Trypanosoma cruzi infection. HLA-G and its murine functional homolog Qa2 have well-recognized immunomodulatory properties. We evaluated the HLA-G 3′ untranslated region (3′UTR) polymorphic sites (associated with mRNA stability and target for microRNA binding) and HLA-G tissue expression  in patients presenting Chagas disease, stratified according to the major clinical variants. Further, we investigated the transcriptional levels of Qa2 and other pro- and anti-inflammatory genes in affected mouse tissues during T. cruzi experimental acute and early chronic infection induced by the CL strain. Chagas disease patients exhibited differential HLA-G 3′UTR susceptibility allele/genotype/haplotype patterns, according to the major clinical variant (digestive/cardiac/mixed/indeterminate). HLA-G constitutive expression on cardiac muscle and colonic cells was decreased in Chagasic tissues; however, no difference was observed for Chagasic and non-Chagasic esophagus tissues. The transcriptional levels of Qa2 and other anti and proinflammatory  genes were induced only during the acute T. cruzi infection in BALB/c and C57BL/6 mice. We present several lines of evidence indicating the role of immunomodulatory genes and molecules in human and experimental T. cruzi infection.Genetic susceptibility factors, parasite strain, and an adequate modulation of the immune system seem to be crucial for disease progression after  Trypanosoma cruzi, which may produce acute and chronic manifestations. During the acute phase, most cases are indeterminate, but some patients may develop severe myocarditis or meningoencephalitis, which can be lethal. The chronic phase exhibits four major clinical variants: (i) the indeterminate form represents 50–70% of cases and develops without evident clinical and pathological signs [Chagas disease or American trypanosomiasis is caused by the parasiteal signs ; (ii) thal signs , 2;  preponderant IFN-γ production by cytolytic natural killer (NK) cells in the acute phase of the disease to control tissue and systemic parasite burden; and (iii) polyclonal B lymphocyte response [In parallel with the chronic parasite infection, the immune response against parasite antigens is associated with the Chagas heart disease pathogenesis. Key features of the immunity in the Chagas disease include (i) predominance of partially activated T CD8 lymphocytes in cardiac inflammatory infiltrates , accomparesponse –13.γ and TNF production. During the chronic phase, a coexistence of the Th1  and Th2 profiles (IL-4 and IL-10) may be observed, and the equilibrium between these profiles may be relevant to disease morbidity [ T. cruzi itself or tissue modifications induced by parasites.Cytokine patterns in Chagas disease are characterized by T-helper (Th)1 polarization in the acute phase, with predominance of IFN-orbidity , 14, 15;orbidity –20. Besiorbidity . In thisAmong immunomodulatory molecules, the human leucocyte antigen- (HLA-) G is a nonclassical histocompatibility class Ib molecule, which has a well-recognized role in controlling several branches of the immune response, inhibiting T cell proliferation, NK, and cytotoxic T lymphocytes, inducing regulatory T cells and tolerizing dendritic cells . These eHLA-G mRNA has restricted expression in nonpathological conditions, for organs such as the placenta, thymus, heart, intestines, brain, and skin [and skin , and theand skin –34. On tand skin , 36. HLA-G gene presents limited polymorphisms compared to classical HLA class I (HLA-A/B/C) genes, exhibiting only 50 alleles , v3.16.0) and coding 16 different membrane-bound full length proteins. The regulatory regions also present several polymorphisms that coincide with or are close to binding sites for transcription factors (promoter region) or are targets for microRNA binding (3′ untranslated region (3′UTR)). At least 29 variation sites have been described at the promoter region [ HLA-G 3′UTR [ HLA-G gene expression, the study of these regions is relevant. In the healthy Brazilian population, at least eight HLA-G 3′UTR variation sites have been described [At the coding region, ther region  and 16 v-G 3′UTR . Consideescribed , 39, andescribed . H2-Q7/Q9 gene, also presents restricted tissue expression, and Qa2 mRNA has been reported in thymus, liver, intestines, spleen, placenta, and brain. Qa2 also modulates the immune response by interacting with as yet unidentified NK cell receptors [The HLA-G murine functional homolog Qa2, encoded byeceptors –45. T. cruzi infection; (ii) Chagas disease is a chronic disorder, in which several mechanisms of immunomodulation have been described in association with its pathogenesis; (iii) clinical variants of Chagas disease may depend on parasite and host genetic factors; (iv) experimental trypanosomiasis may help in the understanding of the human disease counterpart, we designed a study encompassing the HLA-G tissue expression and HLA-G 3′UTR polymorphic site typing in patients presenting Chagas disease, stratified according to major clinical variants. In addition, we evaluated the transcriptional level of the H2-Q7/Q9 (Qa2), and other immunoregulatory H2-T23 (Qa1), CTLA-4, and PDCD1 (PD-1) genes in mouse (BALB/c and C57BL/6) affected tissues during experimental acute and early chronic infection caused by the CL strain of T. cruzi and correlated the expression of these genes with other mediators of inflammation, including INF-γ, inducible nitric oxide synthase (NOS-2), and IL-10.Little information is available regarding HLA-G and the Qa2 murine functional homolog in acute and chronic parasitic diseases. Considering that (i) no information is available regarding the role of these immunoregulatory molecules in T. cruzi antigens followed at the Divisions of Cardiology and Gastroenterology of the Department of Medicine of the School of Medicine of Ribeirão Preto, University of São Paulo, Brazil, and 155 healthy individuals from the same region of patients, exhibiting negative serology for T. cruzi infection, were studied. The Chagas disease patients were submitted to clinical and laboratory examination. Electro- and echocardiography, esophagus and colon contrast X-ray examinations, and esophagus electromanometry were performed to classify patients into cardiac , digestive , cardiodigestive or mixed , and indeterminate (n = 39) variants.The protocol of the study was approved by the local Research Ethics Committee (Protocol number 11237/2009) and written informed consent was obtained from all participants. A total of 177 chronic Chagas disease patients exhibiting positive serology for HLA-G 3′UTR was performed by sequencing analyses as previously described [Genomic DNA was extracted from peripheral blood leucocytes using a standard salting-out procedure. Genotyping of the variation sites atescribed .Fifty-four specimens of tissues obtained from Chagas disease deceased patients were analyzed, of which 30 were derived from heart, 13 from colon, and 11 from esophagus. In parallel, 20 tissue specimens  of deceased individuals without Chagas disease and exhibiting no histopathological tissue alteration were analyzed. Three specimens of trophoblast tissue (positive control for HLA-G expression) were also analyzed. The immunohistochemical analysis was performed using the primary antibody MEM/G9 (HLA-G1 and sHLA-G5 specific mouse IgG) , diluted 1 : 200, as previously described . Briefly T. cruzi, using three infected and three noninfected animals in two independent experiments. The mice were cared for according to the institutional guidelines on ethics in animal experiments and all protocols were approved by the local Ethics Committee on Animal Care and Research (process number 172/2009).Male eight-week-old C57BL/6 and BALB/c mice were intraperitoneously (IP) injected with bloodstream CL strain forms of3 bloodstream forms were IP injected. Mouse survival was verified daily for 30 days and the parasitemia was quantified microscopically by counting the parasites in 5 μL of citrated blood obtained from the tail lateral vein from day 7 until day 29 after infection. Parasite load was analyzed in the heart at day 24 after infection [2 bloodstream forms of T. cruzi were IP injected and mouse survival was verified daily for 60 days. Parasite load was analyzed in the heart at day 60 after infection.For the acute infection, 10nfection . For the H2-Q7/Q9, H2-T23, and PDCD1 genes, respectively, to facilitate understanding we will refer in this text to Qa2, Qa1, and PD-1 expression as synonyms of their respective gene expressions.Gene expression was detected in the heart and esophagus at 24 and 60 days after infection (dpi), for acute and early chronic infections, respectively. Although Qa2, Qa1, and PD-1 are the encoded molecules of theGADPH) housekeeping gene and relative expression of each mRNA was calculated with the 2−∆∆Ct method [ H2-Q7/Q9-forward 5′-ATG GCG ACC ATT GCT GTT GT-3′, H2-Q7/Q9-reverse 5′-TCC AAT GAT GGC CAC AGC T-3′; (ii) H2-T23-fwd 5′-GCA CAA GTC AGA GGC AGT CG-3′, H2-T23-rev 5′-TGC AGG TAT GCC CTC TGT TG-3′; (iii) CTLA-4-fwd 5′-ACC TCT GCA AGG TGG AAC TCA-3′, CTLA-4-rev 5′-CCA TGC CCA CAA AGT ATG GC-3′; (iv) PD-1-fwd 5′-TTC AGG TTT ACC ACA AGC TGG-3′, PD-1-rev 5′-TGA CAA TAG GAA ACC GGG AA-3′; (v) INF-γ-fwd 5′-GCA TCT TGG CTT TGC AGC T-3′, INF-γ-rev 5′-CCT TTT TCG CCT TGC TGT TG-3′; (vi) NOS-2-fwd 5′-CGA AAC GCT TCA CTT CCA A-3′, NOS-2-rev 5′-TGA GCC TAT ATT GCT GTG GCT-3′; (vii) IL-10-fwd 5′-TGG ACA ACA TAC TGC TAA CC-3′, IL-10-rev 5′-GGA TCA TTT CCG ATA AGG CT-3′; (viii) GAPDH-fwd 5′-TGC AGT GGC AAA GTG GAG AT-3′, GADPH-rev 5′-CGT GAG TGG AGT CAT ACT GGA A-3′.Total RNA was extracted from tissues using the TRIzol reagent  and was reverse transcribed to obtain cDNA with the SuperScript III (Invitrogen) transcriptase reverse. SYBR Green Mix-based real-time quantitative PCR assays were performed using a StepOnePlus system . Data were normalized according to the expression of the glyceraldehyde phosphate dehydrogenase  and for the whole group of patients. Two distinct computational methods that do not take any prior information into account were used for this purpose: (1) the EM algorithm [Allele and genotype frequencies were estimated by direct counting, and adherences of phenotypical proportions to expectations under the Hardy-Weinberg equilibrium (HWE) were tested by the complete enumeration method using the GENEPOP 3.4 software . Linkagesoftware . HLA-G 3lgorithm  implemenlgorithm  and (2) lgorithm . Therefot-test or Mann-Whitney tests and the relationship between immunomodulatory and mediators of inflammation genes was performed using the Spearman Correlation, both with the aid of the GraphPad Prism 5 v5.0b software. To facilitate data presentation, variables were expressed as mean ± SEM.For the HLA-G tissue expression, gene expression, and parasitism load, statistical analyses were performed using Student's P < 0.05.For all analyses, significance was defined at HLA-G 3′UTR variation sites were compared between (i) patients (considered as whole) with controls ; (ii) patients presenting clinically detectable disease (CDM) (cardiac (C) plus digestive (D) plus mixed (M) forms) with controls; (iii) patients presenting CDM with indeterminate patients (indeterminate form—I); and (iv) patients stratified according to the four clinical variants with each other and with controls. Association tests were performed for alleles and genotypes of each variation site. HLA-G 3′UTR variation sites were observed in this study: 14-base pair insertion/deletion ; +3001C/T (rs not available); +3003T/C (rs115689421); +3010C/G (rs116152775); +3027A/C (rs115810666); +3035C/T (rs115100128); +3142G/C (rs115928989); +3187A/G (rs114317070); and +3196C/G (rs115045214). Overall, these variation sites adhered to the Hardy-Weinberg Equilibrium, except +3010C/G in cardiac and whole group of patients and +3142G/C in digestive and whole group of patients. The following14-base pair I/D and +3001C/T: no significant differences were observed when the five types of comparisons were performed. The +3001T allele was observed in only one Chagas disease patient presenting exclusively the digestive form.P = 0.0131) and in the digestive form (P = 0.0462); (ii) the frequency of the +3003C allele was decreased in the whole group of patients (P = 0.0101) and in patients exhibiting the digestive form (P = 0.0448); (iii) +3003TT genotype frequency was overrepresented in the whole group (P = 0.0091) and in digestive (P = 0.0321) patients; and (iv) +3003T allele frequency was increased in whole group (P = 0.0101) and in the digestive group (P = 0.0448) of patients.+3003T/C: the +3003CC genotype was not observed in Chagasic patients. Compared to controls, (i) the +3003TC genotype frequency was decreased in the whole group of patients (P = 0.0322) and in those presenting the cardiac variant (P = 0.0098).+3010C/G: compared to controls, the +3010CG genotype was underrepresented in the whole group of patients (P = 0.0069) and to patients with the indeterminate (P = 0.0026), cardiac (P = 0.0076), and mixed (P = 0.0050) forms. The frequency of the +3027CC genotype was increased in patients exhibiting the digestive form compared to indeterminate (P = 0.0026), cardiac (P = 0.0076), mixed (P = 0.0050), and control (P = 0.0071) groups. The +3027AA and +3027AC genotypes were not observed in patients with the digestive form. As consequence, the digestive group exhibited a decreased frequency of the +3027A allele compared to controls (P = 0.0046) and to patients with the indeterminate (P = 0.0029), cardiac (P = 0.0083), and mixed (P = 0.0055) forms. The +3027C allele presented the opposite association pattern.+3027A/C: a decreased frequency of the +3027AC genotype was observed in the digestive form compared to control (P = 0.0024) and to patients with cardiac (P = 0.0247) and mixed (P = 0.0404) forms. A decreased frequency of the +3035CT genotype was observed in patients exhibiting the digestive form compared to mixed (P = 0.0404) and control (P = 0.0095) groups. The +3035C allele was overrepresented in the group of patients with the digestive form compared to control (P = 0.0020), indeterminate (P = 0.0346), cardiac (P = 0.0118), and mixed (P = 0.0489) groups. The +3035T allele presented the opposite association pattern.+3035C/T: the +3035CC genotype was overrepresented in patients with the digestive form compared to control (P = 0.0449), cardiac (P = 0.0363), and mixed (P = 0.0346) forms. The +3142GC genotype frequency was lower in cardiac patients compared to digestive (P = 0.0211) and control (P = 0.0061) groups. In addition, +3142GC genotype frequency was decreased in the whole group of patients group compared to the control group (P = 0.0459).+3142G/C: the +3142GG genotype frequency was significantly lower in digestive patients compared to indeterminate (P = 0.0169) and in the group of patients with the cardiac form (P = 0.0459).+3187A/G: compared to controls, an increased frequency of the +3187GG genotype was observed in the whole group (P = 0.0499) and an increased frequency of +3196GC genotype (P = 0.0258). In addition, +3196CG genotype was increased in the mixed group compared to the indeterminate variant (P = 0.0188).+3196C/G: patients exhibiting the mixed form presented a decreased frequency of the +3196CC genotype (P = 0.0128); (ii) the frequency of the +3003TT genotype was increased in patients (P = 0.0088); (iii) +3003C allele frequency was decreased in patients (P = 0.0097) and +3003T allele presented the opposite association pattern (P = 0.0097); (iv) the +3187GG genotype frequency was overrepresented in patients (P = 0.0282); (v) +3196CC genotype frequency was decreased (P = 0.0259) and +3196GC genotype frequency was increased (P = 0.0325) in patients.When CDM patients (excluding the indeterminate group) were compared to controls, the following results were observed: (i) the +3003CT genotype frequency was decreased in patients (P = 0.0440). On the other hand, when indeterminate patients were compared to healthy controls, no significant results were observed.When CDM patients were compared to the indeterminate form, only the +3196GC genotype was increased in CDM patients , we analyzed the frequency of theP = 0.0005), CDM (P = 0.0008), digestive (P = 0.0274), and cardiac (P = 0.0043) forms; (ii) UTR-13 was overrepresented in the indeterminate group (P = 0.0320); (iii) UTR-14 was overrepresented in cardiac patients (P = 0.0135). A decreased frequency of the UTR-7 haplotype was observed in the digestive group compared to indeterminate (P = 0.0056), mixed (P = 0.0053), and control (P = 0.0048) groups. In addition, UTR-13 was also increased in patients presenting the indeterminate form when compared to CDM patients (P = 0.0415).Compared to controls, (i) UTR-4 was underrepresented in the whole group of patients , compared to controls and early chronically infected animals  and Qa2 and NOS-2  in heart specimens of both mouse strains during acute T. cruzi infection.n BALB/c . During n BALB/c . When we T. cruzi infection. HLA-G gene polymorphism and HLA-G tissue expression have been evaluated in several diseases; however, little is known about the role of these molecules in the human or experimental T. cruzi infections.The present study provides consistent data regarding the involvement of HLA-G and its Qa2 murine functional homolog in the T. cruzi and have not developed clinically detected forms (indeterminate). When patients as a whole were compared to healthy individuals, some HLA-G 3′UTR alleles/genotypes were overrepresented , while others were underrepresented  in Chagas disease patients. When symptomatic patients (CDM) were compared to healthy controls, similar results were obtained, indicating that these polymorphic sites were indeed associated with the clinically detected Chagas disease. In contrast, when indeterminate patients were compared to controls, the frequency of the HLA-G 3′UTR alleles/genotypes was closely similar. This result corroborates the role of the aforementioned HLA-G 3′UTR alleles and genotypes in the clinically recognized Chagas variants.The association of genetic markers with infectious disease has posed intriguing questions regarding the comparisons of gene frequencies between individuals with the disease with healthy individuals who had never been exposed to the specific infectious agent. Due to the myriad of chronic clinical forms observed in Chagas disease, part of these concerns may be circumvented by the possibility of comparing patients presenting well recognized clinical variants with patients who were infected by CTLA-4 [ in silico study, we reported several miRNAs that may target the +3027A/C and +3035C/T polymorphic sites [The stratification of Chagas patients into clinical variants showed that the +3027CC and +3035CC genotypes and the +3027C and +3035C alleles were strongly associated with the digestive form of Chagas disease. In addition, the +3142GC genotype was associated with a decreased risk to cardiac form development, when compared to the digestive form. These results indicate that genetic susceptibility to digestive or cardiac forms may be distinct. Indeed, previous studies conducted by our group evaluating classical histocompatibility  antigens/alleles  and othe CTLA-4 , also shic sites . HLA-G 3′UTR polymorphic sites were considered as haplotypes, decreased UTR-4 and UTR-7 frequencies were associated with the clinically detectable Chagas disease and with the digestive form, respectively. On the other hand, UTR-14 was associated with the cardiac variant and UTR-13 with the indeterminate form. Noteworthy, particular posttranscription elements that are present in these UTRs also exhibited a similar differential behavior when their frequencies were individually evaluated, further corroborating the role of HLA-G 3′UTR sites in disease susceptibility. Our group demonstrated that specific HLA-G 3′UTR haplotypes are associated with differential soluble levels of HLA-G (sHLA-G), being UTR-1 associated with high sHLA-G and UTR-5 and UTR-7 with decreased sHLA-G levels [ HLA-G 3′UTR haplotypes with tissue HLA-G expression.WhenG levels . However TNF [ IL-1β [ IL-10 [ IL-4 [ INF-γ [ TGF-β1 [ IL-12B [ CXCL9 [ CXCL10 [ CCR5 [ CCL2 [ CTLA-4 [Considering that cytokines, chemokine, and immunomodulatory molecules play an important role in the pathogenesis of many diseases, including Chagas disease, and gene polymorphisms may influence the expression of these molecules, different studies have reported the association between different clinical forms of Chagas disease and polymorphisms at TNF –61, IL-1 [ IL-1β , IL-10 [ [ IL-10 , IL-4 [60 [ IL-4 , INF-γ [ [ INF-γ , TGF-β1 [ TGF-β1 , IL-12B [ IL-12B , CXCL9 [ [ CXCL9 , CXCL10 [ CXCL10 , CCR5 [60 [ CCR5 –70, CCL25 [ CCL2 , and CTL T. cruzi. On the other hand, myocardium infiltrating lymphomononuclear cells also exhibited HLA-G expression, which could decrease their functional properties. Since myocardium damage has been primarily attributed to fiber losses due to the action of immune cytotoxic cells, one may consider that HLA-G lymphomononuclear cell expression may not be the only regulatory surface cell molecule. In addition, it is important to emphasize that all these findings were observed in heart specimens of deceased patients, and the temporal HLA-G expression during the chronic phase has not been evaluated as yet.Regarding HLA-G expression on tissue cells, patients with the digestive form exhibited decreased expression in colon but not in esophagus specimens, contrasting with patients exhibiting the cardiac variant who presented a decreased expression on myocardium cells. Many segments of the digestive tract and normal myocardium fibers  may cons T. cruzi infection may depend on several factors, including (i) the inoculated strain, (ii) the amount of parasites, and (iii) the genetic background of the animal. Several of these combinations have been used to induce acute or early chronic infection. BALB/c mouse has been considered to be susceptible to acute T. cruzi infection induced by the Y and Tulahuén strains, due to the predominance of a Th2 immune response, characterized by high production of IL-4 and IL-10 [ T. cruzi infection induced by Y and Tulahuén strains, since the mouse produces a Th1 immune response with the production of INF-γ, IL-12, TNF, and NO [ T. cruzi CL strain that depending on the dose may produce severe acute or lead to the development of early chronic infection. In BALB/c mice, the acute infection by CL strain induced higher heart parasitism and parasitemia with increased production of proinflammatory mediators [ T. cruzi infection, chronic infection is difficult to be induced by more virulent strains. On the other hand, little is known about the acute and early chronic infection induced by CL strain in C57BL/6 mice. With the use of the CL strain we did not observe a differential susceptibility in BALB/c and C57BL/6 mice in terms of mouse survival; however, BALB/c showed higher parasitemia, whereas C57BL/6 presented higher heart parasitism. Therefore, the C57BL/6 controlled CL strain parasitemia but not heart tropism, and the inverse occurred with the BALB/c mouse.Overall, the outcome of the experimentalnd IL-10 , 76. On , and NO , 14, 15.ediators . Since B T. cruzi infection, the transcriptional level of Qa2 was increased in heart and esophagus in both BALB/c and C57BL/6 mouse strains, and the increased expression was not related to resistance or susceptibility to the acute infection. Although little attention has been devoted to the role of immunomodulatory nonclassical MHC molecules in experimental infection, Qa2 expression may be induced to counterbalance the action of proinflammatory and other anti-inflammatory or immunomodulatory molecules. Indeed, the transcriptional levels of the Qa1, CTLA-4, PDCD1, INF-γ, NOS-2, and IL-10 genes were also induced in the acute T. cruzi infection, as shown in this study. Additional literature findings corroborate this idea. Tissue damage observed in the affected tissues is due to the inflammatory process generated by an exacerbated immune response triggered against the parasite, and the high production of CTLA-4, PD-1, and IL-10 is related to control inflammatory process during T. cruzi infection [ Qa2 gene, may be associated with the control of tissue damage caused by inflammatory process, cytolysis, and fibrosis.Similarly to HLA-G, the Qa2 molecule may also control the immune response by inhibiting NK cells . During nfection , 24, 78. T. cruzi in the heart. Considering that mice were exposed to decreased number of the less virulent CL strain, it is reasonable to conclude that both BALB/c and C57BL/6 mice had time and chance to mount an adequate immune response against the parasites.Regarding the early chronic infection, the expression of immunomodulatory, proinflammatory, and anti-inflammatory genes analyzed in this study was closely similar to noninfected mice in both BALB/c and C57BL/6 strains, and both animal groups were able to evolve into the early chronic infection, presenting only a little amount of T. cruzi infection. In terms of Chagas disease susceptibility, HLA-G 3′UTR alleles/genotypes/haplotypes exhibited differential frequencies in infected/diseased patients when compared to only infected patients and healthy controls. The observation of a decreased HLA-G expression on cardiac muscle and colonic cells associated with the increased expression of HLA-G on myocardium infiltrating lymphomononuclear cells may reflect a lack of protection of these tissues that constitutively express HLA-G. In experimental infection, the mouse genetic background exerted only a mild influence on the overall response against the T. cruzi CL strain. The transcriptional levels of Qa2, Qa1, CTLA-4, PDCD1, INF-γ, NOS-2, and IL-10 genes were induced only during the acute T. cruzi infection in BALB/c and C57BL/6 mice, indicating a fine balance between pro- and anti-inflammatory genes.We present several lines of evidence pointing to the participation of the immunomodulatory molecules HLA-G and its functional murine homolog Qa2 on human and experimental


In order to understand how important their findings are, a comparison was made regarding the reinforcing effects of DA D2-like R full agonists with those of DA uptake inhibitors and of a DA D1-like receptor  full agonist (±)-SKF 82958.A series of studies by Drs. Barak Caine, James Woods, Gregory Collins, Jonathan Katz and Takato Hiranita demonstrated a novel and unique reinforcing effect using dopamine (DA) D2-like receptor [D2-like R: D A drug self-administration (SA) procedure has been used to directly assess reinforcing effects of various drugs across pharmacological classes including cocaine under various schedules of reinforcement in various species because of its relative high face and predictive validities –3. UsingAs with the DA uptake inhibitor methylphenidate , the DA E). In addition, a more recent study using the DA D3R full agonist PF-592,379 demonstrated a lack of reinforcing effects of the DA D3R full agonist when substituted for cocaine with response-dependent presentations of a cocaine-paired stimulus [In contrast to the DA D1-like R full agonist (±)-SKF 82958, reinforcing effects of the DA D2-like R full agonists are not appreciable in naïve subjects. For example, none of doses of the DA D2-like R full agonist quinpirole maintained SA responding above vehicle levels in experimentally naïve rats  and in rstimulus ,26 and mstimulus , and thestimulus ,17.B), it appears that DA D1-like Rs are undoubtedly more critical than DA D2-like Rs in the reinforcing effects of cocaine and perhaps another DA uptake inhibitors.Studies using a genetic disruption of the DA receptor subtypes in mice also have revealed the distinct role of each DA receptor subtype in cocaine SA under an FR schedule of reinforcement. Genetic disruptions of each DA receptor subtype could cause a developmental impairment and/or a functionally compensatory mechanism other than their physiological functions. However, consistent with results of assessment of a capacity of direct DA receptor full agonists to induce or maintain SA responding mentioned above  mice, cocaine was virtually ineffective at maintaining SA responding in DA Dfor food . However KO mice . On the 1Rs [2R WT and KO mice, with only a modest rightward/upward shift of the descending limb of the DEC for cocaine SA observed in the DA D2R KO mice [2Rs [3R did not alter the reinforcing effects of cocaine as assess by either FR or progressive ratio (PR) schedules of reinforcement [4R KO mice and their WT controls [1R KO mice, and suggest that in that DA’s effects at DA D1Rs, but not DA D2Rs, are required for cocaine reinforcement.In marked contrast to DA D1Rs , it appe KO mice . Similarice [2Rs , geneticorcement . In addicontrols . NonetheThe reinforcing effects of cocaine under an FR schedule of reinforcement in rats have been demonstrated to be substantially affected by transient pharmacological manipulations using direct DA receptor ligands given as a pre-session treatment. The results further indicate quite distinct role of DA D1-like and D2-like receptors in cocaine reinforcement , the effects of DA D2-like R full agonists may be to enhance the conditioned reinforcing effects of cocaine-pared stimuli rather than the primary reinforcing effects of cocaine [As with standard indirect DA receptor agonists ,10–14 bu cocaine –23.S (−)-eticlopride dose-dependently shift the DECs of cocaine SA to the right, suggesting a competitive and typical antagonism of the reinforcing effects of cocaine [2R antagonist L-741,626 was more effective than either compound alone to antagonize cocaine SA [3/4R antagonist L-745,829 and the DA D4R antagonist L-745,870 were ineffective against cocaine SA [2 receptors.The DA D1-like R antagonist SCH-39166 and the DA D2-like R antagonist  cocaine ,24,26,312-likeR full agonists [As mentioned above in a section D2, the potentiation of cocaine SA with the DA Dagonists ,20 was iagonists ,10–14. Tagonists . Althougagonists –23, (±)-agonists , (±)-7-Oagonists , and praagonists ,21–23. Nagonists , but remagonists . Similaragonists ,21–23. Tagonists ,21–23 anagonists ,20 resulagonists . In addiagonists .per se. These findings are useful in understanding how DA D2-like R antagonists can antagonize several effects of various compounds across pharmacological classes including methamphetamine, opioids, ethanol, nicotine, and non-competitive NMDA glutamate receptor antagonist. Given a lack of effects of DA receptor antagonists on SA of (−)-heroin and (±)-ketamine , DA D2-like Rs might likely contribute to maintenance of D2-like R-mediated conditioning effects of (−)-heroin and (±)-ketamine, but not to their primary reinforcing effects since (−)-heroin and (±)-ketamine can increase DA levels in brains. Further these findings could have a substantial impact on a psychiatric research field since DA is involved in a wide range of various psychiatric diseases and symptoms.The reinforcing effects of cocaine are initiated by its inhibition of DA uptake, resulting in an enhancement of dopaminergic transmission in the central nervous system ,35. The


Escherichia coli (STEC) O104, including H4, H7, H21, and H¯, have been associated with sporadic cases of illness and have caused food-borne outbreaks globally. In the U.S., STEC O104:H21 caused an outbreak associated with milk in 1994. However, there is little known on the evolutionary origins of STEC O104 strains, and how genotypic diversity contributes to pathogenic potential of various O104 H-antigen serotypes isolated from different ecological niches and/or geographical regions.Various H-serotypes of the Shiga toxin-producing eae) gene, involved in the attaching-effacing phenotype of diarrheagenic E. coli, was not found in either strain. Examining various O104 genome sequences, we found that two “complete” left and right end portions of the locus of enterocyte effacement (LEE) pathogenicity island were present in 13 O104 strains; however, the central portion of LEE was missing, where the eae gene is located. In O104:H4 strains, the missing central portion of the LEE locus was replaced by a pathogenicity island carrying the aidA (adhesin involved in diffuse adherence) gene and antibiotic resistance genes commonly carried on plasmids. Enteroaggregative E. coli-specific virulence genes and European outbreak O104:H4-specific stx2-encoding Escherichia P13374 or Escherichia TL-2011c bacteriophages were missing in some of the O104:H4 genome sequences available from public databases. Most of the genomic variations in the strains examined were due to the presence of different mobile genetic elements, including prophages and genomic island regions. The presence of plasmids carrying virulence-associated genes may play a role in the pathogenic potential of O104 strains.Two STEC O104:H21 (milk outbreak strain) and O104:H7 (cattle isolate) strains were shot-gun sequenced, and the genomes were closed. The intimin (E. coli O104 strains is proposed.The two strains sequenced in this study (O104:H21 and O104:H7) are genetically more similar to each other than to the O104:H4 strains that caused an outbreak in Germany in 2011 and strains found in Central Africa. A hypothesis on strain evolution and pathogenic potential of various H-serotypes of The online version of this article (doi:10.1186/s12866-015-0413-9) contains supplementary material, which is available to authorized users. E. coli (EAHEC) O104:H4 infections in Europe and North America in 2011 with prior reporting of similar strains in patients with severe infections and hemolytic uremic syndrome (HUS) in Asia, Norway, and the Republic of Georgia raises questions about the origin, evolution, genetic diversity, and virulence of these pathogens [E. coli (EAEC); however, it carried the gene that encodes Shiga toxin 2a (stx2a), and thus can also be classified as a Shiga toxin-producing E. coli (STEC). Bielaszewska et al. [E. coli O104:H4 strains and O104 strains with other H-types showed genetic and phenotypic differences, including the presence of specific virulence and antibiotic resistance genes, the types of Shiga toxin genes, and their pulsed-field gel electrophoresis profiles [The occurrence of a large outbreak involving enteroaggregative hemorrhagic athogens . The Gera et al.  analyzedprofiles ,9,10.¯, H2, H7, H8, H11, H12, H16, H19, H21, H25, and H28 [E. coli O104:H12 and O104:H21 were isolated from young cattle in Austria in 2009, O104:H7 from sheep and wild boar (O104/O127) in Spain, O104:H7 from sheep in India, from young cattle in Argentina, and from unspecified meat and sheep meat in New Zealand [¯ [Some of the most common flagellar H antigens associated with STEC strains include H and H28 ,11. Shig and H28 . Accordi Zealand -19. To d Zealand , O104:H2aland [¯ .stx-negative O104:H4 strains have been isolated from human patients in Central Africa [eae gene; however they can attach to intestinal cells. The EAHEC O104:H4 outbreak strain 2011C-3493 also lacked eae; however, this strain possessed a plasmid (pAA) that encodes for adherence fimbriae [aggR, which encodes a regulator of virulence plasmid and chromosomal genes including aggregative adherence fimbriae and aaiA-P (aggR-activated island encoding a type VI secretion system), a characteristic of EAEC. EAHEC O104:H4 colonizes the human bowel through aggregative adherence fimbriae encoded by the EAEC plasmid (pAA). In the European O104:H4 outbreak strains, the intimin (eae) function was substituted by the plasmid-encoded aggregative adherence fimbrial colonization mechanism, and once attached, the cells were able to produce and deliver Shiga toxin 2a, resulting in severe illness and HUS. Recently, Beutin et al. [stx2-phage P13374 (from the German EAHEC O104:H4 strain) by EAEC strains of different serotypes. In this study, the authors concluded that the stx2-phage P13374 had a restricted host range, and its spread was dose-sensitive. One of the bovine-specific phages (P13803) was found to be capable of lysogenizing a stx-negative EAEC O104:H4 strain and converted it into an EAEC-STEC producing Stx2a [Differences in virulence and antibiotic resistance genes linked to disease severity and HUS among various O104 serotype strains, including O104:H11, O104:H21, O104:H7, O104:H2, O104:H12, and O104:H16 have been observed . The difl Africa ,27 at dil Africa , and thel Africa -32. In cl Africa , feces ol Africa . FacilitE. coli O104:H4 strains isolated from different geographical locations. The resultant sequence analysis revealed notable differences among O104 strains belonging to H-serotypes H21, H7, and H4, including the presence of different plasmids and other mobile genetic elements.Previously published sequence data on O104:H4 isolates revealed variations in gene content among strains from different hosts and geographical sources ,4,6,9,35E. coli O104 strains. Information on all strains analyzed in this study, including genome size, GC content, and number of plasmids, is shown in , or partially missing  in O104:H7 and O104:H21 strains. Region #3 , O104:H21 (strain 94–3024), O104:H4 , and O104:H4 . Sequence alignment and the linkage pattern analysis  but not to Africa strain C734-09. There were at least four different types of stx bacteriophages found in the O104 strains analyzed in this study . STEC strains that carry specific stx2 subtypes, especially stx2a, stx2c, and stx2d tend to be more virulent [stx2d was found to be the sole stx gene in our sequenced O104:H21 and O104:H7 strains. Sequence analysis also revealed that none of the O104 strains harbored the stx2e, stx2f, or stx2g alleles.Shiga toxins Stxiophages ,39. The ironment ,41. Geneironment ,42-44. Tvirulent . In thiseae is another important virulence gene of EPEC, EHEC, and STEC strains. The three prominent LEE integration sites selenocystyl-tRNA (selC), pheU, and pheV were previously described [eae genes. It has been reported that serotype O104:H− carried an eae-TAU (GenBank Accession: FM872416.1) variant. Strains expressing eae-TAU were generally restricted to and efficiently colonized Peyer’s patches in human intestinal mucosa [− has been found only in humans [E. coli O91:H− that occurs exclusively in a subgroup of STEC strains that are eae negative and contain the variant stx2d gene, similar to O104:H7 and O104:H21 in this study.escribed . The difl mucosa ; so far,n humans . Schmidtn humans  reportedtir) effector gene from serotype O104:H12 strain 4051–6 was sequenced (GenBank Accession# AB288103.1); however tir was missing in all O104:H4, O104H7, and O104:H21 strains examined in the current study  and eae (downstream) although the whole genome sequence of O104:H12 strain 4051–6 is not available currently. The functional Tir protein is translocated into the epithelial cell and is integrated into the cell membrane where it functions as a receptor for eae. Oswald and coworkers [eae were associated with differences in tissue and host specificity. Thus, it is possible that O104 strains belonging to H7 or H21 serotype possess the EAEC or EHEC genetic background to acquire functional genes of various PAIs or eae variants  and/or PAIs that may have been introduced into the genome by horizontal gene transfer (HGT). By directly applying IslandViewer for the identification of GIs in the genomes of O104:H21, O104:H7, and the O104:H4 reference strain (NC_018650), we found that the percentage of horizontally transferred genomic islands varied from 9.3%, 7.6%, and 12.3% among O104:H21, O104:H7, and the O104:H4 reference strain. The conserved GIs are identified by aligning all GIs in the reference strain O104:H4 to the GIs in other O104 strains. We found the conserved GI pools represented 70.9%, 65.7%, 65.5%, 49.1%, 44.3%, 19.3%, 15.3%, and 13.6% of the “reference genomic islands” from NC_018650 in O104:H4 strains LB226692, TY2482, C734-09, C754-09, C777-09, C760-09, O104:H21 94–3024, and O104:H7 RM9387, respectively. O104:H7 and O104:H21 appeared to be closer to each other and the O104:H4 C760-09 strain from Africa than to other O104:H4 strains, which is consistent with the phylogenetic tree in Figure − strain which is inserted into the selC site between yicI and nlpA . The proteins encoded in this island include a novel serine protease EspI (ORF#15); an adherence-associated locus, similar to iha of E. coli O157:H7 (ORF#18); an E. coli vitamin B12 receptor (ORF #20); an araC-type regulatory module (ORF #22-26); and several other important and/or unknown function proteins. The remaining sequence consists largely of complete and incomplete insertion sequences, prophage sequences, and an intact phage integrase gene that is located directly downstream of the chromosomal selC [selC-tRNA loci between yicI and nlpA to this typical O103 PAI shows some interesting genomic structural differences among O104 H-serotypes, O103:H−, O157:H7, non-O157 STEC, and other serotypes including E. coli K-12  and C-terminal (yicL and nlpA), but none of these serotypes are genetically similar in the central portion of this typical PAI structure.PAIs belong to a specialized class of GIs. They carry multiple virulence loci, may take up a large chromosomal region (>10 kb) of pathogens, are absent from non-pathogens, and usually have a different GC content from that of the core genome. A PAI from an O103:Hmal selC . A pairwORF #22-2; and sevspI ORF#1; an adheORF #22-2; and sevselC site in O104:H7 and O104:H21 is 7775 bp and it encodes no known functional proteins. Here we propose the concept of “pseudo PAI”, a genomic region less than 10 kb in size that lacks key functional gene products typically found in O157:H7 and O103:H−  was found in strains of serotypes O104:H7, O104:H21, O111:H−, O113:H21, O121:H19, O91:H21, O103:H25, O103:H2, O26:H11, and even non-pathogenic E. coli K12. These differences in the size of the functional PAIs  or “pseudo PAI” among the same serogroup was also observed for O104 and O103:H− . These observations are suggestive of the acquisition of the different PAIs by the same serotype, including O104:H4 strains due to their different ecological niches and/or the geographical regions where they were found. The selC site can be targeted by bacteriophage carrying LEE or LEE-like PAIs , bacteriophage harboring resistance genes (e.g. O104:H4)  or eae-TAU carried by a prophage/PAI (O104:H−) and other genetic components, such as the plasmid carrying aggR gene. Various O104 strains were also shown to carry stx2 [E. coli in diverse environments, which may be relevant regarding its host specificity, as well.Based on our analyses, we propose an evolutionary model , stx1, stx2, efa1, ent/espL2, tir, nleB and nleE (OI-122 specific), espK (prophage CP-933 N), tehB, iha, and O104- and O157-specific lpfa, tccp, tccp2, IS629 integrase, and the CRISPR2 sequence were used to define serogroup and pathogroups of O104:H7, O104:H21, and other O104:H4 strains. Based on the genotypic differences, E. coli pathotypes can be categorized as atypical EPEC (eae only), typical EPEC (eae and bfpA), STEC (stx1 and/or stx2 with or without eae), EHEC (eae and stx1 and/or stx2), aggR-positive EAHEC , and aggR-negative EAHEC/STEC (absence of eae and aggR but with stx1 and/or stx2). These virulence factors and O- and H-antigen specific genes are useful for molecular characterization of EPEC, STEC, EHEC and EAHEC strains at the genomic level (Table E. coli O104:H4 European outbreak strain contains a stx-encoding phage similar to 933 W with only one nucleotide polymorphism in each of the subunits (stx2a and stx2b) compared to 933 W. The German outbreak O104:H4 strains , sigA (Shigella IgA protease-like homologue), pic , aap (dispersin), aatPABCD (dispersin transporter), and others; however, none of these were found in the sequenced O104:H7 and O104:H21 strains (data not shown). Aside from C760-09, O104:H4 strains from Africa did not carry noteworthy virulence factors other than the iha gene; however, C777-09 possessed the stx2 gene, thus indicating potential virulence (Table lpf (long polar fimbriae) and iha (IrgA homologue adhesin) that are associated with EHEC and colonization of the gut, but lacked the aggR, aggB-C-D modular genes  gene due to frameshift- and nonsense-mutations. After transposition, there is only one IS629 left in O104:H4. IS629 insertion is highly biased toward prophages and prophage-like integrative elements. Both IS629 and IS602 are not present in O104:H7 and O104:H21 but exist in all O104:H4 strains  resistance transposons [629 and CRISPR distribution among O104 strains might be useful for identification and detection of specific O104 strains and population genetic analysis, as well as molecular epidemiology studies. The presence of IS629, CRISPR, and prophage regions (data not shown) may potentially contribute to the larger genome size of O104:H4 , saa, ecf cluster, ehx cluster, etp cluster (type II secretion system apparatus), toxB (putative adhesion), katP , espP, espA, sepA (serine protease sepA precursor), subAB, typeIV pilus gene cluster, mabB, stcE , and excA (exclusion-determining protein), in agreement with other previous reports [subAB (a potent toxin involved in inducing cell death), the ehx cluster, espP, and excA were found in O104:H7, O104:H21, and LEE-negative O113:H21 strains, but not in O104:H4 strains. Therefore, it is suggested that virulence of the O104:H21 outbreak strain may be due in part to plasmid-associated virulence genes, including subAB, the ehx cluster, and espP  between bacteria. In this study, a “pseudo s Figure . Our finstx2, was found in all European O104:H4 outbreak strains, but only in one of the strains of African origin (C777-09)  and from Dr. Robert Mandrell at the USDA Agricultural Research Service, Western Regional Research Center , respectively. Ion Torrent libraries were prepared following the manufacturer’s recommended library construction procedures. Ion Torrent 316-chips with the 200-bp OneTouch kit was used for the generation of sequencing data on the Ion Torrent Personal Genome Machine (PGM). High molecular weight DNA for PacBio sequencing was extracted using Qiagen Genomic-tip 100/G columns and a modified manufacturer’s protocol as previously described . Ten mico Prokka  to predio Prokka .The Artemis comparison tool, ACT version 10  was usedhttp://trace.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?view=search_obj) and our two newly sequenced genomes were re-assembled and ordered based on reference genome. In general, progressive Mauve was applied for whole genome comparison. Homologs and/or orthologous relationship of the nucleotide sequences for profiling of virulence- and resistance-associated genes between genomes were determined using NCBI Basic Local Alignment Search Tool (BLAST) with the following criteria: identity >80%, e-value <1e − 10, and coverage >90%. The identified homologs and/or orthologous genes were further manually curated and confirmed.Mauve was used for comparing and visualizing a number of genomes. By applying O104:H4 strain 2009EL-2050 (NC_018650.1) genome as the “reference” strain, all FDA draft raw sequences for O104:H21 (Table Prophages and putative phage-like elements in the O104:H4 reference strain 2009EL-2050 (NC_018650.1) and the newly sequenced O104:H7 and O104:H21 genomes were analyzed using prophage-predicting PHAST Web server . Regionseae, among all O104:H serotype strains listed in this study. The presence of the LEE carrying eae and its flanking genes was confirmed by manual inspection of the annotation for this region, and/or by detection of the coordinates of genes encoded within the LEE of O157:H7 strain EDL933.IslandViewer  was usedrpoB, β-subunit of RNA polymerase, can result in antibiotic resistance) was also performed (data not shown).Only bacterial genes experimentally confirmed to be involved in bacterial pathogenesis and antibiotic resistance were collected through an extensive literature mining in PubMed. This was followed by reference mapping to confirm their existence with corresponding raw sequencing reads. SNP discovery for RpoB , CP009105, KM085451, KM085452, KM085453 , CP009106 (O104:H21 94–3024 chromosome) and CP009107 (O104:H21 plasmid pO104_H21).


Tomato rugose mosaic virus (ToRMV) and Tomato severe rugose virus (ToSRV) are often found in tomato fields. The complete sequence of the DNA-B components of ToSRV and ToRMV show an identity of 98.2%. Additionally, the high nucleotide identity (96.2%) between their common regions indicates that these two viruses may share the same DNA-B.Begomoviruses are dicot-infecting, whitefly-transmitted viruses with a genome comprised of one or two molecules of circular, single-stranded DNA. In Brazil, tomato-infecting begomoviruses have emerged as serious pathogens since the introduction of a new biotype of the insect vector in the mid-1990’s. Tomato seedlings were biolistically inoculated with ToSRV (DNA-A and DNA-B) and ToRMV (DNA-A and DNA-B) infectious clones in every possible combination of single or mixed infection. Symptom expression was evaluated for up to 35 days post-inoculation (dpi). DNA was extracted at 28 dpi and the presence of each viral genomic component was examined by rolling circle amplification (RCA) followed by digestion, as well as by quantitative, real-time PCR. Sequence comparisons, recombination and phylogenetic analyzes were performed using EMBOSS needle, RDP program and maximum likelihood inference, respectively.Symptoms in tomato plants inoculated with the different combinations of ToRMV and ToSRV DNA-A and DNA-B components consisted of a typical mosaic in all combinations. Pseudorecombinants were formed in all possible combinations. When two DNA-A or two DNA-B components were inoculated simultaneously, the ToRMV components were detected preferentially in relation to the ToSRV components. The combination of minor changes in both the Rep protein and the CR may be involved in the preferential replication of ToRMV components. Recombination and phylogenetic analyzes support the exchange of genetic material between ToRMV and ToSRV.Solanum lycopersicum) and share the same DNA-B. ToRMV DNA components are preferentially replicated over ToSRV components. These results indicate that the emergence of ToRMV involved both recombination and pseudorecombination, further highlighting the importance of these mechanisms in the emergence and adaptation of begomoviruses.ToRMV and ToSRV form viable pseudorecombinants in their natural host ( Begomovirus (family Geminiviridae) contains viruses with a genome comprised of circular, single-stranded DNA, transmitted by the whitefly Bemisia tabaci (Homoptera: Aleyrodidae) to dicot plant species and ToSRoculated using twXhoI and BglII to detect ToRMV-A and -B, respectively; HindIII and BglII + SacII to detect ToSRV-A and -B, respectively.Inoculated plants were evaluated for symptom expression for up to 35 days post-inoculation (dpi). DNA from all plants was extracted at 28 dpi as described and the http://www.idtdna.com/scitools/Applications/RealTimePCR: ToRMV-A(For), 5′CAT CGG GCC TCT GTT GG3′ and ToRMV-A (Rev), 5′GTT ATG CAA CTT GGG CGT TAA G3′; ToSRV-A(For), 5′AAA GTA AAG TGA TTG TCT GTG G3′ and ToSRV-A(Rev), 5′GCC GTT CAA CAA ATT GGG3′. Primer specificity was tested by conventional PCR using positive samples for each virus, followed by electrophoresis in 2.0% agarose gel. Reactions were prepared in a final volume of 10 μL, using Fast SYBR Green Master Mix (Applied Biosystems) following the manufacturer’s instructions, and were analyzed in a CFX96 Real-Time PCR System (Bio-Rad). The PCR protocol included an initial denaturing step at 95°C for 20s, followed by 39 cycles of 95°C for 3 s and 60°C for 30s, followed by a dissociation stage.Based on the results obtained from RCA-RFLP assays, control plants  and plants in which the DNA-A’s of both viruses were co-inoculated in different combinations  were selected for quantitative, real-time PCR (qPCR) assays. Primers were designed based on the sequence of the common region of the DNA-A (in the most divergent region between the two viruses) using the qPCR tool implemented in 6 copies of the viral genome per reaction). Standard curves were obtained by regression analysis of cycle threshold (Ct) values of each one of the three replications of a given dilution in relation to the log of the amount of DNA in each dilution. For absolute quantification of the number of viral DNA molecules in the different treatments, 100 ng of total DNA was extracted as described previously and used in reactions containing virus-specific primers. Each sample was analyzed in triplicates, and three different plants of each treatment described above were assayed. DNA-B accumulation was not evaluated due to the high sequence similarity between the two viruses, which prevented the design of virus-specific primers for the qPCR assay.Standard curves were prepared using serial dilutions of plasmid DNA containing an insert corresponding to the complete ToSRV DNA-A , using the EMBOSS needle  with default settings. Nineteen New World begomovirus DNA components . The begomovirus Macroptilium mosaic Puerto Rico virus (MaMPRV) [GenBank: AY044133] was used as an outgroup.Multiple sequence alignments were performed for the DNA-A sequences of Brazilian begomoviruses Table  based onThe authors declare that they have no competing interests.FMZ, FNS and ATML designed the study. FNS, ATML, GPCU and MAJ performed the experiments. FNS, ATML and CSR analyzed the data. FNS and FMZ wrote the manuscript. All authors have read and approved the final version of the manuscript.A. Digestion with Hind III, generating a fragment of approximately 2600 nucleotides (nt) corresponding to ToSRV DNA-A (the other three components are not cleaved by this enzyme). B. Digestion with Xho I generating a fragment of approximately 2600 nt corresponding to ToRMV DNA-A (the other three components are not cleaved by this enzyme). C. Digestion with Bgl II + Sac II, generating fragments of 1800 and 800 nt corresponding to ToSRV DNA-B, and approximately 2600 nt corresponding to ToRMV DNA-B (the two DNA-A components are not cleaved by these enzymes). Underlined means DNA-A + DNA-B. (–) negative control. M, 1 kb plus DNA ladder (Invitrogen), in bp.Detection by rolling circle amplification followed by restriction digests (RCA-RFLP) of each DNA component from ToRMV-[BR:Ub1:96] and ToSRV-[BR:PG1:Pep:03] after biolistic inoculation of tomato plants. Click here for fileP ≤ 0.05.ToSRV-[BR:PG1:Pep:03] DNA-A accumulation in single or dual infections with ToRMV-[BR:Ub1:96] in tomato plants. Total DNA was extracted from systemically infected leaves at 28 days post-inoculation and used as a template for quantitative, real-time PCR (qPCR) using ToSRV DNA-A primers. 1. Mock-inoculated plants; 2. ToRMV DNA-A + DNA-B; 3. ToSRV DNA-A + DNA-B; 4. ToRMV DNA-A + DNA-B and ToSRV DNA-A + DNA-B; 5. ToRMV DNA-A + DNA-B and ToSRV DNA-A; 6. ToRMV DNA-A and ToSRV DNA-A + DNA-B. In treatment 2, the ToSRV primers weakly amplified ToRMV DNA-A. Values are given as the mean ± confidence interval of three independent tomato plants. Asterisks indicate statistically significant differences by the t test at Click here for file


The structures of the new compounds were revealed through 1D and 2D NMR and mass spectroscopic studies. Three new physalin steroids, physalin III (Supplementary material is available for this article at 10.1007/s13659-013-0021-z and is accessible for authorized users. Supplementary material, approximately 1.54 MB.


TIFA (TNF receptor associated factor (TRAF)-interacting protein with a Forkhead-associated (FHA) domain), also called T2BP, was first identified using a yeast two-hybrid screening. TIFA contains a FHA domain, which directly binds phosphothreonine and phosphoserine, and a consensus TRAF6-binding motif. TIFA-mediated oligomerization and poly-ubiquitinylation of TRAF6 mediates signaling downstream of the Tumor necrosis factor alpha receptor 1 (TNFaR-I) and interleukin-1/Toll-like receptor 4 (TLR4) pathways. Examining TIFA expression in hepatocellular carcinoma (HCC) tissues microarrays, we noted marked decreases TIFA reactivity in tumor versus control samples. In agreement, we found that HCC cell lines show reduced TIFA expression levels versus normal liver controls. Reconstituting TIFA expression in HCC cell lines promoted two independent apoptosis signaling pathways: the induction of p53 and cell cycle arrest, and the activation of caspase-8 and caspase-3. In contrast, the expression of a non-oligomerizing mutant of TIFA impacted cells minimally, and suppression of TIFA expression protected cells from apoptosis. Mice bearing TIFA overexpression hepatocellular xenografts develop smaller tumors versus TIFA mutant tumors; terminal deoxynucleotidyl transferase dUTP nick end labeling staining demonstrates increased cell apoptosis, and decreased proliferation, reflecting cell cycle arrest. Interestingly, p53 has a greater role in decreased proliferation than cell death, as it appeared dispensable for TIFA-induced cell killing. The findings demonstrate a novel suppressive role of TIFA in HCC progression via promotion of cell death independent of p53. Chronic liver inflammation is associated with increased incidence of liver cancer. Hepatocellular carcinoma (HCC), the most common liver cancer, is an end product of chronic liver disease typically requiring decades to evolve. HCC is the fifth most common cancer worldwide, with a doubling incidence in the United States alone during the last two decades.2 TLR4,4 TREM-1,6 have been linked to the development of HCC; other proteins, such as TIFA (TRAF-interacting protein with a Forkhead-associated (FHA) domain), are upregulated in response to hypoxia or other acute stress.7 The most frequent cause for chronic hepatic inflammation in humans is infection with hepatitis B virus or hepatitis C virus, which currently persist in approximately 500 million people worldwide, and fosters an increasing HCC patient population. Effective means to eradicate these chronic viral infections have been elusive, although new treatments for hepatitis C virus are poised to make a significant impact if economic barriers can be overcome.An increased incidence of inflammatory mediators, such as ATF4,8 but may also bind TRAF2.9 In addition to the FHA domain, a phosphothreonine and phosphoserine-binding motif,10 TIFA contains a consensus TRAF6-binding motif.12 Elevated TIFA expression activates nuclear factor (NF)-κB and c-JUN N-terminal kinase in a manner dependent on TRAF6,13 and links TRAF6 to NF-κB in the interleukin-1/TLR4 pathway.8 TIFA is increased after cell stress such as hypoxia; the increase in TIFA level appears to feed forward into TLR4/MyD88-dependent signaling, leading to NF-κB activation and HMGB1 release.7 The data are consistent with a pro-tumorigenic role for TIFA.Analysis of fundamental inflammatory signaling pathways may therefore reveal targets or markers to identify and treat patients with chronic liver inflammation, particularly those predictive of HCC. TIFA, also called T2BP, was identified as a TRAF6-interacting protein in a yeast two-hybrid screen,5 In agreement, TIFA reconstitution induced the expression of p53, promoting apoptosis while suppressing proliferation among surviving cells. The studies implicate TIFA as a previously unappreciated suppressor of liver carcinogenesis via p53-dependent and -independent mechanisms, and provide insight into a vulnerability of HCC.Here, we studied the role of TIFA in normal liver and HCC. Unexpectedly, we find that TIFA expression is suppressed during tumor progression, in contrast to other inflammatory mediators.The expression of TIFA was examined in microarrays containing liver biopsies from 150 patients . As shown , a relatwww.proteinatlas.org). Despite using a different antibody to detect TIFA, HPA data were in agreement with our observations; the detection of TIFA was decreased in liver cancer relative to controls  , but not TRAF2, a component of (apoptotic) complex II. Nonetheless, apoptosis was suggested by Annexin-V staining, and was further supported by detection of increased levels of the cleaved form of caspase-3. Similar results were observed with the HepG2 cell line  TIFA, or an oligomerization defective   into HCCell line , supportin vivo, we next performed tumor xenograft studies in nude mice. Consistent with the results obtained using in vitro assays, a significantly diminished tumor size was observed selectively among tumors from SK-Hep1 TIFA-expressing cells, relative to controls or SK-Hep1 TIFAΔ6-expressing cells . As p53 activation fosters cell cycle arrest and apoptosis, Ser 15; . The obsTo further test whether p53 may be a key element downstream of TIFA, we next used an shRNA approach to stably suppress p53 expression among cells reconstituted for TIFA expression. Perhaps surprisingly, the loss of p53 did not rescue apoptosis induced by TIFA expression . HoweverWe next examined the mechanism by which these cells undergo apoptosis via an examination of the initiator and downstream caspases. In addition to the executioner caspase-3, we detected cleavage of procaspase-8 (but not 9) associated with its maturation and activation. The addition of caspase inhibitors blocked caspase-8 cleavage and rescued HCC cell apoptosis , but did15 In some African and Asian countries, HCC has become the most prevalent cause for cancer-related death.16 Considering the effort invested, the clinical success to pharmacologically treating HCC patients has been limited, although the development of antivirals, such as Sofosbuvir, is promising. A better understanding of tumorigenesis, and the subsequent analysis and identification of new targets or predictive markers for HCC therapy is timely and, for many, urgent.HCC is the third most common cause for cancer-related death worldwide.17The roles shown for here for TIFA are unexpected. Most inflammatory mediators tend to promote HCC development. TIFA, by contrast, is not observed to be upregulated in HCC. In fact, this is independent of tumor stage, supporting the notion that loss or suppression of TIFA may be an early step toward tumor development. Indeed, TIFA can foster HCC apoptosis, and loss of apoptotic sensitivity is a hallmark of cancer.20 and caspase inhibitors targeting these are in clinical development.22 Suppression of inflammation may, however, carry an oncogenic risk, if early events fostering tumor progression are already in process, and the caspase-dependent killing we observe is prevented. This may be particularly important in the generation of the p53-compromised subgroup of HCC (30–40%) where caspase activity may be a final barrier to malignancy.Caspase-1 and 2, in particular, are implicated in inflammation,13 which promotes oncogenesis and tumor progression via nuclear translocation and activation of target genes. TIFA expression can also promote activation of the mitogen-activated protein kinase, extracellular signal-regulated kinase, c-JUN N-terminal kinase and p38. Activation of c-JUN N-terminal kinase and p38 in particular can trigger downstream cascades that lead to inflammation, differentiation or cell death.28 Although we observed some activation of these pathways in both the SK-Hep1 and the HepG2 cell lines following TIFA expression (unpublished observations), the patterns and role of these activations were not simple or clear, and further manipulation of these pathways is ongoing to determine if they are required or even relevant to the processes described here.TIFA has been associated with other signaling pathways, most notably, the NF-κB pathway,13 can induce apoptosis and thus may have a novel role as a regulator of HCC development by fostering removal of transformed cells. This function of TIFA requires the six C-terminal amino-acid residues that bind TRAF6. The second is that this activity proceeds concordant with p53-mediated induction of cell cycle arrest, but occurs even when p53 expression is suppressed. We propose that the oligomerization-competent form of TIFA is poised to promote caspase-dependent cell death as a counterpoint to ongoing inflammation. Caution may be warranted in the development of caspase inhibitors as a mechanism of combating chronic liver inflammation.The studies here present two unexpected findings. The first is that the protein TIFA, which fosters NF-κB signaling,TIFA gene was searched and blasted using RNAi designer from the Invitrogen website (https://rnaidesigner.invitrogen.com/rnaiexpress/index.jsp). ShRNA targeting human TIFA and scrambled control sequence were designed and chemically synthesized as TIFA–shRNA1 (5′-AAAAGGTCAGATTCGGAGAGTATCATTGGATCCAATGATACTCTCCGAATCTGACC-3′) TIFA–shRNA 2 (5′-AAAAGGTGAAATTTGGCCGAAATTCTTGGATCCAAGAATTTCGGCCAAATTTCACC-3′) and TIFA-shRNA3 (5′-AAAACCAAGATCACTCTTGCAAGAATTGGATCCAATTCTTGCAAGAGTGATCTTGG-3′). The control, TIFA-sc, was 5′-AAAAGCTACACTATCGAGCAATTTTGGATCCAAAATTGCTCGATAGTGTAGC-3′. The palindromic DNA oligos were annealed to each other to form a double-strand oligo and ligated to the linearized pLV-H1-EF1α-puro  vector to generate circled pLV-EF1α-shRNA-TIFA-Puro.The complementary DNA of human TIFA was cloned by reverse transcription PCR using total RNA from L02 cells, and using the specific primer pairs for TIFA . The amplified fragments were first ligated into pCMV-Tag 2B labeled with FLAG tag and then cloned using the primer pairs for Flag-TIFA . The amplified fragments were finally ligated into pLV-EF1α-MCS-IRES-Bsd  expression vector. The mutant TIFA fragments were cloned from the overexpression vector with using the primer pairs for TIFAΔ6. The amplified fragments were also finally ligated into pLV-EF1α-MCS-IRES-Bsd  expression vector. The shRNA sequence for silencing mice Human normal liver cell line, L02, was obtained from the Chinese Academy of Sciences . L02 cell line was maintained in RPMI-1640 media supplemented with 10% fetal bovine serum, 100 U/ml penicillin/streptomycin. Human HCC cell line, wt of SK-Hep1 cell line was kindly provided by Dr Ralph A Reisfeld . WT of HepG2 was obtained from the Chinese Academy of Sciences. SK-Hep1-Wt and HepG2-Wt cells were infected with lentivirus carrying pLV-EF1α-Flag-TIFA-IRES-Bsd and pLV-EF1α-Flag-TIFAΔ6-IRES-Bsd plasmids, followed by clonal selection using Blasticidin (5 μg/ml for SK-Hep1 and HepG2) to generate polyclonal cell populations with stable overexpression of Flag-TIFA and TIFAΔ6 . Alternatively, SK-Hep1-Wt cells were infected with lentivirus carrying pLV-H1-shp53-puro or pLV-H1-shTIFA-puro plasmid, followed by clonal selection using 1 μg/ml puromycin to generate polyclonal cell populations with stable expression of shp53 or shTIFA. For control purposes, SK-Hep1-Wt and HepG2-Wt were infected with lentivirus carrying the empty vector plasmid, SK-Hep1-Wt was infected with lentivirus carrying scrambled shRNA plasmid (see below for sequence) and subjected to identical clone selection procedures to generate the stable control cell lines SK-Hep1-Ctrl, HepG2-Ctrl and SK-Hep1-SC. SK-Hep1 and HepG2 cells were maintained in Dulbecco's modified Eagle's media supplemented with 10% fetal bovine serum, 100 U/ml penicillin/streptomycin and 1% nonessential amino acids.−ΔΔCt method was used to determine the relative mRNA folding changes. Statistical results were averaged from three independent experiments performed in triplicate.Total mRNAs from different cell lines were isolated by TRIzol reagent  and reverse transcribed into complementary DNAs with MMLV reverse transcriptase . Real-time PCR was performed on Opticon  in 20 μl reaction volumes by using TransStart Green qPCR SuperMix Kit . The 2Cell lysates from different cell lines were prepared with RIPA buffer in the presence of protease inhibitor cocktails and Phosphatase Inhibitor Cocktail 2 and 3 . Protein (30 μg) was loaded onto 5–12% Tris-acrylamide gels and blotted with antibodies that included: anti-TIFA, , β-actin , caspase-3 , p53 , p-p53 , caspase-8  and horseradish peroxidase-conjugated secondary antibodies. Blotting results were detected by an ECL chemiluminescence kit .Following culture of cells in the absence of fetal bovine serum for 12 h, cells were ‘pulsed' with 10 mM 5-bromo-2-deoxyuridine for 24 h, and cell cycle assay was performed by using the Cytofix/cytoperm kit  following the manufacturer's instructions. For apoptosis assay, apoptotic cells were stained with propidium iodide and Annexin-V-FITC (BD Biosciences). Flow cytometry analysis was performed by FACS Calibur cytometer (BD Biosciences), in which a minimum of 10 000 events were recorded. Three independent assays were conducted in such experiments and the mean values were expressed as mean±s.d.n=5 for each group based on minimal 30% decrease from 1 g tumors with 250 μg s.d. (standard deviation), α error of 0.05 and a β error of 0.8). In all, 3 × 106 SK-Hep1 cells  were inoculated subcutaneously into each mouse at right axilla. Tumor volume (mm3) was measured with calipers two times per week and calculated by using the standard formula: length × width2/2. The individual measuring the mice was unaware of the identity of the group measured. Animal use complied with Nankai University Animal Welfare Guidelines.Male NOD/SCID mice at 6–8 weeks of age were separated randomly into three groups . Expression levels of TIFA in the tissue microarray were scored according to the percentage of TIFA-positive cells in each liver tissues. The images were recorded by Olympus BX51 Epi-fluorescent microscopy under a 10 × or 40 × objective .Paraffin-embedded tissue slides were prepared from the tumor xenografts DeadEndTM Fluorometric TUNEL System kit (Promega) was applied for TUNEL staining. Experiment procedure was performed according to the manuscript instruction. 4,6-Diamidino-2-phenylindole was used to stain the nuclei, and the tissue slides were subjected to Olympus BX51 Epi-fluorescent microscopy under a 40 × objective .2 test in t-test. A value of P<0.05 was used as the criterion for statistical significance. *Indicates significant difference with P<0.05, **indicates significant difference with P<0.01.Values were expressed as means+s.e.m. Significance was determined by χ


The short half-life (<2 h) of the radionuclide requires efficient syntheses of these intermediates considering that multistep syntheses are often time consuming and characterized by a loss of yield in each reaction step. Recent examples of improved synthesis of 18F-labelled intermediates show new possibilities for no-carrier-added ring-fluorinated arenes, novel intermediates for tri[18F]fluoromethylation reactions, and 18F-fluorovinylation methods.This brief review gives an overview of newer developments in  Thus, it finds wide application as noninvasive, quantitative, and versatile modality in medical diagnosis, research, and drug development fluorine into organic molecules can be divided into two groups, namely, direct and indirect. The direct method entails introduction of [18F]fluorine without changing the carbon skeleton structure of the molecule. However, in many cases this necessitates the protection of functional groups or requires other transformations like reduction or oxidation of functional groups after introduction of radiofluorine fluoride acceptor groups fluoride or gaseous [18F]F2 is used, both of which are generally produced at a cyclotron via the 18O18F nuclear reaction [18F]fluoride ion is available in no-carrier-added (n.c.a.) form which allows the synthesis of radiotracers with high specific activity. In contrast, in-target produced [18F]F2 is available only in carrier-added (c.a.) form which leads to radiotracers with low specific activity.As starting material for all chemical syntheses either aqueous [reaction . The nuc18F]fluoro-2-deoxy-D-glucose ([18F]FDG) and 6-[18F]fluoro-L-dopa only electrophilic 18F-fluorination was available. Today this method is rarely used because of the need of carrier for [18F]F2 production. Thus, the use of electrophilic 18F-fluorination is limited to nontoxic compounds as well as to those that can be applied with a low specific activity. Also, since [18F]F2 is very reactive and 18F-labelled side products are formed, less reactive electrophilic 18F-agents were developed [18F]fluorobenzenesulfonimide (NFSi) was described, which is a highly stable, reactive and selective electrophilic 18F-labelling agent and allows the synthesis of 18F-labelled allylic fluorides and α-fluorinated ketones from allylsilanes and silyl enol ethers, respectively F2 leads to moderate specific activity of up to 24.7 GBq/μmol, starting from n.c.a. [18F]fluoride [18F]selectfluor bis(triflate), the 18F-labelled form of (1-chloromethyl-4-fluorodiazonia-bicyclo[2.2.2]-octane bis-(tetrafluoroborate)), an easy to handle and stable electrophilic fluorinating reagent , [18F]fluoro(m)ethylcholine, and O-2-[18F]fluoroethyl-L-tyrosine ([18F]FET) (cf. 18F]Fluoro(m)ethylcholine is an example for 18F-labelled endogenous compounds, whereas [18F]FDG and [18F]FLT are 18F-labelled deoxy derivatives of the corresponding endogenous substances. In all cases a proton is replaced by a fluorine atom without changing the carbon skeleton of the original compound. In contrast, [18F]FET is an example of an endogenous 18F-labelled compound where the introduction of the radionuclide is performed by an 18F-fluoroalkylation reaction. Here, the 18F-label is introduced into the molecule by addition of further C-atoms which means that the skeleton of the molecule is significantly changed. Other examples of this kind of reaction are the 18F-fluoroacylation and 18F-fluoroamidation reactions which are widely used for labelling of macromolecules fluoropropane, due to its very high boiling point, thus eliminating the risk of formation of pseudocarrier fluoromethylation reactions  available from difluoroiodomethane and [18F]fluoride [μmol [18F]fluoroform intermediate [18F]fluoroarenes were determined with up to 90%, but no information on the specific activities was given.Aromatic tri[r groups . Two newfluoride  is couplde [μmol . This mermediate . The RCY18F]CF3Cu, generated in situ from methyl chlorodifluoroacetate, CuI, TMEDA, and [18F]fluoride, is coupled to (hetero)aryl iodides in RCYs ranging from 17 to 87% [μmol exemplified so far only for 4-tri[18F]fluoromethyl nitrobenzene. However, the method enables an efficient tri-[18F]fluoromethylation of complex molecules like [18F]fluoxetine. N-Boc protected [18F]fluoxetine was readily prepared in 37% RCY. The subsequent N-Boc deprotection delivered [18F]fluoxetine with 95% yield. A more detailed review on the scope and limitations of the radiosynthesis of tri[18F]fluoromethyl groups is provided as part of this special issue [An alternative method is used in a one-pot process. The trifluoromethylation agent [7 to 87% . A drawbal issue .18F-chemistry a palladium catalyzed allylic fluorination reaction was developed and transferred to n.c.a. conditions yielding 18F-labelled cinnamyl fluoride starting from [18F]TBAF, cinnamyl methyl carbonate, [Pd (dba)2], and triphenylphosphine in anhydrous acetonitrile [Transition metal catalyzed allylic substitution is a powerful method for carbon–carbon and carbon–heteroatom bond formation  reaction using the [18F]fluoride anion to displace a suitable leaving group from an electron deficient benzene ring. As leaving groups serve halides, the nitro and the trimethylammonium function. The activation of the aromatic ring is usually achieved by suitable functional groups with a–M effect like the carbonyl, carboxyl, cyano, and nitro group [18F]fluoride into aromatic rings to label small 18F-intermediates for build-up syntheses. The activating functionality is then converted by reduction, oxidation, or hydrolysis to nucleophilic groups for subsequent coupling reactions. The n.c.a. intermediates 4-[18F]fluoroaniline, 4-[18F]fluorobenzylamine [18F]fluorobenzoic acid, or 4-[18F]fluorophenol  which hd  reacted rapidly (~3 min) to give the expected major [18F]fluoroarene product in useful, albeit moderate radiochemical yields even when the solvent had a water content of up to 28%. Iodonium salts bearing electron-withdrawing groups in metaposition  or an electron-donating substituent (p-OMe) gave low radiochemical yields under the same conditions. The finding that [18F]fluoroarenes, that having an ortho-alkyl substituent or an ortho- or a para-electron withdrawing group, can be synthesized without the need to remove irradiated water or to add a cryptand, could be attractive in some radiotracer production settings, particularly as this method saves time, avoids any need for automated drying of cyclotron-produced [18F]fluoride, and also avoids substantial loss of radioactivity through adsorption onto hardware surfaces [An interesting aspect here is that the reaction of diaryliodonium salts with [surfaces .18F]fluoride ion on the diaryliodonium salts it is important that one arene ring be more electron-rich than the ring to be labelled with fluorine-18. Here, the use of symmetrically substituted diaryliodonium salts fluorohalopyridines and in useful RCYs, including the otherwise difficult to access 3-[18F]fluorohaloisomers [Halopyridinyl-(4′-methoxyphenyl)iodonium tosylates were used to rapidly produce [oisomers .18F-labelling of nonactivated aromatic compounds makes use of triarylsulfonium salts. The method is applicable to a wide range of substituted aryl systems including amides [Another newer method for the g amides .18F]fluoroarenes is based on the reactions of diaryl sulfoxides bearing electron-withdrawing paragroups with the [18F]fluoride ion. These reactions are relatively mild, rapid, and efficient. However, this reaction is limited to aromatic rings bearing an electron withdrawing function like the nitro, cyano, or trifluoromethyl group [A new radiosynthetic method for producing n.c.a. [yl group .18F-labelled aromatic rings try to achieve fluoride-derived electrophilic n.c.a. fluorination reagents by fluoride umpolung [18F]fluoride capture by a Pd(IV) complex to form an electrophilic 18F-fluorination reagent followed by a subsequent n.c.a. 18F-fluorination of palladium aryl complexes [18F-labelled 1-[18F]fluoronaphthalene in 33% RCY but only in the presence of fluoride carrier Fluoride can also be introduced into organic molecules by electrochemical oxidative fluorination via an aryl cation that undergoes rearomatization by loss of a proton. Oxidation of benzene in an electrolysis cell, using Et3N·3HF and Et3N·HCl in acetonitrile as the electrolyte, gave c.a. [18F]fluorobenzene in 17% RCY [18F]fluorophenylalanine in 10.5% RCY with a specific activity of 1.2 GBq/mmol Fluorophenol is a versatile structural unit for the synthesis of more complex radiopharmaceuticals bearing a 4-[18F]fluorophenoxy moiety. Former syntheses of n.c.a. 4-[18F]fluorophenol were made either by a modified Balz-Schiemann reaction or by hydrolysis of a 4-[18F]fluorobenzene diazonium salt with radiochemical yields of only 10–15% and 15–33% within 35 and 60 min, respectively [18F]fluoroaniline and were not established for radiotracer production.4-[ectively . These m18F]fluorophenol was achieved using the Baeyer-Villiger reaction on 18F-labelled benzaldehyde, acetophenone, or benzophenone derivatives. Total radiochemical yields of about 25% were received using m-chloroperbenzoic acid as oxidant in the presence of trifluoroacetic acid [18F-labelled benzophenone derivatives containing further electron withdrawing groups yielded up to 65% of 4-[18F]fluorophenol within 60 min with a high radiochemical purity. However, a considerable disadvantage of this method is the somewhat cumbersome work-up of the aqueous reaction mixture in order to isolate the product for its further use fluoro-L-dopa [A more reliable preparation of n.c.a. 2- and 4-[tic acid . The Baether use . The foro-L-dopa .18F-fluorination of tert-butylphenols uses the concept of an aryl umpolung  iodonium salts  and bis(18F]fluorohalobenzenes has comprehensively been described [18F]fluorobenzene or 4-[18F]fluoro-1-iodobenzene serves as intermediates for C–C coupling reactions using Grignard-, lithium- [18F]fluorohalobenzenes are illustrated. The use of symmetrically substituted diaryliodonium salts enables an efficient one-step synthesis of n.c.a. 1-bromo-4-[18F]fluorobenzene [18F]fluoro-1-iodobenzene [18F]fluoro-1-iodobenzene [18F]fluoro-1-iodobenzene is, however, the use of triarylsulfonium salts [18F]fluorohalobenzenes, is the formation of other nonradioactive halobenzene derivatives which are normally not separated from the 18F-labelled product and thus could hamper the final product separation.Recently, the synthesis of 4-[escribed ; here a lithium- , or palllithium- , 153. Inobenzene  as well obenzene . For theobenzene . The preobenzene  are easiobenzene . The mosum salts , 159 whi18F-labelled intermediates for the build-up synthesis of radiotracers. Nevertheless, multistep build-up syntheses of 18F-labelled radiotracers are confronted with several fundamental challenges, which often hamper a remotely controlled, large scale production by this type of reactions. Time consuming separation steps and the use of moisture or even air sensitive reagents complicate the automation of these build-up syntheses. Their application is therefore limited to specialized laboratories with the suitable equipment and experienced staff. The use of build-up reactions then often enables the only way to achieve the synthesis of new radiotracers. Once proven that a radiotracer has the potential to be a useful radiopharmaceutical for molecular imaging, most often ways can be found to establish its routine production via an alternative, simpler synthetic concept and/or by optimisation. Here, the novel developments in umpolung reactions or the improvements in iodonium chemistry in 18F-labelling of arenes are promising methods, which might also be effective for the late-stage 18F-fluorination of complex precursors. However, their suitability for daily routine GMP-production of radiopharmaceuticals remains to be elucidated.The lack of universally useful methods for direct n.c.a. radiofluorination of complex molecules causes the wide use of


All h(A)PC variants significantly reduced myocardial infarct area (p<0.05) following I/R injury. IL-6 levels in heart homogenates did not differ significantly between sham, placebo and treatment groups in I/R injury. None of the h(A)PC variants decreased number and size of atherosclerotic plaques in apoE−/− mice. Only hS360A-APC slightly affected phenotype of plaques. IL-6 levels in plasma were significantly (p<0.001) decreased in hwt-APC and hS360A-PC treated mice. In the last group levels of monocyte chemotactic protein 1 (MCP-1) were significantly increased (p<0.05). In this study we show that both hwt and hS360A-(A)PC protect against acute myocardial I/R injury, which implies that protection from I/R injury is independent of the proteolytic activity of APC. However, in the chronic atherosclerosis model hwt and hS360-(A)PC had only minor effects. When the dose, species and mode of (A)PC administration will be adjusted, we believe that (A)PC will have potential to influence development of chronic inflammation as occurring during atherosclerosis as well.Activated protein C (APC) is a serine protease that has both anticoagulant and cytoprotective properties. The cytoprotective effects are protease activated receptor 1 (PAR-1) and endothelial protein C receptor (EPCR) dependent and likely underlie protective effects of APC in animal models of sepsis, myocardial infarction and ischemic stroke. S360A-(A)PC, a variant (A)PC that has no catalytic activity, binds EPCR and shifts pro-inflammatory signaling of the thrombin-PAR-1 complex to anti-inflammatory signaling. In this study we investigated effects of human (h)wt-PC, hS360A-PC, hwt-APC and hS360A-APC in acute  injury) and chronic inflammation (apoE Protein C (PC) circulates as an inactive zymogen that can be activated by thrombin bound to the endothelium associated transmembrane receptor thrombomodulin. When PC is bound to the endothelial protein C receptor (EPCR), its activation is enhanced by 20-fold APC has initially been identified and characterized as an anticoagulant protein; however recent interest has focused more on the cytoprotective functions of APC. APC has anti-inflammatory, anti-apoptotic and endothelial barrier stabilizing effects in different cell types, that are protease activated receptor 1 (PAR-1) and EPCR-dependent q and G12/13 mediated signaling in vivo via EPCR-dependent modulation of PAR-1 signaling.The active site of APC contains the conserved catalytic triad: residues H211, D257 and S360. Mutation of the active site serine to alanine (S360A) yields a protein that is not able to cleave substrates, but still retains some anticoagulant activity through its ability to compete with activated coagulation factors X and IX for binding to FVa and FVIIIa, respectively In the present study we have investigated the cytoprotective effects of human hwt-PC, hS360A-PC, hwt-APC and hS360A-APC in two different cardiovascular relevant mouse models: myocardial ischemia/reperfusion (I/R) injury and atherosclerosis. hAPC is very similar to murine (m) APC (69% sequence identity) Inflammation plays a key role in the pathogenesis of both myocardial I/R injury and atherosclerosis models in the current study, although the sequence of immune cell infiltration and the characteristics of the immune cell infiltrate are markedly different. After acute myocardial I/R, neutrophils migrate into the infarct zone within 24 hours, where they release granular enzymes and reactive oxygen species and cause vascular plugging Recombinant hS360A-PC was obtained by PCR-based site-directed mutagenesis of hwt-PC cDNA. The cDNA of hwt-PC and hS360A-PC were inserted into the eukaryotic expression vector pRc/CMV (Invitrogen), transfected into HEK293 cells (CRL-1573 ATCC) and recombinant h(S360A)-PC was purified and characterized as described earlier hwt-PC and hS360A-PC were activated with a 1 ml HiTrap NHS-Activated HP column  to which 50 Units of carrier-free Protac (Pentapharm) had prior been conjugated. Up to 0.5 mg of h(S360A)-PC was incubated for 2 hours on the column at room temperature, and was next eluted and concentrated with a 10 K Macrosep Advance Centrifugal Device . Complete activation of the hwt-PC was ascertained by measuring the amidolytic activity of APC with S-2366 (Chromogenix) directly and after 60 minutes incubation in the presence of additionally added  Protac. We confirmed the absence of Protac from the APC preps generated. For both hwt- and hS360A-PC, completion of activation was verified by SDS PAGE analysis . APC con−/− mice were used (Charles River Laboratories/in house breeding facility Maastricht University). Treatment, twice weekly, with intraperitoneal (i.p.) injections of saline (n = 20) or 0.2 mg/kg of hwt-PC (n = 13), hS360A-PC (n = 12), hwt-APC (n = 16) or hS360A-APC (n = 16) started at week 21 and was continued for 8 weeks. The 0.2 mg/kg dose has been proven protective in models of myocardial I/R injury All animal experiments were approved by the Animal Ethics Committee of Maastricht University. All mice were housed and bred according to institutional guidelines and were fed a normal chow diet. For the myocardial I/R experiment male C57BL/6 mice (Charles River Laboratories) of 8 weeks old and 25 g were used (n = 13 per group). For the atherosclerosis experiments, 21-week old male ApoEMyocardial I/R was performed as described earlier Infarct size was determined by Evans Blue/triphenyl tetrazolium chloride (TTC) staining Hearts of mice were excised, lyophilized and the resulting dry heart powder was dissolved in glucopyranoside. After determination of protein levels with DC Protein Assay Kit II (Bio-Rad), tissue homogenates were diluted to 5 mg/ml protein and IL-6 levels per mg heart homogenate were determined with mouse IL-6 ELISA Ready-set-go (eBiosciences).At the end of the experimental period, at 29 weeks of age, mice were anaesthetized with pentobarbital . Blood was taken in EDTA via cardiac puncture, followed by perfusion of the arterial tree under physiological pressure with PBS containing sodium nitroprusside (Sigma-Aldrich) and subsequently by 1% paraformaldehyde. The aortic arch including its main branch points was removed, fixed overnight in 1% paraformaldehyde, longitudinally embedded in paraffin, and sectioned. Twenty consecutive sections (4 µm) representing the central area of the aortic arch with an intact morphology of the arch and branch points were selected. For histological analysis of atherosclerosis, 4 sections (20 µm apart) were stained with hematoxylin and eosin (H&E) and the atherosclerotic lesions were classified as “initial” or “advanced” according to the Virmani classification Blood and spleen were collected, processed and fluorescently stained with either antibody mix 1  or antibody mix 2  or isotype control IgG . Cells were analysed using a FACS-Canto II (BD Biosciences) flowcytometer.Aortic arch sections were immunolabeled with anti-Mac3 monoclonal antibody (mAb)  to detect macrophages, anti-CD3  to detect T lymphocytes and anti-CD45 mAb  to detect leukocytes. Antibody staining was visualized by diaminobenzidine or Vectastain red. Sirius red staining was performed for analysis of collagen content.Enzyme-linked immunosorbent assays (ELISAs) were used to quantify concentrations of cytokines IL-6, IL-10 and MCP-1  in plasma. Cholesterol levels in plasma were measured using the Cholesterol CHOD-PAP method (Roche).Data analysis was performed with GraphPad Prism 5. Data were presented as mean +/− SEM. Differences between groups were tested using One-way ANOVA and Dunnett’s multiple comparison post hoc test. P<0.05 was considered statistically significant.Agkistrodon contortrix, the heavy chain isoforms shifted to a slightly lower migration position, consistent with full activation (95%) as judged by SDS-PAGE  of hwt-PC, hS360A-PC, hwt-APC, hS360A-APC or placebo  15 minutes after induction of ischemia and a second dose of the same treatment 5 minutes after induction of reperfusion. Next, infarct areas in the different groups were determined by Evans Blue/TTC staining . Figure Interleukin 6 (IL-6) is a cytokine with pro-inflammatory and pro-coagulant properties that stimulates hepatocytes to produce several acute-phase proteins, like C reactive protein. Increased levels of IL-6 were found in coronary heart disease patients and were associated with prognosis −/− mice were treated twice weekly with placebo, or hwt-PC, hS360A-PC, hwt-APC or hS360A-APC (0.2 mg/kg) for 8 weeks, after which atherosclerotic plaque development in the aortic arch was determined. Representative images of hematoxylin and eosin (H&E) stained initial and advanced plaques are shown in Having observed that all hPC and hAPC forms conferred cytoprotection in the acute inflammatory myocardial I/R model, we subsequently investigated the effect of hAPC administration on plaque development in a chronic inflammatory mouse model of atherosclerosis. ApoESubsequently the plaque phenotype was further characterized. Collagen content /E and inhigh/l°w/−) and T cells   –S4.We next determined the levels of cytokines IL-6, IL-10 and MCP-1 and of cholesterol in the plasma from the animals included in this study and to this end prepared plasma pools by mixing equal volumes of plasma for each of the animals studied per group. Mice treated with hS360A-PC had significantly lower IL-6 levels (P<0.001) and increased MCP-1 levels (P<0.05), see q and/or G12/13) to anti-inflammatory signaling (pertussis toxin–sensitive Gi-protein). In this caveolin-unbound state, cleavage of PAR-1 by thrombin or APC and resulting Gi activation, initiates a barrier protective and anti-inflammatory response, which includes activation of Rac1 and inhibition of RhoA and NFκB signaling cascades. We speculate that these mechanisms likely contributed to the protective effects observed for the hPC and hAPC forms tested by us, including the proteolytically inactive hS360A-(A)PC variant. Despite the mutation of the active site, S360A-(A)PC is still able to bind EPCR via its Gla-domain Several elegant studies have previously provided experimental evidence that wt-APC protects against acute myocardial I/R injury via both anti-inflammatory and anti-apoptotic effects . In thisin vitro antioxidant activity implies that the antioxidant properties are at least partly independent of the catalytic side and in line with our finding that both hwt- and hS360A-(A)PC have protective properties in acute myocardial I/R injury. There are several possible explanations for the observation that zymogen PC induced a similar reduction in infarct size as APC, while in fact its antioxidant capacities are lower than those of APC in vivoin vivo thrombomodulin-mediated activation by thrombin.Additionally it was shown that both PC and APC significantly decrease LPS-induced reactive oxygen species (ROS) generation by macrophages. Remarkably these effects were EPCR-independent and only in APC-treated cells accompanied by inhibited NFκB activation in vivo thrombin inhibition by heparin administration or the selective thrombin generation inhibitor dansyl glutamyl-glycyl-arginyl chloromethyl ketone-treated activated factor X (DEGR-FXa), failed to reduce myocardial I/R injury and I/R-induced spinal cord injury. Likewise, another study by Wang and coworkers Although S360A-(A)PC still has residual anticoagulant activity because of its ability to limit thrombin formation through competition with FXa and FIXa for resp. FVa and FVIIIa, it is unlikely that these anticoagulant properties of S360A-(A)PC have contributed to the reduced myocardial infarct area observed in S360A-(A)PC treated mice. It has been shown before In contrast to the study of Loubele in vivo bioavailability after injection. Previously it was shown that i.p. administration of 0.1 or 0.8 mg/kg mAPC produces a transient rise in APC levels in circulation for 3 hours, with a plateau after around 20 min While we hypothesized that administration of h(A)PC would also influence plaque development in the long term chronic atherosclerosis mouse model, we found that none of the h(A)PC variants significantly protected against the development of atherosclerosis in this mouse model of chronic inflammation. hwt-APC and hS360A-APC even slightly increased plaque area of advanced plaques, but the differences between these groups and the other groups were relatively small. Probably the lack of effect of h(A)PC on reducing plaque development can be explained by the −/− mice are more atherogenic than control mice, so it is not clear what the exact contribution of IL-6 to atherogenesis is We have however observed a clear effect of hS360A-PC and hwt-APC administration on IL-6 levels in plasma. The decrease in concentration of the pro-inflammatory cytokine IL-6 after hS360A-PC and hwt-APC treatment can possibly be explained by inhibited IL-6 release by neutrophils as a result of h(A)PC treatment in vivo effects of (A)PC in a cardiovascular context PC variants in mouse models for cardiovascular disease: in an acute I/R injury model and in a chronic atherosclerosis model. We have shown that treatment with both hwt and hS360A-(A)PC decreases infarct area in our acute murine myocardial I/R injury model. However, no significant effects were observed for the development of atherosclerotic plaques, a chronic slow process. Trends were observed that indicate that h(A)PC may be able to reduce in particular IL-6 and collagen levels, but under the conditions used in our experiments these did not lead to significant differences in total plaque area or plaque phenotype. Comparison of our data with those in literature learns that the (A)PC dose, mode of administration and species of administered (A)PC varied among different studies. We therefore propose that for future experiments in chronic disease models using mice, it will be preferable to include at least mAPC, which is administered preferably intravenously and more frequently than in the current study. If possible a slow-release device would be a preferred method, but this will require that the (A)PC preparations are stable for extended periods of time at body temperature. Nonetheless, while recognizing that such controlled study has not been provided yet, we regard our current study as valuable information that will enable the needed further study. In conclusion, this study showed that both hwt and hS360A-(A)PC protect in an acute murine myocardial I/R injury model and have the potential to influence development of chronic inflammation as occurring during atherosclerosis as well.Figure S1SDS page gel of human wt- and S360A-(A)PC. Reduced samples of human wt-PC (lane 1), wt-APC (lane 2), S360A-PC (lane 3) and S360A-APC (lane 4) were loaded on a 10% SDS PAGE gel and afterwards stained with Coomassie Brilliant Blue.(TIF)Click here for additional data file.Figure S2Effect of wt- and S360A-(A)PC treatment on immune cells in blood. Percentages of A) NK cells , B) NKT cells , C) Granulocytes  and D) monocytes  were determined by flow cytometric analysis of mice blood. E) Monocytes were further characterized as Ly6C high (♦), Ly6C low (♦) or Ly6C− (◊). Graphs A–D show mean +/− SEM.(TIF)Click here for additional data file.Figure S3Effect of wt- and S360A-(A)PC treatment on immune cells in blood. Percentages of A) total T cells (CD3+), B) CD8+ T cells, C) CD4+ T cells, D) regulatory T cells  and E) B cells (B220+) were determined by flow cytometric analysis of mice blood. All graphs show mean +/− SEM.(TIF)Click here for additional data file.Figure S4Effect of wt- and S360A-(A)PC treatment on immune cells isolated from spleen. Percentages of A) total T cells (CD3+), B) CD8+ T cells, C) CD4+ T cells, D) regulatory T cells  and E) B cells (B220+) were determined by flow cytometric analysis of cells isolated from mice spleen tissue. All graphs show mean +/− SEM, statistical significance was tested using one-way analysis of variance with Dunnett post hoc test, *P<0.05.(TIF)Click here for additional data file.Table S1Overview experimental APC studies in cardiovascular diseases. Overview of studies that used different models to study effects of (A)PC on cardiovascular diseases. For each study the species, APC type, dose and injection method and effects on outcome measurements were shown. Abbreviations: ALT: alanine aminotransferase; APC: activated protein C; BBB: blood brain barrier; BCAO: bilateral carotid artery occlusion; CBF: cerebral blood flow; ERK: Extracellular signal-regulated protein kinases; h: human; IL: interleukin; ip: intraperitoneal; I/R: ischemia/reperfusion; iv: intravenous; JNK: c-Jun N-terminal kinase; m: mouse; MAP: mean arterial pressure; MCAO: middle cerebral artery occlusion; MMP: matrix metalloproteinase; MOD: multiple organ dysfunction; MPO: myeloperoxidase; NFκB: nuclear factor kappa B; pd: plasma derived; r: recombinant; TNFα: tumor necrosis factor α; wt: wild type.(TIF)Click here for additional data file.