The eukaryotic F-box protein family is characterized by an F-box motif that has been shown to be critical for the controlled degradation of regulatory proteins. We identified agene encoding an F-box protein from a cDNA library of silkworm pupae, which has anORF of 1821 bp, encoding a predicted 606 amino acids. Bioinformatic analysis on theamino acid sequence shows that BmFBXO21 has a low degree of similarity to proteinsfrom other species, and may be related to the regulation of cell-cycle progression. Wehave detected the expression pattern of BmFBXO21 mRNA and protein and performedimmunohistochemistry at three different levels. Expression was highest in the spinningstage, and in the tissues of head, epidermis, and genital organs. Caenorhabditis elegans with most also conserved in yeast, but only about half of the human FBXO class of proteins are conserved in nematodes or yeast [F-box proteins are an expanding family of eukaryotic proteins characterized by an amino-acid motif called an F-box , 2. The or yeast , 7. Feweor yeast –11.Present studies have found that F-box proteins play an important role in mediating special degradation to ubiquitylated substrates, associating a phosphorylation signal pathway in the cell with ubiquitin-mediated proteolysis, and participating in many important biological processes, such as cell cycle progression, differentiation and development, signal transduction and transcriptional regulation, and silencing –19. F-boF-box proteins have important regulatory roles, and the identification of novel F-box genes and their function has vital significance to the study of the regulative role of this family. In this study, we identified an F-box gene from a silkworm pupae cDNA library . The GenEscherichia coli strains TG1 and BL21 (DE3) were grown at 37°C in LB medium. The Bombyx mori strain was the offspring of Jingsong × Haoyue. Silkworms were reared on mulberry leaves under standard conditions. Heads, silk glands, guts, malpighian tubules, testes, ovaries, fat bodies, and the epidermis were dissected from spinning silkworms, frozen immediately in liquid nitrogen and stored at −70°C. Whole spinning silkworms were also frozen in liquid nitrogen and stored at −70°C.http://www.expasy.org/cgi-bin/protscale.pl and http://au.expasy.org/prosite/, respectively. NetPhos (http://www.cbs.dtu.dk/services/NetPhos/) was used to predict the serine, threonine, and tyrosine phosphorylation sites.Similarity analyses for nucleotide and protein sequences were carried out using GenBank BLASTN and BLASTP algorithms. Multiple alignments and homology analysis of BmFBXO21 were conducted using the program CLUSTAL-W from the software Bioedit. The hydrophobicity analysis and the functional sites prediction were carried out on ExPASy websites, Bam HI and Xho I, respectively. The PCR products were purified using the PCR Rapid Purification Kit , and after digestion by Bam HI/Xho I (Promega) the fragments were cloned into the expression vector pET-28a and transformed into E. coli TG1 competent cells. A positive colony with the BmFBXO21 plasmid was identified by PCR and double digestion of the plasmid.The 687 bp 3′ fragment of BmFBXO21 cDNA, previously isolated and cloned from metaphase pupae by our laboratory , was useE. coli BL21 (DE3) competent cells, which were incubated at 37°C in liquid LB culture media containing 50 μg/mL kanamycin. Expression of the His-tag fusion protein was induced at an A600 of 0.6 followed by adding IPTG  to a final concentration of 1 mM before another 5-hour incubation. A 5 mL Ni2+-SephadexTM G-25 Superfine column (Amersham) was used to purify the expressed recombinant BmFBXO21 (rBmFBXO21) protein, as instructed by the manufacturer. The eluted target protein was subsequently concentrated and desalted by dialysis. The presence and purity of BmFBXO21 were evaluated by 12% SDS-PAGE and quantified by the Bradford method [The recombinant plasmid pET-28a-BmFBXO21 was transformed into d method . The purThe polyclonal antisera were divided and purified using His-Trap Chelatings HP (Amersham) following the manufacturer's instructions. Indirect ELISA was used to detect the titer of antibody, with negative rabbit sera as control. Western blot analysis was used to evaluate the specificity of polyclonal antibodies with extracts of recombinant plasmid and silkworm pupa, respectively .μg/mL Aprotin (Sigma)), then incubating for 30 minutes on ice. The homogenates were centrifuged at 12 000 × g for 15 minutes at 4°C. The supernatants contained whole proteins and were quantified by the Bradford method. Samples were equalized and electrophoresed by 10% SDS-PAGE and electro-transferred onto polyvinylidene difluoride (PVDF) membranes. After blocking with 3% nonfat milk in phosphate-buffered saline (PBS) at 4°C overnight, the membranes were probed with rabbit anti-BmFBXO21 antibody at room temperature for 2 hours. After washing with PBST (PBS + 0.05% Tween-20) the membranes were incubated with antirabbit IR Dye 700 and detected with Infrared Laser Imaging System 9201F (Licor).Embryos, larvae, spinning silkworms, pupae and moths were collected. Spinning silkworm organs samples were ground to powder in liquid nitrogen followed by suspending in buffer M  under the following conditions: an initial cycle at 50°C for 3 minutes, one cycle at 95°C for 5 minutes, followed by 40 cycles of 95°C for 15 seconds, 59°C for 15 seconds, and 72°C for 30 seconds. Each reaction was performed in triplicate in 96-well plates, with the endogenous 18S rRNA control gene. Dissociation curves were performed to check for the presence of nonspecific dsDNA SYBR Green hybrids, such as primer dimers. Data analysis was performed using ABI Prism 7300 SDS Software V1.3.1 . Expression levels of the target genes were normalized against the expression level of the 18S rRNA gene. The relative expression level was calculated using 2−ΔΔCT, where ΔCT = CT(target gene)－CT(18S rRNA), ΔΔCT = ΔCT(target gene)－ΔCT(maximum) [Total RNA was extracted from tissues of spinning silkworms using Trizol reagent (Invitrogen) according to the manufacturer's instructions. Contaminating genomic DNA (gDNA) was removed by DNase I (Invitrogen). The purity of extracted RNA was determined by UV spectrophotometer. Ratios of UV 260/280 were between 1.8 and 2.1 for all RNA samples analyzed. The concentration of total RNA was determined by measuring the absorbance at 260 nm using SPECTRA max PLUS384 (Molecular Devices). RT-PCR primers were designed using the Primer Select program of the DNA STAR software. The primer pairs were as follows: FBXO21 Forward CAATGCCCTTCACTGTTCTCA, Reverse TGTGTCCAAATTCTAATCTGTTCA;18S rRNA Forward CGATCCGCCGACGTTACTACA, Reverse GTCCGGGCCTGGTGAGATTT. SuperScript III Platinum SYBR Green One-Step RT-PCR Kit with ROX (Invitrogen) was used. 15 maximum) .μm thick) were attached to microscope slides, treated with cold acetone, washed three times in PBS for 15 minutes, treated with 5% BSA in PBS plus Triton X-100 (PBST) for 1 hour, and incubated for 2 hours with purified anti-BmFBXO21 (1 : 500). After three washes in PBST, sections were incubated with Alexa Fluor 488 conjugated antirabbit IgG (1 : 500) for 45 minutes, washed three times in PBST and twice in PBS, and then observed by fluorescence microscopy at 488 nm.Following liquid nitrogen treatment, spinning silkworms were embedded with OCT compound at temperatures below −20°C. Sections  encoding a protein identified as an F-box only protein by Genbank BLAST. Through BLASTP comparison, a conservative structural domain of the YccV-like superfamily was discovered in at amino acids 470–560. YccV is a hemimethylated DNA binding protein that regulates the expression of dnaA [Apis mellifera, Mus musculus, Canis lupus familiaris, Sus scrofa, Pongo abelii, and Homo sapiens revealed that BmFBXO21 had a low degree of similarity to the F-box only proteins of other species. The homology with Apis mellifera was 24%, while with others it was 23%. Nonetheless, several amino acid positions, specifically position 57 (L), position 58 (P), position 65 (I), position 66 (L), position 76 (I), position 81 (S) (Comparison of the deduced amino acid sequence of the BmFBXO21 protein with those of six F-box only proteins from n 81 (S)  were theWe predicted the hydrophobicity of the target protein BmFBXO21 using the ProtScale tool of ExPASy. The results showed that the maximum hydrophobicity value of BmFBXO21 protein was 2.489, while the minimum was −3.078; amino acids 250 ~ 300 amino acid were the most hydrophobic region. The potential function tool of PROSITE predicted one F-BOX domain in this region, at residues 4–51. We predicted the phosphorylation sites using NetPhos tool, with the following results: S6, S95, S119, S204, S229, S233, S292, S343, S350, S352, S412, S469;T172, T234, T251, T327, T480;Y82, Y99, Y145, Y158, Y228, Y261, Y413, Y428, Y444, Y457, Y470, Y486, Y593.E. coli, and after Ni2+ affinity chromatography, recombinant BmFBXO21 was highly purified as detected by electrophoresis  ≥ 2.1 was positive; 1.5 ≤ P/N < 2.1 was suspicious expression; P/N < 1.5 was negative. From this, the titer of the antibodies was greater than 1 : 12800 at a concentration of 10 E. coli, that had a molecular weight of only 30 kD , spinning silkworm, pupa, and moth. We performed Western blot analyses on protein extracts to determine BmFBXO21 expression levels. The highest amount of expressed BmFBXO21 was in the spinning silkworm, decreasing in the pupa and egg; no BmFBXO21 was expressed in larval instars or moth .In order to detect BmFBXO21 protein expression levels in each tissue, we extracted protein from epidermis, head, silk gland, gut, ovary, testis, Malpighian tubule, trachea, and fat body of spinning silkworms and used Western blots to determine the levels of BmFBXO21 in each tissue. Immunoblots of these protein extracts revealed that anti-BmFBXO21 serum reacted with a 70 kD protein in extracts from the testis, ovary, gut, head, and epidermis . To determine transcription levels of BmFBXO21 in these tissues, we isolated total RNA from the spinning silkworm tissues and used it to perform RT-PCR. From the melting curves (data not shown), we established, there were no overt primer dimers, indicating good primer specificity. The amplification curves (data not shown) indicated excellent reproducibility. Our results showed that BmFBXO21 mRNA was highest in head, lower in testis, epidermis, gut, ovary, trachea, fat body, Malpighian tubule, and lowest in silk gland .B. mori (insect outline) has spontaneous green fluorescence, we observed no obvious difference in the epidermis between the samples and the negative control.To determine the tissue-specific localization of BmFBXO21, frozen sections were prepared from fresh spinning silkworms quick-frozen in liquid nitrogen and subjected to immunohistochemical analysis with purified anti- BmFBXO21 IgG as the primary antibody. We found that BmFBXO21 was expressed in the posterior part of head, periphery of gut, and periphery of testis . BecauseSequence analysis and homology comparisons with the predicted BmFBXO21 amino acid sequence indicated amino acid residues that are highly consistent with the conserved sites of the F-box motif. In addition, an F-BOX domain was found in the amino-terminal part of the protein by ExPASy tool. This suggests that BmFBXO21 is a protein of the F-box family, specifically an F-box only protein. Homology analysis showed that BmFBXO21 had a low degree of similarity, generally 20–30%, to proteins from other species. Some of the conserved sites were in the F-box motif, and others in the carboxy-terminal part of the protein, where they may be necessary for binding specific substrates.4) to target proteins by covalent bond. This may occur at many kinds of amino acids, mainly serine, threonine, and tyrosine. Phosphorylation is a major mechanism for signal transmission, and phosphorylation and dephosphorylation participate in many cell processes, such as signal transformation, gene expression, and the cell cycle [The prediction of phosphorylation sites using NetPhos tool showed BmFBXO21 had 30 potential phosphorylation sites: 12 serines, 5 threonines, and 13 tyrosines. Phosphorylation is the reaction of attaching a phosphate group  cell fission proliferation (cell number increase), in which growth mainly depends upon cell fission to increase the number of congener cells, although the sizes of the cells are essentially invariable, such as blood cells and sperm cells; (2) increasing cell size, in which cells that divide during the embryonic development begin in the larval stage to increase in volume to achieve tissue growth; examples are silk gland, salivary gland, glandula exuviae, and vasa mucosa; (3) both cell fission and size increase, with most tissues belonging to this kind of growth, including fat body, body wall dermis, and epithelial cells –39. In oB. mori is an important and widely used model organism, so study in this organism will contribute to understanding the physiological functions of this F-box only protein, and the broader F-box family.This study analyzes the molecular characterization and tissue location of an F-box only protein, providing important evidence to support further study of BmFBXO21.


Multiple sclerosis (MS) remains without an effective treatment in spite of intense research efforts. Interferon-beta (IFN-β) reduces duration and severity of symptoms in many relapsing-remitting MS patients, but its mechanism of action is still not well understood. Moreover, IFN-β and other available treatments must be given parenterally and have a variety of adverse effects. Certain naturally occurring flavonoids, such as luteolin, have anti-oxidant and anti-inflammatory effects, including inhibition of activated peripheral blood leukocytes from MS patients. Luteolin also inhibits mast cells, as well as mast cell-dependent T cell activation, recently implicated in MS pathogenesis. Moreover, luteolin and structurally similar flavonoids can inhibit experimental allergic allergic encephalomyelitis (EAE), an animal model of MS in rodents. An appropriate luteolin formulation that permits sufficient absorption and reduces its metabolism could be a useful adjuvant to IFN-β for MS therapy. This issue includes an interesting article by Sternberg et al. showing that the flavonoid luteolin inhibits IL-1, TNF and metalloproteinase-9 (MMP-9) release from activated peripheral blood mononuclear cells (PBMCs) from multiple sclerosis (MS) patients, and that the effect of luteolin is augmented by concurrent administration of interferon-beta (IFN-β). This paper extends previous similar results with quercetin that required higher concentrations of the flavonoid [Luteolin with or without IFN-β, could be helpful in MS by not only inhibiting PBMC release of cytokines, but also by inhibiting T cells, which we recently showed can be superstimulated by mast cells, an action also inhibited by luteolin . In addiBrain MS plaques also contain activated mast cells ,10, whic2 [Flavonoids such as quercetin have potent anti-oxidant and anti-inflammatory activity . Quercet2 , as well2 ,29. More2  leading 2 , and fro2 . Flavono2 , as well2 -36.Quercetin and its structurally related luteolin are safe . The facTCT has been awarded US patents No 6,689,748; 6,984,667 and EPO No 1365777 that cover the use of flavonoids in inflammatory diseases; he has also filed (3/30/04) US patent applications No. 10/811,826; 11/214,831; 11/999,991; 12/151,268 specifically covering combinations of flavonoids, including luteolin with INF-β, for the treatment of MS.


Cytoplasts and karyoplasts were obtained by ultracentrifugation of Hepatoma D23 cells on a Ficoll gradient containing cytochalasin B. Their nuclear and protein content and their metabolic activity were determined. Three i.p. injections of 2.3 x 10(7) cytoplasts were unable to protect syngeneic WAB/Not rats against an s.c. challenge of 10(4) D23 cells, whereas a similar amount of karyoplasts, or 3 injections of 10(6) irradiated D23 cells, were fully protective. Ability of cytoplasts to act as primary or secondary immunogen were also studied, and compared to that of 0.01% glutaraldehyde-treated cells, 43 degrees C heat-treated cells and 3M KCl-soluble extracts, these preparations also being of weak immunogenicity. Only heat-treated cells behaved as a primary immunogen, whereas none of the preparations provided a secondary stimulation. Moreover, when these preparations were fed in vitro to peritoneal-exudate cells before their injection into rats, cytoplasts and glutaraldehyde-treated cells showed no immunogenicity, whereas heat-treated cells induced full protection against tumour challenge. Therefore, in this tumour model, the in vivo persistence of immunogen and the presence of a nucleus are likely to be crucial in inducing transplantation resistance to tumour.


There is increasing evidence to suggest that COPD is also characterized by systemic inflammation. The primary goal of this study was to identify soluble proteins in plasma that associate with the severity of airflow limitation in a COPD cohort with stable disease. A secondary goal was to assess whether unique markers associate with diffusion impairment, based on diffusion capacity of carbon monoxide (DLCO), independent of the forced expiratory volume in 1 second , granulocyte- colony stimulating factor (G-CSF) and IL-13 showed inverse relationships with the % DLCO.Plasma concentrations of CCL2/monocyte chemoattractant protein-1 (CCL2/MCP-1), CCL4/macrophage inflammatory protein-1β (CCL4/MIP -1β), CCL11/eotaxin, and interleukin-13 (IL-13) were inversely associated with the % FEV1.Systemic inflammation in a COPD cohort is characterized by cytokines implicated in inflammatory cell recruitment and airway remodeling. Plasma concentrations of IL-13 and chemoattractants for monocytes, T lymphocytes, and eosinophils show associations with increasing severity of disease. Soluble Fas, G-CSF and CXCL9/Mig may be unique markers that associate with disease characterized by disproportionate abnormalities in DLCO independent of the FEV Chronic obstructive pulmonary disease (COPD), while defined by the presence of incompletely reversible airflow obstruction, represents a syndrome of various physiologic impairments ,2. COPD The best characterized systemic marker is C-reactive protein (CRP) ,4, but i1 or % DLCO, during stable disease and plasma concentrations of 25 different cytokines and growth factors. We adjusted for current cigarette smoking and corticosteroid use because others have shown that these factors may be potential modifiers of systemic inflammation in this cohort [We conducted an exploratory analysis to determine the associations between increasing physiologic severity of COPD, as defined by the % predicted FEVs cohort . We alsoSeventy-three individuals enrolled in the Emphysema/COPD Research Center (ECRC) of the University of Pittsburgh gave informed consent for the study. Inclusion criteria included clinically stable COPD at the time of the examination, tobacco exposure of at least 10 pack years, and no clinical diagnosis of rheumatologic, infectious or other systemic inflammatory disease. Exclusion criteria included dominant restrictive spirometric impairment, a significant allergic history, completely reversible airflow obstruction or a history of clinical asthma. The study was approved by the University of Pittsburgh Institutional Review Board.1 and % DLCO were used [Spirometry was performed on 73 subjects using standard methodology at the time of entry into the study -10. Fiftere used ,13.Plasma samples were obtained from subjects upon enrollment into the ECRC registry. Blood was collected into acid citrate dextrose (ACD) cell preparation tubes (CPT tubes). Samples were processed immediately, and plasma was isolated and stored immediately at -80°C until analyzed. A detailed methods of the multiplex assay performed at the University of Pittsburgh Cancer Institute Luminex Core Facility has been previously described . We haveH related cytokines were also of considerable interest, given recent findings regarding the role of the TH phenotype in COPD [H1 and TH2 cytokines interferon-gamma (IFN-γ), interleukin-2 (IL-2) and its soluble receptor IL-2R, interleukin-4 (IL-4), and IL-13 were chosen on this basis. Inflammation related proteins included TNF-α [Selection of specific cytokines in the study was based upon two main criteria: 1) availability of reagent using the Luminex platform, and (2) prior published data to suggest biological plausibility of a cytokine or soluble protein in either systemic or local inflammation observed in COPD. We chose six broad classes of soluble proteins and measured representative markers  × 100. Concentrations for the unknown samples were calculated based upon a 5 parametric curve fitting program . The 5 parametric curve fitting program yields extrapolated values beyond the concentrations for a given standard curve as determined by conventional linear regression, and is the preferred mathematical modeling for multiplex immunoassays ,42. ThisWe defined the lower limit of detection (LLD) for each analyte as the lowest observed concentration in pg/mL. This was, in some instances, an extrapolated value that was lower than the lowest standard curve concentration. Unknown sample concentrations, below the LLD for a given analyte, were assigned a value set just below the LLD using the following equation: undetectable value = LLD of analyte/squared root 2. This method of assigning a value for unknown sample concentrations with undetectable levels has been previously used to examine the relationship of impaired lung function to circulating levels of C-reactive protein and fibrinogen . This al1 and the % predicted DLCO. The dependent variable of interest, plasma cytokine concentration, was not normally distributed; thus, values were log transformed to meet the assumption of normality for linear regression. Standard regression diagnostics were performed to ensure the assumptions for linear regression were met. Covariates previously published as associated with the outcomes of interest (e.g. current smoking and corticosteroid use) were identified a priori and also included [We performed univariate and multivariate linear regression analysis to test the association between the concentration of each plasma cytokine and the physiologic indices of interest: percent (%) predicted FEVincluded -7. We alincluded . SAS 8.2Seventy-three individuals were recruited for analysis. Table Fifty-three of the 73 individuals from the cohort received DLCO measurements  related cytokines interferon-γ (IFN-γ), interleukin-2 (IL-2), interleukin-4 (IL-4) and IL-13 were associated with increasing severity of airflow obstruction, as characterized by decreasing % predicted FEV1 .We did not observe significant associations between plasma CRP concentrations and the % predicted FEVP levels . In cont1  and G-CSF associated with increasing severity of diffusion impairment. This is in contrast to soluble Fas where lower concentrations were associated with increasing severity of diffusion impairment. Systemic markers such as CRP, IL-6 and MPO did not show significant associations with the % predicted DLCO.We examined the association between systemic cytokines and the % predicted DLCO Table . Increas1, age, gender, pack years smoking history, current smoking, inhaled corticosteroid use, systemic corticosteroid use and statin use . An additional 15 out of the 73 subjects did not have bronchodilator reversibility testing at the time of study entry, although 10 of these subjects had emphysema by CT scan and/or abnormally low % predicted DLCO. Further excluding these 15 individuals with unknown bronchodilator response from the cohort, the point estimates for the association between IL-13 and % FEV1 in the remaining 46 subjects was essentially unchanged but did not reach significance due to greater variation .Of the 25 cytokines examined, increasing plasma concentrations of IL-13 showed inverse relationships with both % FEVresponse . Excludi1, was associated with increasing systemic concentrations of IL-13, and the inflammatory chemokines CCL2/MCP-1, CCL4/MIP-1β, and CCL11/eotaxin after adjusting for age, gender, pack years smoking history, current smoking, inhaled corticosteroid use, systemic corticosteroid use and statin use. Furthermore, increasing severity of diffusion impairment, as defined by the % DLCO, was associated with increasing IL-13, CXCL9/Mig, and G-CSF concentrations and decreasing soluble Fas concentrations.We examined the association between 25 different plasma markers of inflammation and two physiologic parameters of COPD in a well-defined clinical population. The main observation was that increasing severity of airflow limitation, as defined by the % FEVH2) type cytokine IL-13 was associated with increasing severity of airflow obstruction, suggesting that IL-13 may be an important mediator in human COPD. The association between increasing IL-13 concentrations and increasing severity of airflow obstruction could not be accounted for by a subset of the cohort with bronchodilator reversibility. This finding suggests that the association is unlikely due to misclassification of asthmatic patients in our COPD cohort.In both univariate and multivariate analysis, increasing plasma concentration of the T helper 2 , may limit the ability to detect significant associations between systemic markers and lung function impairment. Furthermore, we included age, gender, pack years smoking history, current smoking, inhaled corticosteroid use, systemic corticosteroid use and statin use in the multivariate model. It is uncertain whether adjustment for these covariates is appropriate. Thus, we present both univariate and multivariate analysis. We also recognize that the observed associations between plasma concentrations of a protein and lung function severity do not necessarily invoke a cause-effect relationship. However, the findings of this study can serve as the basis for a larger prospective cohort study examining a narrower profile of cytokines on a longitudinal basis.1 or % DLCO. Increasing severity of diffusion impairment is also associated with increasing G-CSF and decreasing soluble Fas concentrations. We speculate that disease characterized by disproportionate abnormalities in DLCO may be associated with peripheral markers independent of the FEV1. The biological plausibility of IL-13 and the discrete repertoire of inflammatory chemokines identified in our model underscore the possibility to more precisely characterize systemic inflammation of COPD.Systemic inflammation has been increasingly recognized in patients with COPD. CRP has been shown to be increased in COPD ,4, yet mCCL2/MCP-1, CC chemokine ligand 2/monocyte chemotattractant protein-1; CCL3/MIP-1α, CC chemokine ligand 3/macrophage inflammatory protein-1α; CCL4/MIP-1β, CC chemokine ligand 4/macrophage inflammatory protein-1β; CCL5/RANTES, CC chemokine ligand 5/regulated on activation normal T cell expressed and secreted; CCL11/eotaxin, CC chemokine ligand 11/eotaxin; CRP, C-reactive protein; CXCL8/IL-8, CXC chemokine ligand 8/interleukin-8; CXCL9/Mig, CXC chemokine ligand 9/monkine induced by interferon-γ; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; FasL, Fas ligand; FGFβ, fibroblast growth factor β; G-CSF, granulocyte-colony stimulating factor; HGF, hepatocyte growth factor; IFN-γ, interferon-γ; IL-1β, interleukin-1β; IL-2, interleukin-2; IL-2R, interleukin-2 receptor; IL-4, interleukin-4, IL-6, interleukin-6; IL-10, interleukin-10; IL-13, interleukin-13; MPO, myeloperoxidase; TNF-α, tumor necrosis factor α, TNFRI, tumor necrosis factor receptor 1, TNFRII, tumor necrosis factor receptor 2; VEGF, vascular endothelial growth factor;Frank C. Sciurba has received funding from GlaxoSmithKline and AstraZeneca in 2005 through 2006 for participation in multi-center clinical trials. He has served on advisory boards for GlaxoSmithKline and AstraZeneca. None of the other authors has any competing interests to declare.JSL, VK, YZ, JM, RAB, AMC and FCS participated in the design of the study. JSL contributed to the statistical analysis, interpretation of the data, and wrote the manuscript. MRR performed portions of the statistical analysis, contributed to the interpretation of the data, and revised the manuscript for important intellectual content. VK performed the statistical analysis. YZ participated in the collection of data. JM participated in the analysis of the data. RAB contributed to the analysis and interpretation of data, and revised the manuscript for important intellectual content. AMC and FCS conceived the study, contributed to the acquisition of the data, and provided important intellectual content to the manuscript. All authors read and approved the final manuscript.


To the Editor: Dengue infection is an endemic and epidemic urban disease  45 U/L, alanine aminotransferase (ALT) 31 U/L, AP 73 U/L, and lactate dehydrogenase (LDH) 198 U/L. On the fifth day of illness, platelet count was 50 × 109/L. Because viral hemorrhagic fever was suspected, the patient was referred to a specialized hospital in Zagreb. Chest radiograph and abdominal ultrasound scan showed bilateral pleural and peritoneal effusions.A 33-year-old Spanish woman who had worked in Anantapur, India, for 180 days, returned to Spain on August 1, 2007; on August 3, she traveled to Dubrovnik, Croatia, on holiday. She also had visited Thailand 45 days before August 1 and Brazil 2 years ago. Two months previously, she experienced a 3-day episode of fever that spontaneously resolved but without laboratory evidence of dengue. On August 6, she exhibited a high fever, chills, headache, arthralgia, and myalgia, with hypotension and was admitted to the hospital. Three days later, a confluent maculopapular rash developed. Dubrovnik hospital laboratory values were hemoglobin (Hb) 143 g/L, packed cell volume (PCV) 41.6%, mean corpuscular volume (MCV) 84.6 fL, platelet count 97 × 10The patient was treated with fluid and plasma replacement, antipyretics, and ceftriaxone plus doxycycline to counteract bacterial and other possible tick-borne infections. She was placed under strict isolation measures while awaiting final diagnosis. The patient was transferred to Barcelona (Spain) University Hospital on August 14; on the basis of her clinical symptoms, hemorragic fever was suspected. She exhibited headache, arthralgia, and myalgia. The fever subsided 9 days after the onset of symptoms. Clinical examination showed a maculopapular rash involving the face, thorax, limbs, and palms and soles, with diffuse petechiae and bruising . BarceloSerologic tests on day 3 and day 11 after the onset of symptoms were not reactive for Crimea-Congo hemorrhagic fever (CCHF), chikungunya, yellow fever, Hantaan, Puumala, and Dobrava viruses; HIV 1 and 2; parvovirus B19; cytomegalovirus; Epstein-Barr virus; or rickettsial diseases. Immunoglobulin (Ig) M tests on day 3 for all 4 dengue virus serotypes were negative. Positive IgG were 1:320 (type 1) and 1:100 (type 3 and 4). A second sample on day 11 showed all 4 IgG serotypes >1:10,000, and IgM >1:10,000 for serotypes 1, 2, and 4. Results of real-time PCR for CCHF were negative but reverse transcription–PCR multiplex for dengue virus was positive for dengue type 1 virus. The patient recovered and was monitored for 2 months.9/L) and 4) increased capillary permeability  and several outbreaks around the world, even during the nondengue season (In dengue-endemic areas, despite the higher disease incidence, many cases still fail to meet WHO criteria (Ae. albopictus (Although our patient was not infected in Europe, lessons from the recently described chikungunya outbreak in Italy indicate the possibility of new arbovirus outbreaks in previously non–disease-endemic areas due to the increasingly established presence of vectors like Dengue virus infection should therefore be considered in the differential diagnosis of fever in returning travelers. DHF diagnosis, although unusual, could become more frequent in the future.


A critical issue with nanomaterials is the clear understanding of their potential toxicity. We evaluated the toxic effect of 24 nanoparticles of similar equivalent spherical diameter and various elemental compositions on 2 human pulmonary cell lines: A549 and THP-1. A secondary aim was to elaborate a generic experimental set-up that would allow the rapid screening of cytotoxic effect of nanoparticles. We therefore compared 2 cytotoxicity assays  and analyzed 2 time points (3 and 24 hours) for each cell type and nanoparticle. When possible, TC50  was calculated.The use of MTT assay on THP-1 cells exposed for 24 hours appears to be the most sensitive experimental design to assess the cytotoxic effect of one nanoparticle. With this experimental set-up, Copper- and Zinc-based nanoparticles appear to be the most toxic. Titania, Alumina, Ceria and Zirconia-based nanoparticles show moderate toxicity, and no toxicity was observed for Tungsten Carbide. No correlation between cytotoxicity and equivalent spherical diameter or specific surface area was found.Our study clearly highlights the difference of sensitivity between cell types and cytotoxicity assays that has to be carefully taken into account when assessing nanoparticles toxicity. For example, titanium dioxide nanoparticles possess photocatalyst activity and are used as antibacterial coatings and in sunscreens  * TC50 where f: percentage that needs to be calculated, H: hillslope, *: multiply, **: to the power.The authors declare that they have no competing interests.SL, FR, JB, GL, JG and PH designed the study. FR, JG, AD, and EMM performed the cytotoxicity assays. SL drafted the manuscript, and GL and PH helped in the final version. All authors read and approved the final manuscript.This study had the financial support of the European Commission through the Sixth framework programme for research and technological development NMP2-CT-2005-515843 contract "NANOSAFE2".cell viability after 3 hours incubation on A549 cells, measured with Neutral Red assay. TC50, TC25 and TC75 values (μg/ml) obtained with NR assay, after 3 hours exposure of A549 cells, for each laboratory.Click here for filecell viability after 24 hours incubation on A549 cells, measured with Neutral Red assay. TC50, TC25 and TC75 values (μg/ml) obtained with MTT assay, after 24 hours exposure of THP-1 cells, for each laboratory.Click here for filecell viability after 3 hours incubation on A549 cells, measured with MTT assay. TC50, TC25 and TC75 values (μg/ml) obtained with MTT assay, after 3 hours exposure of A549 cells, for each laboratory.Click here for filecell viability after 24 hours incubation on A549 cells, measured with MTT assay. TC50, TC25 and TC75 values (μg/ml) obtained with MTT assay, after 24 hours exposure of A549 cells, for each laboratory.Click here for filecell viability after 3 hours incubation on THP-1 cells, measured with Neutral red assay. TC50, TC25 and TC75 values (μg/ml) obtained with NR assay, after 3 hours exposure of THP-1 cells, for each laboratory.Click here for filecell viability after 24 hours incubation on THP-1 cells, measured with Neutral red assay. TC50, TC25 and TC75 values (μg/ml) obtained with NR assay, after 24 hours exposure of THP-1 cells, for each laboratory.Click here for filecell viability after 3 hours incubation on THP-1 cells, measured with MTT assay. TC50, TC25 and TC75 values (μg/ml) obtained with MTT assay, after 3 hours exposure of THP-1 cells, for each laboratory.Click here for file


Therefore, additional prognostic indicators are needed. Here, we evaluated the prognostic value of cyclin H expression in GIST.Risk estimation of gastrointestinal stromal tumours (GIST) is based on tumour size and mitotic rate according to the National Institutes of Health consensus classification. The indication for adjuvant treatment of patients with high risk GIST after RIn order to identify prognostic factors of GIST we evaluated a single centre cohort of ninety-five GIST patients. First, GISTs were classified with regard to tumour size, mitotic rate and localisation according to the NIH consensus and to three additional suggested risk classifications. Second, Cyclin H expression was analysed.Of ninety-five patients with GIST  risk classification revealed: 42% high risk, 20% intermediate risk, 23% low risk and 15% very low risk GIST. In patients with high risk GIST, the expression of cyclin H was highly predictive for reduced disease-specific survival (p = 0.038). A combination of cyclin H expression level and high risk classification yielded the strongest prognostic indicator for disease-specific and disease-free survival (p ≤ 0.001). Moreover, in patients with tumour recurrence and/or metastases, cyclin H positivity was significantly associated with reduced disease-specific survival (p = 0.016) regardless of risk-classification.Our data suggest that, in addition to high risk classification, cyclin H expression might be an indicator for "very-high risk" GIST. Gastrointestinal stromal tumours (GIST) display a wide range of clinical and pathological features and represent the largest group of mesenchymal tumours of the gastrointestinal tract. They are mostly characterised by a gain-of function mutation of the c-kit gene encoding a receptor tyrosine kinase -3. The oGenes involved in cell-cycle regulation, such as cyclins and cyclin-dependent kinases (CDKs), are among such potential markers ,12. The In this study, we investigated the expression pattern of cyclin H in a single-centre population of 95 GIST and evaluated its prognostic value, since our gene expression analysis in normal and tumour tissue of a high-risk GIST patient revealed a 10 fold upregulation of cyclin H in tumour tissue.Medical records as well as paraffin-embedded and frozen specimens of 95 gastrointestinal stromal tumours were included in the study. The clinocopathological features are outlined in Table 2 First Strand Kit . Gene profiling was done as described by the manufacturer using the RT2 profiler PCR array for the human p53 signalling pathway . For CCNH (gene encoding cyclin H), housekeeping gene HPRT1 (hypoxanthine Phosphoribosyltransferase 1) was used for normalisation. The reactions were carried out in a 7500 Fast Real-Time PCR System . The results were analysed with the 7500 Fast System SDS Software 1.4 .In a pilot study, total RNA was isolated from frozen normal jejunal tissue and from a clinically very aggressive tumour relapse which occurred 1.91 years after the initial diagnosis of a jejunal high risk GIST in a 53-year old female patient  using the RNAeasy Kit . Total RNA (2 μg) was reversely transcribed into complementary DNA using the RTOriginal haematoxylin and eosin-stained tumour sections were used to evaluate cell type features and to determine the mitotic rate in 50 high power fields . GISTs were classified according to Fletcher et al.  - see T. AdditioFor immunohistochemical analysis the following antibodies were used: anti cyclin H , anti KIT , anti smooth muscle actin , anti CD34 , anti desmin , anti vimentin , anti NSE , anti S100 , anti Ki-67 .In brief, deparaffinised and re-hydrated tissue sections (3 μm) were pretreated in a microwave in CitraPlus solution . After blocking the endogenous peroxidase activity , the sections were incubated with the monoclonal mouse anti cyclin H antibody followed by incubation with anti-mouse immunoglobulins conjugated with peroxidase-labeled dextran polymers . Staining was detected with 3, 3'- diaminobenzidine  as chromogen and counterstained with hematoxylin before being cover slipped. Cyclin H positivity was defined as positive staining of ≥10% of the tumour cell nuclei according to the modified classifications of Bondi et al. and Kayaselcuk et al. ,23. AsseFor investigation of the obtained data, an exploratory data analysis was performed using SPSS 16  and Excel 2007 . All criteria were rated equally important, without adjustment of p-values for multiple testing. Tumour size, mitotic rate and age were considered to be continuous variables, while others, such as positivity for immunohistochemical markers, initial symptoms and sex, are treated as categorical variables.2), or (if one or more cells contain less than 5 respondents) Fisher's exact test was performed. Testing was always done two-sided. For statistical analysis of timeline-dependent parameters such as disease-specific survival, a Kaplan-Meier estimation was created and significance was tested using log-rank test. For the calculation of disease-specific-survival (DSS), non GIST-related death-events were censored. P-values < 0.05 were considered statistically significant (α = 0.05). No correction for multiple testing was done.To analyse hypotheses regarding the independence of variables, a contingency table was created and either a chi-square test (XA single-centre population of ninety five patients with a mean age of 64.3 years (median 66.9 ± 13.5) ranging from 17 to 94 years and a male/female ratio of 42/53  underwent surgical resection for a GIST via laparotomy . Clinical manifestations, pathological findings, treatment options, and clinical outcome were evaluated, and statistical analyses were carried out with regard to a potential predictive value of cyclin H.Major clinical symptoms, tumour location, histomorphology, immunohistology and risk classification are summarised in Table 0 in 83 (88%), R2 in 7 (8%) and R1 in 4 (4%) of 94 patients; one 94-year-old patient with a huge gastric GIST was not resected and only biopsies were performed due to a coexisting metastatic obstructive colonic cancer. 16 out of 27 patients who had tumour recurrence and/or metastases were treated with imatinib (200-800 mg/d) in addition to surgery. Of these 15 patients, 8 (50%) achieved stable disease, 4 (25%) attained a partial remission and 4 (25%) had tumour progression. Imatinib did not lead to a complete remission in any of our patients.Dependent on the primary tumour site and the extent of tumour growth, final resection state was RMedian follow-up time of the surviving patients was 5.37 years (64.39 months) . At the time of diagnosis, 15% (n = 14) of all GIST patients showed metastatic disease. 16% (n = 15) of the patients died due to GIST-related causes ) and the rate of patients with metastases or tumour recurrence increased to 27% (n = 26) during the time of observation. Disease-specific 1-, 3-, and 5-year survival probability (DSS) was 96%, 87%, and 84%. Respectively, disease-free survival (DFS) was 80%, 76% and 72%. The median disease-free interval after primary diagnosis of patients with primary unifocal disease and later developed metastases or tumour recurrence was 2.1 years (25 months), mean 2.6 years (31.5 months), range 0.5 to 6.1 years (5.6 to 73 months). Interestingly, 32% (n = 30) of the patients exhibited additional malignant neoplasms. Concurrent benign neoplasias were found in 17% (n = 16). Results of the survival analysis are summarised in Tables low risk, 1 as very low risk and 1 as high risk GIST. Further interpretation of strongly positive or moderate positive cannot be assessed because of the limited number of cases. Analysis of the relationship between nuclear cyclin H positivity and risk of malignancy according to Fletcher and co-workers [Quantification of cyclin H expression by Real time PCR in normal intestinal tissue and in a relapse of a jejunal high risk GIST indicated that cyclin H transcription is increased by 10 fold in the tumour tissue. Based on this high transcription level of cyclin H in one GIST, we analysed cyclin H expression at a protein level in the tumour tissue of the same patient. A high nuclear staining of cyclin H was detected. Consequently, immunohistochemical analysis of cyclin H expression in the tumour tissue of 95 GIST patients was undertaken and revealed nuclear positivity of cyclin H in 24% n = 23)  Figure . In the  , sex p = 0.935), and routine immunohistochemical markers such as CD34 (p = 1.000), smooth muscle actin (p = 0.479), desmin (p = 0.564) and Ki-67 (p = 0.227); Table 5, and roThe aim of the present study was to evaluate the prognostic value of cyclin H expression in GIST. To this end, the expression of cyclin H was analysed at the mRNA  and consecutively at a protein level of the whole cohort. The cyclin H immunostaining pattern of tumours of 95 patients with GIST was characterised and correlated to clinicopathologic features and clinical outcome.Due to the absence of reliable genetic or immunohistochemical predictors, tumour size and mitotic rate are still the determining NIH criteria for risk estimation in GIST . AlthougQuantification of cyclin H mRNA revealed a 10.2 fold increased transcription of cyclin H in a high risk jejunal GIST compared to normal tissue, suggesting an important role of cyclin H and the cyclin-CDK-system in GIST pathogenesis. By immunohistochemical analysis of cyclin H expression in 95 tumour specimens of a single centre population we found a high expression of cyclin H (≥ 10% reactive cells) in 24% of the tumours, which correlated well with the risk of malignancy (p = 0.176). Among all Cyclin H positive patients enrolled in our study there is a tendency for a poor prognosis, although there was no statistical significance (p = 0.692 for DFS and p = 0.189 for DSS). In patients with high risk GIST the expression of cyclin H was highly predictive for the reduction of DSS (p = 0.038). Accordingly, cyclin H expression differentiated high risk and "very-high risk" GIST with regard to disease-specific mortality and might be a valuable tool for further treatment decisions. Moreover, with regard to the whole population, the combination of cyclin H positivity and high risk classification according to Fletcher was strongly predictive of a poor DFS as well as DSS (p = 0.001 and p < 0.001) [Although the predictive value of Cyclin H is limited with regard to the whole population, our results suggest for the first time a predictive value of cyclin H expression in high risk GIST patients, underlining the importance of the cyclin-CDK system for GIST pathogenesis. Therewith, our data strengthen previous reports on the predictive value of cyclin A, cyclin B, cyclin D1, cyclin E, cdc2, p27 and p21 -20,25-27The efforts to identify parameters that clearly correlate with the clinical outcome of GIST are not restricted to the cyclin-CDK-system. Similarly, the prognostic value of various other factors, such as p53, p16, p21, pRb, E2F1, p27KIP1, Mdm2, Bcl-2 and Bax is not yet entirely clarified. Although changes in their expression have been evaluated in regard to risk ranking, in most cases their correlation with the clinical outcome  has not been validated in detail. Furthermore, the conclusions drawn are still in some cases contradictory ,18,28,290-resected high risk GIST with a tyrosine kinase inhibitor remains to be further investigated.In conclusion, conventional risk estimations including tumour size, mitotic rate and tumour location are useful to differentiate high risk and non-high risk GIST. The combination of positivity for cyclin H and high risk classification predicts highly significant poor prognosis in GIST. Also in patients with recurrence or metastases, the expression of cyclin H is the only relevant clinical predictor. Therefore, protein expression of cyclin H may allow subclassification of "very-high risk" (cyclin H-positive high risk GIST) from high risk GIST. Whether cyclin H alone or in combination with any other factor will be an indicator for the necessity of adjuvant treatment of RThe authors declare that they have no competing interests.Study concepts: JD, HS, UK, KK; Study design: JD, HS, MS, UK, KK; Data aquisition: JD, HS, MS, TFEB, UK, KK; Quality control of data and algorithms: JD, HS, MS, TFEB, UK, KK; Data analysis and interpretation: JD, HS, MS, AB, TFEB, DHB, UK, KK; Statistical analysis: JD, MS, AB, UK, KK; Manuscript preparation: JD, HS, UK, KK; Manuscript editing: JD, HS, AB, TFEB, DHB, UK, KK; Manuscript review: JD, HS, MS, AB, TFEB, DHB, UK, KKThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/350/prepubTable S1. Table 1: Suggested risk classificationsClick here for fileTable S2 & S3 and Figure S1. Table S2: Combined Cyclin H and p16 positivity - Results of the Survival Analysis Table S3: P values for Combined Cyclin H and p16 positivity Figure S1: Disease specific survival in high-risk GIST with combined positivity for cyclin H and p16 (p < 0.001).Click here for file


Understanding hub genes involved in gastric cancer (GC) metastasis could lead to effective approaches to diagnose and treat cancer. In this study, we aim to identify the hub genes and investigate the underlying molecular mechanisms of GC.three expression profiles  of the genes were extracted from the Gene Expression Omnibus (GEO) database. The GEO2R online tool was applied to screen out differentially expressed genes (DEGs) between GC and normal gastric samples. Database for Annotation, Visualization and Integrated Discovery was applied to perform Gene Ontology (GO) and KEGG pathway enrichment analysis. The protein-protein interaction (PPI) network of these DEGs was constructed using a STRING online software. The hub genes were identified by the CytoHubba plugin of Cytoscape software. Then, the prognostic value of these identified genes was verified by gastric cancer database derived from Kaplan-Meier plotter platform.To explore potential therapeutic targets for GC,A total of 85 overlapped upregulated genes and 44 downregulated genes were identified. The majority of the DEGs were enriched in extracellular matrix organization, endodermal cell differentiation, and endoderm formation. Moreover, five KEGG pathways were significantly enriched, including ECM-receptor interaction, amoebiasis, AGE-RAGE signaling pathway in diabetic complications, focal adhesion, protein digestion and absorption. By combining the results of PPI network and CytoHubba, a total of nine hub genes including COL1A1, THBS1, MMP2, CXCL8, FN1, TIMP1, SPARC, COL4A1, and ITGA5 were selected. The Kaplan-Meier plotter database confirmed that overexpression levels of these genes were associated with reduced overall survival, except for THBS1 and CXCL8.Our study suggests that COL1A1, MMP2, FN1, TIMP1, SPARC, COL4A1, and ITGA5 may be potential biomarkers and therapeutic targets for GC. Further study is needed to assess the effect of THBS1 and CXCL8 on GC. Gastric cancer (GC) ranks as the second most common malignancy and is the second leading cause of cancer–related deaths, following lung and bronchus cancer, in China . Poor 5-In recent years, the Gene Expression Omnibus (GEO) database has provided a powerful tool to elucidate key genetic alternations in carcinogenesis and has extensively used to discover promising biomarkers for cancer diagnosis, new therapeutic target, and prognosis prediction . MoreoveIn this work, we firstly performed an integrated analysis and identified differentially expressed genes (DEGs) by using three gene expression profiles between human GC and normal gastric tissue samples from GEO database. Secondly, Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were further conducted to analyze the major biological functions of co-modulated DEGs. Then, we constructed protein–protein interaction (PPI) network to identify hub genes related to GC by STRING and Cytoscape. Lastly, the overall survival analyses of the hub genes were validated via “Gastric cancer” database on the online Kaplan–Meier platform.https://www.ncbi. nlm.nih.gov/geo/), a total of 8,443 series about human gastric cancer (GC) were retrieved from the database. After a careful review, three gene expression profiles on the GEO Datasets database  were downloaded. Among them, GSE54129 was based on Agilent GPL570 platform ([HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array). GSE33651 was based on platform GPL2895 . GSE81948 was based on platform GPL6244 ([HuGene-1_0-st] Affymetrix Human Gene 1.0 ST Array [transcript (gene) version]). All data were freely accessible online, and no experiment on animals or humans was implemented by any of the authors.Using the keyword “gastric cancer” to search on the GEO Datasets database (http://www.ncbi.nlm.nih.gov/geo/geo2r), and the corrected P-value and —logFC— were calculated. Genes that met the cutoff criteria, corrected P-value <0.05 and —logFC— ≥1.0, were considered DEGs. The Venn diagram was carried out for the intersection part via Funrich software (http://funrich.org/).The DEGs analysis between GC and normal tissues was performed using the GEO2R online analysis tool (http://amp.pharm.mssm.edu/Enrichr/) is an essential application for the success of any high-throughput gene function analysis. Gene functions can be classified into biological process (BP), molecular function (MF), and cellular component (CC). KEGG is a widely used database which stores a lot of data about genomes, biological pathways, diseases, chemical substances, and drugs. In current study, we analyzed the DEGs that were significantly up- and downregulated as determined from integrated microarray gastric cancer data, P<0.05 were considered statistically significant.The newly powerful enrichment analysis online tool  to generate the PPI network. The PPI pairs were extracted with a combined score >0.4 and repeated interactions were removed. Subsequently, the PPI network topology was analyzed by using Cytoscape software (http://www.cytoscape.org/). CytoHubba, a plugin tool in Cytoscape, was applied to calculate the degree of each protein node. Gene with 10 or more gene degree in the PPI network were deemed as hub genes.The DEGs were online introduced to the Search Tool for the Retrieval of Interacting Genes (STRING) database  is a web-based tool applied to evaluate the effect of 54,675 genes on survival using 10,461 cancer samples, including 5,143 breast, 1,816 ovarian, 2,437 lung, and 1,065 GC  were selected in this study. Among them, GSE54129 contained 21 normal samples and 111 GC samples, and GSE33651 included 12 normal samples and 40 GC samples, and GSE81948 included 5 normal samples and 15 GC samples , biological process (BP), cellular component (CC) ontologies. The results of GO analysis indicated that DEGs were mainly enriched in BP, including extracellular matrix (ECM) organization, endodermal cell differentiation, endoderm formation, wound healing, spreading of epidermal cells and negative regulation of chemotaxis . MF analThe results of KEGG pathway analysis showed that DEGs were mainly enriched in pathways in ECM-receptor interaction, amoebiasis, AGE-RAGE signaling pathway in diabetic complications, focal adhesion, protein digestion and absorption .Proteins interactions among the DEGs were predicated with the STRING online software. A total of 129 nodes and 224 edges were involved in the PPI network, as presented in To assess the prognostic values of the nine potential hub genes, we subsequently drew overall survival curves . A totalIn the present study, 85 upregulated DEGs and 44 downregulated DEGs between GC and normal human gastric tissues were identified after integrating and screening three gene expression profile datasets from the GEO database. These 129 DEGs were divided into groups by GO functional annotation, including MF, BP, and CC groups. The results of functional enrichment analysis indicated that significant DEGs in GC patients were involved in the GO BP terms such as ECM organization, endodermal cell differentiation, endoderm formation, wound healing, spreading of epidermal cells and negative regulation of chemotaxis. By performing KEGG pathway analysis, 129 DEGs were enriched in the signaling pathways such as ECM-receptor interaction, amoebiasis, AGE-RAGE signaling pathway in diabetic complications, focal adhesion, protein digestion and absorption. A DEG-encoding proteins PPI network was constructed to screen the most closely related genes, and nine hub genes were identified, including COL1A1, THBS1, MMP2, CXCL8, FN1, TIMP1, SPARC, COL4A1, and ITGA5. All of these nine genes were upregulated in GC patients. In addition, the Kaplan–Meier plotter was applied to assess the effect of nine key DEGs on survival in GC patients. Based on the Kaplan–Meier plotter, overexpression of COL1A1, MMP2, FN1, TIMP1, SPARC, COL4A1, and ITGA5 was related to unfavorable prognosis of GC patients. Interestingly, overexpression of THBS1 and CXCL8 was favorable prognostic factors in GC patients, which provide new insights for GC intervention strategy.Carcinogenesis is a complex process driven by specific genetic alterations and involving multiple signaling cascades. COL1A1, a major component of collagen type I, serves a key role in the tumor cell adhesion and invasion. COL1A1 is overexpressed in a series of cancers including gastric cancer, breast cancer, cervical cancer, non-small lung cancer, and hepatocellular carcinoma. Moreover, COL1A1 overexpression is also related to tumor aggressiveness and poor clinical outcomes . The mecTHBS1 is an adhesive glycoprotein that mediates cell-to-cell and cell-to-matrix interactions. It has been shown that THBS1 plays essential role in platelet aggregation, angiogenesis, and tumorigenesis . Huang aMMP2 belongs to the matrix metalloproteinase (MMP) gene family, which are major extracellular enzymes involved in cancer initiation, progression, and metastasis . The derCXCL8, a member of the CXC chemokine family, is originally described as a mediator of the inflammatory response. Recently, it has been reported that CXCL8 exerts potent pro-tumoral effects in malignant context, such as angiogenesis, cancer stem cells proliferation, and attraction of immunosuppressor cells . NotablyFN1 encodes fibronectin, a glycoprotein present in a dimeric form or multimeric form at the surface and in extracellular matrix. The available reports regarding FN1 have mainly focused on cell adhesion and migration processes, blood coagulation, and host defense . RecentlTIMP1 belongs to the TIMP gene family, which are natural inhibitors of the MMPs. TIMP1 is also involved in the cell proliferation, antiapoptotic function, angiogenesis, and oncogenesis . MoreoveThe SPARC gene encodes a cysteine-rich acidic matrix-associated protein, which is required for the collagen and is also involved in extracellular matrix synthesis and promotion of changes to cell shape . In GC, ITGA5, a member of the integrin alpha chain family, is known to participate in cell–surface mediated signaling. ITGA5 overexpression has been reported to be in a series of tumors . IncreasAlthough we identified several hub genes essential for the progression of GC using comprehensive bioinformatics analysis, several limitations existed in this study. First, this study lacked experimental validation of genes and their functions in GC carcinogenesis. Second, we cannot eliminate the possibility that these key genes may be involved in non-developmental aspects of GC. Finally, the sample size for the RNA-Seq experiments was small, which may have led to potential misleading outcomes. Hence, further bioinformatics analysis of larger samples is warranted, and experimental studies are necessary to corroborate our findings.In conclusion, we identified some key genes and systematically presented the biological processes and signaling pathways closely related with GC initiation and development. Many of these genes had not previously been reported but could play imperative roles in GC. Notably, compared to other international studies, analyzing prognostic data on CXCL8 and THBS1 separately in our study yielded inconsistent conclusions. Nevertheless, results were inconsistent due partly to unequal sample sizes. Further rigorous studies on CXCL8 and THBS1 are warranted. By sharing genotype data on individuals with GC with fellow investigators, specialists, and other health care professionals, common underlying molecular pathways that may be amenable to potential therapeutic interventions are likely to emerge.10.7717/peerj.6092/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.6092/supp-2Supplemental Information 2NOClick here for additional data file.10.7717/peerj.6092/supp-3Supplemental Information 3Click here for additional data file.


Since the inflammation process plays an important role in several diseases, TLRs have been considered potential therapeutic targets, including treatment for cancer. However, TLRs’ role in cancer remains ambiguous. This study aims to analyze the expression levels of plasmatic cell membrane TLRs  in human astrocytomas the most prevalent tumors of CNS different grades (II-IV). We demonstrated that TLR expressions were higher in astrocytoma samples compared to non-neoplastic brain tissue. The gene and protein expressions were observed in GBM cell lines U87MG and A172, proving their presence in the tumor cells. Associated expressions between the known heterodimers TLR1-TLR2 were found in all astrocytoma grades. In GBMs, the mesenchymal subtype showed higher levels of TLR expressions in relation to classical and proneural subtypes. A strong association of TLRs with the activation of cell cycle process and signaling through canonical, inflammasome and ripoptosome pathways was observed by  For the past decade, the inflammatory response in the tumor microenvironment has been in the spotlight due to the controversy regarding its ultimate effect. Despite the expectation that inflammation would wield a positive effect against tumorigenesis, relevant studies about the tumor-promoting effects of immune cells have been published. The resulting bioactive molecules from inflammation can provide the tumor microenvironment with growth factors that sustain proliferative signaling, with survival factors that impair apoptosis, along with proangiogenic and extra-cellular matrix-remodeling factors that facilitate invasion, metastasis, and the formation of new blood vessels .Toll-like receptors (TLRs) act as the first line of defense, recognizing nonself-molecules and activating inflammatory processes . TherefoThere are two subgroups of TLRs, based on the subcellular location of these proteins and their respective pathogen-associated molecular pattern (PAMP) ligands. In humans, cell surface TLR1, TLR2, TLR4, TLR5, and TLR6 comprise one group, recognizing mainly microbial membrane components such as lipids, lipoproteins, and proteins. The other group consists of TLR3, TLR7, TLR8, and TLR9, which are expressed exclusively in intracellular vesicles, including endoplasmic reticulum (ER), endosomes, lysosomes, and endolysosomes, and recognizing microbial nucleic acids. TLR2 and TLR4 also identify endogenous molecules rising upon tissue injury, the damage-associated molecular patterns (DAMPs), such as heat-shock proteins, including HSP70, HSP60 Gp96, HSP22, and HSP72, and high-mobility group box-1, protein, as well as extra-cellular matrix (ECM) molecules such as biglycan, tenascin-C, versican, and fragments of ECM molecules  [Structurally, TLRs are integral membrane receptors, presenting a N-terminal ligand recognition domain, with a leucine-rich repeat motif, a single transmembrane helix, and a C-terminal cytoplasmic signaling domain—known as Toll IL-1 receptor (TIR) domain due to the homology of signaling domains of IL-1R family members .TLR1, TLR2, TLR4, TLR5, and TLR6 are involved in a diversity of cellular responses, ranging from cell proliferation to cell death, and as such they are the targets of the current study in astrocytomas. Cell surface TLRs may present distinct signaling pathways. Below is a brief summary of their signaling pathways:Canonical signaling pathway of plasmatic membrane TLRs involves myeloid differentiation primary response 88 (MYD88), which activates nuclear factor-kappa B (NF-κB) by a protein complex with kinases that recruit tumor necrosis factor receptor associated factor 6 (TRAF6) and leads to proinflammatory cytokines production, such as interleukin-6 (IL-6), interleukin-1β (IL-1β) and α, tumor necrosis factor (TNF), interleukin-8 (IL-8) and interleukin-18 (IL-18). The final result of this pathway is a cell survival profile [ profile . NF-κB c profile . Several profile , c-Myc,  profile , 12, c-J profile , and ser profile , 15.Pathway through TLR2 dimerization with TLR1 or TLR6. TLR2 can form a heterodimer complex with both TLR1 and TLR6 when activated. The heterodimerization of TLR2 with TLR1 or TLR6 expands the ligand spectrum, enabling the innate immune system to recognize different structures of pathogens associated molecular patterns. The downstream signal pathways remain the same for both heterodimers [rodimers .Pathway through TLR4 endocytosis, activating TIR-domain-containing adapter-inducing interferon-β (TRIF), with interferon type 1 response [response .Pathway through TLR4 activating ripoptosome complex formation, composed of receptor-interacting serine/threonine kinase 1 and 3 (RIPK1/3) and mixed lineage domain kinase like pseudokinase (MLKL), tumor necrosis factor receptor type 1-associated death domain protein (TRADD), and fas-associated protein with death domain (FADD). Ripoptosome activation can lead to apoptosis when caspase 8 (CASP8) is present, or to necroptosis when it is absent. TLR5 and TLR2 are indirectly involved in ripoptosome activation by TNF secretion [ecretion –20.Pathway through TLR4, TLR2, and TLR5 forming inflammasome complex, with the production of Caspase-1 and the proinflammatory cytokines IL-1β and IL-18. The inflammasome complex is constituted by NLRP3, PYCARD, and procaspase 1 [aspase 1 , 22.The present study was performed in astrocytoma, a brain tumor with an astrocytic phenotype. The 2007 World Health Organization (WHO) Classification of Tumors of the Central Nervous System (CNS) divided astrocytomas in four malignant grades, I to IV. Glioblastoma  is the most frequent malignant CNS tumor, with a median overall survival of 15 months under current standard of care treatment, which consists of total macroscopic surgical resection combined with radiotherapy and chemotherapy with the alkylating agent, temozolomide , 24.With the onset of next generation sequencing, it was possible to assess the molecular landscape characterizing GBMs. In an attempt to better elucidate the involved molecular tumorigenic processes, and to better guide the adjuvant therapeutic strategies, three main molecular patterns, associated to somewhat distinct clinical outcomes, were identified: 1) the proneural subtype with molecular markers related to progenitor neuronal cells; 2) the classical subtype with markers of proliferative cells; and 3) the mesenchymal subtype with markers of epithelial-mesenchymal transition. Patients exhibiting molecular markers for the mesenchymal subtype presented poor prognosis and the worst response to standard of care, and proneural markers showed better prognosis –28. The 1) TLR  expressions in WHO grades II to IV (diffusely infiltrative) human astrocytomas and in GBM molecular subtypes, 2) correlation of TLRs expression levels, and 3) the presence of these receptors in GBM cell lines.This study aims to analyze: The studied cases were composed of 140 astrocytoma grade II-IV samples, collected during therapeutic surgical intervention in the Neurology Department of the Hospital das Clinicas of University of Sao Paulo School of Medicine, by the institutional neurosurgery group. The cases were stratified according to the WHO classification as: 22 non-neoplastic (NN) cases from epilepsy surgery, 26 astrocytoma grade II (AGII) cases, 18 astrocytoma grade III (AGIII) cases, and 96 astrocytoma grade IV (GBM) cases, this cohort has been previously published , 31. TheThe samples were macrodissected and frozen in liquid nitrogen right after the surgical removal, then cryosectioned for RNA extraction. For the tumor tissue analysis, a 6 µm thick section and hematoxilin-eosin staining was made to guarantee the absence of necrotic, gliosis, non-neoplastic areas, and more than 80% of tumor cells in all tumor tissue specimens , 33.The RNA extraction was accomplished by the RNeasy Mini Kit  following the manufacturer instructions. The RNA concentration and purity were evaluated by NanoDrop, and 1.8–2.0 values for 260 nm and 280 nm absorbance ratios were considered satisfactory. RNA quality was checked by electrophoresis in agarose gel. Reverse transcription was performed with 1 µg of RNA treated with DNase I , and amplified with random primers and oligodT oligonucleotides, RNase inhibitor, and the SuperScript III reverse transcriptase , following the manufacturer instructions. Finally, the cDNA was treated with RNase H , diluted in TE (Tris/EDTA) buffer and stored at -20°C for posterior use by quantitative real-time PCR (qRT-PCR) analysis.TLR1, TLR2, TLR4, TLR5, and TLR6 mRNA levels were evaluated by qRT-PCR, using Power SYBR Green. The results were normalized with the geometrical mean of three reference genes for each sample, as previously described [HPRT), glucuronidase beta (GUSB), and TATA box-binding protein (TBP). The primers were designed to amplify 80-120pb, with melting temperature around 60°C and synthesized by Exxtend  and IDT . The primers were designed as described in escribed : hypoxan-ΔCt as ΔCt is: the Ct of analyzed gene–geometric mean Ct of the reference genes.Primers were optimized to the minimum concentration for the minor cycle threshold (Ct), the maximum amplification efficiency, and minimum nonspecific amplifications. The mix was composed by cDNA (3 µl), Power SYBR Green PCR Master Mix  (6 µl), and the reverse and forward primers (3 µl of each). The qRT-PCR was done in duplicate using the ABI Prism 7500  following the protocol: 2 minutes at 50°C, 10 minutes in 95°C, and 40 cycles of 15 seconds at 95°C, and 1 minute at 60°C. The expression values were assessed by the formula 2The presence of TLR proteins in tumor cells was analyzed by immunofluorescence. A172 and U87MG human GBM cell lineages were acquired from ATCC and authenticated by short tandem repeats (STR) analysis using GenePrint 10 System . Cells were cultured in monolayer with DMEM medium , 10% fetal bovine serum and 100 µg/ml streptomycin and 100 IU/ml penicillin.Cells were fixed with methanol and acetone (1:1), the membrane was permeabilized with Triton-X-100 (0.1%), and, to avoid unspecific reactions, the cells were treated with 2% bovine serum albumin. The primary antibodies anti-TLR1 , anti-TLR2 FITC-conjugated , anti-TLR4 , anti-TLR5 , and anti-TLR6   were incubated overnight at 4ºC. The secondary antibodies  goat anti-Rabbit IgG H&L (Alexa Fluor 568) and goat anti-Mouse IgG H&L (Alexa Fluor 568 and 488) were incubated for one hour, and nuclei were stained with DAPI . The preparations were analyzed in confocal microscopic Zeiss 510 LSM META and Zeiss 780-NLO . The retrieved images were analyzed by Image J/Fiji [Paraffin embedded tissue of five representatives cases of GBM, and five non-neoplastic brain tissue from surgical epilepsy cases were stained for the five TLRs, Iba1, CD68 and GFPA by immunohistochemistry using the Novolink kit , following the manufacture guide. The sections were processed to antigen retrieval, by citrate buffer  for 3 minutes at 122ºC, using an electric cooker . After protein blocking, the tissue was incubated with antibodies: anti-TLR1, anti-TLR2 , anti-TLR4 , anti-TLR5 , anti-TLR6  , anti-CD68 , anti-IBA1 , anti-GFAP  at 20° for 16 hours. The reaction performed by the kit uses diaminobenzidine, and for nuclear staining Harris hematoxylin was used. To obtained optimal dilution tonsil sections was used. The immunoreactions for the five TLRs were analyzed according to a semi-quantitative score system considering the intensity of staining , and percentage of immune-positive cells . An immunolabeling score (ILS) was obtained by the product of the intensity of staining and the percentage of stained cells, by two independent investigators , and simultaneous revision were performed to obtain the final score in case the concordance was not achieved. Digital photomicrographs of representative fields were captured and processed using Adobe Illustrator CS6 .r ≥ 0.7, moderate when 0.4 < r > 0.7, and low when r ≤ 0.4. Differences were considered significant for p < 0.05.The statistical analysis was performed in the program SPSS version 23.0 . The data distribution was evaluated by Kolmogorov-Smirnov test. The non-parametric results were evaluated by Kruskal-Wallis test, followed by post hoc Dunn’s test. The correlations between each two receptors gene expression levels were assessed by Spearman-rho. The associations are considered strong when The Cancer Genome Atlas (TCGA - http://cancergenome.nih.gov/) was downloaded from *.rsem.genes.normalized_results address. The data was analyzed by DE-seq and the RPKM values were normalized by the z-score.The GBM RNAseq dataset from TLR1, 2, 4, 5, and 6, presented higher expression levels in astrocytoma samples compared to non-neoplastic brain tissues, with statistical significance (p < 0.05) . TLR1, TLR2, and TLR6 mRNA expression median values showed an increase in parallel to the increase of malignancy. On the other hand, the median TLR4 expression in GBM cases was lower than in the other astrocytoma grades (AGII-AGIII).Genes coding the five receptors,  < 0.05) . Only TLp < 0.05 .All TLRs expressions were correlated between themselves in GBM, with statistical significance of p < 0.05 . ExpressTLR4 and TLR6 mRNA median levels than other molecular subtypes in our cohort  missense mutation in A172, and neurofibromin (NF1) missense and deletion mutation in U87MG [TLR mRNA levels by qRT-PCR corroborated their presence in both cell lineages; TLR5 mRNA expression level was not detected in U87MG cells by qRT-PCR : RB transcriptional corepressor  by Spearman-rho test with expression levels of cell proliferation genes: JUN (r = 0.422 and r = 0.536) and SRF (r = 0.462 and r = 0.577) and in mesenchymal and classical subtypes, respectively. In contrast, the gene expressions related to interferon type I response presented low or undetermined z-score values (not shown). Interestingly, expression levels of genes involved in ripoptosome signaling, such as RIPK3 and MLKL, were also comparatively increased in mesenchymal GBM subtype, and their values correlated to TLR2 , and to TLR4 . Of note, PYCARD, NLRP3 and CASP1, which participate in the inflammasome pathway, presented higher expression levels in mesenchymal subtype of GBM compared to the other two subtypes  in comparison to non-neoplastic brain tissue. Moreover, an associated expression of these receptors were observed in GBM cases, suggesting their role in tumor aggressiveness. The presence of TLR4 has been previously described in GBM, particularly in CD133+ tumor stem cells [TLR1, TLR2, and TLR4 compared to A172, in contrast to lower expression of TLR5  and genes related to cell proliferation , were upregulated, suggesting that the activation of these TLRs leads to tumor growth. In fact, activation of the TLR canonical pathway through TIRAP-MYD88 was related to NF-κB (RELA) upregulated expression levels and positive correlation with JUN, and SRF, transcription factors implicated in cellular proliferation. The role of NF-κB in tumor growth has been demonstrated in a mouse model of GBM [IL1B and IL18 are also end targets of the inflammasome pathway, which were upregulated in the mesenchymal subtype. On the other hand, the increased expression of RIPK3 and MLKL may suggest the upregulation of the ripoptosome pathway, and may allow the link with the presence of necrosis, one of the characteristics of GBM. In a rat glioma model, apoptosis was induced by activation of CASP8 and inhibition of RIPK1/RIPK3 complex [In the present study, higher expression levels of em cells , 39, andem cells , 41, andem cells . Such TLem cells , 42. Furem cells . We furtLR5 Figs . Such heLR5 Figs . To analLR5 Figs . Interesl of GBM  and in pl of GBM . Additiol of GBM . Therefol of GBM , 47–49.  complex , 50, 51.CASP8 was increased in the mesenchymal subtype of GBM; however, increased apoptosis is not expected and, therefore, avoidance of apoptosis should be under additional modulation. Moreover, according to the present in silico analysis, the interferon type I response by TLR4 endocytosis was unlikely.The expression of PTEN deletion, and 59.45% and 66.66% of PTEN phosphorylation in classical and mesenchymal subtypes, respectively . Interestingly, a negative correlation was found between them in the proneural subtype , which presents better prognosis among the three analyzed GBM subtypes.Another hypothesis for the proliferative profile and TLR signaling in GBM is the pathway involving PTEN, which has been shown to regulate TIRAP and TLR4 internalization , 53, in ectively . [36, 55er TIRAP  and conser TIRAP . ActuallTLR1, TLR2, TLR4, TLR5, and TLR6 expressions were demonstrated in astrocytomas compared to non-neoplastic brain tissue, mostly in GBM group, and particularly in the mesenchymal subtype. In silico exploration of the putative activation pathways by these upregulated TLRs, the canonical and inflammasome pathways in the mesenchymal subtype and the PTEN-TIRAP signaling in the classical subtype demonstrated their increase and their possible contribution to tumor growth. According to the TCGA dataset the microglia compartment based on AIF1 expression level did not parallel the TLRs upregulated expressions in mesenchymal subtype, suggesting that the tumor cells themselves may contribute to the increased expression of these receptors.In summary, increased cell membrane Further understanding of the particular TLR responses from each tumor compartment, which includes inflammatory cells in addition to tumor cells, will be essential to designing new therapeutic strategies involving TLRs.S1 FigHorizontal bars indicate the mean value of each group.(TIF)Click here for additional data file.S2 FigTLRs is observed between the cell lines, although both GBM cell lines present somatic mutation profiles of mesenchymal subtype. TLR4 expression is high in both cell lines, whereas TLR5 is undetectable in U87MG cell line.A heterogeneity in the distribution of the five (TIF)Click here for additional data file.S3 FigImmunohistochemistry for a representative non-neoplastic case stained for TLR1, TLR2, TLR4, TLR5, TLR6 and GFAP for glial cell identification. Presence of few microglia in the same GBM sample of (TIF)Click here for additional data file.S4 Figp < 0.05 when compared to proneural cases and (†) p < 0.05 when compared to classical cases by Kruskal-Wallis and Dunn’s test. The values were normalized in DEseq.The dataset is composed of 37 proneural (PN), 40 classical (CS), and 55 mesenchymal (MES) subtype cases, wherein (*) (TIF)Click here for additional data file.S5 FigPTEN, and in orange the Y240-phosphorylated PTEN.The GBM cohort from our lab was previously analyzed for PTEN mutational and phosphorylation status. The number of cases for each status of PTEN and GBM subtype are presented. In green is the amount of cases presenting wild-type PTEN, in black the deleted (TIF)Click here for additional data file.


GPR68 (OGR1) belongs to the proton-sensing G protein-coupled receptors that are involved in cellular adaptations to pH changes during tumour development. Although expression of GPR68 has been described in many tumour cell lines, little is known about its presence in human tumour entities. We characterised the novel rabbit monoclonal anti-human GPR68 antibody 16H23L16 using various cell lines and tissue specimens. The antibody was then applied to a large series of formalin-fixed, paraffin-embedded normal and neoplastic human tissue samples. Antibody specificity was demonstrated in a Western blot analysis of GPR68-expressing cells using specific siRNAs. Immunocytochemical experiments revealed pH-dependent changes in subcellular localisation of the receptor and internalisation after stimulation with lorazepam. In normal tissue, GPR68 was present in glucagon-producing islet cells, neuroendocrine cells of the intestinal tract, gastric glands, granulocytes, macrophages, muscle layers of arteries and arterioles, and capillaries. GPR68 was also expressed in neuroendocrine tumours, where it may be a positive prognostic factor, in pheochromocytomas, cervical adenocarcinomas, and endometrial cancer, as well as in paragangliomas, medullary thyroid carcinomas, gastrointestinal stromal tumours, and pancreatic adenocarcinomas. Often, tumour capillaries were also strongly GPR68-positive. The novel antibody 16H23L16 will be a valuable tool for basic research and for identifying GPR68-expressing tumours during histopathological examinations. Unlike other receptors, the pH window of GPR68 is quite narrow. The receptor seems to be inactive at pH 7.8 and fully active at pH 6.8 [Under many pathophysiological circumstances, such as inflammation, ischaemia, and tumour development, acidosis occurs in the local microenvironment, and proton-sensing GPCRs (amongst others) are involved in cellular adaptations to these changes ,6,7. In Studies have shown that GPR68 can act as either a tumour suppressor or a tumour promoter. In HEY human ovarian cancer cells, MCF-7 breast cancer cells, and Caco-2 colorectal adenocarcinoma cells, increased GPR68 expression is associated with reduced cell proliferation and migration ,11,12, sUnfortunately, other than its expression in tumour cell lines or in animal models, little is known about the presence of GPR68 in human tumour samples. This limited knowledge may be due to a lack of specific antibodies suited for use in formalin-fixed, paraffin-embedded tumour samples from routine pathological studies. Therefore, we have developed a novel rabbit monoclonal antibody, 16H23L16, which is directed against the carboxyl-terminal tail of human GPR68. In the present study, we demonstrate that this antibody is well suited both for Western blot analyses and immunocytochemistry in basic research and for immunohistochemical staining of routine clinical pathology samples. The antibody was further tested in a large series of formalin-fixed, paraffin-embedded normal and neoplastic human tissue samples, revealing GPR68 expression in glucagon-producing islet cells of the pancreas, specific endocrine cells of the intestinal tract, and pancreatic neuroendocrine tumours. Based on these findings, we expanded the number of neuroendocrine tumours studied and ultimately evaluated a broad panel of bronchopulmonary and gastroenteropancreatic neuroendocrine neoplasms of different origins for GPR68 expression. The staining results were then correlated with clinical data, such as grading, staging, glucagon or insulin secretion, and patient overall survival.r 56,000–72,000 from both cell lines . Accordingly, Kaplan–Meier analysis revealed a slightly better outcome for patients with GPR68-positive tumours (IRS ≥ 3) compared to those with GPR68-negative neoplasms , somatostatin receptor (SST) 2A , SST3 , and SST5 ). Furthermore, significant differences regarding patient sex were observed, with lower GPR68 IRS values in males than in females .When data from all bronchopulmonary and gastroenteropancreatic neuroendocrine tumours were analysed together, significant differences were noted between patients with and without lymph node metastases, with higher GPR68 IRS values in patients with no lymph node metastases  and a negative correlation with levels of the proliferation marker Ki-67 . Also here, Kaplan–Meier analysis revealed a slightly better outcome for GPR68-positive tumour cases , SST2 , and SST5 . Again, lower GPR68 IRS values were observed in males than in females .If only bronchopulmonary neuroendocrine tumours were considered, a positive association was detected between GPR68 expression and patient overall survival . Additionally, in gastroenteropancreatic neuroendocrine neoplasms, a tendency towards lower GPR68 levels was observed in the metastases as compared to the primary tumours . However, Kaplan–Meier analysis could not demonstrate statistically significant differences between patients with GPR68-positive or -negative tumours, likely due to too few positive cases . Corresponding to the findings in all tumours and in bronchopulmonary neoplasms alone, gastroenteropancreatic neuroendocrine tumours alone yielded slightly lower values in male patients than in female patients, though without reaching statistical significance . If only gastroenteropancreatic neuroendocrine tumours were included in the analysis, significantly higher GPR68 levels were again noted in patients without lymph node metastases  or tumour grade , while a positive association between GPR68 and SST2 expression  was observed. Because double-labelling experiments revealed the presence of GPR68 in glucagon-producing, but not insulin-producing, pancreatic islet cells, pancreatic neuroendocrine tumours were additionally evaluated for insulin or glucagon expression. As expected, a strong positive relationship was found between IRS values for GPR68 and glucagon , but not insulin .If considering only the tumour entity with the highest percentage of GPR68-positive cases (pancreatic neuroendocrine neoplasms), a negative correlation was found between GPR68 expression and Ki-67 levels endocrine tumour entities with a high prevalence of GPR68, such as pheochromocytomas, paragangliomas, and medullary thyroid carcinomas, but also with cervical adenocarcinomas and endometrial cancer will be of interest in this respect.Independent of tumour cells, capillaries, granulocytes, and macrophages within some tumours were strongly GPR68-positive. Similar findings have been described in the literature ,16,37, aA rabbit monoclonal antibody (16H23L16) that recognizes the carboxyl-terminal tail of human GPR68 was generated in collaboration with and obtained from Thermo Fisher Scientific . The identity of the peptide used for immunisations of the rabbits was Cys-TKLHPAFQTPNSPGSGGFPTGRLA, corresponding to residues 342–365 of human GPR68. This sequence is unique for human GPR68 and, because of dissimilarities in the carboxyl-terminal amino acid sequence, 16H23L16 does not cross-react with rat or mouse GPR68 .Human embryonic kidney 293 cells , either mock-transfected or transfected with human GPR68, or BON-1 cells  endogenously expressing GPR68 were seeded onto poly-L-lysine-coated 60-mm dishes and grown to 80% confluency. Cells were lysed in detergent buffer (20 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.2 mM phenylmethylsulfonylfluoride, 10 mg/mL leupeptin, 1 mg/mL pepstatin A, 1 mg/mL aprotinin, and 10 mg/mL bacitracin). GPR68 was enriched using wheat germ lectin agarose beads. Samples were subjected to 7.5% SDS-polyacrylamide gel electrophoresis and immunoblotted onto polyvinylidene fluoride (PVDF) membranes. Blots were incubated with the rabbit monoclonal anti-GPR68 antibody 16H23L16 at a dilution of 1:1000 followed by 1:5000-diluted peroxidase-conjugated secondary anti-rabbit antibody  and enhanced chemiluminescence detection .When indicated, expression of endogenous GPR68 in BON-1 cells was silenced using chemically synthesised double-stranded GPR68 siRNA duplexes  according to the manufacturer’s instructions. As negative control, scrambled siRNA was used .BON-1 cells were grown on coverslips overnight and then exposed to different pH conditions  for 60 min. When indicated, cells were additionally stimulated with different concentrations of lorazepam  for 30 min at pH 7.8. Cells were fixed with 4% paraformaldehyde and 0.2% picric acid in phosphate buffer (pH 6.9) for 20 min at room temperature, washed with phosphate buffer, and incubated with 16H23L16 (1:1000) at room temperature for 2 h, followed by an Alexa Fluor 488-conjugated secondary antibody  overnight at 4 °C. Samples were mounted and examined using a Zeiss LSM 510 META laser scanning confocal microscope . n = 6–15 each), obtained from the Department of Pathology of the Ernst-Moritz-Arndt-University  and the Laboratory of Pathology and Cytology Bad Berka , were also evaluated. Staining patterns were compared to those seen in the non-malignant tissues surrounding the tumours.For the initial evaluation of GPR68 expression in different human tumour entities, 265 formalin-fixed and paraffin-embedded tumour samples  were included in the present investigation. From some patients, both primary and metastatic samples were available. Of the tumours, 91 (32.7%) originated from the lung , 27 atypical carcinoids (AC), 39 small-cell lung cancer (SCLC), and 1 large-cell neuroendocrine carcinoma), 18 (6.5%) from the stomach, 15 (5.4%) from the duodenum/jejunum, 58 (20.9%) from the ileum, 5 (1.8%) from the appendix, 9 (3.2%) from the colon, 15 (5.4%) from the rectum, and 47 (16.9%) from the pancreas. Localisation of 20 (7.2%) primary tumours was unknown. The samples were provided by the Institute of Pathology and Cytology Bad Berka , and had been surgically removed between 1998 and 2016 at the Department of General and Visceral Surgery, Zentralklinik Bad Berka . Clinical data were gathered from patient records.Permission was obtained from the local ethics committee  for this retrospective analysis. All data were recorded and analysed anonymously.The neuroendocrine tumours evaluated in the present investigation were from 140 male (50% of the cases) and 121 female (44%) patients. The sex of 17 patients (6%) was unknown. The overall mean age of the patients at diagnosis was 58.8 years . Forty-eight of the (corresponding) primary tumours (17%) were classified as T1; 41 (15%) as T2; 58 (21%) as T3, and 24 (9%) as T4. In 107 cases (38%), the extent of the primary tumour was unknown. Lymph node metastases were confirmed in 120 cases (44%), while seventy-nine patients (28%) showed none. For 79 tumours (28%), the patient’s lymph node status was not known. Eighty-one patients (29%) had no distant metastases, 113 patients (41%) had distant metastases, and 84 patients (30%) lacked any report of distant metastases in their medical files. At diagnosis, 17 of the 167 patients with gastroenteropancreatic neuroendocrine neoplasm (GEP-NEN) (10%) had UICC stage I disease, 12 patients (7%) had stage II disease, 22 patients (13%) had stage III disease, and 99 patients (60%) had stage IV disease. For 17 GEP-NEN patients (10%), the disease stage was unknown. For the 91 bronchopulmonary tumour patients and the 20 patients with unknown tumour origin, the stage of the disease was not determined. Regarding histological grading, 102 patients (37%) had grade 1, 86 patients (31%) had grade 2, and 63 patients (23%) displayed grade 3 histology. Tumour grading was unknown for 27 (9%) patients. p = 0.016). The median follow-up time was 52.5 months overall. One hundred sixty-three patients were alive at the end of the follow-up period, 35 patients had no survival data available, and 80 patients died of tumour-related causes; of these, the median survival time was 26.5 months, which differed according to patient sex , SCLC cell lines , a lung adenocarcinoma cell line (A549), colon carcinoma cell lines , hepatocellular carcinoma cell lines , a renal cell carcinoma cell line (A498), a neuroendocrine tumour cell line (BON-1), urinary bladder carcinoma cell lines (T24 and RT-112), prostate cancer cell lines , breast cancer cell lines , cervical cancer cell lines , ovarian carcinoma cell lines (SK-OV-3 and A2780), and an epidermoid carcinoma cell line (A431)  were grown in 75-cm² culture flasks to 80% confluency. For preparation of cytoblocks, cells were washed once with phosphate-buffered saline and transferred into 10% buffered formalin for 2 h. After centrifugation for 10 min at 3500× N-terminal tail of GPR68 . Detection of the primary antibodies was performed using biotinylated anti-rabbit IgG followed by incubation with peroxidase-conjugated avidin . Binding of the primary antibody was visualised using 3-amino-9-ethylcarbazole in acetate buffer . Sections were rinsed, counterstained with Mayer’s haematoxylin, and mounted in Vectamount™ mounting medium . For immunohistochemical controls, 16H23L16 was either omitted or adsorbed for 2 h at room temperature with 10 µg/mL of the peptide used for immunisations.From the paraffin blocks, 4-µm sections were prepared and floated onto positively-charged slides. Immunostaining was performed by an indirect peroxidase labelling method as described previously . BrieflyFor dual immunofluorescence, sections were incubated overnight at 4 °C with 16H23L16 together with either a mouse monoclonal anti-insulin antibody , a mouse monoclonal anti-glucagon antibody , a mouse monoclonal anti-CD68 antibody , a mouse monoclonal anti-smooth muscle actin antibody  or a mouse monoclonal anti-CD31 antibody . Sections were incubated with Cy3-conjugated anti-rabbit and Alexa Fluor 488-conjugated anti-mouse antibodies. Specimens were mounted and examined using a Zeiss LSM 510 META laser scanning confocal microscope.Staining of GPR68 in the tumour slides was scored with the semiquantitative Immunoreactivity Score (IRS) according to Remmele and Stegner . The perp values ≤0.05 were considered statistically significant.For statistical analysis, SPSS 25.0.0.0  was used. Because the data were not normally distributed (Kolmogorov–Smirnov test), Kruskal–Wallis test, Mann–Whitney test, Chi-square test, Kendall’s τ-b test, and Spearman’s rank correlation were performed. For survival analysis, the Kaplan–Meier method with a log-rank test was used. We have generated and characterised a novel rabbit monoclonal anti-human GPR68 antibody that is well suited for Western blot and immunocytochemical analyses in basic research and for visualising human GPR68 in formalin-fixed, paraffin-embedded tissues during pathological examinations.This antibody enabled detection of GPR68 expression in a wide variety of non-neoplastic and neoplastic tissues as well as cell populations. Our results also point to a possible involvement of GPR68 in many important physiological and pathophysiological processes, such as hormone secretion, glucose homeostasis, regulation of bicarbonate and mucus secretion in the gastrointestinal tract, modulation of inflammatory processes, angiogenesis, and tumour growth. However, further experiments are clearly necessary to substantiate these findings.