Citrobacter rodentium, an attaching and effacing enteric pathogen that causes colitis. Induction of SLC7A2 was upregulated in colitis tissues, and localized predominantly to colonic epithelial cells. Compared to wild-type mice, Slc7a2–/–mice infected with C. rodentium had improved survival and decreased weight loss, colon weight, and histologic injury; this was associated with decreased colonic macrophages, dendritic cells, granulocytes, and Th1 and Th17 cells. In infected Slc7a2–/–mice, there were decreased levels of the proinflammatory cytokines G-CSF, TNF-α, IL-1α, IL-1β, and the chemokines CXCL1, CCL2, CCL3, CCL4, CXCL2, and CCL5. In bone marrow chimeras, the recipient genotype drove the colitis phenotype, indicative of the importance of epithelial, rather than myeloid SLC7A2. Mice lacking Slc7a2 exhibited reduced adherence of C. rodentium to the colonic epithelium and decreased expression of Talin-1, a focal adhesion protein involved in the attachment of the bacterium. The importance of SLC7A2 and Talin-1 in the intimate attachment of C. rodentium and induction of inflammatory response was confirmed in vitro, using conditionally-immortalized young adult mouse colon (YAMC) cells with shRNA knockdown of Slc7a2 or Tln1. Inhibition of L-Arg uptake with the competitive inhibitor, L-lysine (L-Lys), also prevented attachment of C. rodentium and chemokine expression. L-Lys and siRNA knockdown confirmed the role of L-Arg and SLC7A2 in human Caco-2 cells co-cultured with enteropathogenic Escherichia coli. Overexpression of SLC7A2 in human embryonic kidney cells increased bacterial adherence and chemokine expression. Taken together, our data indicate that C. rodentium enhances its own pathogenicity by inducing the expression of SLC7A2 to favor its attachment to the epithelium and thus create its ecological niche.Solute carrier family 7 member 2 (SLC7A2) is an inducible transporter of the semi-essential amino acid L-arginine (L-Arg), which has been implicated in immune responses to pathogens. We assessed the role of SLC7A2 in murine infection with  Citrobacter rodentium. SLC7A2 is induced in colonic epithelial cells during the infection and facilitates the intimate attachment of the bacteria, thus initiating the inflammatory response of the infected mucosa. These data were confirmed in vitro using C. rodentium-infected mouse cells and human colonic epithelial cells infected with enteropathogenic Escherichia coli. Our work describes a mechanism by which A/E bacteria manipulate host response to favor their colonization, thereby positioning SLC7A2 as an unrecognized therapeutic target to limit infection with enterobacteria.Intestinal infections by attaching and effacing (A/E) bacteria widely impact human health, with major social and economic repercussions. Mucosal immunity plays a critical role in determining the outcome of these infections. The amino acid L-arginine regulates inflammatory responses to bacterial pathogens. We studied the role of the L-arginine transporter solute carrier family 7 member 2 (SLC7A2) during infection with the A/E pathogen  Tissues were then washed three times with PBS, lysed with RIPA buffer, and supernatants were collected. Tissue lysates were mixed with scintillation fluid and the [14C] content was determined in a scintillation counter. Protein was measured in tissue lysates by the bicinchoninic acid method as described previously [14C]-L-Arg/min/mg protein.Freshly collected colonic tissues from uninfected and eviously . L-Arg tSlc7a2–/–mice at the time of euthanasia via cardiac puncture and serum was separated as described [Blood was collected from WT and escribed , 10. Serescribed .t test was performed for comparison between two groups. Analysis of variance with the Student-Newman-Keuls post-hoc multiple comparisons test was performed to compare multiple groups. Survival was analyzed by the Log-rank (Mantel-Cox) Test.Quantitative data are shown as the mean ± SEM. Statistical analyses were performed with Prism version 5.0c (GraphPad Software). Student’s S1 FigA) Swiss-rolled colons from Slc7a2–/–animals that were uninfected (Ctrl) or infected with C. rodentium (C. rod), were probed for Slc7a2 RNA, and visualized using DAB. Scale bar, 50 μm. Representative images of 3 mice. (B) Slc7a1 mRNA levels were analyzed in colon tissues by real-time PCR . (C) Western blot for SLC7A2 in isolated CECs. (D) Ex vivo measurement of L-Arg uptake by colonic tissues and L-Arg concentration in serum. **P < 0.01, ***P < 0.001.((TIF)Click here for additional data file.S2 FigC. rodentium (C. rod) or with the isogenic mutants for 4 h. The expression of Slc7a2 (A) as well as Cxcl1 and Cxcl2 (B) mRNA was analyzed. *P < 0.05 compared to Ctrl, §P < 0.05 compared to C. rodentium-infected cells; n = 3 independent experiments.YAMC cells were infected or not with (TIF)Click here for additional data file.S3 FigA) Colons from WT and Slc7a2–/–mice, that were uninfected (Ctrl) or infected with C. rodentium (C. rod), were stained for goblet cells. Representative images are shown. Scale bar, 200 μm (B) Percent of mucosa with loss of goblet cells; **P < 0.01. For (A) and (B), n = 5–7 mice for Ctrl and n = 5–9 for C. rodentium-infected mice.((TIF)Click here for additional data file.S4 FigSlc7a2 and neo. Representative PCR gel of two animals in each condition. The predominant band is that of donor mice.After bone marrow transplantation, DNA from spleen of recipient animals was analyzed by PCR for the genes (TIF)Click here for additional data file.S5 FigSlc7a2 in HEK 293 cells expressing pLX304 or pSlc7a2 (A), and in YAMC cells transduced with Ctrl or Slc7a2 shRNA and then infected with C. rodentium (C. rod) (B). **P < 0.01, ***P < 0.001; n = 3–5 independent experiments. (C) Levels of SLC7A2 by Western blotting.mRNA levels of (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


The E2 protein cage derived from the Geobacillus stearothermophilus presents 12 pores, 6 nm in diameter, and an empty core of 13 nm in diameter. We engineered the E2 protein by site-directed mutagenesis with oligohistidine sequences exposing them into the cage’s core. Dynamic light scattering and electron microscopy analysis show that the structures of E2 protein cages mutated with bis- or penta-histidine sequences are well conserved. The surface of the gold nanoparticles was passivated with a self-assembled monolayer made of a mixture of short peptidols and thiolated alkane ethylene glycol ligands. Such monolayers are found to provide thin coatings preventing non-specific binding to proteins. Further functionalisation of the peptide coated gold nanoparticles with Ni2+ nitrilotriacetic moieties enabled the specific binding to oligohistidine tagged cages. The internalisation via affinity binding was evaluated by electron microscopy analysis. From the various mutations tested, only the penta-histidine mutated E2 protein cage showed repeatable and stable internalisation. The present work overcomes the limitations of currently available approaches and provides a new route to design tailored and well-controlled hybrid nanoparticles.Porous protein cages are supramolecular protein self-assemblies presenting pores that allow the access of surrounding molecules and ions into their core in order to store and transport them in biological environments. Protein cages’ pores are attractive channels for the internalisation of inorganic nanoparticles and an alternative for the preparation of hybrid bioinspired nanoparticles. However, strategies based on nanoparticle transport through the pores are largely unexplored, due to the difficulty of tailoring nanoparticles that have diameters commensurate with the pores size and simultaneously displaying specific affinity to the cages’ core and low non-specific binding to the cages’ outer surface. We evaluated the specific internalisation of single small gold nanoparticles, 3.9 nm in diameter, into porous protein cages  Protein cages and virus-like particles are highly structured and stable homo- and hetero-supramolecular protein self-assemblies. All protDuring the last two decades, materials scientists have intensively investigated the manipulation of protein cages for the encapsulation of single inorganic nanoparticles into their core to design novel hybrid bioinspired nanoparticles.,10 As suvia electrostatic interactions.[A general concern in the synthesis of protein cage-based hybrid nanoparticles is the ability to control the interactions at the protein/inorganic nanoparticle interface. Such conractions.,26–28 Thractions. Biominerractions. A typicaractions. In this via nanometre size pores presents an attractive opportunity also for the internalisation of inorganic nanoparticles. Direct internalisation of nanoparticles inside porous protein cages could provide an alternative approach for the preparation of hybrid nanoparticles. However, to our knowledge this strategy remains largely unexplored. Ishii et al. published the first instance of internalisation of non-coated inorganic nanoparticle, i.e., quantum dots, into preformed tubular chaperonin proteins GroEL (from Escherichia coli) and T.th (from Thermus thermophilus HB8).[The unique feature of porous protein cages which allow for the internalisation of materials lus HB8). The studlus HB8).,33 or inlus HB8). Direct ilus HB8). In additlus HB8). However,via affinity binding of single small gold nanoparticles into porous protein cages. As a porous protein cage, we used the E2 protein cage derived from the Geobacillus stearothermophilus that presents 12 pores of 6 nm and an empty core of 13 nm in diameter. We engineered the E2 protein by site-directed mutagenesis with oligohistidine sequences of 4 different repeats exposed in the cages’ core. Dynamic light scattering and electron microscopy analysis confirmed well-conserved structure of the E2 protein cages mutated with bis- or penta-histidine sequences. To facilitate the internalisation, and to prevent non-specific binding of 3.9 nm diameter gold nanoparticles, their surface was passivated with a self-assembled monolayer (SAM) made of a mixture of short peptidols and thiolated alkane ethylene glycol ligands.[2+ nitrilotriacetic (Ni-NTA) moieties enabled the specific binding to oligohistidine tagged cages. Finally, the internalisation via affinity binding of Ni-NTA-functionalised peptide coated gold nanoparticles into the core of E2 protein cages was evaluated for the different E2 mutants and wild-type E2 and validated by electron microscopy analysis.In this investigation, we evaluate the specific internalisation  ligands.,37 The f2)11-EG3-NTA and HS-(CH2)11-EG4 ligands were purchased from Prochimia . Gold nanoparticles of 3.9 nm diameter stabilised in citrate buffer were purchased from Nanopartz Inc. (Canada). PBS buffer, Sephadex G25 superfine resin, dimethylsulfoxide (DMSO), Tween 20, nickel (II) nitrate hexahydrate and sodium phosphotungstate tribasic hydrate were purchased from Sigma-Aldrich Ltd. . Nanosep filters 10 kDa cut-off were purchased from PALL . Affi-Gel 10 resin and Tris-HCl were obtained from Bio-Rad Laboratories Pte Ltd (Singapore). UV spectra were acquired with a Spectra Max Plus spectrophotometer  using a 384 well plates from Corning . E. coli strains DH5α (Zymo Research) and BL21(DE3) (Stratagene) were used as host cells. The vector pET-11a was purchased from Novagen. The oligonucleotides were synthesised by 1st BASE (Singapore). Restriction enzymes (BamH I and Nde I), T4 DNA ligase, and Pfu Ultra High-Fidelity DNA polymerase, and isopropyl β-D-thiogalactopyranoside (IPTG) were obtained from Fermentas.The peptidol H-CVVVT-ol was purchased from Peptide and Protein Research . HS-(CH2O milliQ 25:75 (v:v). Solutions of HS-(CH2)11-EG3-NTA and HS-(CH2)11-EG4 (both 2 mM) were prepared in DMSO and ethanol respectively. To prepare coated gold nanoparticles, 1 volume of 2 mM solution containing 60% of H-CVVVT-ol, 30% of HS-(CH2)11-EG4 and 10% HS-(CH2)11-EG3-NTA ligand was mixed with 1 volume of PBS 10X  and 0.005 volume 1% (v:v) Tween 20 in milliQ H2O. Then, 9 volumes of colloidal gold solution were added and mixed overnight at room temperature.A 2 mM solution of H-CVVVT-ol was prepared in DMSO:HSephadex G25 superfine (10 mL) was prepared, loaded into a column and kept in 0.02% (w:v) sodium azide. The column was equilibrated with 30 mL of 100 mM NaCl aqueous solution with 0.002% (v:v) Tween20. Coated gold nanoparticles (1.1 mL) were concentrated to approximately 150 μL by centrifugation on a 10 kDa cut-off Nanosep filter. Then, nanoparticles were loaded on the column and eluted under gravity. Approximately 1.4 mL coloured fractions corresponding to nanoparticles were collected in the excluded volume.3)2.6H2O aqueous solution was added to 1 volume of purified coated gold nanoparticles in 100 mM NaCl with 0.005% (v:v) Tween20. The reaction was mixed overnight at room temperature. The Ni2+ excess, not chelated to the NTA groups, was removed by size exclusion chromatography on Sephadex G25. The purified nanoparticles were finally dispersed in PBS pH 8.0 with 0.005% (v:v) Tween 20.One-tenth volume of 2.8 M Ni(NOAffi-Gel 10 resin slurry (2 mL) was washed with 10mL of cold (4°C) 1 mM HCl, and then twice with DMSO, to give, 1 mL of resin. To this 950 mL DMSO, and 50 mL H-CVVVTGGGHHHHHH-OH peptide  were added. The reaction was stirred overnight at room temperature before adding 6 mL 100 mM Tris-HCl, pH 8.5 for 4 h to block unreacted N-hydroxysuccinimide groups. The resin was washed three times alternately with 50 mM sodium acetate, 0.5 M NaCl, pH 4.5, and then with 100 mM Tris-HCl, pH 8.5. The resin was stored at 4°C in 20% (v/v) ethanol/water until further use.Mutant plasmids were generated using site-directed mutagenesis. Oligonucleotides in st Base.To construct the expression mutant vectors, the vector pET-11a and the PCR products were digested with NdeI and BamHI restriction enzymes. Both vector and target E2 genes were purified by 0.8% (w/v) agarose gel electrophoresis and ligated with T4 ligase. The sequences of the mutant genes were confirmed by the DNA sequencing service provided by 1E. coli strain BL21(DE3) following a previously described protocol.[E. coli cells were cultured in LB medium supplemented with 100 μg/ml ampicillin. Gene expression was induced by addition of 1 mM IPTG at an optical density (OD600) between 0.6 and 0.8. Cells were harvested 3 h after induction by centrifugation at 4500 ×g for 40 min and stored at -80°C. Subsequently, the cells were suspended in HEPES buffer  and disrupted by ultra-sonication. The supernatant after centrifugation at 40000 ×g for 1 h was heat-treated at 72°C for 20 min to denature the majority of E. coli proteins. Re-centrifugation at 40000 ×g for 1 h was then used to remove the denatured proteins.Wild-type E2 (E2-WT) and mutant proteins were produced in protocol. The E. cThe supernatant after ultra-centrifugation was filtered and loaded onto an anion exchange chromatography column , which had been equilibrated with HEPES buffer (pH 8.0) on an ÄKTA liquid chromatography system . The E2 protein was eluted with HEPES buffer containing 1 M NaCl at elution concentration gradient set over 5 column volumes. Fractions containing the pure E2 protein were pooled. The concentration of protein was determined with a Micro BCA protein assay kit (Pierce) using bovine serum albumin (BSA) as the standard.The self-assembly of mutant E2 protein cages in 20 mM Tris pH 8.7 was characterised by dynamic light scattering (DLS) . DLS was used to assess the hydrodynamic diameters of the proteins (1 mg/ml) at 25°C with 5 min equilibration. Results were an average of 3 measurements.2PO4, 0.5 M NaCl pH 8.0 (phosphate buffer). Final concentrations of the cages were about 1 μM.All protein cages were washed twice in milliQ water on Nanosep 10 kDa filters and dispersed in 50 mM NaH2)11-EG4:HS-(CH2)11-EG3-NTA 60:30:10 (mole/mole/mole) coated gold nanoparticles (3.9 nm) at 0.4 μM were mixed with 50 μL of E2 protein cages at 1 μM overnight at room temperature. Samples were then stored at 4°C for a few days and analysed by electron microscopy.In 500 μL centrifuge tubes, 50 μL H-CVVVT-ol:HS-(CHGold nanoparticles were concentrated by ultrafiltration of stock solutions with Nanosep 30 kDa filters. Concentrated solutions were deposited on Ultrathin Carbon (< 3 nm) on Carbon Holey support film TEM grids . Gold nanoparticles and protein cages were analysed with a Philips CM300 high resolution analytical TEM/STEM. Typically, protein cage mixtures were analysed at 0.1 mg/mL stained with 1% (w/v) sodium phosphotungstate tribasic hydrate. Obtained images were not digitally modified, such as, for example, to remove the carbon grids. The size determination of the gold nanoparticles was done with ImageJ v1.47e software using the macro ‘ParticleSizeAnalyzer’.Geobacillus stearothermophilus as the porous protein cage model. The E2 protein cage consists of 60 identical subunits self-assembled into a dodecahedral protein complex.[We used the E2 protein, the core unit of the pyruvate dehydrogenase multi-enzyme complex found in  complex. The thre complex. and ther complex.,41The method proposed here is developed in three steps . We firsIn this investigation, we considered two major factors that would drive the successful internalisation of small gold nanoparticles functionalised with Ni-NTA groups into oligohistidine mutated E2 protein cages: the stability of the protein cages and the binding strength between the oligohistidine sequences and the Ni-NTA groups.We studied the formation of modified E2 protein cages presenting various oligohistidine sequences on the protein cages’ inner surface. To ensure the correct self-assembly and stability of the protein cages while providing sufficient access to the oligohistidine sequences, we chose to modify the RDGE loop present on the inner surface of the E2 protein cages. We synthesised four different variants of the E2 protein cage, i.e., E2-LH2, E2-LH5, E2-LH6 and E2-H3LH3, presenting an increasing number of histidine residues at the RDGE loop sequence by substitution and/or addition of histidine residues . We evalThe correct formation and stability of the E2 protein cage mutants was confirmed by dynamic light scattering (DLS) measurement  and by t2)11-EG4 at a 60:30 ratio (mol:mol). The utilisation of such SAM has previously been shown to increase the stability[2)11-EG3-NTA. The NTA-functionalised peptide coated gold nanoparticles were then loaded with Ni2+ for binding of the nanoparticles to oligohistidine sequences present on the inner surface of the protein cages. The functionalisation with HS-(CH2)11-EG3-NTA ligands and the specific binding to a oligohistidine tagged biomaterial were evaluated by the immobilisation of Ni-NTA-functionalised peptide coated gold nanoparticles on a hexa-histidine loaded agarose resin. All the Ni-NTA-functionalised peptide coated gold nanoparticles bound to the oligohistidine tagged resin, showing that 100% of the gold nanoparticles were functionalised with NTA moieties and activated with the Ni2+ Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig2+ NTA-functionalised gold nanoparticles and 25 nm diameter E2-LH5 protein cages by Superdex 200 size-exclusion chromatography.Electron microscopy analysis of (A-B) the fraction 4 and (C-D) the fraction 6 after purification of mixture of 3.9 nm diameter Ni(PDF)Click here for additional data file.


We used a bacterial artificial chromosome (BAC) transgenic approach and TLR4/MD-2 knockout mice to specifically examine the role of human TLR4 variants in recognition of LPS. Using  D299G+T399I allele is ~7% [D299G polymorphism in Africa , whereas the TLR4D299G+T399I allele was found to be prevalent in Europe  [D299G variant initially arose to protect against cerebral malaria in Africa and that this allele was hyperresponsive to LPS, in contrast to the previous study. They proposed that the compensatory T399I mutation that reduced LPS responsiveness arose in Europe to protect from sepsis. That study also showed that the single TLR4T399I variant is very rare. Other in vitro studies also demonstrated reduced responsiveness of TLR4D299G+T399I to hexa- or penta-acylated LPS [Two coding human Toll-like receptor 4 (TLR4) single nucleotide polymorphisms  resulting in Asp→Gly or Thr→Ile substitutions at amino acids 299 and 399, respectively, have been described [D299G variant is protective against increased systolic blood pressure with increased obesity as well as against periodontitis, though both failed to examine the co-segregating rs4986791 SNP and thus actual genotype cannot be determined [Multiple association studies have linked these SNPs to a wide variety of diseases (early studies reviewed in ). Publistermined , 19. SinD299G or the TLR4D299G+T399I human allele and compared them to mice expressing human TLR4WT. Although our comparisons were limited by the copy number of lines randomly generated, we found that the expression level of human TLR4 was the largest determinant of LPS response and that the actual sequence of human TLR4 did not reveal obvious differences in LPS recognition.Thanks to the success in generating mice that display human-like responses to LPS through the expression of human TLR4 and MD-2 from human genomic BACs , we geneUltrapure O111:B4 EC LPS was purchased from InvivoGen , and ODN1826 from Coley Pharmaceuticals . Monophosphorylated lipid A (MPL) was purchased from Avanti Polar Lipids Inc . Antibodies for flow cytometry were purchased from Becton Dickinson , eBioscience  or BioLegend . Anti-huTLR4 clones HTA125 and TF901 as well as anti-muTLR4 clone MTS510 were used for surface expression analysis.P. aeruginosa (PAO-1) and Y. pestis (KIM6-). P. aeruginosa was grown in Lysogeny broth (LB) supplemented with 1mM MgCl2 at 37°C with shaking and Y. pestis was grown in brain heart infusion (BHI) supplemented with 1mM MgCl2 at 37°C with shaking.The strains used in this study are g at room temperature for 30 minutes. The aqueous phase was collected and an equal volume of endotoxin-free water was added to the organic phase. The extraction was repeated and aqueous phases were combined and dialyzed against Milli-Q purified water to remove residual phenol and then freeze-dried. The resultant pellet was resuspended at a concentration of 10 mg/ml in endotoxin-free water and treated with DNase  at 100 μg/ml and RNase A  at 25 μg/ml and incubated at 37°C for 1 hour in a water bath. Proteinase K  was added to a final concentration of 100 μg/ml and incubated for 1 hour in a 37°C water bath [LPS was extracted by the hot phenol/water method . Freeze-ter bath . The solter bath  and furtter bath . For MasLipid A isolated by small-scale lipid A isolation procedures was analyzed on a AutoFlex Speed MALDI TOF mass spectrometer . Data was acquired in reflectron negative and positive modes with a Smartbeam laser with 1 kHz repetition rate and up to 500 shots were accumulated for each spectrum. Instrument calibration and all other tuning parameters were optimized using Agilent Tuning mix . Data was acquired and processed using flexControl and flexAnalysis version 3.3 .This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All protocols were approved by the Institutional Animal Care and Use Committee of the University of Washington.2 narcosis, followed by a secondary method, when only bones were collected. However, for experiments using splenocytes , mice were euthanized by sharp cervical dislocation. Finally, for experiments where mice had received intraperitoneal LPS prior to euthanasia, mice were injected IP with an overdose of Beuthanasia-D .All mice were bred in-house at the University of Washington. C57BL6/J mice were originally obtained from the Jackson Laboratory . Mice were maintained in a specific pathogen free (SPF) facility in standard ventilated cages on autoclaved corn cob bedding with an automatic watering system and fed irradiated standard rodent chow with a 12 hr on/12 hr off light cycle. Several methods of euthanasia were used, depending on experimental manipulation. Mice were euthanized by COgalK positive/negative selection to generate the D299G and T399I variants as described [Human genomic BAC RP11-150L1 was modified by introducing point mutations using lambda Red mediated recombination and escribed . Primersescribed .Copy number of the TLR4 BAC was determined using real-time PCR as previously described . PrimersBone-marrow-derived macrophages (BMDM) were generated and stained as described for surface TLR4 expression using anti-human TLR4 clones HTA125 and TF901 and anti-mouse TLR4 clone MTS510 and appropriate isotype controls  . Whole sRelative TLR4 expression levels were determined by real-time PCR as described  using anSerum samples were filtered in a 96-well 1.2 μm membrane HTS plate  and diluted 1:4 or 1:12 in serum diluent. Cytokines  were quantified using a Bio-Plex 200 system with Bio-Plex Pro Assay Kits  following the manufacturer’s instructions as described [Prism software  was used for all statistical analyses. Multiple comparisons were performed using 1-way ANOVA followed by Bonferroni’s Multiple Comparison test.http://depts.washington.edu/compmed/transgenic/). Chimeric pups were identified by coat color and backcrossed to B6 mice and subsequently to TLR4/MD-2 double-KO mice, such that mice differed only in the sequence of human TLR4. The mice were also bred to humanized MD-2 mice [Initially, we modified the human TLR4 BAC to introduce the D299G mutation using BAC recombineering methods . We thenD-2 mice . Thus, iD299G and 1 and 8 copies of huTLR4D299G+T399I, as compared to our previously generated 2- and 4-copy lines of huTLR4WT. While increasing BAC copy number from 1 to >48 correlates with increased expression , when independent lines of same copy number were examined differences in staining intensity could be seen [We first examined copy number using methods as described in  and foun be seen . TherefoE. coli (EC) LPS responsiveness in the splenic macrophage/monocyte population expressing the variant huTLR4 alleles, we used primary splenocyte intracellular cytokine staining (D299G+T399I line.To determine whether we had restored staining  as descrstaining . The absstaining . This coPseudomonas aeruginosa (PA) LPS [Yersinia pestis (YP) LPS [WT, showed reduced activity in response to hypoacylated LPS, as compared to WT B6 mice. In addition, following correction by the response to CpG, all lines except the 1-copy TLR4D299G+T399I line showed significantly increased response to PA LPS or MPL compared to the KO responses. The 1-copy TLR4D299G+T399I line did not respond to either PA or YP LPS even though we observed a response to EC LPS  LPS , as well(YP) LPS . Unlike o EC LPS .D299G and 8-copy TLR4D299G+T399I lines compared to lower copy numbers. All humanized lines and the KO line were significantly reduced for IL-6 production, as compared to WT B6 cells, when stimulated with EC LPS. However, all humanized lines with the exception of the 1-copy TLR4D299G+T399I line had increased IL-6 production compared to the KO. In contrast to the previous assays, the 1-copy TLR4D299G+T399I line did not respond to EC LPS.We next generated bone-marrow-derived macrophages (BMDM), and stimulated cells with either PA, YP LPS, or EC LPS, and measured IL-6, CCL5, and IL-10 cytokines in the supernatants 24 hr later by Luminex . All humin vivo, we extended our studies into mice, and injected mice intraperitoneally (IP) with either EC or PA LPS , a known vaccine adjuvant that results in a selective decrease in MyD88-dependent responses while TRIF-dependent responses are maintained [Pseudomonas aeruginosa contains lipid A that is penta-acylated although cystic fibrosis isolates produce hexa-acylated molecules [Yersinia pestis produces hexa-acylated lipid A at room-temp but a tetra-acylated molecule at 37°C [There is a large body of literature that continues to expand examining the association of TLR4 SNPs with disease. However, no study has clearly, reproducibly, and mechanistically linked the common coding TLR4 SNPs (rs4986790 and rs4986791) with disease. TLR4 and MD-2 are coreceptors that bind directly to the lipid A component of LPS , 34. Theintained , 38. Theolecules . Similar at 37°C , 41. The at 37°C , 31. Sim at 37°C .D299G+T399I in complex with E. coli LPS demonstrated conformational changes at the site of the D299G polymorphism though not at the site of LPS binding [in vitro study that showed equivalent LPS binding between the D299G variant and WT TLR4 suggested that decreased inflammatory cytokine production mediated by huTLR4D299G is due to decreased recruitment of MyD88 and TRIF [D299G+T399I variant is impaired in dimerization in response to MPLA [in vitro and in mouse studies [The crystal structure of TLR4 binding . One in and TRIF . Our stu studies –48, thatS1 Fig(A) BMDM from each of the genotypes indicated above plots were stained with anti-huTLR4 clone TF901 or isotype control. Boxed number in each plot shows ΔMFI of TLR4  vs. isotype control (filled grey histogram). Histograms from 2 separate experiments are shown. (B) B6 mouse BMDM stained with anti-muTLR4. (C) Each symbol represents a separate BMDM preparation. All cells express huMD-2; red squares depict huTLR4WT, light blue triangles huTLR4D299G (299), and dark blue inverted triangles huTLR4D299G+T399I (399). X (KO) does not express TLR4. Open symbols have lower copy numbers than closed symbols. Brackets show significant pair-wise comparisons using 1-way ANOVA followed by Bonferroni’s Multiple Comparison test. *P<0.05.(TIF)Click here for additional data file.S2 FigData from a single experiment shown in (TIF)Click here for additional data file.S3 FigTNF histograms of the macrophage/monocyte population stimulated with indicated ligands. The % of macrophages/monocytes producing TNF is shown in each histogram.(TIF)Click here for additional data file.


Hepatitis C virus (HCV) genotype (GT)3 is associated with increased risk of steatosis, development of cirrhosis and hepatocellular carcinoma. Limited data are available regarding genetic variability and use of direct-acting antiviral agents in these patients. non-structural protein 5A (NS5A) and non-structural protein 5B (NS5B) sequencing was performed on 45 HCV GT3-infected Italian patients subsequently treated with sofosbuvir ± daclatasvir (SOF ± DCV). Novel GT3a polymorphisms were observed by Sanger sequencing in three NS5A  and three NS5B  baseline sequences in patients who achieved sustained virological response (SVR). Baseline NS5A resistance-associated substitutions (RASs) A30K and Y93H were detected in 9.5% of patients; one patient with A30K did not achieve SVR. Phylogenetic analyses of sequences showed no distinct clustering. Genetic heterogeneity of NS5A and NS5B was evaluated using ultra-deep pyrosequencing (UDPS) in samples longitudinally collected in patients not achieving SVR. Some novel NS5A and NS5B polymorphisms detected at baseline may not impact treatment outcome, as they were not enriched in post-failure samples. In contrast, the clinically novel GT3 NS5A-L31F RAS emerged in one treatment failure, and I184T, G188D and N310S, located on the same NS5B haplotype, became predominant after failure. These findings suggest a potential impact of these novel substitutions on the treatment outcome; however, their significance requires further investigation. HCV GT3During the era of pegylated interferon (pegIFN) + ribavirin (RBV) therapy, GT3 was considered an “easy-to-treat” genotype, with high cure rates compared with other viral genotypes ; most faIn GT3 patients with cirrhosis, SOF + DCV treatment for 12 weeks resulted in 63% rate of sustained virologic response (SVR), compared with 96% without cirrhosis . AdditioAt least 10 GT3 subtypes have been identified , and druIn this study, genetic variability of NS5A and NS5B in 45 GT3a Italian patients, who received SOF ± DCV treatment with/without pegIFN + RBV, was evaluated. In particular, the type and frequency of amino acid substitutions, as well as the DAA selective pressure on the targeted gene regions was assessed. In addition, the RAS dynamics was analyzed by ultra-deep pyrosequencing (UDPS) in patients not achieving SVR.Plasma samples from 45 HCV GT3a-infected patients receiving DAA-based therapies were obtained from our clinical center in Italy. The use of patient samples was approved by the Ethics Committee of the National Institute for Infectious Diseases “L. Spallanzani” (statement n20/2015). Written informed consent was obtained from all patients.All patients were DAA treatment-naïve. Baseline characteristics including HCV-RNA, age, coinfection with human immunodeficiency virus (HIV) and cirrhosis status are reported in Hepatitis B serum surface antigens (HBsAg), anti-hepatitis virus surface antibodies (anti-HBs), HIV sero-status, serum alanine aminotransferase (ALT) levels, serum total bilirubin level, and serum platelet counts were also recorded (data available upon request).All patients received SOF-based treatment ± DCV . Plasma HCV RNA was assessed using the Abbott real time HCV RNA assay (Abbott Molecular) with a lower limit of quantitation (LLOQ) and of detection (LLOD) of 12 IU/mL. Time points for virological monitoring were: between 4 weeks pre-treatment and baseline visits, on-treatment every month, and post-treatment every month to post-treatment week 24. A tolerance of one week was accepted in the intervals of post-treatment virological monitoring. On-treatment, virological response was defined as HCV RNA < LLOQ with target not detected (HCV RNA < LLOQTND). Post-treatment virological response was defined as HCV RNA < LLOQ with/without target detected (HCV RNA < LLOQTD/TND).Virological failure was defined as relapse: any confirmed HCV RNA measurement ≥LLOQ during post-treatment follow-up subsequent to an on-treatment response <LLOQ without target RNA detected (<LLOQTND) at the end-of-treatment visit.Resistance testing was performed on plasma samples at baseline and at virological rebound. Detailed analysis of RAS was performed by UDPS in three of four patients experiencing therapy failure for whom available samples had sufficient HCV RNA levels (see below).Viral RNA was extracted from 600 µL plasma using QIASYMPHONY  and reverse transcribed by Moloney Murine Leukemia Virus (M-MULV) reverse transcriptase  using random hexamers.The substitutions were identified using the strain NZL1 (accession number D17763) as GT3a reference, according to Smith et al. [The HCV NS5A and NS5B regions were amplified with nested PCR using GT3-specific primers for NS5A and pan-genotypic primers for NS5B, as described ,15. The The same amplicons were used for UDPS. In the second round PCR, the primers were conjugated with multiplex identifiers for sample barcoding and to adaptors for UDPS.Sanger sequencing of second round amplicons was performed using the ABI PRISM 3100 genetic analyzer . The cutoff frequency for detecting variants with Sanger sequencing was around 15% .Ultra-deep pyrosequencing was carried out with the 454 Life Sciences platform , according to the manufacturer’s instructions.Ultra-deep pyrosequencing was performed on NS5A and NS5B regions from Pt42 and Pt43 at baseline (T0), as well as at the first (T1) and second (T2) treatment failure visits. For Pt45, UDPS was performed on the NS5B region at T0 and T1. For T1 and T2, the first sample with HCV RNA ≥10,000 IU/mL was used for UDPS to avoid target re-sampling bias .Sanger-derived sequences were aligned with MUSCLE software  and manu.HCV sequences obtained by Sanger sequencing in this study can be accessed in the GenBank database (accession numbers KY474420-KY474510)For the analysis of UDPS results, all reads underwent a correction pipeline with in-house-developed scripts that excluded reads <450 nt in length or with a mean quality score <20. Moreover, the sequences were clustered with CD-HIT software , using aThe presence of haplotypes was observed using a script developed in-house.Phylogenetic trees were built using sequences of study patient as well as HCV GT3a sequences from the Los Alamos database, for which sampling year and geographic location was reported. For the Sanger sequences, nucleotide mixtures observed in the electropherogram were reported when representing ≥15% of the sequence population. The best-fit model (K2 + G + I) was selected according to the Akaike Information Criterion and trees were reconstructed using the maximum likelihood method implemented in MEGA6 . Bootstrn = 35, 78%) had cirrhosis; 58% were treatment-naïve and 42% had been previously treated with pegIFN + RBV. Males represented 84% of patients with a median age of 56 years, 74% were cirrhotic and 60% were treatment-naïve. All women  were cirrhotic and had a median age of 54 years; 43% (n = 3) were treatment-naive. The median baseline HCV RNA level for all patients was 5.3 Log10 IU/mL (range 1.1–7.0). Forty patients completed the treatment and achieved SVR; one (Pt13) died just after the end of treatment (EOT); four patients  did not achieve SVR and were retreated with a SOF-containing regimen. Specifically, Pt42, a transplanted patient, was first treated with SOF + DCV for 12 weeks and relapsed four weeks after EOT. He was retreated with SOF + DCV in association with RBV for 24 weeks, but relapsed eight weeks after EOT. Pt43, in waiting list for transplantation, was treated with SOF + DCV for 24 weeks, but relapsed four weeks after EOT. He was retreated with SOF + RBV for 48 weeks, and relapsed three weeks after EOT. Pt44 and Pt45 were treated with SOF + RBV for 24 weeks, but relapsed four weeks after EOT. They were retreated with SOF + DCV + RBV for 24 weeks and achieved SVR12 .Sequences from 42 patients were available for analysis; the frequency of polymorphic sites, established by comparison with the GT3a reference sequence D17763 [n = 3) with these polymorphisms achieved SVR.In NS5A, 42 amino acid positions showed polymorphism, with 13 positions harboring multiple substitutions . Three oNS5A polymorphisms reported to be associated with DCV resistance in GT3a include M28T, A30K, L31F/I/M, and Y93H ,26. M28Ln = 4) who experienced SOF-based treatment failure. A62T (n = 5) and A62P (n = 1) were also detected.The presence of NS5A polymorphisms at positions 24, 32, 58, 62 and 92, which can reduce susceptibility to some NS5A inhibitors in other genotypes ,27, wereThe frequency of substitutions at NS5A positions not associated with DAA resistance was consistent with the frequency observed in the Los Alamos database .In NS5B, 32 amino acid positions had polymorphisms that differed from D17763, with eight positions harboring more than one polymorphism .Three substitutions  were not observed in available GT3 NS5B sequences in the Los Alamos database ; all thrNS5A and NS5B amino acid positions under drug selective pressure were evaluated by calculating the mutation rates (dN-dS) in GT3a sequences present in the Los Alamos HCV database . For NS5For NS5B, results indicated that positions 159, 244, 282, 289, 316, 320, 321, involved in drug resistance, were under negative pressure while positions 309 and 310 appeared to be under neutral pressure B. The onA total of 42 NS5A sequences derived from our Italian study patients and 47 GT3a sequences from North America, Europe and Asia were included in the phylogenetic analysis. Overall, several clusters of sequences appeared on the phylogenetic tree, showing apparent paraphyly, but there was no statistical support. Overall, sequences from Italian study patients were inter-dispersed with database sequences, with a tendency to group with sequences of European origin, although not supported by a significant bootstrap value A. BootstFor NS5B, Italian study patient sequences were less inter-dispersed between the database sequences B.Sanger sequencing results for the four patients who failed treatment are shown in NS5A and NS5B nucleotide diversity in the three treatment failures was calculated  and a prThe dynamics of substitutions potentially involved in DAA resistance, as evidenced by the UDPS analysis, are shown in For Pt43, known RAS were detected in NS5A  and NS5B  at T1. After re-treatment (T2), NS5A-Y93H was still the predominant substitution (99.5%), while NS5B-S282T was no longer detected. No additional RAS were observed. With respect to other NS5A positions potentially involved in DAA resistance, substitutions at positions 58 and 62 were also observed in nearly all viral quasispecies at the tested time points.In Pt45, no substitutions were detected at positions known to reduce susceptibility to DAAs in both drug target regions.When assessing substitutions not reported to be associated with drug resistance in Pt42, substitutions at NS5A positions 34 and 52 emerged at T1 and were also detected at T2 . The NS5For Pt43, enrichment of NS5B-Q273P was observed: Q273P was detected at 3.0%, 99.8% and 98.9% at T0, T1 and T2, respectively.In Pt45, the NS5B polymorphisms I184T, G188D and N310S represented minor viral populations at baseline (ca. 0.7%) and were enriched at T1 (>97%). These substitutions were linked on the same haplotype in various combinations . The frequency of the triple substitution haplotype was below the UDPS threshold (0.2%) at baseline and 94.6% after virological failure.In this study, NS5A and NS5B genetic characteristics in 45 HCV GT3a Italian patients with advanced fibrosis or cirrhosis, who were treated with SOF-based therapies were analyzed. Twenty-three of these patients were treated with SOF + DCV ± RBV while 22 were treated with SOF + RBV ± IFN.NS5A and NS5B polymorphisms were identified at amino acid positions not previously reported: three were in NS5A  and three were in NS5B . However, none of these substitutions were observed in patients who failed DCV + SOF treatment.The number of patients harboring known RAS at baseline was 11.9% for NS5A and 0% for NS5B. These findings are in agreement with data reported by Chen et al., where only 0.1% of sequences from all genotypes included in the Los Alamos database harbored RAS to SOF .Among NS5A and NS5B positions associated with selective pressure in our study , T7 was Phylogenetic analysis revealed no distinct grouping of NS5A sequences. Only GT3 sequences harboring A30K tended to cluster together A, suggesCombination of DCV + SOF + RBV may overcome the presence of baseline NS5A RAS in cirrhotic patients, since two patients Pt19, Pt28) achieved SVR despite harboring A30K, a substitution with reduced (44-fold over wild-type) susceptibility to DCV . In cont achievedBaseline polymorphisms at position 58 (P58A/S/T) were detected in three patients, and only NS5A-P58S was observed in Pt43 who subsequently failed treatment with DCV + SOF . HoweverNS5A-A62 substitutions (A62P/S/T) did not seem to impact therapy response, since seven patients with these polymorphisms achieved SVR.NS5A-Y93 substitutions (Y93D/N) were observed in one patient (Pt28) who also harbored A30K. This patient was cirrhotic, received SOF + DCV + RBV, and achieved SVR. The novel Y93D substitution, detected in 2 of 42 patients, has not been reported previously, and although its DCV susceptibility is unknown, it did not seem to impact treatment response.The longitudinal UDPS analysis of patients who did not achieve SVR  highlighted the dynamics of viral quasispecies. The diversity was lower after failure as expected , but theIn one patient with virological failure (Pt42), NS5A-L31F emerged; this substitution has only been reported to emerge under DCV selection in vitro . In our No minor substitutions were observed in addition to NS5A-A62P/S/T . Y93C anThe signature SOF RAS NS5B-S282T was observed in Pt43 at first virological failure to SOF + DCV, but was not detected (by Sanger sequencing or UDPS) at baseline or at failure to re-treatment with SOF + RBV .Further studies are required to address possible significance of the combination of NS5A and NS5B substitutions observed at failure to SOF + DCV treatment, as well as their fitness and possible selective advantage. In addition, possible linkage of NS5A and NS5B to substitutions within distant genomic regions, such as NS3, may play a role in the future, considering the upcoming GT3 therapeutic regiments, including new protease inhibitors.Overall, high concordance between Sanger sequencing and UDPS was observed; however, UDPS allowed investigating the presence of minor substitutions, and their frequency changes after therapy failure. In fact, some NS5B polymorphisms, like G188D, Q273P, L285F, A306V, N310S, not previously reported in the public databases of HCV sequences, represented minor substitutions at baseline (<1%), that were enriched after virological failure. However, their significance to SOF failure is unclear and would need further investigation. In Pt45, who failed treatment without known RAS, three baseline polymorphisms, present at low-frequency , were observed on the same haplotype, and became the predominant species at virological failure. Studies on GT3 replicon harboring NS5B I184T-G188D-N310S showed that the SOF activity was similar against these substitutions as wild-type (data not shown).The main limitation of this study is the small number of virological failures analyzed. Furthermore, analysis of minor viral populations at baseline in patients achieving SVR was not performed. Overall, a full understanding of the role of some of these minor substitutions may be hard to determine, given the high rate of virological success of SOF-based regimens and the rare occurrence of RAS emergence in patients not achieving SVR.


PTEN via down-regulation of miR-21, in vitro and in tumour xenografts. A2M increases the expression of CD29 and CD44 but did not evoke EMT. Transcriptome analysis of A2M-treated tumour cells, xenografts and mouse liver demonstrated a multifaceted regulation of tumour promoting signalling pathways indicating a less tumorigenic environment mediated by A2M. By virtue of these multiple actions the naturally occurring A2M has strong potential as a novel therapeutic agent.Cancer resistance is a major cause for longevity of the naked mole-rat. Recent liver transcriptome analysis in this animal compared to wild-derived mice revealed higher expression of alpha2-macroglobulin (A2M) and cell adhesion molecules, which contribute to the naked mole-rat’s cancer resistance. Notably, A2M is known to dramatically decrease with age in humans. We hypothesize that this might facilitate tumour development. Here we found that A2M modulates tumour cell adhesion, migration and growth by inhibition of tumour promoting signalling pathways, e.g. PI3K / AKT, SMAD and up-regulated  Heterocephalus glaber), a subterranean rodent, tolerates hypoxia, hypercapnia, avoids many physiological characteristics associated with aging and, most importantly, exhibits pronounced resistance to cancer [in vitro by inhibition of members of the WNT/ß-catenin pathway [PTEN), CD29 (ITGB1) and CD44 but does not evoke epithelial-mesenchymal-transition (EMT). Furthermore, A2M* was found to down-regulate microRNA-21 (miR-21), which is a dominant inhibitor of PTEN expression. Notably, A2M* inhibits growth of tumours in nude mice independent of their origin, and induces tumour necrosis in tumour tissue and tumour slices cultures. Transcriptome analysis displayed fundamental and unexpected insights in regulatory power of this ancient and highly conserved human plasma protein. The unique features of using A2M* as a novel anti-tumorigenic therapeutic in cancer patients prompted us to performing this study.The naked mole-rat (NMR), . All animal studies were carried out in strict accordance with the recommendations for the care and use of animals and were approved by the”Regierungspräsidium Sachsen”, Germany (reference number: 24–9168.11/18/11).A2M was purified from human citrated blood obtained from healthy donors  as described earlier . The purLRPAP1) inhibition. The final product used for in vitro and in vivo stimulation experiments exhibited insignificant stimulatory effects on white blood cells indicating the absence of lipopolysaccharide (LPS), and factors accidentally co-purified with A2M could not be detected by mass spectroscopy .Human A2M purified from pooled plasma preserved its native structure and can be transformed to its active form, A2M*, by thiol ester disruption . This inLRP1 (2 μg/mL)  was coated onto 96-well plates, followed by incubation with a standard solution of A2M/A2M* and the test samples. Bound A2M/A2M* was detected by Horseradish (HRP)-labelled polyclonal anti-A2M-imunoglobulin (Ig).6 /mL in RPMI 1640 medium, containing penicillin (100 U/mL), streptomycin (100 μg/mL), glutamine (2 mM) and 10% fetal calf serum (FCS). Cells were seeded 48h and the medium was replenished 24h prior adding of A2M*. In case of IGF-1 and TGFß1 studies, FCS was omitted after cells had settled. Cultures were incubated in a moist atmosphere with 5% CO2 at 37°C.Cell lines used for this study are prostate cancer cell line PC-3 , astrocytoma cell line 1321N1 , lung cancer cell line A549 , breast cancer cell line MDA-MB 231 , colon rectal cancer cell line HT29 , and melanoma cell line LOX (DCTD TUMOR REPOSITORY). Cells were cultured at a density of 10Cytosolic and nuclear cell extracts were prepared according to the Abcam guidelines (ww.abcam.com). All lysis buffer contained 0.3% protease inhibitor cocktail (P-8340) and 2 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM sodium orthovanadate, 10 mM sodium fluoride  in case of phosphorylation detection. Determination of protein concentration was performed according to Bradford .(Isolated proteins (5–10 μg) and cell/tissue lysate (20–200 μg) were subjected to SDS-PG-PAGE (4–20%), homogeneous SDS-PAGE (8%) under reducing or non-reducing conditions or to native PG-PAGE (4–20%). Proteins were blotted to nitrocellulose membranes , detected by specific antibodies in conjunction with HRP-labelled goat anti-mouse/anti-rabbit-Ig. Band visualization was performed by chemiluminescence detection and densitometric analyses were performed by means of the E.A.S.Y. Win32 software . To determine the amount of immunoreactive protein all values were normalized to the control. Dilution of primary/secondary antibodies are tabulated in .×g, 20min, 4°C), the supernatant was incubated with appropriated amount of Protein A-agarose for 1h at 4°C to reduce non-specific binding. Then, the supernatant was incubated with respective monoclonal antibodies (20 μg/mL) overnight at 4°C. Protein A-agarose was again added and incubated under shaking for 2h. The agarose beads were washed with PBS-T  and finally subjected to SDS-PG-PAGE followed by Western blotting.Tumour cells were lysed in lysis buffer  freshly supplemented with 1 μg/mL aprotinin, 1 μg/mL leupeptin, 1 mM PMSF, 1 mM NaF and 1 mM Na3VO4. After centrifugation  using the WST-1 assay .t-test was employed.1321N1 astrocytoma cells were seeded in petri dishes and cultured until complete adherence and confluence. Cell monolayers were scratch with a p10 pipet tip, and fresh medium was added containing increasing concentration of A2M* , and culturing was continued. The distance of gap closure was measured under phase-contrast microscope daily. Each experiment was performed 3 times with 8 replicates for each treatment. The data were expressed as mean ± s.d.; Student 2 for 24h. Cell detachment was accomplished by 0.05% trypsin for 3 min. Remainder cells were fixed with para-formaldehyde (4%) and stained with Gentiana violet followed by intensive washing to remove excess dye. Finally, cells were treated with acetic acid and the liberated dye was measured spectrophotometrically at 590 nm. The absorbance represents the number cells left attached to the bottom of the well (n = 3) .5,000 cells were seeded into 6-well plates and cultured in the absence or presence of A2M* or RAP  at 37°C, 5% CO6 tumor cells  were resuspended in 100μL RPMI-1640 medium and 100μL matrigel , before injected subcutaneously. Therefore the mice were anaesthetized and analgesia was done by ketamine and xylazine 10 minutes before the tumor cell injection. Tumors were grown for 7 days and treated with 5.6 mg A2M* i.p. three times per week for 31 days. Animals were sacrificed by neck dislocation after CO2/O2 anesthesia. Tumors were then removed, weighted and fixed either in 4% formalin for histology and immunohistochemistry or were shock frozen in liquid nitrogen for molecular analyses.Subcutaneous tumor xenografts were generated in 8 weeks old male athymic nude mice . The animals were kept in a 12h day and 12h night cycle and had unrestricted access to drinking water and food. Briefly, 10Whole blood assay was applied to trace for bacterial by-products in the A2M preparations as described earlier . Three d.Impedance spectroscopy was performed in 2D and 3D cell cultures using Multiwell-Multiel-Ectrode-Arrays (MMEA)  and prot2 and 37°C for 15h. Cells were washed with PBS and treated by trypsin/EDTA for dissolution. After washing with PBS cells were immune-stained with mouse anti-human CD44-PE. Intracellular labelling of CD44 was performed using the BD Cytofix/Cytoperm Plus fixation/permeabilization kit according to the manufacturer’s protocol. The PE labelled isotype IgG1 antibody was used for control .Adhered A549 cells were cultured in the presence of various concentrations of A2M* at 5% CO.After sacrificing the animals, tumours were either fixed by 4% (v/v) paraformaldehyde buffered in 0.2 M phosphate-buffer, pH 7.0, for at least 24h, or tumour slice cultures (TSC) (350 μm) were prepared immediately using a vibratome. The slices were cultured, A2M* was added and incubation was continued for 48h followed by fixation. Prepared paraffin-embedded tissue sections (4 μm) were stained for HE, dehydrated and mounted on a microscope slide with Entellan. Ki67 nuclear labelling index was calculated as the percentage of Ki-67 immunoreactive nuclei divided by the total number of analysed tumour cells. Vascular and blood cells were excluded. Tumour cells were counted in five representative areas of the sections at a magnification of x 400. Between 500 and 2000 cells were counted per section from each specimen. Used primary and secondary antibodies are presented in .For the analysis of gene expression, total RNA was isolated using the peqGold total-RNA kit . Isolation was performed according to manufacturers’ instruction. RNA integrity  of total RNA was assessed by running an aliquot on a 1.5% agarose gel stained with ethidium bromide. For miR-21 analysis standard Trizol RNA isolation protocol was used. Reverse transcription was done using Revert Aid first stand kit  with random hexamer primers. For mature miR-21 the stem loop primer was used. Target genes were amplified pipetting 25 ng cDNA, 0.5 μM of each primer, 100 μM dNTPs, 1 x GoTaq buffer ready to load and 0,025U/μl GoTaq polymerase (Promega). PCR (3 step) was performed using the PTC 200 Multicycler (Biozym). Amplicons were analysed and visualized on 1.5% agarose gels under an ultraviolet transilluminator. For quantitative PCR SYBR-Green Mix (Roche) was used. Amplification and analysis was done using the Stratagene MX3000p and a standard SYBR green amplification program  with melting curve. Specific Primers are listed in A549 cell cultures, both control and treatment  were pelleted and suspended using 50 μl RNase-free water. RNA isolation was performed using the QIAamp RNA Blood Mini Kit, according to manufacturer specifications. The resulting RNA was quality controlled using Qubit RNA HS Assay and NanoDrop 2000c. Library preparation was done using ScriptSeq™ Complete Gold Kit with the required additional FailSafe™ PCR Enzyme Mix following the Epicentre® manual. The library preparation was executed on a Caliper Life Sciences Sciclone G3, (Perkin Elmer). To validate the quality of the cDNA library, we performed both a Qubit™ (Qubit dsDNA HS Assay) and LabChip GX® (DNA 1K / 12K / Hi Sensitivity Assay LabChip and DNA High Sensitivity Reagent Kit) measurement. We used Angencourt RNAClean XP and Agencourt AMPure XP beads in the respective washing steps.The library quality was checked by sequencing the multiplexed libraries on a MiSeq using a MiSeq Reagent Kit v2 (300 cycle) in 2x150 cycles mode. The afore prepared pool was then sequenced in two runs on a NextSeq 500 using a NextSeq® 500/550 High Output Kit v2 (300 cycles) in 2x150 cycles mode. The NextSeq was controlled by NextSeq Control Software v1.4. Reads were extracted into FastQ files using Illumina’s BCL2FastQ v2.16.0.10, on default setting, arriving at an average of 54 million reads per sample (ranging from 49 to 67 million).For each sample (six with A2M*treatment and fives PBS controls), reads were aligned to the Ensembl Genome GRCh38.p2 Release 80 using a two-step approach. Firstly, reads were mapped with default parameters with the splice-aware alignment program Hisat v2.0.0b . Secondlwi according to their p-values pi provided by the Cancer RNASeq Nexus to distinguish between each stage and the adjacent normal, using the following formula.Finally, for calculating the phenotype correlation of the A2M* treatment and the PBScontrol group against the lung adenocarcinoma stages of the Cancer RNASeq Nexus , we calcTotal RNA was extracted using the prepGold total RNA kit. Isolation was performed according to manufacturer’s instruction. Library preparation was done as described in . DeviatiFor separating the mice and human tissue derived reads in the xenografted samples they were introduced into the tool xenome . As grafReads obtained from liver samples were also mapped using TopHat v2 using the murine reference sequence as listed above.Counting of reads per gene was done based on the resulting sam files using the tool featureCounts  and the http://www.sabiosciences.com/pathwaycentral.php) was used to determine structures of intracellular pathways, which were used for OncoFinder, as described previously [Differential pathway analysis was performed with OncoFinder . In ordeeviously . For theeviously . It enabP < 0.05 was considered to be significant. The numbers of animals and individual samples used are described in the appropiate figure legend.Unless stated otherwise, student’s two-tailed t-test was used to compare differences between 2 groups. One- or two-way ANOVA were used to analyze differences between more than 2 groups. All data are presented as means s.e.m. using GraphPad Prism 6.0 . . The observed increase in cell adhesion might be causative of the inhibition of cell motility as analysed by the wound scratch assay .A2M* is a ligand of LRP1, which has been shown to regulate cytoskeleton organization, interaction with the extracellular matrix and affects migration and invasion of various non-tumour and tumour cells , 9. SpecCD29 and CD44 in 1321N1 and A549 cells . A2M* increased the expression of both CD29 and CD44 in 1321N1 cells and A549 cells  similarly to the receptor associated protein (RAP)  in A549 cells. The latter effect was previously reported for thyroid carcinoma cells and supports the assumption of a tight interaction between the hyaluronan receptor CD44 with LRP1 [CD44-isoform shift, which is essential for cells undergoing EMT [in vitro and in vivo and to modulate CD44 transcript level in A549 cells. Because, A549 cells showed a stronger increase in CD44 and CD29 increase after A2M* treatment, FACS analysis of these cells demonstrated an enhanced total cellular as well as plasma membrane-anchored CD44 upon A2M* treatment . Similar results were obtained by immunocytochemistry indicating accumulation of CD44 immunoreactivity at adjacent cell membranes . These data indicate that A2M* might regulate CD44 expression at a translational or post-translational rather than transcriptional level.To examine the contribution of membrane adhesion molecules, we analysed the A2M* dependent expression of ith LRP1 . The autith LRP1 , we lookoing EMT , in vitr. Less immunostaining for CD44 was seen in non-treated cells indicating that the CD44 co-precipitation signal may depend on A2M* levels. In accordance with the recent literature [. Using purified LRP1, we were able to perform ligand blots with A549 cell lysates, which surprisingly disclosed the interaction of CD44 with RAP rather than with subunits of LRP1 . Similar results were obtained by ligand binding studies using recombinant RAP .Treatment of A549 cells with 30 nM A2M* followed by precipitation of cell lysates by an A2M*-specific monoclonal antibody (mab) showed co-precipitation of cellular CD44 that was not observed in the presence of the control antibody (anti-GLO1 mab) of the same isotype terature , immunopThis is in contrast to the work of Perrot et al. , who fouCD29 and CD44 in different tumour cell lines . Integrins such as CD29 (ß1-integrin) bind to the extracellular matrix (ECM) and thus connect the cytoskeleton to ECM, which is accompanied by signal transduction [CD29 knockdown has been shown to reduce cell adherence [CD29 and regulates its surface expression [CD29 has to be elucidated.Here we found that A2M* increases the expression of sduction . Adhesiodherence . Recentl. After seeding 1321N1 cells on collagen-coated microelectrode arrays A2M* (1 to 300 nM) was applied and impedimetrically monitored for up to 20h. The statistical analysis of selected time  revealed a continuous increase of relative impedance even at the lowest concentration of 3 nM A2M* up to 8h. Afterwards, this effect diminished for concentrations up to 30 nM A2M*. Cellular alterations that could reflect this impedimetric effect course could be among others an increased cell-cell and/or cell substrate adhesion [.Recently, we investigated the impact of A2M and LRP1 on cellular properties of 1321N1 astrocytoma cells . Therefoadhesion . This co. Additionally, microscopic images were taken from each spheroid at each time point , showing no morphological alterations. Furthermore, the spheroid size was statistically analysed by determination of the cross section area . In comparison to the control group, a slight but not significant reduction of spheroid size was observed (3–7% at 8h). As nonspecific effects of A2M* like proliferation on cell size in 3D cultures could be excluded, there is strong evidence for an A2M*-induced transient strengthening of cell-cell contacts in a small time window of 4 to 12h (In 3D-cultures of single spheroids (d = 150 μm) A2M* treatment significantly increased the impedance at 4h and 8h and 30 nM A2M*, respectively, but disappeared after 12h 4 to 12h .CD44 was found to correlate with EMT, which describes the transition of cells from an epithelial to a mesenchymal phenotype that plays a crucial role in tumour proliferation, invasion, and metastasis. EMT promotes expression of CD44 that is under control of several regulatory factors such as TWIST, SNAIL (SNAI1), ZEB1 and SLUG (SNAI2). In contrast, the epithelial cadherin (E-cadherin), an epithelial marker, participates in the negative regulation of EMT [Fig 4A). This finally could explain the inhibition of tumour cell proliferation shown in Fig 4B. In addition, A2M* inhibited also phosphorylation of BAD (BCL2-associated death promotor). Phosphorylation of BAD by AKT at Ser112 or Ser136 normally results in the loss of its pro-apoptotic activity [High expression of n of EMT . It is wn of EMT  and trann of EMT  are stron of EMT . For thin of EMT . Targetin of EMT . To analactivity . Non-phoCD44. In contrast, A2M* displayed no significant effect on transcription level of CD44, vimentin and E-cadherin indicating that A2M* is not inducing EMT. This is all the more important as A2M* increased the cellular E-cadherin protein level in A549 and HT29 cells  and antagonized the TGF-ß1 induced phosphorylation of SMAD2/3 transcription factors .The EMT inducing property of TGF-ß1 is confirmed by increased RNA expression of the mesenchymal marker vimentin and decrease expression of E-cadherin, while no effect was observed on the expression of in vivo effects of A2M*, nude mice were inoculated with 3 different cancer cell lines subcutaneously and treated intraperitoneally (i.p.) with A2M* (5.6 mg/20g body weight). A2M* treated mice showed a reduced growth of A549, 1321N1 and PC-3 tumours compared to control groups (n = 10) . Injected human A2M* could be detected in the blood of animals as indicated by the presence of the fast-moving A2M* form in treated mice .To reveal . This chemokine is known to recruit monocytes, dendritic and memory cells to sites of injury [A2M* did not affect the animals’ body weight, but caused alterations of some blood cell parameters such as an increase of neutrophils and elevation of the monocyte chemotactic protein 1  level in isolated spleen cells of A2M*-treated animals f injury .. The high accumulation of tumour-infiltrating macrophages in A2M*-treated mice may arise from induction of MCP1 by A2M* as shown above.In contrast to controls, tumours of A2M*-treated animals showed a shrunken capsule together with multiple necrosis in different stages together with damaged tumour cells. The overall picture of the tumour was dominated by infiltrating macrophages in areas of tumour cell degeneration and at tumour margins . A2M*-treated TSCs showed a decline in the number of tumour cells with large areas of scar formation at all A2M* concentrations investigated. Immunostaining for activated caspase-3 showed only few cells within the tumour with a random distribution pattern. Staining for Ki-67 was higher in control TSCs than in A2M*-treated slices.Tumour tissues slice cultures (TSC) (350 μm) were prepared to eliminate the influence of remote organs on tumour behaviour in A2M*-treated animals. In control TSCs, the tumours were bordered by a fibrous capsule composed of tight connective tissue showing only few cells presenting signs of mitosis or apoptosis PTEN function is sufficient to down-regulate both PI3K and MAPK signalling and triggers dramatic tumour regression [. Particularly in 1321N1 astrocytoma cells, where PTEN expression is absent due to a mutation [ and mRNA level  and expectedly an increase of the expression of E-cadherin [-miR-21 level . MiR-21 is overexpressed in almost all solid tumours, where it is involved in the genesis and progression of human cancer [/PTEN/AKT signalling cascade.PTEN is a negative regulator of the intracellular levels of phosphatidylinositol-3,4,5-trisphosphate. Loss of this tumour suppressor gene is implicated in breast cancer progression and resistance to targeted therapies, and is thought to promote tumorigenesis by activating PI3K signalling . Restorigression . Here, wmutation , we founcadherin . To eluccadherin . We obsen cancer  and tumon cancer . Thus, A(P < 0.05) and GSK3B (P < 0.01) phosphorylation in tumour xenografts of A2M* treated mice . Analysis of PTEN expression revealed an increase of PTEN protein level by A2M* treatment although not reaching significance (P = 0.06) . These findings were in accordance with a decline in tumour cell proliferation measured by the number of Ki-67 positively stained nuclei .Analogously, we found significant inhibition of AKT 1/2) of 4 min [n = 3; P < 0.001)  In addition, the endogenous expression of liver A2M mRNA was inducible by zinc orotate  . We have re-enacted the animal studies in vitro using primary mouse hepatocytes and found that only transformed A2M* but not native A2M is able to up-regulate its own expression by positive feed-back activation .A2M* is rapidly removed from circulation by endocytosis through LRP1 with a half-life time .These findings raised the basic question, whether high concentration of mouse A2M on its own might be effectually enough for provoking anti-tumour effects. Therefore, we treated A549-xenotransplant bearing mice with zinc orotate (0.5 mg/g body weight) every second day. No effect in tumour growth was observed indicating that zinc substitution (GEO GSE106261) for completion of the in vitro analyses of A2M* treatment. Enrichment analysis  showed 55 DETs  down- and 74 up-regulated. Among those, the top five up-regulated DETs are transcripts of UBAP2L, ZNF786, DAPK1, EIF2B3 and WIPI2. The top five down-regulated transcripts belong to LRP12, miR-1296, PREP, LIN9 and NED1. DETs from genes such as RMI1 (up) and NDE1, LIN9, ANXA10 (down) involved in mitosis and cell cycle are regulated in a manner consistent with the induction of cell cycle arrest. Modulation of transcripts of genes such as ZPR1 (down) and THTPA (up) support the anti-proliferative effects of A2M*. Apoptosis related genes such as transcripts of DAPK1 (up) and NT5E (down), matched with the histochemical findings showing increased amounts of apoptotic cells in A2M* treated tissue slices and tumor xenografts. Obviously, the down-regulation of miR-1296 in A2M*-treated cells may not follow the overall anti-tumour activity of A2M* as this miR is known to up-regulate oncogene MCM2 [PTEN in most tumours, that might override the effect of A2M* at miR-1296. An interesting aspect concerns the modulation of transcripts of EIF2B3 and PREP. Both are involved in longevity and aging [EIF2B3 are known to make cells less tolerant to endoplasmic reticulum stress [EIF2B3 and down-regulation of PREP in samples treated with A2M* might probably explain some of the anti-aging effects observed in naked-mole rats [ARHGAP29 that belongs to the Rho GTPase activating proteins (ARHGAP) which are involved in brain development [The impact of A2M* 30 nM, 24h) on the transcriptome of cultured A549 cells was analysed by RNA-seq  nM, 24h nd aging . Malfuncm stress , and higm stress . The sigGEO GSE06261 forelopment , modulatelopment  and showelopment .Overall, the correlation of the A2M*-treated samples is on average 9.8% higher than the correlation of the control samples when comparing against the adjacent normal transcriptome of each Stage-vs-Normal pair of the TCGA lung adenocarcinoma samples of Cancer RNASeq Nexus , and on , we decided to perform transcriptome analysis of tumors from these xenografts (GEO GSE106989). Because no significant changes in PC-3 tumour tissues (adjusted P < 0.01) were observed, we decided to discriminate between human tumour PC-3 cells and mouse stroma. Considering the mouse specific reads, using the tool xenome for separation, we could identify 216 DEGs in A2M* treated mice compared to controls (FDR < 0.01), of which 156 were up- and 60 were down-regulated . Up-regulated genes primarily clustered in an induced immune-phenotype, being associated with the induction of the innate immune system. The top five fold-change wise up-regulated DEGs are ILDR1, ICOS, TOX, TIGIT, IFNG. This coincides with the occurrence of masses of macrophages in PC-3 xenograft slices and the up-regulation of IFNG . The most abundantly down-regulated DEGs in the tumour stroma cells are more heterogeneous. The top five fold-change wise were CRABP1, ELOVL3, PTPRZ1, PRG4 and LUM.Due to the strongest effects of A2M* at growth of PC-3 xenografts (GEO GSE107195). We identified 189 DEGs (FDR < 0.01) of which 86 were higher and 103 were lower expressed in A2M*treated mice liver compared to controls. The most fold-change wise up- and down-regulated liver DEGs were USP2 and CDKN1A, respectively . USP2 is associated with the clock proteins in the liver [p21  is down-regulated in the liver of A2M*-treated animals. The p21 is a negative prognostic marker in human hepatocellular carcinoma suggesting a role for A2M* in liver regeneration and cancer risk control [WEE1, which blocks CDK1 and CIART, PER2 and PER3 involved in the circadian rhythm [As systemic A2M is mainly synthesized in the liver, we analysed how A2M* regulates the liver transcriptome of tumour mice he liver . On the  control . Other sn rhythm , 40. Modn rhythm  and indun rhythm . Collectn rhythm  and trean rhythm . The senn rhythm . ImplemeP < 0.02) characteristic molecular pathways.Additionally, we built pathway activation profiles  for 388 in vitro and in vivo. Due to its fast endocytosis mediated by LRP1, it comprises one of the most important clearance systems of the extracellular space. Above all, it is involved in cell adhesion by modulation of the expression of cell surface molecules at protein and transcript level. Unexpectedly, A2M* interfered with one of the most important tumour suppressor, PTEN and its upstream modulator miR-21. A2M* was found to affect important genes not only of the tumour stroma but perturbs also important cell signalling pathways such as AKT that regulates diverse cellular processes including cell proliferation and survival, tissue invasion and angiogenesis. Many oncoproteins and tumour suppressors converge with AKT signalling transduction in an equilibrium that is altered in many human cancers by activating and inactivating mechanisms. Our results suggest that A2M* may guard this equilibrium by intervening at several cellular levels simultaneously. This is supported by exploring the effect on liver genes showing the up-regulation of branches of the AKT and RAS signalling pathways promoting cell survival and cell cycle progression, which may be considered as compensatory pro-survival mechanism, which is in contrast to its effect on tumour cells.A2M showed an extensive history as a protease inhibitor, immune modulator, and scavenger of growth factors, hormones and cytokines . Here, wBMAL1 (ARNTL) was recently characterized as tumour suppressor that increases sensitivity of cancer cells to chemical drugs  [For the first time we disclosed the implication of A2M* in the regulation of circadian clock genes . The mecbstract) .The overall observed strong tendency to induce cell cycle arrest in A2M*-treated cells together with the regulation of circadian clock genes in liver of A2M*-treated mice is an indicator for the known coupling of the two oscillating systems internal clock and cell cycle .All data indicate that A2M fulfils important regulatory functions at the cellular level and among organs. As we showed earlier the blood level of A2M decreases significantly with age . AssuminA2M gene promotor may explain the effect of external zinc supplementation on its expression [Here we found that the degree of supplementation with zinc might obviously monitor the A2M level in blood. This is all the more important as A2M is the main zinc transporter in blood  deliveripression .A2M in the blood and only minute amount forms complexes with proteases (A2M*) in tissue or blood [Our study strongly accentuates the requirement of activated A2M* for inhibition of tumour growth. In humans, native A2M represents approximately 99% of total or blood . As A2M or blood .S1 Fig(a) A2M was purified from human plasma and separated by native PAGE (4–20). (b) Purified native and transformed A2M (A2M*) were separated by Rate-PAGE into native (slow-migrating) and transformed (fast-migrating) A2M. (c) Three purified A2M preparations were separated by SDS-PAGE. The A2M* band was subjected to trypsin digestion and analysed by mass spectrometry to confirm the presence of A2M and to identify further co-migrating proteins. (d) Evidence for receptor binding of A2M*. LRP1 was coated to 96-well plates and incubated with increasing concentrations of A2M and A2M*. Bound A2M/A2M* was detected by HRP-labelled polyclonal anti-A2M-Ig. (e) Receptor-associated protein (RAP) (0–50 nM) inhibits binding of A2M* (10 nM) to immobilized LRP1. (f) Analysis of three A2M preparations for their stimulatory effect on human blood monocytes using the whole blood assay. Heparinized blood was incubated with medium (control), 10 ng/mL LPS and three purified A2M samples , 2respectively, at 5% CO, 37°C for 8h. Cells were centrifuged and the supernatant was analysed for TNF-alpha using cytometric bead array (CBA) (n = 3). Alb = albumin; Trf = transferrin, A2M = native A2M, A2M* = transformed A2M, RAP = receptor-associated protein.(DOCX)Click here for additional data file.S2 Fig(a) Coarse of body weight of tumour-bearing A549 mice treated with A2M* (n = 10) compared to control (n = 9). (b) EDTA blood was withdrawn from A549 tumour bearing mice and analysed in a ScilVet apparatus . Blood cells were counted at day 7 after tumour induction (control) and day 31 after A2M* treatment. WBC–white blood cells, RBC–red blood cells, HGB—Hemoglobin, HCT–Hematocrit value, MCV–mean corpuscular volume, MCH–mean corpuscular hematocrit, PLT—platelets, MPV–mean platelet volume, RDW–red cell distribution width, LYM–Lymphocytes, MO—Monocytes, GRA—Granulocytes, (n = 9), . (c), Effect of A2M* on mouse spleen cells. Spleen cells from A549 tumour-bearing mice treated with A2M* were isolated, stimulated with 10 nM lipopolysaccharide (LPS) or PBS (control) and cytokines were measured by cytokine bead arrays (CBA). (n = 10) (**P < 0.01). Error bars represent mean ± s.d.(DOCX)Click here for additional data file.S3 Fig(a) Peripheral compartment of PBS treated tumour in overview. (b) Compact tumour organization with a few cells yielding apoptotic signs. (c) Tumour cells in a small area of tumour destruction (+) and cells with signs of apoptosis (arrow). (d) Dispersed vital A549 cells with few cells showing signs of degradation. (e) Peripheral compartment of an A2M*-treated tumour in overview. (f) Necrotic area (*) with macrophage accumulation the tumour tissue (arrow). (g) Low number of vital tumour cells paralleled by massive loss of tumour cytoarchitecture. (h) Loss of tumour tissue (*) accompanied by accumulation of macrophages (arrow). Scale bar: 300 μm (a and e), 100 μm .Hematoxilin-eosin (HE) stained A549 tumour slices obtained from PBS-treated animals  and A2M*-treated animals (e-h). (DOCX)Click here for additional data file.S4 Fig(a) Peripheral compartment of PBS treated tumour in overview. (b) Compact tumour organization with a few cells yielding apoptotic signs. (c) Tumour cells in a small area of tumour destruction (+) and cells with signs of apoptosis (arrow). (d) Dispersed vital A549 cells with few cells showing signs of degradation. (e) Peripheral compartment of an A2M*-treated tumour in overview. (f) Necrotic area (*) with macrophage accumulation the tumour tissue (arrow). (g) Low number of vital tumour cells paralleled by massive loss of tumour cytoarchitecture. (h) Loss of tumour tissue (*) accompanied by accumulation of macrophages (arrow). Scale bar: 300 μm (a and e), 100 μm .Hematoxilin-eosin (HE) stained A549 tumour slices obtained from PBS-treated animals  and A2M*-treated animals (e-h). (DOCX)Click here for additional data file.S5 Fig(a-c) Liver of scarified mice were homogenized and analysed for A2M protein content and RNA by qRT-PCR and Western blotting. (d) Balb/c mice were injected with A2M* (5.6 mg/20g body weight), sacrificed after indicated times and the expression of mice A2M in the liver was analysed by qRT-PCR (n = 3 for each time point). (e) Balb/c mice were given a bolus injection of zinc orotate (0.5 mg/kg) (SigmaAldrich), and mouse A2M gene expression in the liver was determined by qRT-PCR. (f) Primary murine hepatocyte cultures from Balb/c mice were stimulated with native and transformed human A2M* (0–100 nM) for 24h followed by qRT-PCR for mouse A2M (n = 3).(DOCX)Click here for additional data file.S1 TableP < 0.01) and additional (P > 0.01). Full list of regulated transcript can be found at GSE 106261.TPM counts for regulated transcripts in A2M*-treated cells; explicitly mentioned in the text ((DOCX)Click here for additional data file.S2 Table(A) Correlation between the individual stages I through IV of the lung adenocarcinoma samples of the CRN and both sample groups (A2M* and PBS). (B) Correlation against the adjacent normal of the CRN against both sample groups.The Pearson correlation coefficients between the average transcript expressions of the A549 A2M*-treated sample groups against the Cancer RNASeq Nexus (CRN), as well as the correlation between the average transcript expressions of the A549 controls (PBS) and the CRN. (DOCX)Click here for additional data file.S3 TableRPKM counts for regulated genes in tumour stroma samples of A2M*-treated mice; explicitly mentioned in the text, including the top 10 up- and down-regulated genes. Full list of regulated genes can be found at GSE 106989.(DOCX)Click here for additional data file.S4 TableRPKM counts for regulated genes in livers of A2M*-treated mice; explicitly mentioned in the text, including the top 10 up- and down-regulated genes. Full list of regulated genes can be found at GSE 107195.(DOCX)Click here for additional data file.S5 TableObserved Pathway Activation Strength (PAS) for regulated pathways in livers of A2M*-treated mice.(DOCX)Click here for additional data file.S6 TableList of primary and secondary antibodies used for Western blotting, immunohistochemistry and flow cytometry.(DOCX)Click here for additional data file.S7 TableList of forward and reverse primer pairs used for analysis of gene expression by RT-PCR and qPCR.(DOCX)Click here for additional data file.S1 Protocol(DOCX)Click here for additional data file.


CYP4F2 (Cytochrome P450 Family 4 Subfamily F Member 2) polymorphisms and the susceptibility to cardiovascular and cerebrovascular diseases.Inconsistent conclusions have been reported for the genetic relationship between CYP4F2 in the risks of cardiovascular and cerebrovascular diseases. The retrieval of four databases, including PubMed, Web of Science (WOS), China National Knowledge Infrastructure (CNKI) and WANFANG DATA, was conducted. Mantel-Haenszel statistics for association test, Cochran’s Q statistic, sensitivity analysis for heterogeneity assessment, and Begg’s/Egger’s tests for publication bias evaluation were performed under allele, homozygote, heterozygote, dominant, and recessive models, respectively.We performed a meta-analysis to assess the potential role of rs1558139 C/T and rs2108622 G/A polymorphisms of P values in association tests < 0.05, odds ratio (OR) < 1]. For rs2108622, a decreased coronary artery disease (CAD) risk was observed in the subgroup meta-analysis based on disease type under all genetic models . Begg’s/Egger’s tests excluded the potential publication bias, while sensitivity analysis data supported the stability of the above results.A total of 597 articles were initially obtained by database searching, and twenty eligible articles were finally included. For rs1558139, a decreased risk of cardiovascular and cerebrovascular diseases was observed in the overall meta-analysis and in “hypertension”, “population-based” and “male” subgroups under models of T vs. C, CT vs. CC, and CT + TT vs. CC [all CYP4AF2 rs1558139 may be linked to the decreased risk of hypertension in the male patients of Asian populations, while CYP4F2 rs2108622 is likely associated with reduced susceptibility to CAD.C/T genotype of The online version of this article (10.1186/s12872-018-0763-y) contains supplementary material, which is available to authorized users. CYP17A1 (Cytochrome P450 Family 17 Subfamily A Member 1) gene or rs2681492 of the ATP2B1 (ATPase Plasma Membrane Ca2+ Transporting 1) gene, are associated with susceptibility to blood pressure or essential hypertension [CYP4F2 (Cytochrome P450 Family 4 Subfamily F Member 2) polymorphism in the risk of cardiovascular and cerebrovascular diseases, such as myocardial infarction (MI), coronary artery disease (CAD), ischemic stroke (IS), cerebral infarction (CI) and hypertension.Single nucleotide change-induced nucleic acid sequence polymorphisms, namely, single nucleotide polymorphisms (SNPs), occur more frequently in the human genome, with an average of approximately one polymorphism per 1000 bases, and functional SNPs may help to guide population genetics research, as well as the mechanistic, epidemiologic or diagnostic study of some clinical genetic diseases . EmerginCYP4F2 gene is located on chromosome 19 and contains 12 introns and 13 exons [CYP4F2 gene, while rs2108622  is located in the exon region of this gene [Human 13 exons . There a13 exons , 9. The his gene . IncreasCYP4F2 SNPs and the risk of cardiovascular and cerebrovascular diseases via meta-analysis based on the publicly published data. To our knowledge, one meta-analysis of hypertension [CYP4F2 rs2108622. However, no meta-analysis for the other SNPs of the CYP4F2 gene have been reported. Additionally, there is no meta-analysis regarding the role of CYP4F2 rs2108622 in the risk other cardiovascular and cerebrovascular diseases.Considering the conflicting conclusions, we quantitatively measured the genetic correlation between rtension  and anotrtension  were preIn accordance with the methodology of “Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)” , we perfCYP4F2 gene in each case/control group. The following exclusion criteria were considered: a) animal or cell experiments; b) other genes/diseases or non-SNP gene variants; c) meeting abstract; d) meta-analysis; e) review or letter; f) undetected variants; g) the absence of genotype frequency data; h) duplicated articles; and i) not in HWE (Hardy-Weinberg-Equilibrium).After screening, eligible case-control studies provided the sufficient genotype frequency data of 2 and P values of HWE, and source of control. The paper quality was assessed by the Newcastle-Ottawa Scale (NOS) system [We extracted the basic information from the eligible articles, namely, first author, year, ethnicity, genotyping assay, SNPs, genotype frequency, disease type, gender, mean age, study number, sample size of the case/control, X) system , which iP value of association test was generated under the allele, homozygote, heterozygote, dominant, and recessive models. A fixed-effect model was used when the P value of heterogeneity from Cochran’s Q statistic was larger than 0.05 or I2 less than 50.0%. Subgroup meta-analyses by ethnicity (Caucasian/Asian), disease type (hypertension/CAD/IS/CI/MI), source of control , and gender , were also conducted through Mantel-Haenszel statistics. For publication bias assessment, Begg’s/Egger’s tests were performed. Stata/SE software version 12.0  was used for the above statistical analyses.Based on Mantel-Haenszel statistics, the value of OR (odds ratio), 95% CIs  and CYP4F2 gene (rs1558139 C/T and rs2108622 G/A) were analyzed. The NOS system data suggested that no included studies showed poor quality, as all NOS scores were larger than five , and fixed-effect model was applied. In pooled data from overall meta-analysis . Moreover, similar results were observed under allele, heterozygote and dominant genetic models in “hypertension” and “population-based” subgroups . We also performed a subgroup analysis based on gender. As shown in Additional file P value in association tests < 0.001, OR = 0.79), homozygote , heterozygote  and dominant  genetic models. Figure CYP4AF2 rs1558139 might be associated with a reduced risk of hypertension in the male patients of Asian populations.We first conducted a meta-analysis to analyze the association between CYP4AF2 rs2108622 G/A polymorphism and the risk of cardiovascular and cerebrovascular diseases was subsequently performed. A total of 17 case-control studies were enrolled [P value of heterogeneity < 0.05). A decreased CAD risk was observed in the subgroup meta-analysis by disease type under all genetic models . However, no significant associations were observed for other factors , suggesting the absence of large publication bias. Fig. CYP4AF2 rs2108622 G/A polymorphism. Furthermore, we observed the stability of the above data based on the sensitivity analysis , partly by CYP4F2 protein, 20-HETE induces the transcription of ACE (angiotensin-converting enzyme) gene through stimulating the NF-ΚB pathway and contributes to the pathophysiology of hypertension [CYP4AF2 rs1558139, located in an intron region, likely affects the enzyme activity of CYP4F2 protein during 20-HETE metabolism through altering the gene transcription level, RNA secondary structure or RNA splicing of the CYP4AF2 gene, which is worth further investigation of the molecular mechanism. In addition, CYP4F2 rs2108622 was tightly associated with systolic blood pressure as well as the excretion of 20-HETE [CYP4F2 rs2108622 in hypertension risk.in India  or Northin India . Howeverin India . In 2015in India , 23, 32 ion risk . Here, eion risk , 29, 32 CYP4AF2 gene rs2108622 polymorphism was associated with the risk of coronary artery disease in a Han Chinese population [CYP4AF2 rs2108622 and coronary heart disease risk was reported in Mongolian cases in China [CYP4AF2 rs2108622 polymorphism is likely associated with the reduced susceptibility to coronary artery disease. Here, the meta-analysis provided evidence of the involvement of CYP4AF2 rs2108622 in susceptibility to coronary heart disease. Given the reported correlation between CYP4AF2 rs2108622 and 20-HETE [CYP4AF2 rs2108622 may mediate the nonsynonymous mutation of a valine (V) located at residue 433 to a methionine (M), namely, V433 M, which then leads to the alteration of CYP4F2 protein function and 20-HETE synthesis, thereby influencing the occurrence of coronary heart disease.Coronary heart disease results from the interplay between polygenic predisposition and complex environmental factors . The CYPpulation . Howeverin China . In the  20-HETE , functioCYP4AF2 SNPs and ischemic stroke risk. For example, no detectable difference was reported by Liao, et al. for the genotype distribution of CYP4AF2 rs2108622 between the control and cases of ischemic stroke [CYP4AF2 rs2108622 SNP as an important risk factor for ischemic stroke in an Indian population [P < 0.0001 [CYP4AF2 gene rs2108622 polymorphism. Different searches and analysis strategies might contribute to this finding.The early evaluation of genetic risk factors is essential for the effective diagnosis and treatment of clinical ischemic stroke , 37. We c stroke . Howeverpulation . In 2015pulation , enrollipulation , 30, 38,< 0.0001 . In the < 0.0001 , and fin< 0.0001 , 26, 30 CYP4AF2 rs1558139 C/T polymorphism and risk of cardiovascular and cerebrovascular diseases was obtained under allele, heterozygote and dominant genetic models. In addition, sensitivity analysis indicated the stability of the statistical results. Nevertheless, the small sample size of the included case-control studies, to a certain extent, limits the statistical power. Limited data were extracted for the CYP4AF2 rs1558139 C/T polymorphism subgroup with the diseases of ischemic stroke or myocardial infarction.We found that not all control data in these studies were population-based. After removing studies with hospital-based control data, the same positive correlation between the CYP4AF2 rs1558139 polymorphism, high heterogeneity was observed in most of the overall meta-analyses of CYP4AF2 rs2108622. Apart from coronary heart disease and hypertension subgroup analyses, high heterogeneity still existed in other “Asian”, “ischemic stroke” and “population-based” subgroups. The complexity of disease feature might be the source of the large publication bias. In addition, unpublished data or articles in other languages may bias the selection of the studies analyzed.Although no high degree of heterogeneity was observed in all meta-analyses of the CYP4AF2 rs1558139 and hypertension risk, while six studies [CYP4F2 rs2108622.Given the complicated etiologies in different types of cardiovascular and cerebrovascular diseases , 39, add studies , 27, 32 CYP4F2 gene, such as rs3093100, rs3093105, rs3093166, and rs3093135. The “G/G/G/T” haplotype of rs2108622-rs3093100-rs3093105-rs3093135 has been associated with an increased risk of coronary artery disease, however the “G/G/T/A” haplotype was associated with a reduced risk of coronary heart disease [CYP4F2 and other genes, such as CYP4A11 (Cytochrome P450 Family 4 Subfamily A Member 11), should be analyzed upon the publication of sufficient data.Adequate data on the genotype frequencies of both case and control studies are necessary to conduct SNP meta-analyses or subgroup analyses. Thus, only two SNPs  were studied in the present meta-analysis based on the available data. We did not examine the role of other variants within the CYP4AF2 rs1558139 polymorphism might serve as a protective factor for male patients with hypertension in Asian populations, and CYP4AF2 rs2108622 may confer reduced genetic susceptibility to coronary heart disease.Overall, the C/T genotype of the Additional file 1: Table S1-S7.Table S1. Details of the search strategy based on four databases. Table S2. Basic information of the eligible studies in the meta-analysis. Table S3. Data of NOS assessment system. Table S4. Genotype frequencies of the eligible studies in the meta-analysis. Table S5. Genotype frequencies of the male/female group in some eligible studies. Table S6. Subgroup analysis by gender for the association between CYP4AF2 rs1558139 and hypertension risk. Table S7. Subgroup analysis by gender for the association between CYP4AF2 rs2108622 and hypertension risk. (DOCX 63 kb)Additional file 2: Figure S1.CYP4AF2 rs1558139 polymorphism and the risk of cardiovascular and cerebrovascular diseases under the allele (T vs. C) model.. (TIFF 887 kb)Subgroup analysis by disease type of the association between the Additional file 3: Figure S2.CYP4AF2 rs1558139 polymorphism and the risk of cardiovascular and cerebrovascular diseases under the dominant (CT + TT vs. CC) model. (TIFF 967 kb)Subgroup analysis by disease type of the association between the Additional file 4: Figure S3.CYP4AF2 rs1558139 polymorphism and the risk of cardiovascular and cerebrovascular diseases under the allele (T vs. C) model. (TIFF 1958 kb)Subgroup analysis by gender of the association between the Additional file 5: Figure S4.CYP4AF2 rs2108622 polymorphism and the risk of cardiovascular and cerebrovascular diseases under the homozygote (AA vs. GG) model. (TIFF 1760 kb)Subgroup analysis by disease type of the association between the Additional file 6: Figure S5.CYP4AF2 rs2108622 polymorphism and the risk of cardiovascular and cerebrovascular diseases under the heterozygote (GA vs. GG) model. (TIFF 1717 kb)Subgroup analysis by disease type of the association between the Additional file 7: Figure S6.CYP4AF2 rs2108622 polymorphism and the risk of cardiovascular and cerebrovascular diseases under the dominant (GA + AA vs. GG) model. (TIFF 1689 kb)Subgroup analysis by disease type of the association between the Additional file 8: Figure S7.CYP4AF2 rs2108622 polymorphism and the risk of cardiovascular and cerebrovascular diseases under the recessive (AA vs. GG + GA) model. (TIFF 1792 kb)Subgroup analysis by disease type of the association between the Additional file 9: Figure S8.CYP4AF2 rs2108622 polymorphism and the risk of cardiovascular and cerebrovascular diseases under the allele (A vs. G) model. (TIFF 1045 kb)Subgroup analysis by gender of the association between the