-diamidino-2-phenylindole (DAPI) was used to stain the cell nuclei (blue) for 5 min at a concentration of 1 μg/ml. After 3 × 5 min washes with PBS, the confocal images were acquired using an Olympus FV1000 confocal microscope . FV10-ASW software  was used to process the images.Cell-free supernatants were saved and aliquots were stored at −70 °C until use. The protein levels of IL-8 in medium were measured with commercially available cytokine specific ELISA kits  according to the manufacturers’ recommendations.Total RNA was isolated from cultured cells by TRIzol and used in reverse transcription and PCR amplification reactions as described . Quantit50 % confluent cells were first preincubated with Actinomycin D (5 μg/ml) for 2 h; cells were then treated with TNF-α (10 ng/ml) for 2 h in the presence of actinomycin D. Total RNA was prepared as described . HuR, IC7cells/ml. We used this neutrophilic suspension in PBS within 4 h.Neutrophil polymorphonuclear granulocytes (PMN) were isolated from the peripheral blood of healthy human volunteers from Lianyungang First People’s Hospital using Ficoll-Hypaque  density gradient centrifugation, as previously described . ResiduaPMN were labeled for 1 h at 37 °C with 10 mM 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein,acetoxymethyl ester BCECF/AM . HPMECs were grown in 12-well plates and transfected with siRNA or negative control siRNA as described above. After treatment with TNF-α to HPMEC, the labeled PMN were added to each HPMEC containing well and incubated for 1 h. Non-adherent PMN were removed by two gentle washes with PBS; The adhesion PMN cells were counted in five high-power fields per well under fluorescence microscope  and expressed as adhesion PMN per high-power fields.t test two tailed, analyzing the differences between the different time points within the same treatment group and between different groups, respectively. P values < 0.05 was consideredstatistically significant. Statistical analysis was performed by SPSS 13.0 .Data were expressed as either a representative experiment or the mean ± standard error (SE) of three independent experiments. Statistical analysis to determine significance was calculated using variance (ANOVA) and an unpaired via IL-1β and TNF-α is implicated in HuR-mediated stabilization of mRNAs coding for key inflammatory mediators including IL-6, cyclooxygenase-2 (COX-2) and TNF-α [It was reported that activation of p38 MAPK nd TNF-α . MK2 hasnd TNF-α , 25, butIt has been observed that the active MK2 can induce cytoplasmic accumulation of HuR in HeLa cells . AnotherIt has been previously reported that HuR plays an important role in the post-transcriptional regulation of inflammatory factors expression such as vascular endothelial growth factor (VEGF) and IL-8 in malignant glioma cells and HCT8 cells, respectively , 27. It We next investigated whether HuR regulates the mRNA of ICAM-1 and IL-8. The real-time quantitative PCR results showed that ICAM-1 mRNA gradually increased and reached summit at 4 h of TNF-α stimulation, then went to baseline after 8 h in negative control siRNA-transfected cells. In sharp contrast, silencing of HuR significantly reduced ICAM-1 mRNA levels, and that was one fifth of controls 4 h after treatment with TNF-α Fig. . In contP <0.05) . These results indicates a TNF-α-induced, HuR-dependent stabilization of ICAM-1 mRNA in HPMECs.Subsequently, we explored whether HuR affects ICAM-1 and IL-8 mRNA stability. TNF-α stimulated HPMECs which were transfected with HuR or negative control siRNAs for 2 h, and then Act D (5 μg/ml) was added to stop gene transcription. After 0, 1, 2, and 4 h, total cellular RNA was harvested for real-time PCR to detect ICAM-1 and IL-8 mRNA levels. In control cells, the half-life of ICAM-1 mRNA after TNF-α stimulation was 118 min while was significantly reduced to 52 min in HuR-silenced HPMECs (05) Fig. . The halP <0.05)  Fig. . These rIn this study, we found that knockdown of MK2 by siRNA did not affect the expression but significantly impaired the cytoplasmic accumulation of HuR after TNF-α stimulation in HPMECs. We further revealed that HuR knockdown decreased the mRNA and protein levels of ICAM-1 in HPMECs after TNF-α stimulation. Most importantly, silencing of HuR resulted in destabilization of the mRNA of ICAM-1 and inhibition of TNF-α-induced PMN adhesion to HPMECs. Our results suggest that MK2 regulates TNF-α-induced ICAM-1 expression by altering the cytoplasmic localization of HuR in HPMECs.HuR is a member of the embryonic lethal abnormal visual family and can bind to the ARE motif such as AUUUA in the 3′-UTR of mRNAs. The cytoplasmic localization of HuR increases its binding to the AREs and stabilizes the ARE-containing mRNAs , 31, 32.HuR has been reported to regulate the expression of several factors such as VEGF, IL-8 and survivin by stabilizing these ARE-bearing mRNAs in other cells , 27, 33.To date, the exact mechanisms of ALI/ARDS are not completely known. The present results demonstrated that knockdown of HuR inhibited neutrophil adherence to the HPMECs. Our findings suggest that the MK2/HuR pathway may play an important role in acute inflammatory response in HPMECs and may become an effective treatment target for ALI/ARDS in future.Although the latest research indicates that the MK2/HuR pathway regulated ICAM-1 expression after TNF-α stimulation in HPMECs, the mechanisms by which MK2 regulates HuR translocation are not completely understood. Doller et al.  have revIn summary, our results showed that the MK2/HuR signaling axis regulated TNF-α-induced ICAM-1 expression by promoting the stabilization of ICAM-1 mRNA in HPMECs, and silencing of HuR impaired TNF-α-induced adhesion of neutrophils to HPMECs. Our findings suggest that the MK2/HuR pathway play an important role in acute inflammatory response in HPMECs and targeting MK2/HuR signaling is a promising strategy for the development of an effective treatment for ALI/ARDS.


Virus-like particles (VLPs) can be used as powerful nanoscale weapons to fight against virus infection. In addition to direct use as vaccines, VLPs have been extensively exploited as platforms on which to display foreign antigens for prophylactic vaccination and immunotherapeutic treatment. Unfortunately, fabrication of new chimeric VLP vaccines in a versatile, site-specific and highly efficient manner is beyond the capability of traditional VLP vaccine design approaches, genetic insertion and chemical conjugation. In this study, we described a greatly improved VLP display strategy by chemoenzymatic site-specific tailoring antigens on VLPs surface with high efficiency. Through the transpeptidation mediated by sortase A, one protein and two epitopes containing N-terminal oligoglycine were conjugated to the LPET motif on the surface of hepatitis B virus core protein (HBc) VLPs with high density. All of the new chimeric VLPs induced strong specific IgG responses. Furthermore, the chimeric VLPs with sortase A tagged enterovirus 71 (EV71) SP70 epitope could elicit effective antibodies against EV71 lethal challenging as well as the genetic insertion chimeric VLPs. The sortase A mediated chemoenzymatic site-specific tailoring of the HBc VLP approach shows great potential in new VLP vaccine design for its simplicity, site specificity, high efficiency, and versatility. As natural nanoparticles, VLPs are widely applied in nanotechnologies, including bioimagingDisplaying antigens on the surface of VLPs is of great importance for applications in prophylactic and immunotherapeutic vaccination. There are currently two classic chimerical vaccine design approaches based on VLPs, genetic insertion and chemical conjugation1416E. coli exhibited a high neutralizing capacity against HCMVIn this study, we developed a simple and site-specific vaccine design approach with high efficiency. Antigens are simply covalently linked to HBc VLPs by chemoenzymatic site-specific coupling mediated by sortase A, sortagging for short, in one reaction. The HBc VLP platform, one of the most advanced VLP display platforms, is used as a scaffoldThrough sortase A mediated tagging, a protein domain and two epitopes are conjugated to VLPs surface with high density. All of the chimeric VLPs induced a strong and specific IgG response. Our results demonstrate that our platform is versatile and convenient for coupling of different antigens, which is beyond the capacity of genetic insertion. In addition, our platform is simpler, more efficient, and more site specific than chemical conjugation. In general, the novel vaccine design strategy proposed in our study surpasses traditional approaches and can be used to tailor VLP surfaces for further applications.We designed a strategy for efficient and versatile coupling of foreign antigens to HBc VLPs in a site-specific manner . In thisE. coli and purified by immobility metal affinity chromatography (IMAC) and anion exchange. The aggregates of sortase A-4 were removed by sucrose gradient density ultracentrifugation  were immunized to mice with PBS and N-LPETGG-C VLPs as control. All six mice in each group received the first intraperitoneal injection at week 0 and boosters at weeks 2 and 4. Serum samples were collected 2 weeks after each immunization. Only GGGS-AD-4 and N-LPETGGGS-AD-4-C induced an anti–GGGS-AD-4 IgG response. After the first injection, GGGS-AD-4 sortagged to VLPs stimulated a greater average specific IgG response than the third booster of GGGS-AD-4. Second booster of N-LPETGGGS-AD-4-C elicited very strong specific IgG response and further booster would not enhance IgG response . Then weStructural modeling by MODELLER predicted that genetic inserted or sortagged epitopes (SP55 and SP70) were located at the c/e1 loop, and exposed on the surface of the N-LPETGG-C VLP scaffold . To testin vivo challenge assay. To do this, 7-day-old ICR mice were intraperitoneally injected with 100 μl anti-N-LPETGG-C, anti-HBc-SP70 or anti-N-LPETGGG-SP70-C serumsWe then assessed the immunogenicity between VLPs sortagged with GGG-epitopes and VLPs genetically fused with epitopes by immunization study. The vaccination schedule was the same as above. No-split genetic insertion of HBc-SP55 and HBc-SP70 VLPs was prepared  and 5 anAccording to our results, we propose a novel chimeric VLPs design strategy . Our newVLPs are used as universal vaccine delivery system due to their intrinsic characteristic of enhancing immune responses. By genetic insertion, epitopes and proteins are displayed on VLPs with high efficiency and in proper orientation and induce strong immune responses30313233In this study, we have developed a strategy that possesses the major advantages of genetic insertion and chemical conjugation. Through chemoenzymatic site-specific conjugation mediated by transpeptidase, antigens are displayed on the VLP surface in proper orientation and with high density. There is no need to activate VLPs with excess heterobifunctional cross-linker, which makes coupling a simple process. By chemoenzymatic site-specific coupling, interested molecules are more easily displayed on the VLP surface at a high density. The only drawback of our approach is the requisition of excess substrates. The excess of antigens can be reduced by modification of LPETGG to depsipeptide, which makes sortagging irreversibleOur results demonstrate that our proposed vaccine design strategy constitutes a great advance in the bioengineering production of new vaccines. In our new coupling system, antigens are conjugated to the VLP surface in a simple, fast, site-specific, and highly efficient manner. Our new system is unique for its integration of the advantages of genetic fusion and chemical conjugation in the modification of VLPs. New vaccine production engineering will be facilitated by our strategy. In summary, interested molecules will be displayed on the VLP surface in a simple, fast, convenient, and versatile way by our proposed technology.The split N-LPETGG-C molecule contained N-LPETGG, a ribosome binding site (RBS) and C-core to form VLP structure with 1:1 N-LPETGG and C-core. A stop codon was introduced to N-LPETGG. The N-LPETGG-C molecule was constructed by overlap-PCR. The discontinuous GGGS-AD-4, aa 112–132 and 344–438 with Ile-Ala-Gly-Ser-Gly linker were cloned from cDNA of HCMV strain AD 169 by overlap-PCR. Other clones were constructed according to standard protocol of molecular cloning. All of the clones used in coupling system were inserted to pET28a vector and expressed in BL21 (DE3) plysS.2. All reactions were achieved at room temperature between 20 °C and 26 °C. The reaction was terminated by sucrose gradient ultracentrifugation of sortagged products from sortase A-4 and GGG- antigens. Fractions 7-10 were collected and dialyzed against PBS. Sortagged products were concentrated to 1mg/ml by a 100-kD cutoff Amicon Ultra centrifugal filter. The target products were stable at 4 °C for a couple of weeks. For more than one year store, −80 °C condition was essential. Different batches of sortagged products for immunization study were monitored by Tricine-SDS-PAGE analysis. This reaction was highly reproducible. The sortagging efficiency was estimated by the decrease intensity of N-LPETGG band. The recovery rates were calculated by comparing the sortagged VLPs products with initial N-LPETGG-C VLPs. More than 50% recovery rates were obtained.The split HBc N-LPETGG-C VLPs, HBc-SP55 and HBc-SP70, were sequentially purified by ammonium sulfate precipitation and sucrose gradient ultracentrifugation as mentioned in our previous studyThe VLP samples  that were negatively stained with 1% uranyl acetate were monitored with a Tecnai G2 Spirit microscope at 120 kV at 67,000 × magnification.The 6- to 8-week-old female BALB/c mice were vaccinated with 10 μg proteins or VLPs in PBS (100 μl) with 1:1 v/v aluminium hydroxide wet gel adjuvant (Invivogen). All six mice in each group received their first injection at week 0 and boosters at weeks 2 and 4. Blood samples were taken 2 weeks after each immunization. The immunization studies were approved by the Institutional Animal Care and Use Committee at the Institut Pasteur of Shanghai. All the experiments were carried out according to approved guidelines.ELISA analysis and neutralization against EV71 was carried out as described before, except that the serum samples in the ELISA assay were diluted to 1:10005 cells/ml RD cells were added to each well. The initial dilution of the neutralizing assay was 8-fold, and the PBS and N-LPETGG-C control groups were defined as 8-fold. After incubation at 37 °C CO2 incubator for 3 days, the cytopathic effects (CPE) were monitored. All immunization studies of EV71 epitope-based VLPs were repeated two times. The neutralizing analysis against HCMV strain AD 169 (GFP insertion after IE2 for monitor) was done at similar condition with 800 PFU HCMV strain AD 169 and 1.5 × 105 cells/ml MRC5 cells in each well. The CPE were assayed by fluorescence microscopy after 5 days post infection. No inhibitions of HCMV entry were observed. The neutralizing results were analyzed by graphpad prism 5.The neutralizing analysis was carried out by 96-well plates. Equal volumes of 100 PFU EV71 strain G082 and 2-fold serially diluted serum samples (50 μl/well) were incubated at 37 °C for 1 h. Then 100 μl of 1.5 × 10in vivo assay against EV71 in ICR mice with slight modification according to previous studyin vivo assay was carried out at biosafety level 2 laboratories of Institut Pasteur of Shanghai. This assay was approved by the biosafety core facility and the Institutional Animal Care and Use Committee at the Institut Pasteur of Shanghai. All the experiments were done according to approved guidelines. Three groups of 7-day-old ICR mice received intraperitoneal injection of 100 μl anti-N-LPETGG-C, anti-HBc-SP70 or anti-N-LPETGGG-SP70-C serum/mouse. The mice were intraperitoneally challenged with 2.1 × 104 TCID50 of EV71/MAV-W  24 h later. All mice were daily monitored for 14 days after EV71 lethal challenging. The survivals of mice were recorded.To evaluate the effect of anti-EV71 epitope antibodies, we performed To evaluate the statistic difference, two-tailed student’s t-test was performed. The survival curve of EV71 lethal challenge was compared with Log-rank (Mantel-Cox) test. All the statistic analysis was done by GraphPad Prism version 5.All the protein models were generated by MODULLER in UCSF Chimera. Protein sequences were added and models were automatically produced by comparing with known structure of proteins. N-LPETGG-C protein was adapted to 3D structure of HBc (1QGT)How to cite this article: Tang, S. et al. A Modular Vaccine Development Platform Based on Sortase-Mediated Site-Specific Tagging of Antigens onto Virus-Like Particles. Sci. Rep.6, 25741; doi: 10.1038/srep25741 (2016).


A disastrous form of pedestrian behavior is a stampede occurring in an event involving a large crowd in a panic situation. To deal with such stampedes, the possibility to increase the outflow by suitably placing a pillar or some other shaped obstacles in front of the exit has been demonstrated. We present a social force based genetic algorithm to optimize the best design of architectural entities to deal with large crowds. Unlike existing literature, our simulation results indicate that appropriately placing two pillars on both sides but not in front of the door can maximize the escape efficiency. Human experiments using 80 participants correspond well with the simulations. We observed a peculiar property named tangential momentum, the escape speed and the tangential momentum are found to be negatively correlated. The idea to reduce the tangential momentum has practical implications in crowd architectural design. Crowd stampedes happen not rarely in religious gatherings, music concerts, earthquakes or in other panic situations Architectural design can either facilitate or hamper the escape process in panic situations. To do this, some interesting obstacle placements were studied Helbing et al. People with an experience of running to catch a bus during rush hour in large and crowded cities such as Beijing may have the experience that people pushing from the middle are comparatively less successful to get on board the bus than those people pushing from the sides. This experience provides us with the heuristics required to study the property that benefits the people who push from the sides. Intuitively stated, the people pushing from the middle are “blocked” by the force coming from aside, which we call here the tangential momentum that will be defined in Simulation Results section. This is like the case when you pull out two balls from a bottle. When you pull out the balls one by one, the force is along the pulling rope and you can finish the task easily. When you pull them out simultaneously, the two balls interact with tangential force that is perpendicular to the pulling rope that gets them stuck. We make an assumption that the property that reduces the escape speed in a panic situation is the tangential momentum. If this assumption is true, to increase the escape speed is to find a better layout design of a room that can reduce the tangential momentum.In this work, a genetic algorithm is used to provide the layout design of the obstacles (pillars) that can reduce the tangential momentum, the settings and parameters for the simulation are extracted from human experiments. The human experiments are used as a test of the simulated optimal layout to see whether the layout can speed up the escape. The model, on the other hand, is a prediction of the experiments, provided the simulated escape time corresponds well with the experiment outcomes.We consider the evacuation of a room of size We used an Genetic Algorithm to optimize the setting of the obstacles. The simulation is run on a AMD 6172 based 2.1 GHz server. A typical optimization using 80 participants costs approximately 1,250 CPU core hours. The total computation time for our nine optimizations was 11,242 CPU core hours.The optimized obstacle settings when there are 80 participants in the room are shown in The optimization results indicate that the obstacle settings near the two sides of the gate are better than the obstacle settings studied in other literature, which are in front of the gate We examined the “tangential momentum” in the simulations. The tangential momentum To obtain various escape speed value vs. tangential momentum samples, we need different obstacle placement configurations. During the evolutionary process of the GA, it gives plenty obstacle placement configurations which we can use. For each individual obstacle configuration, the average escape speed was calculated for over 100 escape runs. To compare the effect of the GA-optimized obstacle setting, which is “at the side of the door”, and the “in front of the door” setting wildly used in existing literature, we used the data in the last generation of our genetic algorithm. As seen from To confirm our simulation results, we performed a series of human experiments. As shown in To clearly present the results, we show the average escape time vs. the number of escaped participants in The simulation and the experiment results are compared in We hypothesized in this paper that the property that reduces the escape speed in a panic situation is the tangential momentum. To test this hypothesis, we simulated escapes in a panic situation using genetic algorithms to optimize the settings of the obstacles considered in the simulations, including the number, the size and the position of the obstacles. We found that obstacles at the two sides of the door is the best configuration to increase the escape speed, which is different from the configurations of existing literature. The escape speed and the tangential momentum were found to be negatively correlated, as shown in The previous works got various kind of settings, some of them are more or less complicated To better understand that the two pillars at the two sides of the door can reduce the tangential momentum we can refer back to the catching bus example in Introduction section. The two pillars near the door actually “block” the people pushing from the sides, and this leads to the fact that the majority people pushing from the middle get released and can pass the relatively weakened bottleneck. This may act as a guidance for further researches in the layout design to focus on reducing tangential momentum rather than trying other approaches.The simulations in this work are based on the social force model The detailed simulation parameters are available as Genetic algorithms have been fairly successful at solving problems of the form that are too ill-behaved  for the more conventional hill-climbing and derivative based techniques to solve. In this paper, we must optimize the position of the obstacles, which is defined in two-dimensional real number coordinates. We used a real-coded GA because it is better than binary-coded for solving this stampede problem We use the escape speed (persons/sec) as the fitness index and perform computer simulations of the panic escape process. Normally 80 people can escape from the room in less than 40 sec when there is no obstacle, if pedestrians didn′t escape from the room in 45 sec, it indicates that the obstacle setting is bad, thus there is no need to continue the escape simulation. Therefore, if the entire crowd is out of the room in less than 45 sec .To simulate the panic condition, the participants were divided into 10 groups, with each group receiving a score according to their escape speed for each evacuation; a prize was awarded according to the ranking of the groups determined by their cumulative scores. To minimize the effect of the participants selecting their positions near the gate for each of the experimental runs, all of the participants were organized into a moving round circle, with the participants rushing to the gate when prompted by an acoustical signal. Two test runs were allotted to enable the participants to become familiar with the experiment. Subsequently, the experiment was performed for the cases of zero obstacle, one obstacle and two obstacles, with three runs for each case. Each of the 3 runs for no obstacle, one obstacle and two obstacles cases are to get rid of the fluctuations due to the initial configuration of participants location and fluctuations created in the escape process. S1 DataAll the simulation parameters and detailed experiments data are reported in the MS word file R1_SI_clean.docx.(DOCX)Click here for additional data file.S1 FigThe GA results of the tournament selection and the roulette wheel selection. It shows the results of the 2 pillar case with R = 0.5 m. The coordinates for the two pillars with the tournament selection are  and , respectively. The coordinates for the two pillars with the roulette wheel selection are  and , respectively. The results are almost the same, which means that for the current optimization problem both selection operators work well.(EPS)Click here for additional data file.S1 VideoA video clip of the human experiment. It shows that the evacuation is quite close to a panic escape scenario.(AVI)Click here for additional data file.


Expression of a subset of late phase genes was mediated by autocrine IL-10, which activated STAT3 with delayed kinetics. Hypoxia, which occurs at sites of infection or inflammation where TNF is expressed, suppressed this IL-10-STAT3 autocrine loop and expression of late phase genes. TNF-induced expression of IL-10 and downstream genes was also dependent on signaling by mTORC1, which senses the metabolic state of cells and is modulated by hypoxia. These results reveal an mTORC1-dependent IL-10-mediated late phase response to TNF by primary human macrophages, and identify suppression of IL-10 responses as a new mechanism by which hypoxia can promote inflammation. Thus, hypoxic and metabolic pathways may modulate TNF responses during chronic inflammation.Tumor necrosis factor (TNF) is best known for inducing a rapid but transient NF-κB-mediated inflammatory response. We investigated later phases of TNF signaling, after the initial transient induction of inflammatory genes has subsided, in primary human macrophages. TNF signaling induced expression of late response genes, including inhibitors of NF-κB and TLR signaling, with delayed and sustained kinetics 6–24 hr after TNF stimulation. A subset of late phase genes was expressed in rheumatoid arthritis synovial macrophages, confirming their expression under chronic inflammatory conditions  TNF-α (referred to hereafter as TNF) signals through receptors TNF-R1 and TNF-R2 and initiates a classic pro-inflammatory immune response by activating NF-κB, AP-1, and MAPK2361Leishmania major were capable of clearing the parasites but were unable to resolve the ensuing sterile inflammatory lesionsCorynebacterium parvum infectionDespite our understanding of proximal and early phase TNF signaling (1–120 minutes after TNF stimulation)in vivo, with tissue tension ranging from 2.5–9% O2 but decreasing to levels <1% O2 in wounds and inflammatory and necrotic tissue sites2. Oxygen demand can outstrip supply, which is termed hypoxia, during a pro-inflammatory response, when the activation, proliferation, and recruitment of cells to a confined area consumes available oxygen and diffusion is restricted. For example, hypoxia is present in the inflamed synovium of RA patients and is associated with synovial macrophage number, growth factors, and angiogenesis131416Another key regulator of inflammatory and immune responses is oxygen tension, which varies Although HIF is the best known and extensively studied mediator of the hypoxic response, the metabolic stress of hypoxia induces additional signaling pathways that enable cells to adapt to hypoxic conditions. These include activation of the ER stress response and inhibition of mechanistic target of rapamycin complex 1 (mTORC1) signaling1919mTOR is a serine/threonine kinase that signals through two distinct large, multi-protein complexes, mTORC1 and mTORC2, to regulate protein and lipid synthesis, autophagy, lysosome biogenesis, cytoskeletal organization, and metabolism2322mTORC1 regulates innate immune functions in part by regulating translation222831TNIP3 , CLU (encodes clusterin), and SIGLEC10, suggesting a homeostatic component to this late response. This late phase gene induction was partially dependent on mTORC1 and was abrogated by hypoxia. These results identify a new component of the TNF response and demonstrate its regulation by hypoxia. The results highlight a role for TNF-mediated feedback inhibition and the importance of metabolic and stress pathways in cross-regulating immune signaling pathways.We are interested in the later phases of macrophage responses to TNF, which occur after the initial transient induction of canonical inflammatory genes such as IL-6 and IL-8 has subsided. Previous work in our laboratory had shown that TNF induces a late phase interferon (IFN) response (apparent 1–2 days after TNF stimulation) that is mediated by autocrine IFN-βA20, ABIN3, CLU, SIGLEC10) and genes strongly induced with markedly delayed kinetics  physiological importance. The data indicate that TNF signaling induces late phase expression of genes that are expressed during TNF-driven inflammation in RA, but are not expressed during the early and transient classic pro-inflammatory TNF response that occurs 1–3 hours after TNF stimulation.In previous work we found that activation of human macrophages with various factors, including TNF, activated a subset of genes whose expression was not apparent until 6–24 hours after stimulation and was sustained for several days11363738nd IL7R) . These g, CLU (P < 0.01) and TNIP3 (P < 0.05) . The partial suppression of gene expression may be secondary to incomplete blockade of IL-10 or may reflect activation by distinct IL-10-independent pathways. These results show that TNF induces an IL-10-mediated delayed autocrine loop that activates STAT3 and contributes to late phase gene expression. As clusterin (encoded by CLU) and ABIN-3 (encoded by TNIP3) are negative regulators of NF-κB and inflammatory signaling46We wished to test whether STAT3 activation downstream of TNF was predominantly mediated by autocrine IL-10, as STAT3 can be activated by other TNF-inducible factors such as IL-6, leptin, and VEGF < 0.05) ; express2, reflects inflammatory sites) strongly induced canonical hypoxia response HIF-1α target genes GLUT1, VEGF and GAPDH relative to normoxia (20% O2) or physiological tissue oxygen tension (10% O2)  . Accordi(10% O2) ; levels (10% O2) . Likewis(10% O2) , as was (10% O2) .ENPP2, CLU, and TNIP3, were strongly suppressed by hypoxia  and ARNT2 by hypoxia at the protein level was confirmed using immunoblotting CLU, TNIP3)47versus the late phase of a TNF response are distinct, and mediated by, respectively, transcriptional repression and inhibition of signaling. This inhibition of inflammatory signaling may contribute to the ‘tolerized’ state induced by prolonged TNF stimulation, in which inflammatory signaling in response to challenge by TLR ligands is attenuatedAutocrine loops that feed back to regulate macrophage or DC phenotype have been well described in cell responses to TLR stimulation. TLR stimulation induces at least two important autocrine feed back loops that become rapidly operational, within 1–3 hours after stimulation. One is mediated by IFN-β, which induces expression of interferon-stimulated genes (ISGs), including chemokines and molecules important for antigen presentation that are overall pro-inflammatory. At the same time, TLRs induce an IL-10-STAT3-mediated autocrine loop that serves as a potent feed back inhibitor of the early inflammatory phase of TLR-induced macrophage activationH17 and Treg cells, with glycolysis necessary for development of TH17 cells and inhibition of glycolysis skewing towards Treg developmentMetabolic pathways regulate cell fate and function of immune cells. For instance, HIF-1α and mTOR mediated glycolysis is an important checkpoint for the differentiation of TThe important role of hypoxia-regulated pathways and factors, like the HIFs, in innate immune cell function is well established1860ex vivo TNF exposure, variations in oxygen tension, and rapamycin. It is possible that responses could be different between healthy and RA macrophages, and these differences could yield insights into disease pathogenesis.The inflamed synovium of RA patients is characterized by chronic exposure to TNF and also by hypoxia1314in vitro studies because of practical limitations, into investigation of inflammatory signaling pathways. In addition to our work, it was recently shown that differentiating monocyte-derived, primary human macrophages under 5–10% O2, among other factors, resulted in cells that better mimicked tissue macrophages in the context of Mycobacterium tuberculosis infectionThis study highlights the importance of incorporating the role of low oxygen tension, which is rarely done in mechanistic In summary, TNF induces a late phase response characterized by mTORC1-dependent induction of an IL-10-STAT3-mediated autocrine loop and a late phase gene response that differs markedly from the early inflammatory gene response. Late-phase TNF signaling is regulated by environmental and metabolic pathways, including those activated by hypoxia, that are sensed and integrated by mTOR. In macrophages, hypoxia overrides the induction of feedback signaling in response to TNF and thereby alters the balance between activating and suppressive TNF signaling that determines the potency of TNF as a promoter of inflammation.2 and 5% CO2 at 37 °C in a chamber flushed with N2 under the control of a PRoOX sensor and ProCO2 regulator (BioSpherix). Cells were incubated in the 1% oxygen chamber for 3 hours prior to TNF or IL-10 stimulation, as 3 hours was sufficient to detect HIF1α protein stabilization in our system. Recombinant human TNF-α (10 ng/mL) and IL-10 (10–100 ng/mL) were purchased from Peprotech. Rapamycin  and desferoxamine (200 uM) were purchased from EMD Millipore, Billerica, MA, USA. Polymyxin B (14 ug/mL) was purchased from Sigma Aldrich, St. Louis, MO, USA. IL-10 and IL-10R blocking antibodies were purchased from R&D Systems, Minneapolis, MN, USA, and used at 5 ug/mL.Peripheral blood mononuclear cells (PBMC) obtained from blood leukocyte preparations purchased from the New York Blood Center were separated by density gradient centrifugation with Ficoll  using a protocol approved by the Hospital for Special Surgery Institutional Review Board that adheres to NIH guidelines and regulations. Human monocytes were purified from PBMCs immediately after isolation by positive selection with anti-CD14 magnetic beads, as recommended by the manufacturer and previously described67For immunoblotting, whole cell lysates were fractionated on 7.5% or 10% polyacrylamide gels using SDS-PAGE and transferred to polyvinylidene difluoride membranes for probing with Ab. ECL was used for detection. The following Abs were used: pY-STAT3 (Y705) , STAT3 , HIF-1 alpha , p38α (C-20) , and beta-tubulin .Sandwich ELISA using paired human IL-10 Abs was performed according to the manufacturer’s instructions .RNA was extracted using the RNeasy Mini Kit  and reverse-transcribed using the RevertAid First Strand cDNA Synthesis Kit  following the manufacturer’s instructions. Quantitative real-time PCR was performed in duplicate using Fast SYBR Green Master Mix and a 7500 Fast Real-Time Cycler . Expression was normalized relative to levels of GAPDH or HPRT (as indicated in figures). Primer sequences are available on request.t tests. The Mann-Whitney test was used instead of the Student t test. The Wilcoxon matched-pairs signed-rank test was used instead of the paired Student t test.All statistical analyses were performed with Graphpad Prism 5.0 software. Statistical tests included: Shapiro-Wilk normality test, nonparametric Wilcoxon matched-pairs signed-rank test, and nonparametric Mann-Whitney test. P values less than 0.05 were considered significant. Because data sets were not normal , nonparametric tests were used instead of the parametric Student How to cite this article: Huynh, L. et al. Opposing regulation of the late phase TNF response by mTORC1-IL-10 signaling and hypoxia in human macrophages. Sci. Rep.6, 31959; doi: 10.1038/srep31959 (2016).