Tinospora cordifolia stem aqueous extract in the specific pathogen free (SPF) chicks, experimentally infected with very virulent IBDV (vvIBDV). Both of these agents  showed significant increase in the IFN-γ, IL-2, IL-4, and IL-1 levels in the peripheral blood mononuclear cells (PBMCs) (p < 0.05) of chickens in the treatment groups following IBD infection. Further we found significant reduction in mortality rate in vvIBDV infected chicks treated with either, or in combination, compared with the birds of control group. Additionally, the adjuvant or immune enhancing potential of these two immunomodulatory agents with the commercially available IBDV vaccine was determined in chicks. The augmentation of vaccine response in terms of an enhanced antibody titer after vaccination, along with either or a combination of the two agents was noticed. The findings provide a way forward to counter the menace of IBDV in the poultry sector through use of these herbal or synthetic immunomodulatory supplements.Infectious bursal disease (IBD), caused by infectious bursal disease virus (IBDV), is characterized by severe immunosuppression in young chicks of 3 to 6 week age group. Although vaccines are available to prevent IBD, outbreaks of disease are still noticed in the field among vaccinated flocks. Further, the birds surviving IBD become susceptible to secondary infections caused by various viral and bacterial agents. This study assessed the immunoprophylactic potential of Cytosine-guanosinedeoxynucleotide (CpG) oligodeoxynucleotides (ODN) and  Avibirnavirus of the family Birnaviridae [Infectious bursal disease (IBD), caused by IBD virus (IBDV), is an acute, highly contagious, and immunosuppressive disease particularly affecting chicks of 3 to 6 weeks age worldwide. The disease results in severe economic loss to poultry farmers. IBDV is a non-enveloped, double stranded RNA virus belonging to the genus aviridae . This viaviridae , and fataviridae . IBDV inaviridae . Althougaviridae , thus exAugmentation of innate immune responses through immunomodulatory supplements may help to overcome such challenges in the management of IBDV induced infection and associated secondary complications. Amid several agents known to augment the innate immune response, Toll like receptors’ (TLR) agonists and some herbs can further improvise an appropriate adaptive immune response, countering the invading pathogen efficiently ,9. TLRs,Astragalus sp, Withania somnifera, Tinospora cordifolia, Azadirachta indica, and Phyllanthus emblica. [T. cordifolia  has been well established for potent immunomodulatory properties [Herbs have been used widely in India under the ayurvedic system of medicine since time immemorial. Several studies have proven the immunomodulatory potential of various herbs; to name few, emblica. ,21,22. Aoperties ,24. Guduoperties . GuduchiIn this study, we established the interaction of CpG ODN (TLR21 agonist) and guduchi aqueous extract in providing protection against IBDV, and their mechanism of interaction. Furthermore, adjuvant action of CpG ODN and guduchi aqueous extract with commercially available IBDV vaccine was also evaluated. To the best of our knowledge, this is the first study demonstrating the interaction of any TLR agonist and herbal extract for providing protection against a viral infection in chicken.n = 170) were procured from Venky’s Pvt. Ltd.,  and hatched at the Central Avian Research Institute (CARI), Izatnagar. The chicks were maintained at the experimental animal shed facility of the Avian Disease Section, ICAR-IVRI, Izatnagar, maintaining required pathogen free settings. The experiment had prior approval from the institute animal ethics committee (number F. 26-1/2015-16/JD(R)).Specific pathogen free (SPF) eggs (A very virulent strain of IBDV (vvIBDV) (GenBank accession: KM205793.1 (VP2 gene)), maintained in the viral laboratory of the Avian Disease Section, was used for the experimental infection after passaging in three-week-old SPF White Leghorn (WL) chicken. At 72 h post-inoculation, the bursae were harvested, chopped, and homogenized in peptone broth containing penicillin and streptomycin (1000 mg/mL each). The filtered supernatant obtained from the bursal homogenate was used as a source of the vvIBDV for further work. The virus suspension was titrated in 10 day-old SPF embryonated chicken eggs by inoculation into the chorioallantoic membrane route .Chicken TLR21 agonist (CpG ODN) was procured from Alpha Diagnostic Intl Inc, San Antonio, TX, USA. (Cat. #ODN2007-1), and was used in the study after making appropriate dilutions.T. cordifolia stem powder was procured commercially  and was used for preparing an aqueous extract for further use in the experiment. Aqueous extract was prepared by boiling stem powder of this plant in water at a 1:5 ratio, at 100 °C for 30 min [r 30 min , followeCommercially available IBDV (Intermediate and Intermediate Plus) vaccines  and Newcastle disease virus (NDV) vaccine, lentogenic ‘F’ strain  were used in the present study.The ELISA kit for IBDV was procured from IDEXX Laboratories, Westbrook, MA, USA.The published oligonucleotide primers selected for chicken specific genes; viz., β-actin, IL-2, IFN-γ, IL-4, IL-1β, and vvIBD-VP2 , were usn = 20/group). Group A was kept as a negative control and was given water in a pulsating manner (over a period of eight hours), 10 mL/bird/day orally, to mimic the T. cordifolia extract treatment (control for group C). Group B (positive virus control) was given phosphate buffered saline  via intramuscular route at 4 weeks of age. Birds in group C were given T. cordifolia aqueous extract from day one until 4 weeks of age in a pulsating manner (over a period of eight hours), 10 mL/bird/day orally. Group D was given CpG ODN, 50 µg/bird via intramuscular injection at 4 weeks of age. Group E was given both aqueous extract, prepared as above, from one day old, until 4 weeks of age, in a pulsating manner, 10 mL/bird/day; and CpG ODN, 50 µg/bird via intramuscular route at 4 weeks of age. Birds in group B, C, D, and E were challenged with 105 ELD50 of vvIBDV at 24 h post-injection of CpG ODN through the ocular route and observed for the clinical signs and mortality until 10 days post-challenge.A total of 100 SPF WL birds, three-weeks old, were randomly divided into five groups; viz. A, B, C, D, and E (n = 6/group) were collected in heparinized vials (20 IU/mL) at the 4th , 5th ), and 6th (14 DPC) week of age. On day four post-challenge, three birds from each group (A–E) were sacrificed for the collection of bursa. A piece of bursal tissue was stored in 10% neutral buffered formalin for histopathology (HP), and the remaining half of the bursal tissue was stored in an RNA later  solution at −80 °C for cytokine expression studies.Blood samples  at −80 °C until RNA extraction.PBMCs were isolated from the blood, as described previously . BrieflyTotal RNA was extracted from the PBMCs and bursal tissue, using RiboZol reagent as per manufacturer’s guidelines. Briefly, bursal tissue was homogenized in RiboZol (1:10) and 250 µL PBMCs mixed with 750 µL RiboZol, followed by addition of chloroform (200 µL) and centrifuged at 12,000 rpm for 15 min at 4 °C for phase separation. Aqueous phases were precipitated with 500 µL isopropanol followed by centrifugation , supernatants were removed, and the RNA pellets were washed carefully with 75% ethanol and centrifuged (7500 rpm for 5 min at 4 °C). After removing ethanol, RNA pellets were dissolved in RNase free water, warmed at 55–60 °C and then stored at −20 °C, until further use. RNA purity and quantity were determined by Nano-drop spectrophotometer analyzer  at A260/280.Reverse transcription (RT) for the first strand synthesis was carried out by using Revert-Aid Reverse Transcriptase  in a standard 20 µL reaction mixture. To a sterile nuclease free 0.2 mL PCR tube, the reaction mixture was prepared using: RNA template (1 μg), Random Hexamer Primer 100 µM , dNTP mix (10 mM each) (2.0 μL) and nuclease free water (NFW) (to make up to 20 µL volume). The mixture was incubated at 70 °C for 5 min, followed by immediate snap chilling in ice. Then reagents were added as follows, 5X RT buffer (4.0 μL), RiboLock RNase inhibitor  and RevertAid Reverse Transcriptase . RNA was subsequently reverse-transcribed at 25 °C for 10 min, and 42 °C for 60 min, followed by 10 min at 70 °C to denature the enzyme. All these steps were carried out in a thermal cycler . The cDNA samples were cooled at 4 °C and stored at −20 °C.SYBR green-based quantitative real-time PCR was performed using Stratagene MX300 5P for studying the expression of different immune response genes. β-actin was used as the internal control gene. Each sample was run in duplicate and the total reaction volume was 20 µL, containing SYBR green master mix , 10 µL, template cDNA, 1 µL, gene specific forward and reverse primers, 0.5 µL each, and NFW, 8 µL. Real-time PCR conditions were one cycle at 95 °C for 10 min, 40 cycles of 95 °C for 15 s, 60 °C for 15 s, 72 °C for 15 s and one cycle 95 °C for 1 min; and a 65–95 °C ramp for melt curve analysis to check the amplicon specificity.The humoral immune response against vvIBDV was assessed using an ELISA kit  following the manufacturer’s protocol on collected sera samples. Briefly, antigen coated plates were taken, and sample positions were recorded, followed by the addition of 100 µL of undiluted negative control (NC), 100 µL of undiluted positive control (PC), and 100 µL of respective diluted samples (1:500) into appropriate wells. Plates were incubated for 30 min at 18–26 °C, followed by removal of the solution in each well and thrice washing with distilled water (350 µL/well). 100 µL of conjugate was dispensed into each well, incubated for 30 min at 18–26 °C, and followed by washing, as previously explained. 100 µL of TMB substrate was dispensed into each well, incubated for 15 min at 18–26 °C and finally 100 µL of stop solution was dispensed. Absorbance was recorded at 650 nm using the ELISA reader.The antibody titer was calculated as per the equations provided in the ELISA kit;ControlsSamplesAntibody titer is the antilog of that value.The protection study included observation of clinical signs and mortality up to ten days post-challenge, viral load estimation, and gross and histopathological examination of bursae.Necropsy was done on dead birds to record the gross lesion and post-mortem diagnosis. Bursae from dead as well as sacrificed birds were processed for routine paraffin embedding technique. Tissue sections of 4–5 mm thickness were stained with hematoxylin and eosin (H&E) for microscopic evaluation. Bursal lesions (BLS) were scored on a scale of 0 to 5  by a trained avian pathologist, following the blind folded method, as described elsewhere .RNA samples from bursal tissues collected at 4 DPC of vvIBDV infection from all the groups was used for cDNA synthesis, followed by quantitative real-time PCR for IBDV specific gene (VP2). Real-time PCR conditions included one cycle at 95 °C for 8 min, 40 cycles of 94 °C for 1 min, 57 °C for 50 s, 72 °C for 50 s, and one cycle of 95 °C for 1 min; and 65–95 °C for melt curve analysis to check the amplicon specificity. For evaluating the concentrations of virus in bursal samples, a standard curve was prepared for the VP2 gene amplified in the pDrive Cloning Vector.n = 5/group) for three weeks post-vaccination, and stored at −20 °C until further use.On day 10 post-vvIBDV challenge, the surviving birds were vaccinated with Newcastle disease virus (NDV) vaccine, lentogenic ‘F’ strain  through the ocular route with the recommended dose. Sera were collected at weekly intervals  test, according to the OIE-World organization for animal health recommended protocol , using aThe evaluation of the immune-enhancing effect of T. cordifolia and a TLR21 agonist (CpG ODN) on IBDV vaccine responses in chicks.n = 10/group). Group A was kept as a negative control. Group B was kept as a positive control. Birds in group C were given T. cordifolia aqueous extract from day one until 4 weeks of age in a pulsating manner (over a period of eight hours), 10 mL/bird/day orally. Group D was given CpG ODN, 10 µg/bird via the intramuscular route at 2 weeks, repeated at 4 weeks of age. Group E was given both aqueous extract, prepared as above, from day one until 4 weeks of age in a pulsating manner (over a period of eight hours), 10 mL/bird/day; and CpG ODN, 10 µg/bird via the intramuscular route at 2 weeks, and again at 4 weeks of age. Chicks in group B to E were vaccinated with IBDV intermediate  vaccine at 2 weeks and a booster dose at 4 weeks of age, with IBDV intermediate plus vaccine (each dose of vaccine containing Intermediate Plus strain ≥ 102 EID50), while group A acted as negative control.Fifty SPF WL birds of two-week age were randomly divided into five groups; viz. A, B, C, D, and E (n = 6/group) were collected from birds in all the groups pre-vaccination; and at 7, 14, 21, and 28 days post-vaccination (DPV) in vaccinated birds, for assessing the IBDV specific antibody responses. IBDV specific antibody responses were measured using a commercially available ELISA kit (IDEXX), as described in the earlier procedure.Blood samples without anticoagulant . The control group was considered a calibrator for relative quantification of mRNA expression. Multiple comparisons between groups were done through Tukey’s post hoc test. Values of p < 0.05 were considered significant.Relative expressions in target genes were analyzed based on the 2−fl, 2001 , normaliT. cordifolia aqueous extract to birds at 10 mL/day/bird from one day old to 4 weeks of age) group showed significant increases (p < 0.05) in IL-2 (5.657 ± 0.1663), IFN-γ (15.15 ± 0.2288), IL-4 (8.458 ± 0.277), and IL-1β (13.09 ± 0.2681) expressions in chicken PBMCs compared to the control group  in IL-2 expression in PBMCs at 7 DPC (10.840 ± 0.232) and 14 DPC (10.620 ± 0.723) which was the only increase, and was followed by group D, which showed significant differences from groups A, B, and C at 7 DPC (8.068 ± 0.207) and 14 DPC (7.462 ± 0.507) (The group E showed an increase (± 0.507) A.p < 0.05) in IFN-γ expression in PBMCs at 7 DPC (25.702 ± 0.328) and 14 DPC (26.982 ± 0.345) which was the only increase, and was followed by group D, which showed significant differences from groups A, B, or C at 7 DPC (18.672 ± 0.490) and 14 DPC (19.973 ± 0.094) (The group E showed an increase (± 0.094) B.(p < 0.05) in IL-4 expression in PBMCs at 7 DPC (19.400 ± 0.376) and 14 DPC (20.468 ± 0.207). Group D, followed, but showed significant differences from groups A, B, and C at 7 DPC (15.265 ± 0.162) and 14 DPC (15.397 ± 0.293) (The group E showed an increase ± 0.293) C.p < 0.05) in IL-1β expression in PBMCs at 7 DPC (15.628 ± 0.444) and 14 DPC (15.247 ± 0.169) which was the only increase, followed by group D which showed significant differences from groups A, B, and C at 7 DPC (11.987 ± 0.243) and 14 DPC (11.660 ± 0.123) (The group E showed an increase (± 0.123) D.p < 0.05) in IL-2 expression in PBMCs at 7 DPC (27.550 ± 0.363) and 14 DPC (27.370 ± 0.496), which was followed by group D, which showed significant difference from group A, B and C at 7 DPC (17.397 ± 0.232) and 14 DPC (14.797 ± 0.189) (The group E showed an increase (± 0.189) A.p < 0.05) in IFN-γ expression in PBMCs at 7 DPC (40.892 ± 0.446) and 14 DPC (39.693 ± 2.031) which was the only increase, followed by group D, which showed significant difference from group A, B and C at 7 DPC (31.348 ± 0.298) and 14 DPC (31.232 ± 0.659) (Group E showed an increase (± 0.659) B.p < 0.05) in IL-4 expression in PBMCs at 7 DPC (5.842 ± 0.272) and 14 DPC (5.663 ± 0.211) which was the only increase, followed by group D, which showed significant differences from groups A, B, and C at 7 DPC (3.712 ± 0.172) and 14 DPC (3.310 ± 0.195) (Group E showed an increase (± 0.195) C.p < 0.05) in IL-1β expression in PBMCs at 7 DPC (7.498 ± 0.054) and 14 DPC (7.275 ± 0.413) which was the only increase, followed by group D, which showed significant differences from groups A, B, and C at 7 DPC (5.543 ± 0.128) and 14 DPC (5.345 ± 0.133) (Group E showed an increase (± 0.133) D.p < 0.05) from other groups at 7 DPC (1152.477 ± 8.040) and 14 DPC (2115.643 ± 18.462), with the maximum antibody titer being estimated in this group. Following E, was group D, which differed significantly (p < 0.01) at 7DPC (871.597 ± 7.625) and 14 DPC (1776.421 ± 7.847) from group C and group B. Additionally, in both group D and group E antibody titer significantly increased (p < 0.01) from 7 DPC to 14 DPC. Group C also showed a significant increase (p < 0.01) in antibody titer 7 DPC (699.968 ± 17.517) and 14 DPC (809.135 ± 3.712) from the positive virus control (group B) (Group E showed a significant difference (group B) .(a)Clinical signs and mortality: No clinical signs and mortality were observed in any of the birds for the first 24 h post challenge with vvIBDV. After 48 h, affected birds showed clinical signs, such as drowsiness, depression, ruffled feathers, inability to move, and severe prostration. Group E showed the highest protection (100%), followed by group D (80%), and group C (50%), whereas 100% mortality was observed in group B ( group B .(b)Gross lesions and Histopathology: In group A (negative control), the bursa of Fabricius revealed apparently normal histo-architecture, characterized by intact lining epithelia, lymphoid follicles with sufficient numbers of lymphocytes in the cortex, and medulla with thin connective tissue stroma separating each follicle (follicle a. No patfollicle b. In grofollicle c. In grofollicle d. In grofollicle e. But in(c)Viral load: Real time quantification of viral load from bursa of Fabricius in birds from groups A and C to E was done by using VP2 as the target gene, cloned in a pDrive Cloning Vector . The viral copy number of samples was calculated by interpolating the samples’ Ct values with Ct value of serially diluted VP2 cloned plasmid. The group E showed the lowest (p < 0.05) of copy number of all the groups at 4 DPC (2.647 ± 0.277), followed by the group D (3.977 ± 0.222), and then by group C (5.910 ± 0.262). The highest copy number was found in group A (8.840 ± 0.180) (± 0.180) .(d)Humoral immune response analysis after NDV vaccination: The birds treated with both T. cordifolia and CpG ODN (group E) prior to the vvIBDV challenge showed a highly significant increase in HI titer, more so than those of groups C and D at two and three weeks post-NDV vaccination (p < 0.05) ( < 0.05) . The antThe results of ELISA for serum antibody titer estimation after IBDV vaccination are shown in p < 0.05) from other groups at 7 DPV (784.827 ± 12.559), 14 DPV (979.217 ± 9.894), 21 DPV (2020.827 ± 20.893), and 28 DPV (2509.359 ± 12.774), with the maximum antibody titer being estimated in this group; followed by the group D which differed significantly (p < 0.01) at 7DPV (587.897 ± 11.084), 14 DPV (717.898 ± 11.729), 21 DPV (1217.303 ± 18.584), and 28 DPV (1857.785 ± 4.094) from group C and group B. Furthermore, in both group D and group E, antibody titer significantly increased (p < 0.01) from 7 DPV to 28 DPV. Group C also showed a significant increase (p < 0.01) in antibody titer at 7 DPV (525.834 ± 3.913), 14 DPV (602.523 ± 5.427), 21 DPV (716.618 ± 11.656), and 28 DPV (944.575 ± 12.222), from group B.The group E showed a significant difference  derived from T. cordifolia was found to stimulate murine macrophages leading to up-regulation of IL-2, IFN-γ, IL-4, and IL-1β in vitro [T. cordifolia stem hydro-alcoholic extract in chickens against a 2.4-dinitrofluorobenzene (DNFB) skin sensitization test, as seen in the form of increases in skin thickness [T. cordifolia extract supplementation in chicks has also shown protection against Escherichia coli infection by increasing the E. coli specific antibody titer and lymphocyte proliferation response [T. cordifolia, responsible for its immunomodulatory potential, acts by activating the B cells polyclonally, via an increase in CD69 expression in lymphocytes. TLR4 on B lymphocytes and macrophages acts as a receptor for G1-4A polysaccharide, activating these immune cells via TLR4/MyD88 dependent manner [T. cordifolia stem extract, termed immunomodulatory protein  which is reported to possess lymphoproliferative and macrophage stimulating properties [In this study, the aqueous extract of in vitro . Therefohickness . T. cordresponse . It has t manner ,43. Simit manner . Apart foperties .TLRs are the best characterized component of the innate immune system, and have been reported in wide range of species, from arthropods to humans. Microbes stimulate TLRs with their PAMPs which in turn activates both the innate and acquired immune responses . The sigT. cordifolia aqueous extract (group C) and CpG ODN (group D) were evaluated for their prophylactic efficacy against the vvIBDV challenge in chickens at 4 weeks of age. Moreover, their combined effect to protect against the vvIBDV challenge (group E) was also evaluated. Protection was evaluated based on the cellular immune response, humoral response, histopathological, and the protection study. Cellular immune response was evaluated through the quantitative real-time PCR based expression of cytokines  in chicken PBMCs and bursa of Fabricius. Expression levels of IL-4 and IL-1β was also assessed in chicken PBMCs and bursa of Fabricius using qRT-PCR. Humoral immune response was assessed using an ELISA kit for estimating the antibody titer.In this experiment, T. cordifolia extract in stimulating PBMCs and immune cells in the bursa of Fabricius towards a Th1 response, thereby stimulating a cell mediated immune response in a chicken, when challenged with vvIBDV. IL-2 is expressed exclusively by T-lymphocytes, and it promotes the growth of T-lymphocytes, thereby enhancing cell mediated immunity [H1 cells [T. cordifolia aqueous extract and TLR 21 agonist suggests that they enhance the ability of the immune system to combat various intracellular pathogens in chickens, which is biased towards Th1 response. This biased Th1 immune response is essential to combat intracellular pathogens such as viruses [T. cordifolia aqueous extract in inducing TH1 responses, as observed in the present study, suggests the mechanism of their protective effects against vvIBDV in the treated chickens.The results of the current study indicated highest expression levels of IL-2, IFN-γ, IL-4, and IL-1β in the PBMCs and bursa of Fabricius in group E, followed by group D, and then group C. This indicates that the three groups treated with immunomodulatory agent  showed more expression of cytokines under study than the positive virus control (group B) at both 7 DPC and 14 DPC. Significant increases in the expression levels of IL-2 and IFN-γ cytokines in group E point to the additive influence of CpG ODN and immunity . IFN-γ iH1 cells , produciH1 cells . Thus, i viruses . Since s viruses , the abiH1 and TH2 responses and the polarization of the responses is largely based on antigen-specific TH cells. TH1 cells drive cell-mediated, inflammatory responses, while TH2 cells drive responses against helminthic worms, extracellular bacteria, and some viral infections. TH1 cells typically produce IFN-γ, while TH2 cells typically produce IL-4 [H2 mediated immunity [T. cordifolia aqueous extract can induce a mixed and more balanced TH1 and TH2 response which is vital to protect a host against distinct types of microbes, such as viruses and worms.The immune system is regulated between Tuce IL-4 . IL-4 isuce IL-4 . MHC II uce IL-4 . MHC II immunity . IL4 expT. cordifolia aqueous extract in stimulating macrophages could be evaluated from the expression of the pro-inflammatory cytokine, chicken IL-1β, which is a mammalian homolog produced from avian macrophages [Besides the above described adaptive responses, the influences of CpG ODN and rophages . IL-1β arophages .Humoral immune response stimulation in vvIBDV challenged birds was evaluated through estimating the antibody titer using an ELISA kit. The antibody titer was found to be highest in group E, followed by group D, and then group C. The immunomodulatory agent treated groups showed significantly higher IBDV specific antibody titers than the positive virus control group at both 7 DPC and 14 DPC. This correlates with the level of IL-4 levels estimated by qRT-PCR. Anti-IBDV antibody plays an important role in protecting the chicken against IBDV infection .Bursal damage, as seen histopathologically, was found to be least in group E, followed by groups D and C, respectively. Positive control (group B) showed extensive bursal damage. The least bursal damage in group E might be due to the highest virus clearance in their bursa. This clearance of virus is due to the stimulation of the cell mediated immune response as indicated by high IFN-γ and IL-2 expression levels in both their bursa and PBMCs .T. cordifolia aqueous extract. In groups C and D, 50% and 80% protection was observed, respectively. In the positive virus control (group B), 100% mortality was observed during the observation period. These birds showed characteristics signs of IBDV infection, such as depression, drowsiness, inability to move, ruffled feathers, and severe prostration. On necropsy examination of birds in group B, characteristics of IBDV lesions were found, such as hemorrhages in the thigh muscles, splenomegaly, and swollen and hemorrhagic bursae. The bursal folds were extensively hemorrhagic and contained variable amounts of blood clotting.Moreover, this study provides evidence that the challenging of SPF chickens with vvIBDV in group E induced an obvious protective immune response to avoid an otherwise lethal outcome. The protection of 100% from vvIBD virus was achieved in the group E which received CpG ODN along with Further, immunosuppression, which is the most serious impact of IBDV infection, was found to be diluted in immunomodulatory agent treated groups, with the highest dilution in group E, followed by group D, and then group C. This finding is significant, since vaccination with live vaccines leaves birds protected to some extent, but birds have lowered immunity, and further vaccination against other diseases fails, leaving them vulnerable to various pathogens.Antibody response was highest after IBDV vaccine in group E, followed by group D, and then group C. The immunomodulatory agent treated groups showed significantly higher IBDV specific antibody titers than positive vaccine control group at 7 DPV, 14 DPV, 21 DPV, and 28 DPV. Thus, both the immunomodulatory agents possess adjuvant activity with IBDV vaccine. Such adjuvants with balanced stimulation of the immune response and with less adverse effects are highly desirable to achieve efficient protection against infectious diseases.T. cordifolia and CpG ODN be used as prophylactic agents, and as adjuvants in poultry for viral diseases. They both have immunomodulatory potential via the TLR mediated pathway. There is evidence for using a combination of TLR agonist as an adjuvant in vaccines for a better immune response [To conclude, we suggest that response . However


The compounds 3a–c were found selective for fluoride (F−) with no affinity for other anions, i.e. −OAc, Br−, I−, HSO4−, SO42−, PO43−, ClO3−, ClO4−, CN− and SCN−. Further, upon the gradual addition of a fluoride anion (F−) source (tetrabutylammonium fluoride), a well-defined change in colour of the solution of probes 3a–c was observed. The anion-sensing process was studied in detail via UV–visible absorption, fluorescence and 1H-NMR experiments. Moreover, during the synthesis of acridine probes 3a–c nickel fluoride (NiF2), a rarely explored transition metal fluoride salt, was used as the catalyst. Theoretical studies via density functional theory were also carried out to further investigate the sensing and anion (F−) selectivity pattern of these probes.New thiosemicarbazide-linked acridines  In medicinal chemistry, many acridine derivatives have found applications as drugs such as acriflavine  and quinacrine . Most of the acridine derivatives and their metal complexes also act as DNA intercalating agents. However, the use of acridine-based compounds as chemosensors has not yet been explored for anionic analysis. With this interest, we aimed to synthesize acridine-based compounds functionalized with semicarbazide moieties. Previous studies have provided evidence regarding the role of semicarbazide moiety in anion sensing –4. Chemo−), acetate (AcO−), sulfate, phosphate, pyrophosphate, ascorbate, citrate, etc. [Traditionally, the known techniques for ion detection are spectroscopy, chromatography and ion-selective electrodes which require expensive instrumentation and extensive and destructive sample preparation, and hence cannot be employed for cell or tissue imaging . In recete, etc. ,30,31. I2.2.1.2·4H2O is not well explored as a catalyst in organic synthesis, though such type of reactions have been explored with some traditional fluorides such as CsF or tetrabutylammonium fluoride (TBAF) [2·4H2O (25 mol%) as the catalyst to furnish the acridine scaffold 7 with a simple work-up and washing with n-hexane. The use of transition metal fluoride (NiF2) as a new catalytic source to promote organic reaction could serve as a substitute for common fluoride salts, i.e. CsF, KF, etc. Further, in order to endow the acridine scaffold with a semicarbazide moiety, it was treated with excess of hydrazine hydrate (80%), and subsequently with phenyl isocyanate in acetonitrile at 80–85°C, until the disappearance of the starting materials, as monitored by thin-layer chromatography (TLC). The desired thiosemicarbazide-linked compounds, as anion-sensing probes 3a–c, were obtained in a 55–85% yield (The synthetic layout of targeted thiosemicarbazide-linked acridine is given in e (TBAF) –34. The 5% yield .Scheme 1H-NMR spectrum of acridine-based thiosemicarbazone 3b (as a selected example), characteristic broad signals for two NH groups were observed at δH 10.32 and 9.23 ppm, while the broad signal for NH of the acridine scaffold appeared at δH 8.80 ppm. The peaks of all 4 appeared as doublets at (i) 2.49 ppm (obscured by the dimethylsulfoxide (DMSO) signal), (ii) 2.31 ppm, (iii) 2.23 ppm and (iv) 2.16 ppm. Two singlets for each of two methyl groups at position C-3/6 appeared at δH 1.03 ppm and 0.82 ppm. The structure of 3b was further confirmed with two-dimensional NMR techniques. The heteronuclear single quantum coherence spectrum showed direction correlation of the methine (CH-9) group at 35.6 ppm, while the heteronuclear multiple bond correlation (HMBC) spectrum showed correlations of H-9 with C-1/8, C-2′′/6′′, C-10/13 and C-11/12. The two NHa protons showed correlations with C-1′, C-6′ carbons atoms, while the NHb protons showed correlations with C-1/8 carbon atoms in the HMBC spectrum. The proton of acridine-NH has a correlation with C-10/13 carbon atoms. Further details of NMR signals and correlations have been provided in 3b have been provided in the experimental section. The mass of 3b via electrospray ionization (ESI+) was found at m/z 684.1 (M + H).In the s of all  aromatic2.2.3a–c for different biologically important anions, i.e. F−, −OAc, Br−, I−, HSO4−, ClO3−, CClO4−, CN− and SCN−, has been studied via UV–visible (UV–Vis) spectroscopy. The anion-sensing study with thiosemicarbazide-linked acridines 3a–c showed high selectivity for fluoride when compared with other anions such as acetate, bromide, iodide, bisulfate, chlorate, perchlorate, cyanide and thiocyanate. An increasing amount of different anions  was added to the solution of receptors 3a–c (4 × 10−5 M). The change in colour of the receptor solution from yellow to reddish brown, to dark brown  was only observed upon the addition of fluoride  with a clear shift in the UV spectrum (a). Further the UV–Vis titration experiments of receptor 3a upon gradual increase of fluoride anions (0–30 equiv.) showed a decrease in intensity at 320, 415 and 480 nm with a concomitant appearance of a new red-shifted band at 550 nm (b). The formation of three isobestic points at 332, 375 and 455 nm clearly indicates the formation of new species upon the addition of a fluoride anion source, TBAF. The UV–Vis spectra of receptor 3b having mono-fluoride substituent on the phenyl ring were found to be almost similar in selectivity and detection pattern for fluoride anions as that of probe 3a, when titrated with different anion solutions such as F−, −OAc, Br−, I−, HSO4−, SO42−, PO43−, ClO3−, ClO4−, CN−, SCN− and H2PO4− . Moreover, the titration of di-fluoride-substituted receptor 3c with different anions also showed it to be selective only for fluoride anion . Selectivity of all probes 3a–b towards fluoride anion could be due to the small size and basic nature of fluoride that helps to remove acidic hydrogen atoms from the thiosemicarbazide moiety of receptor 3c, resulting in observed changes in colour and UV–Vis spectrum .The titration of thiosemicarbazide–acridine receptor spectrum a. Furthet 550 nm b. The fo3a and the fluoride anion during titration experiments. The Job's plot of probe 3a and fluoride showed maxima at a mole fraction greater than 0.8 , while for 3b and 3c between 0.7 and 0.8 , which indicates the interaction/abstraction of more than one fluoride anion [2 M−1 with a limit of detection (LOD) of 6.879 × 10−5 M for probe 3a . For probes 3a and 3c, the binding constant and LOD values are given in Moreover, a continuous variation method, known as Job's plot, was used to determine the stoichiometric ratio between receptor de anion  was foun3a receptor–anion solution in methanol, the absorption intensity at 360 nm was restored with a concomitant disappearance of the band at 460 nm (c). These results suggested the degradation of the receptor–anion complex via re-protonation of NH groups in the presence of protic (i.e. MeOH) solvent. The other probes 3b and 3c also showed the same pattern of UV–Vis absorption spectra . The sensitivity of probes 3a–c was further explored via a fluorescence spectral study. The solutions of probes 3a and 3b were prepared followed by gradual addition of fluoride ions (0 to 10 equiv.), and were then subjected to fluorescence spectroscopic studies. The fluorescence spectra of probe 3b upon excitation at 278 nm showed a gradual decease in the peak at 530 nm with a concomitant appearance of a new peak at 610 nm . The pattern of probe 3a was also observed in the same way, with a little difference in that it showed two peaks at 480 and 560 nm (d). Moreover, the applicability of the thiosemicarbazone-linked acridine, i.e. probe 3b, has been explored with toothpaste samples . The variations in UV spectra clearly indicate the fluoride-sensing ability of the synthesized linked acridine–thiosemicarbazone receptors.Interestingly, it has been also observed that upon dilution of t 460 nm c. These d 560 nm d. Moreov3a–c with fluoride anions was further investigated by 1H-NMR study. The receptor 3b was treated with a fluoride anion source (TBAF) in DMSO-d6. The fluoride source was added gradually to the NMR sample of receptor 3b in DMSO-d6. 1H-NMR spectra were recorded before fluoride addition and then after adding different concentrations  , δ 9.23 (2NH) and δ 8.79 (1NH) ppm before addition of fluoride anion (TBAF). Upon addition of 1–2 equiv. of fluoride anions to the solution of receptor 3b, a drastic decrease in the intensity of signals for NH protons (b) was observed. The signals for NH protons completely disappeared after addition of 4 equiv. of fluoride ions to receptor 3b (c). The disappearance of characteristic signal for NH protons of model receptor 3b upon the gradual addition of fluoride anion strongly suggested the abstraction of more than one NH proton by fluoride anions (The interaction of semicarbazide-linked acridines of TABF) . The are protons b was obseptor 3b c. The die anions .Figure 3a and fluoride anions was also studied by 1H-NMR spectroscopy . The amount of TBAF as the fluoride anion source was gradually increased (0‒4 equiv.) to observe the changes in 1H-NMR signals. The signals for all NH groups at δ 10.17, δ 9.48 and δ 8.79 ppm for probe 3a were significantly deceased and completely disappeared after the addition of 4 equiv. of fluoride anions (TBAF). The abstraction of NH protons of 3b occurred due to the strong basic effect of F− with respect to other anions studied. The signal of CH-9 proton at δ 5.26 ppm shifted slightly up-field, which was considered insignificant. The sequential 1H-NMR data showed clearly the selective interaction of the fluoride anion with NH protons due to its strong basic characteristic compared to other anions, i.e. −OAc, Br−, I−, HSO4−, SO42−, PO43−, ClO3−, ClO4−, CN− and SCN−. Moreover, the presence of five NH groups in thiosemicarbazide-linked acridine as receptor 3a bestows the ability to detect more than one fluoride anion during the sensing process.The interaction between a model probe 2.3.Ecomplex is the energy of complex formed between thiosemicarbazide–acridine and anions, whereas Etsc and Eanion are the energies of isolated thiosemicarbazide–acridine and anions, respectively.All calculations were performed with a Gaussian 09 suite of programs . GeometrThe excitation energies were also calculated with time-dependent density functional theory (DFT) at the (TD)CAM-B3LYP/6–311G ,38 level3a is searched. We have previously shown [E-orientation of the N-group  is shown in DFT calculations have been performed to rationalize the selectivity of thiosemicarbazide–acridine-based probes for fluoride ions. In this regard, the calculations have been performed for complexation energies (of anions with the probe molecules) and UV–Vis absorption spectroscopic properties. In the first step, the most stable geometry of the probe molecule ly shown  that acr3a with various anions. Analysis of the probe molecule reveals that there are three positions where anions can interact. These positions include N–H of the central ring, and H–N–C(S)–N–H of both thiosemicarbazide groups. An anion can interact with both N–H group of the H–N–C(S)–N–H moiety simultaneously to form a stable six-membered ring. The computational study is performed for complexes of probes 3a–c with F−, −OAc, Br−, Cl− and CN− anions, whereas other less basic and bulky anions  are not considered. The optimized geometry of the 1 : 3 complex of probe (3a): F− is shown in figure 6b.In the next step, the interactions of various anions with the probe molecule are studied. The experimental results show that the probe molecule-to-anion ratio is more than three. Therefore, we started with the optimization of the 1 : 3 complex of probe 3a shows considerable changes in the geometry after complexation with fluoride ions. Important structural reorganizations during complexation are the changes in the orientations of the central and terminal phenyl rings. The fluoride ion interacts not only with the N–H protons of the thiosemicarbazide but also with the ortho hydrogen atoms of the terminal phenyl ring (b). Owing to this interaction, the terminal phenyl rings become coplanar with the thiosemicarbazide moiety, which causes the thio group to form loose non-bonding interactions with the central phenyl ring. As a result, the central phenyl ring gets rotated to have favourable interaction through its ortho hydrogen atoms with the nitrogen atom (of thiosemicarbazide) at a distance of 2.72 Å (b). The fluoride ion has strong proton affinity; therefore, the N–H bond of the central ring is considerably broken. The N–H and F–H bond lengths are 1.21 Å and 1.18 Å, respectively. The weakening of N–H bond  causes the nitrogen atom to become more electron-rich. The increased electron density causes changes in the electronic and spectroscopic properties of the complex (vide infra). The strong proton abstraction ability of F− can be attributed to the high charge density on fluoride ions. The fluoride ion has −1 charge which is completely localized on it, whereas the negative charge on other ions  is either more delocalized or diffused. The diffused or delocalized negative charges on other anions result in relatively weak interaction with the probe molecule . These anions  interact only with the N–H of the thiosemicarbazide without any involvement of the ortho hydrogen of the terminal phenyl ring. Therefore, the coplanarity of the terminal phenyl ring with the thiosemicarbazide moiety is not seen for these anions.Compound nyl ring b. Owing f 2.72 Å b. The fl3a has the strongest binding affinity for the fluoride ion. The binding of three fluoride ions with a molecule of probe 3a is exothermic by 383.97 kcal mol−1, which corresponds to an exothermicity of −127.99 kcal mol−1 per fluoride ion  per anion. Nitrile anion has a binding energy of −80.82 kcal mol−1 per anion, which amounts to −242.45 kcal mol−1 for three CN− anions. The least binding energy is calculated for the interaction of bromide with the probe molecule, which may be attributed to the low charge density of bromide due to its large size.The binding energies are also calculated for these complexes, which reflect that the probe ride ion . The bin3b and 3c. The binding energies of the fluoride ion with probe 3b and 3c are −383.25 and −386.43 kcal mol−1, respectively, which amount to −127.75 and −128.81 kcal mol−1 per fluoride ion. The probe 3c has highest binding energy for the fluoride ion when compared with 3b and 3a, which may be attributed to a greater electron-withdrawing effect of fluoride ions. Probe 3c has relatively higher binding affinity for other anions compared to probe 3b and 3a but the difference is almost negligible. In general, the electron-withdrawing substituent on the terminal aromatic ring increases the binding affinities for different anions compared to the unsubstituted probe 3a.A similar trend in the binding energies of different anions is observed for other probes probe–anion complexes, and they are compared to the UV–Vis spectrum of a pure probe molecule. The probe 3a has two absorption bands appearing at 355.0 nm and 274.75 nm. The band at 355 nm has a tail which is stretched to 480 nm. The calculated UV–Vis spectrum at CAM-B3LYP is consistent with the experimental UV–Vis spectrum. Complexation of all anions except F− does not cause any shift in the UV–Vis spectrum of the probe  the abstraction of proton by the fluoride ion leaves the nitrogen with more electron density which is now delocalized on the entire skeleton and (ii) the complexation of fluoride with the probe causes the planarization of the skeleton, particularly at the terminal phenyl ring . The other probes 3b and 3c also show similar selectivity for fluoride ions compared to other anions  (99%), hydrazine hydrated (80%) and phenyl isothiocyanate (98%) were purchased from Sigma Aldrich or Merck and used directly for carrying out our reaction unless otherwise stated. Acetonitrile (HPLC grade), DMSO (HPLC grade), methanol (HPLC grade) and distilled or Milli-Q water were used. TLC was carried out with silica gel 60 aluminium-backed plates 0.063–0.200 mm . Analytical grade solvents such as ethyl acetate, diethyl ether, hexane and methanol were used as eluents for purification purpose through column chromatography. Round-bottomed flasks  were used. TLC visualization was carried out using UV lamp radiation at 254 nm. In addition, different spot test mixtures, such as basic potassium permanganate or vanillin, were also used. IR spectra were recorded with a Bruker Vector-22 spectrometer. The 1H-NMR spectra were recorded using Bruker Avance spectrometers at 300, 400, 500 and 600 MHz, while 13C-NMR spectra were recorded at 75, 100, 125 and 150 MHz in the appropriate deuterated solvent. The chemical shifts were recorded on the δ-scale (ppm) using residual solvents as an internal standard . Coupling constants were calculated in hertz (Hz) and multiplicities were labelled as s (singlet), d (doublet), t (triplet), q (quartet) and quint (quintet), and the prefixes br (broad) or app (apparent) were used. Mass spectra (EI+ and FAB) were recorded with a Finnigan MAT-321A, Germany. Melting points of solids were determined using a Stuart™ melting point SMP3 apparatus and are uncorrected.All the starting materials including 5,5-dimethyl-1,3-cyclohexanedione (95%), benzaldehyde, ammonium acetate, nickel fluoride .To a 25 ml round-bottomed flask, equipped with a magnetic stirrer, dimedone , ammonium acetate , benzaldehyde , nickel(II) fluoride tetrahydrate (25 mol%) as the catalyst and ethanol (3 ml) were added. The reaction mixture was heated on an oil bath at 80°C with continuous stirring for 80 min until the complete consumption of the starting material, as monitored using TLC analysis. The resulting mixture was dissolved in methanol, and poured onto crushed ice with stirring to obtain precipitates of the product. The precipitates were washed with 7  was treated with hydrazine hydrate (10 ml) and acetic acid (2 ml) at 120°C for 28 h. Progress of the reaction was monitored by TLC analysis using 30% EtOAc in n-hexane. On completion of the reaction, water was added to get the precipitate of compound 8, which was filtered and dried under vacuum. The yield was 97% (0.36 g) and the colour was golden yellow.The compound 3a–c was carried out by reacting intermediate 8 (1.0 equiv.) and the appropriate substituted aryl isocyanates or isothiocyanates (2 equiv.), which were taken in 15 ml acetonitrile and heated at 98–100°C for 13 h. The progress of the reaction was monitored through TLC analysis using a 0.20 mm thick precoated silica plate, and spots were visualized through UV light. On completion of the reaction, the mixtures were cooled down to ambient temperature, resulting in the formation of precipitates. The precipitates were filtered, washed with warm acetonitrile and dried to obtain the pure compounds 3a–c in a 55–85% yield -3,3,6,6-Tetramethyl-9-phenyl-3,4,6,7,9,10-hexahydroacridine-1,8-diylidene)bis(N-phenylhydrazine-1-carbothioamide)2,2 (3a). Light yellow solid, yield 60% , MP 281–284°C. IR : (KBr disc) 3420, 3272, 3216, 3093, 2956, 2925, 2863, 1645, 1596, 1526, 1479, 1405, 1367, 1316, 1260, 1240, 1193, 1156, 1074, 1001, 755, 697, 661. 1H-NMR : δH 10.17 , 9.48 , 8.79 , 7.46–7.38 , 7.23–7.12 , 6.98–6.91 , 5.35 , 2.39 , 2.32 , 2.24 , 2.16 , 1.04 2), 0.81 2). C13-NMR : δC 193.7 (C), 175.0 (C), 151.9 (C), 148.7 (C), 139.9 (C), 137.8 (C), 128.2 (CH), 128.0 (CH), 127.3 (CH), 125.3 (CH), 124.8 (CH), 124.5 (CH), 123.7 (CH), 106.7 (C), 39.6 (CH2 × 2), 38.3 (CH2 × 2), 35.9 (CH), 30.3 (C), 29.4 (CH3 × 2), 26.4 (CH3 × 2). ESI-MS: m/z 648.1 (M + H).′--3,3,6,6-Tetramethyl-9-phenyl-3,4,6,7,9,10-hexahydroacridine-1,8-diylidene)bis(N-(2-fluorophenyl)hydrazine-1-carbothioamide)2,2 (3b). Yellow solid, yield 85% (over three steps), MP 275–278°C. IR : (KBr disc) 3749, 3446, 3271, 3222, 3093, 2955, 2926, 1646, 1620, 1527, 1478, 1404, 1367, 1239, 1223, 1157, 1077, 1029, 811, 752, 709, 658. 1H-NMR : δH 10.32 , 9.23 , 8.80 , 7.64 , 7.31 , 7.22–7.18 , 7.10 , 6.96–6.91 , 5.27 , 2.49 , 2.31 , 2.23 , 2.16 , 1.03 2), 0.82 2). C13-NMR : δC 193.7 (C), 175.8 (C), 157.2/154.8 (C), 152.1 (C), 148.4 (C), 140.1 (C), 128.2 (CH), 128.0 (CH), 127.2 (CH), 126.9/126.8 (CH), 126.6/126.5 (CH), 125.2 (CH), 123.6 (CH), 115.3/115.1 (CH), 39.6 (CH2 × 2), 29.2 (CH2 × 2), 35.9 (CH), 30.3 (C), 29.2 (CH3 × 2), 26.5 (CH3 × 2). ESI-MS: m/z 684.1 (M + H).′--3,3,6,6-Tetramethyl-9-phenyl-3,4,6,7,9,10-hexahydroacridine-1,8-diylidene)bishydrazine-1-carbothioamide)2,2 (3c). Yellow solid, yield 55% (over three steps), MP 279–281°C. IR : (KBr disc) 3440, 3273, 2957, 1644, 1612, 1529, 1478, 1308, 1237, 1146, 1086, 965, 848, 765, 710. 1H-NMR : δH 10.21 , 9.39 , 8.68 , 7.34–7.29 , 7.04–6.98 , 6.94 , 5.42 , 2.31–2.06 4), 1.01 2), 0.83 2). ESI-MS: m/z 720.1 (M + H).


The purpose of the study is to determine whether the PM2.5 every other day for 4 weeks. Then, 10-month-old and 4-week-old mice were exposed to PM2.5 for 4 weeks and withdrawal PM2.5 1 or 2 weeks. Heart rate and systolic blood pressure were measured using a tail-cuff system. Cardiac function was assessed by echocardiography. Left ventricles were processed for histology to assess myocardial fibrosis. ROS generation was detected by photocatalysis using 2′,7′-dichlorodihydrofluorescein diacetate (DCFHDA). The expression of cardiac fibrosis markers  and possible signaling molecules, including NADPH oxidase 4 (NOX-4), transforming growth factor β1 (TGFβ1), and Smad3, were detected by qPCR and/ or Western blot.Female C57BL/6 mice at different ages  received oropharyngeal aspiration of 3 mg/kg b.w. PM2.5 exposure induced cardiac diastolic dysfunction of mice, elevated the heart rate and blood pressure, developed cardiac systolic dysfunction of 10-month-old mice, and caused fibrosis in both 4-week-old and 10-month-old mice. PM2.5 exposure increased the expression of Col1a1, Col3a1, NOX-4, and TGFβ1, activated Smad3, and generated more reactive oxygen species in the myocardium of 4-week-old and 10-month-old mice. The withdrawal from PM2.5 exposure restored blood pressure, heart rate, cardiac function, expression of collagens, and malonaldehyde (MDA) levels in hearts of both 10-month-old and 4-week-old mice.PM2.5 than adults and suffer from cardiac dysfunction. PM2.5 exposure reversibly elevated heart rate and blood pressure, induced cardiac systolic dysfunction of older mice, and reversibly induced fibrosis in juvenile and older mice. The mechanism by which PM2.5 exposure resulted in cardiac lesions might involve oxidative stress, NADPH oxidase, TGFβ1, and Smad-dependent pathways.Juvenile and older mice are more sensitive to PMThe online version of this article (10.1186/s12989-018-0264-2) contains supplementary material, which is available to authorized users. Even in the same region, PM2.5 from different seasons appears to have different chemical composition. It has been reported that seasonal variation in the association between PM2.5 and cardiovascular hospitalization [2.5 does not affect all people equally. Several studies have suggested that susceptible individuals are at greater risk for PM2.5-associated cardiovascular morbidity and mortality including the elderly, women, and patients with preexisting coronary artery disease and diabetes [Air pollution, mostly by fine particulate matter , the amount of PM2.5 inhalation for each mouse over 2 days is 19.425 μg. Therefore, PM2.5 exposure concentration for mouse (about 20 g b.w.) every 2 days should be 0.97 mg/kg (b.w.). The dose used in the present study was about 3 fold of secondary standards or more than 3.6 fold when considering the deposition fraction [2.5 concentration could exceed 300 μg/m3 during the polluted periods in Beijing [http://113.108.142.147:20035/emcpublish/). The average concentration of PM2.5 in northern China with non-haze weather was 161 μg/m3 [3 [Several previous studies of seasonal patterns of cardiovascular disease (CVD) indicated a peak of CVD in winter months , 14, 15.0.259 m3 . Accordifraction . However Beijing  or otherse about  g b.w. e2.5-induced cardiac dysfunction is age-dependent; (2) to examine whether the above-mentioned effects could be restored after PM2.5 exposure withdrawal; (3) to determine the potential mechanism of susceptibility to PM2.5 exposure.Therefore, the purpose of this study was: (1) to determine whether the PM2.5 exposure caused a significant increase compared with pre-exposure but a non-significant increase compared with age-matched control group  of 10-month-old mice compared to age-matched control group. . The ROS levels in the hearts of 4-week-old and 10-month-old mice exposed to PM2.5 were significantly increased compared to those of the corresponding control mice and 4-month-old mice after PM2.5 exposure , and MDA levels in hearts and lungs. The mRNA levels of TGFβ1 were significantly increased in both lungs and hearts of 4-week-old and 10-month-old mice after PMure Fig. . The higure Fig. . The mRNure Fig. .Fig. 6PM2.5 were significantly higher than in aged-matched controls  in the myocardium. Other studies have documented similar results with exposure to concentrated ambient PM [We found that 10-month-old and 4-week-old mice developed more fibrosis following PMbient PM , 35 and bient PM .2.5. Similar results were observed with secondhand smoke or lipopolysaccharide (LPS) treatment. Secondhand smoke or LPS induce more cardiac dysfunction, fibrosis, inflammation, and oxidative stress in aged mice than in young mice [2.5 on older mice might accelerate the development of aging. Furthermore, our data showed that 4-week-old mice were more susceptible than 4-month-old mice. PM2.5 exposure induced cardiac fibrosis, elevated Col1a1 and Col3a1 expression in 4-week-old mice, but not in 4-month-old mice. One possible reason for the difference in susceptibility to PM2.5 of mice is their differences in estrogen levels. It is well established that the incidence of cardiovascular disease is reduced in females prior to menopause, which may be due to estrogen levels [2.5 elevated non-significantly the estradiol 2 (E2) levels of 4-week-old and 4-month-old mice. The E2 levels of 10-month-old mice were lower than 4-month-old mice in PM2.5 groups  rather than direct effects (translocated carbon nanoparticles) [Different potential mechanisms for PM-associated cardiovascular disease have been hypothesized as either activated by direct effects of pollutants on the cardiovascular system, or by indirect effects due to pulmonary oxidative stress and inflammatory responses . In the rticles) .2.5 exposure. The increase in NOX-4 expression was accompanied by the elevation of TGFβ1 protein expression. ROS stimulates the release and activation of cytokines, such as TGFβ1 [2.5 exposure. This finding implied that PM2.5 induced cardiac fibrosis might involve the TGFβ1-Smad pathway.ROS generation has been detected in rat ventricular myocytes exposed to diesel exhaust, and it is likely mediated through NADPH oxidase-dependent pathways . NADPH oas TGFβ1 . TGF-β1 as TGFβ1 . Cardiacas TGFβ1 . TGFβ1 ias TGFβ1 , and funas TGFβ1 . Howeveras TGFβ1 . Hu and as TGFβ1 . Among nas TGFβ1 . TGF-β1 as TGFβ1 . In our 2.5-bound metals could reach and deposit in the heart with a developmental window-dependent property. The content of Zn increased significantly in hearts of 4-week-old and 10-month-old mice. The contents of Mn and Cd increased significantly in hearts of 10-month-old mice [2.5 induced cardiac toxicity. However, further experiments are needed to confirm the hypothesis.Many studies have documented that Zn and Mn can enter systemic circulation and lead to lung fibrosis and cardiovascular disease –60. In oold mice . It is sThere are several limitations to our study. The first limitation is the exposure method. Although oropharyngeal aspiration has been commonly used for experimental PM exposure –64. Beca2.5 exposure induced cardiac dysfunction in mice at all ages tested, elevated heart rate and blood pressure in 10-month-old mice, and caused cardiac fibrosis in 10-month-old and 4-week-old mice. In combination, these data provide strong evidence that exposure to PM2.5 has significant effects on juveniles and the elderly because they are more susceptible to cardiovascular disease. Furthermore, all the above adverse effects in 10-month-old and 4-week-old mice could be restored after withdrawal PM2.5 exposure for 2 weeks. The mechanisms by which PM2.5 exposure resulted in cardiac lesions might involve the generation of oxidative stress and inflammation in lungs and the activation of TGFβ1-Smad pathway.In this report, we provide evidence that PM2.5 samples were collected onto quartz filters  with PM middle-volume air samplers . Details of sample collection and components analysis of PM2.5 have been previously described [Sampling was performed between 2012 and 2013 in Taiyuan, Northern China. The PMescribed  and provFemale C57BL/6 mice were purchased from the Junke Biological Engineering Co., LTD . Animals were housed in individual stainless steel cages under standard conditions (24 ± 2 °C and 50 ± 5% humidity) with a 12 h light–dark cycle in SPF conditions. The protocol of animal procedures was conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and was approved by the Institutional Animal Care and Use Committee of Shanxi University.study-1, C57BL/6 mice at 4 weeks, 4 months and 10 months were randomized into two subgroups. The mice received oropharyngeal aspiration of 0.9% saline (control groups) or 3 mg/kg. b.w. PM2.5 suspension  15–30 μl depend on the body weight of each animal every other day for 4 weeks. When not being treated, the mice had free access to standard food and water. Mice were sacrificed 24 h after the final exposure.In study-2, C57BL/6 mice at 4-week-old or 10-month-old were received oropharyngeal aspiration of 0.9% saline (controls) or 3 mg/kg. b.w. PM2.5 suspension (PM2.5 groups) every other day for 4 weeks. Mice were sacrificed 24 h, 1 week, or 2 weeks after the final exposure, respectively. The hearts and lungs were removed, and all samples were fixed or quick frozen in liquid nitrogen and stored at − 80 °C.In Heart rate and blood pressure were measured using a standard tail-cuff system . The blood pressure and heart rate data of each mice were measured as the mean of at least 5 successful measurements.Heart function and structure were assessed by echocardiography using a Vevo 770™ ultrasound system , which included a 21-MHz transducer. Mice were anesthetized with 2% isoflurane administered via a nose cone, were shaved from the chest, and were placed in the supine position on a hotplate. Echocardiography and off-line data analysis was performed by a single observer. The observer was blinded as to the age and group of mice being analyzed. Two-dimensional (2D) and M-mode echocardiography images were obtained from the parasternal region and viewed to measure left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVESD), posterior wall thickness in diastole (LVPWd), left ventricular end diastolic volume (LVEDV), and left ventricular end systolic volume (LVESV). Left ventricle filling parameters were assessed from the ends of mitral leaflets in apical quadrilocular image, and peak early (E) and after (A) diastolic flow velocities were measured. Tissue Doppler assessment was conducted on apical quadrilocular image. Peak early motion (E’) and peak after motion (A’) wave values were measured. Ejection fraction (EF), fractional shortening (FS), E/A ratio, E’/A’ ratio, and E/E’ ratio were obtained using defined calculations. Each parameter was measured in at least 3 cycles, and mean figures were utilized. All measurements were performed offline using the Vevo 770™ system software .The cardiac tissue from mice of each group was rapidly removed, washed several times with 0.1 μM phosphate buffered saline , fixed in 10% formalin for 24 h at room temperature, dehydrated by graded ethanol, and embedded in paraffin. Sections (5–6 μm-thick) were deparaffinized with xylene, stained with Masson’s trichrome staining, and observed by microscopy. Quantitative planimetric analyses were performed on three successive sections per slide, and at least 7 sections from 3 consecutive slides per mouse were examined. Each image was digitized using a digital camera and analyzed under a research microscope  using Image-Pro Plus software (version 5.0). The left ventricular cross-section collagen accumulation was quantified by the ratio of blue area to total myocardium area. All analyses were performed by an investigator blinded to the group assignments.w/v) PBS homogenate. After centrifuging at 1000 g for 10 min at 4 °C, the supernatant was collected and used to evaluate reactive oxygen species (ROS) and protein content. In 96-well plates, 190 μL of the supernatant and 10 μL of 1 mM DCFHDA were added to each well. After incubating at 37 °C for 30 min in the dark, the fluorescence was read at 485 nm for excitation and 530 nm for emission with a fluorescent plate reader . The results were calculated as the fluorescence units (FLU)/mg protein. The value of the 4-week-old mice in control group was set to 1.Fresh hearts were used to prepare a 10%  was measured using commercial kits .Total RNA was isolated using TRIzol Reagent  according to the manufacturer’s instructions. Total RNA was then treated with DNase I , and cDNA was synthesized using a First Strand cDNA Synthesis kit  The cDNA product was stored at − 80 °C until used.Real-time quantitative PCR (qPCR) was performed using an IQ5 Real-Time PCR System  and SYBR Green qPCR Master Mix kit . The primers are listed in Additional file Proteins were extracted in ice-cold lysis buffer . The protein concentration was determined according to the Bradford method using BSA as the standard protein . Sodium 2.5 groups were used to compare between groups in others figures. Differences for all tests were considered significant when P < 0.05.All data are expressed as the means ± standard error of the mean (SE). Comparison between groups in Fig. Additional file 1:Figure S1. PM2.5 induces collagen mRNA (A) and protein (B) expression in H9C2 cells. (A) mRNA expressions of Col1a1 and Col3a1 in H9C2 cells were detected by qPCR. (B) The protein levels of Col1a1 and Col3a1 were detected by Western blot. GAPDH was used as the internal control. The mean expression in four seasons PM2.5 treated group was shown as a fold change compared to the mean expression of control group, which had been calculated as target gene or protein /GAPDH and ascribed an arbitrary value of 1. Each column and bar represents the mean ± SE (n=3). Significantly different from control by one-way ANOVA with Tukey's post-test. * P<0.05 vs. control group; a P<0.05 vs. spring PM2.5 group; b P<0.05 vs. summer PM2.5 group; c P<0.05 vs. autumn PM2.5 group. (TIF 440 kb)Additional file 2:Figure S2. Estrogen levels in plasma of different age mice. Each column and bar represents the mean ± SE (n=6). # P<0.05 vs. 4-week-exposure by two-way ANOVA and Bonferroni's post hoc tests. (TIF 106 kb)Additional file 3:Table S1. Contents of inorganic ion in PM2.5 samples from different seasons (unit: μg/m3). Table S2. Element contents in PM2.5 samples from different seasons (unit: ng/m3). Table S3. Contents of polycyclic aromatic hydrocarbons (PAHs) in PM2.5 samples from different seasons (unit: ng/m3). Table S4. Carbon contents in PM2.5 samples from different seasons (unit: μg/m3). Table S5. Sequences of primers used in real-time PCR. (DOCX 35 kb)


However, %Ndfa of soybean was greater in plants grown in Mambuk soil, followed by Pawe with the lowest %Ndfa being in Amaro soil. The amount of N-fixed followed similar pattern as %Ndfa. The significant interaction found between soil type and soybean genotype for plant DM, shoot N concentration, δ15N, %Ndfa, N-fixed and soil N-uptake clearly indicated the effect of soil factors. This study revealed the presence of native rhizobia in Ethiopian soils that are compatible with soybean. The N contribution of the soybean genotypes was variable, and strongly influenced by the soil factors.Achieving food and nutritional security is a major challenge in Ethiopia, especially with increasing human population and low crop productivity. Legumes offer an alternative choice to chemical fertilizers for increasing crop yields. The aim of this study was to assess, under glasshouse conditions, plant growth and symbiotic performance of uninoculated soybean genotypes planted in soils collected from different locations in Ethiopia. The results showed significant differences in plant growth and symbiotic performance among the soybean genotypes planted in different soils. There was a location-specific effect of soil on plant growth and symbiotic N nutrition of soybean. Whole-plant biomass was highest in soil from Amaro, followed by Boricha, Dorebafano, Pawe, and Mambuk. The δ Legumes also have the ability to fix atmospheric N2 when in symbiosis with soil rhizobia. The annual fixed-N has been estimated to be 21.45 Tg from food legumes, and 12–25 Tg from pasture fodder legumes  the distribution of rhizobia in Ethiopian soils. The aim of this study was i) to assess the ability of native rhizobia to nodulate soybean, as well as ii) to assess plant growth and symbiotic performance of soybean genotypes planted in uninoculated soils collected from different locations in Ethiopia under glasshouse conditions.Studies done so far on soybean in Ethiopia have focused on rhizobial inoculation response, genetic variability of soybean for agronomic traits, and intercropping trials with cereals . However, Mambuk and Pawe are located in the Benshangul Gumuz Region (BGR) with coordinates 10° 38′ N and 35° 43′ E in north-eastern Ethiopia. The BGR is close to the Ethiopia-Sudan border, and is characterized by 500 to 1800 mm of annual rainfall, 21 to 35 °C daily temperature and 600 to 2731 m above sea level.The soil samples used in this study were collected from five locations  within the soybean-growing areas of Ethiopia. maro, Boricha and Dorebafano are located in the Southern Nations Nationalities and People’s Region (SNNPR) of Ethiopia, an area found between geographical coordinates 4−1 as triple superphosphate (TSP) before planting.Fields with no history of rhizobial inoculation were particularly targeted for soil collection. Soils were sampled at 20-cm depth, placed in plastic bags and taken to the laboratory, where they were air-dried in a dust-free environment and sieved (5 mm). A sub-sample of the sieved soil was analysed at the Agricultural Research Council laboratory in Pretoria, South Africa, for pH, electrical conductivity (EC), organic carbon, total N, cation exchange capacity (CEC) and plant-available P. At the laboratory, the sieved soil from each location was used to fill 4-L capacity plastic pots that were previously surface-sterilised. All potted soils were supplied with a blanket treatment of 46 kg P haEleven soybean genotypes  obtained from the Awassa and Pawe Agricultural Research Centres in Ethiopia, were used in this study. Seeds of non-legume species included as reference plants were obtained from Hawassa University, Ethiopia. Where there was history of soybean cultivation, the soybean genotypes used by farmers, were planted at those particular locations. The exception was forest soil which had no history of soybean cultivation.Sorghum bicolour (L.), Eragrostis tef, Triticum aestivum) included as reference plants were similarly processed and planted in triplicates to measure soil N uptake. The plants were raised under natural light in the glasshouse.Soybean seeds were surface-sterilized Vincent  and the 15N isotopic analysis.At 58 days after planting (DAP) at R1 stage, the soybean plants were harvested and separated into shoots, roots and nodules. The shoots and roots were oven-dried at 60 °C for 48 h and weighed. The shoots of reference plants were also harvested, oven-dried and weighed. The shoots of both legume and reference plants were each milled separately to a fine powder (0.85 mm size) and stored prior to 15N/14N ratio and %N using mass spectrometry. Plant samples were weighed into Al capsules (2.0–2.5 mg/sample) and each fed into a Carlo Erba NA1500 elemental analyser  coupled to a Finnigan MAT252 mass spectrometer via conflo II open-split device. The 15N abundance, usually expressed in a relative δ (delta) notation, is the ‰ deviation of the 15N natural abundance of the sample from atmospheric N2 (0.3667 atom% 15N). The isotopic composition (δ15N) of sample was measured as (Mariotti The finely ground (0.85 mm) shoot samples of soybean and reference plants were analysed for Mariotti :15N/14Nsample and 15N/14Nstandard are the abundance ratios of the plant and air as standard, respectively.2 (%Ndfa) was quantified as , and B value is 15N natural abundance of soybean plants which were solely dependent on symbiotic N2 fixation for their N nutrition. The mean δ15N values of the reference plants for each location were used to estimate the percent N derived from the atmosphere in soybean plants.The amount of N-fixed was calculated as  package. Plant growth and isotopic data were tested for normality in distribution and then subjected to a one-way analysis of variance (ANOVA) for each location, and a 2-Way ANOVA to compare the means of results between the two regions. Where there was a significant difference, the means were separated using the Duncan’s multiple range test at −1. The organic carbon content ranged from 1.29 to 2.15%. The plant available P ranged from 0.35 to 24.7 mg kg−1. According to the Bray-1 P test rating, the soil from Dorebafano (24.7 mg kg−1) had medium P fertility, Boricha (10.2 mg kg−1) low P, Amaro (6.7 mg kg−1), Mambuk (0.35 and 0.44 mg kg−1) and Pawe (0.60 mg kg−1) very low P   were analysed for pH, organic carbon (OC), electrical conductivity (EC), cation exchange capacity (CEC), total N and plant-available P Table . The pH P Jones, . AccordiP Jones, , soil fe1) Table .Table 1C15N natural abundance technique requires a value for soil N uptake by the legume. In this study, Sorghum bicolour (L.), Eragrostis tef and Triticum aestivum were used as reference plants. The δ15N values varied from +2.21‰ for Triticum aestivum in Pawe soil to +10.46‰ for Eragrostis tef in Mambuk soil. The average δ15N for the 3 reference plants for each site ranged from +2.72‰ for Pawe to +6.33‰ for Dorebafano (Table Estimating the percentage N derived from the atmosphere (%Ndfa) by nodulated legumes using the no Table .Table 2δAt Pawe, a 1-Way ANOVA showed significant differences in plant growth and symbiotic performance between and among the six soybean genotypes.−1 for the genotype Coker-240 to 5.66 g plant−1 for TGx-3326-44 , followed by TGx-3326-44 (108.7 mg plant−1), AGS-71 (107.3 mg plant−1), Wegayen (105.4 mg plant−1), Coker-240 (90.6 mg plant−1) and least in genotype Gishama (90.5 mg plant−1).Shoot N concentration also ranged from 1.92% for genotype TGx-3326-44 to 2.73% for Coker-240. Genotypes AGS-71 and TGx-3326-44 recorded the lowest shoot N concentration, 1.92 and 1.95% respectively  and AGS-71 (+0.25‰) exhibiting the lowest δ15N values, followed by Gishama (+0.56‰) and Wegayen (+0.77‰). Genotype Coker-240 and Nova recorded the highest δ15N values, +1.87‰ and 1.32‰ respectively. As a result, the %Ndfa of TGx-3326-44 and AGS-71 (which showed lower δ15N) were much higher, 57.0 and 54.3%, respectively . By contrast, Coker-240 which had the highest δ15N (+1.87‰) recorded the lowest %Ndfa (18.7%). The soybean genotypes varied in the amounts of N-fixed, which ranged from 16.2 mg plant−1 for Coker-240 to 61.6 mg plant−1 for TGx3326–44. Soil N-uptake was highest for Nova (97.1 mg plant−1) and lowest (47.1 mg plant−1) for TGx3326–44  Nova (4.2 g.plant−1), Gishama (3.87 g.plant−1) and Crowford (38.4 g.plant−1) produced much greater shoot DM compared to Clark-63 K (2.80 g.plant−1). Root DM was highest for Crowford, followed by genotype Clark-63 K  in genotype TGx-3326-44 and highest (+3.41%) in Nova. As a result, TGx-3326-44 recorded the highest %Ndfa (69.9%), followed by genotype Gishama (65.6%), with Nova (33.1%) being the lowest. The highest N contribution was by genotype TGx-3326-44 (58.4 g.plant−1), followed by Gishama and Crowford, 53.8 and 45.5 mg.plant−1, respectively  successfully nodulated with native soil rhizobia. Table .Awassa −95 produced the largest shoot biomass, followed by Wegayen, and lowest in Williams and Coker-240. Root DM was similar for all seven genotypes, but whole-plant biomass was significantly greater in Awassa-95, followed by Wegayen, and AFGAT, and lowest in Williams. Although shoot N concentration was also similar for all genotypes, N content was highest in Awassa-95, followed by AFGAT, and lowest in Williams Table .15N of soybean genotypes ranged from +0.08‰ for AGS-71 to +2.47‰ for Crowford. As a result, AGS-71 and Crowford had the highest (66.5%) and the lowest (24.4%) %Ndfa, respectively. The amount of N-fixed was much greater in genotype AFGAT (69.2 mg plant−1), followed by AGS-71 (62.1 mg plant−1) and Wegayen (59.3 mg plant−1), and lowest in Crowford (23.6 mg plant−1). Soil N-uptake was significantly increased in Awassa-95, followed by Crowford, and lowest in AGS-71 , whole plant biomass was also similar for all the genotypes Table . Shoot N15N of soybean shoots was generally high in Amaro soil, and ranged from +3.67‰ to +4.87‰. As a result, percent N derived from fixation was low, and varied from 5.37% for Awassa-95 to 22.2% in TGx-3326-44. The amount of N-fixed by soybean genotypes in Amaro soil was very low, and ranged from 6 mg plant−1 for Awassa-95 to 19.5 mg plant−1 in TGx-3326-44. Due to the generally high N concentration in Amaro soil, the genotypes took up more N from soil than symbiosis. Soil N-uptake was high in the low-fixing Awassa-95 (101.0 mg plant−1), and much lower in TGx-3326-44, which fixed the most symbiotic N  and Coker-240 (19.7%). These 3 genotypes fixed relatively more symbiotic N than Awassa-95 and Williams  was done Table .−1), root (1.78 g plant−1) and whole-plant (6.68 g plant−1) biomass when compared to soil from BGR , Eragrostis tef and Triticum aestivum] were used in this study, with average δ15N values that varied from +2.72 to +6.33‰, a range suitable for estimating soil N-uptake by legumes  showed moderate levels of soil nitrate and high organic C concentration (Amaro) (Table 2 fixation in nodulated soybean receiving 5 mM NO3− (Mbah and Dakora Soybean plants grown in soils from the BGR region recorded the lowest δo) Table . A recen15N, %Ndfa, N-fixed and soil N-uptake clearly indicate the modulating effect of soil factors on legume N2 fixation , and this could also help to explain the variations found in soybean nodulation and N2 fixation in this study.Although this study did not assess the existing cropping systems at the sites of soil collection, both legume-based and cereal-based rotations have been reported to alter the populations of soybean rhizobia in soil, with the former stimulating more rhizobial growth than the latter  the presence and/or competitiveness of native soybean rhizobia in the soil, ii) the level of strain symbiotic efficacy, iii) mineral concentrations in soil, especially N (Dakora and Keya


The binding affinity for CA-IX ranged from 0.22 to 0.96 μM. We evaluated compound 2 as a diagnostic PET imaging agent. Compound 2 was radiolabeled with 18F in 10 ± 4% decay-corrected radiochemical yield with 85.1 ± 70.3 GBq/μmol specific activity and >98% radiochemical purity. 18F-labeled 2 was stable in mouse plasma at 37 °C after 1 h incubation. PET/CT imaging was conducted at 1 h post-injection in a human colorectal cancer xenograft model. 18F-labeled 2 cleared through hepatobiliary and renal pathways. Tumor uptake was approximately 0.41 ± 0.06% ID/g, with a tumor-to-muscle ratio of 1.99 ± 0.25. Subsequently, tumor xenografts were visualized with moderate contrast. This study demonstrates the use of a cationic motif for conferring isoform selectively for CA-IX imaging agents.Carbonic anhydrase IX (CA-IX) is a marker for tumor hypoxia, and its expression is negatively correlated with patient survival. CA-IX represents a potential target for eliminating hypoxic cancers. We synthesized fluorinated cationic sulfonamide inhibitors  Hypoxia occurs when oxygen level of the microenvironment is unable to sustain the metabolic demands of a growing tumor3An established surrogate marker for hypoxia is carbonic anhydrase IX (CA-IX). CA-IX is the protein that is most strongly upregulated by hypoxia and hypoxia-inducible factor 1α (HIF-1α)18F-labeled trivalent tracers  UltraBasic Benchtop pH meter. Proton NMR spectra were obtained using a Bruker  Avance 400inv Spectrometer, and are reported in parts per million. Mass analyses were performed using an AB SCIEX  4000 QTRAP mass spectrometer system with an ESI ion source. Melting points were taken on a Fisher–Johns melting point apparatus  and were uncorrected. The quaternary methyl ammonium (QMA) anion exchange and C18 light Sep-Pak cartridges  were obtained from Waters . Purification and quality control of 18F-labeled 2 were performed on an Agilent  HPLC system equipped with a model 1200 quaternary pump, a model 1200 UV absorbance detector, and a Bioscan  NaI scintillation detector. The radio-detector was connected to a Bioscan B-FC-1000 Flow-count system, and the output from the Bioscan Flow-count system was fed into an Agilent 35900E Interface, which converted the analog signal to digital signal. The operation of the Agilent HPLC system was controlled using the Agilent ChemStation software. The HPLC columns used were a semi-preparative column  and an analytical column . 18F-Fluoride was produced by the 18O18F reaction using an Advanced Cyclotron Systems Inc.  TR19 cyclotron. Radioactivity of 18F-labeled 2 was measured using a Capintec  CRC®-25 R/W dose calibrator, and the radioactivity of mouse tissues collected from biodistribution studies were counted using using a Perkin Elmer  Wizard2 2480 automatic gamma counter. PET imaging experiments were conducted using a Siemens  Inveon microPET/CT scanner.All chemicals and solvents were obtained from commercial sources, and used without further purification. Triethylammonium phosphate buffer  was prepared by titrating a solution of triethylamine (8 mL) in deionized water (1 L) with N,N-diisopropylethylamine  in dichloromethane (80 mL) was added N,N′-dicyclohexylcarbodiimide . After stirring at room temperature for 27 h, the reaction mixture was filtered, and the filtrate was extracted with 2 M NaOH aqueous solution (100 mL). The organic phase was dried with anhydrous magnesium sulfate, and evaporated under reduced pressure. The residue was dissolved in 1:1 diethyl ether/hexane (100 mL), and the insoluble crystals were removed by filtration. The filtrate was concentrated under reduced pressure to yield 4 as colorless oil . 1 H NMR  δ 8.17 , 7.50 , 7.04 , 3.53 , 2.27 . MS (ESI) calculated for C16H13F4NO2 327.1, found (M + H)+ 328.0.A mixture of 4-[(dimethylamino) methyl] benzoic acid hydrochloride , 2,3,5,6-tetrafluorophenol , and 4 in acetonitrile (6 mL). The reaction mixture was stirred at room temperature for 3 days. The precipitated product was filtered, and washed with diethyl ether (2 mL ×3) to yield 5 as white powder . 1 H NMR  δ 8.33 , 8.05 , 7.89 , 7.67 , 7.38 , 4.74 , 4.65 , 2.93 . MS (ESI) calculated for C23H19F5NO2+ 436.1, found (M)+ 435.8. Melting point: 198 °C.4-Fluorobenzyl bromide  was added to a solution of 5  and 3-(aminomethyl) benzenesulfonamide  in methanol (5 mL) was stirred at room temperature for 2 days. The solvent was removed by heating at 70 °C. Tetrahydrofuran (2 mL) was added to the residue, and the resulting mixture was heated at 70 °C for 2 min. The insoluble product was isolated by filtration to yield 1 as white solid . 1H NMR  δ 9.32 , 8.04 , 7.78 , 7.75–7.68 , 7.68–7.63 , 7.58–7.51 , 7.44–7.29 , 4.65 , 4.64 , 4.57 , 2.89 . MS (ESI) calculated for C24H27FN3O3S+ 456.2, found (M)+ 456.2. Melting point: 226 °C.A solution of 1 by starting with 5  and 4-(2-aminoethyl) benzenesulfonamide  in methanol (5 mL), 153 mg (93%) of 2 was obtained as white solid. 1 H NMR  δ 8.73 , 7.94 , 7.74 , 7.71–7.60 , 7.53–7.33 , 7.30 , 4.63 , 3.55 , 2.94 , 2.88 . MS (ESI) calculated for C25H29FN3O3S+ 470.2, found (M)+ 470.2. Melting point: 215 °C.Following similar procedures as described above for the preparation of 1 by starting with 5  and 4-(aminomethyl) benzenesulfonamide  in methanol (5 mL), 160 mg (99%) of 3 was obtained as white solid. 1 H NMR  δ 9.29 , 8.03 , 7.77 , 7.74–7.59 , 7.49 , 7.37 , 7.31 , 4.65 , 4.63 , 4.55 , 2.88 . MS (ESI) calculated for C24H27FN3O3S+ 456.2, found (M)+ 456.0. Melting point: 228 °C.Following similar procedures as described above for the preparation of 4  and 4-(bromomethyl) benzeneboronic acid pinacol ester  in acetonitrile (15 mL) was stirred at room temperature for 20 h. The resulting precipitate was filtered and washed with diethyl ether (10 mL ×3) to yield 6 as white solid . 1 H NMR  δ 8.33 , 8.15–7.98 , 7.90 , 7.81 , 7.62 , 4.77 , 4.68 , 2.95 , 1.31 . MS (ESI) calculated for C29H31BF4NO4+ 544.2, found (M)+ 544.3. Melting point: 179 °C.A mixture of 6  in a mixture of acetonitrile (6 mL) and methanol (6 mL). The resulting solution was stirred for 4 h. The formed silver bromide precipitate was filtered through celite, and washed with methanol (5 mL ×2). The filtrate was concentrated under reduced pressure to yield 7 as light yellow solid . 1 H NMR  δ 8.23 , 7.87 , 7.79 , 7.50 , 7.16 – 6.94 , 4.95 , 4.75 , 2.99 , 1.34 . MS (ESI) calculated for C29H31BF4NO4+ 544.2, found (M)+ 544.3. MS (ESI) calculated for CF3O3S− 149.0, found (M)− 149.0. Melting point: 108 °C.A solution of silver trifluoromethanesulfonate  in acetonitrile (3 mL) was added dropwise to a solution of 7  and 4-(2-aminoethyl) benzenesulfonamide  in methanol (6 mL) was stirred at room temperature for 20 h. The volatile solvent was removed under reduced pressure. Tetrahydrofuran (6 mL) was added to the residue, and the resulting mixture was sonicated for 5 min. The resulting precipitate was filtered, washed with tetrahydrofuran (1 mL ×2), and dried under reduced pressure to yield 8 as white solid . 1 H NMR  δ 8.77–8.66 , 7.93 , 7.80 , 7.74 , 7.71–7.63 , 7.63–7.51 , 7.43 , 7.30 , 4.75–4.46 , 3.69–3.49 , 3.06–2.90 , 2.89 , 1.31 . MS (ESI) calculated for C31H41BN3O5S + 578.3, found (M)+ 578.4. MS (ESI) calculated for CF3O3S− 149.0, found (M)− 149.0. Melting point: 165 °C.A mixture of 2 hydration activity with Phenol red as indicator, working at the absorbance maximum of 557 nm, following the initial rates of the CA-catalyzed CO2 hydration reaction for 10–100 s. For each inhibitor at least six traces of the initial 5–10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.01 mM) were prepared in distilled-deionized water with 5% DMSO and dilutions up to 0.1 nM were done thereafter with the assay buffer. The inhibition constant (Ki) was obtained by considering the classical Michaelis–Menten equation, which has been fitted by non-linear least squares by using PRISM 3. All CA isozymes used in the experiments were purified human recombinant proteins obtained as reported earlier by our groupA stopped-flow method18F-fluoride in H2[18O]O was passed through the QMA cartridge, and 18F-fluoride was trapped and then eluted out with 0.3 mL aqueous solution of 5 mg tetrabutylammonium triflate into a 4-mL V-shaped reaction vial. Acetonitrile (1 mL) was added and the reaction vial was placed in a heating block and heated at 110 °C under vacuum for 6 min and subsequently under N2 flow for another 6 min. A mixture of Cu(OTf)2 , pyridine , and precursor  solutions in N,N-dimethylformamide was added to the reaction vial. The reaction mixture was incubated at 110 °C for 20 min. The reaction was quenched with water (1 mL) and the resulted mixture was purified by HPLC using the semi-preparative column eluted with 24% CH3CN and 76% TEA-PB at a flow rate of 4.5 mL/min. The retention time of 18F-labeled 2 was 17.6 min. The collected 18F-labeled tracer was diluted with ammonium formate , and trapped on a C18 light Sep-Pak cartridge. The final product was eluted out with ethanol (0.4 mL), and formulated with saline (4 mL) for plasma stability, biodistribution, and PET/CT imaging studies. Quality control was performed by HPLC on the analytical column eluted with 25% CH3CN and 75% PBS at a flow rate of 2.0 mL/min. The retention time of 18F-labeled 2 was 6.9 min. The specific activity of 18F-labeled 2 was measured using the analytical HPLC system. It was calculated via dividing the injected radioactivity of 18F-labeled tracer solution by the amount of the tracer in the injected solution. The amount of the tracer was calculated from the UV absorbance standard curve of non-radioactive 2.The 7.4 value of 18F-labeled 2 was measured using the shake flask method as previously reported18F-labeled 2 was added to a vial containing 3 mL of n-octanol and 3 mL of phosphate buffer . The mixture was vortexed for 1 min and then centrifuged at 3000g for 10 min. Samples of the n-octanol (1 mL) and buffer (1 mL) layers were taken and counted in a well counter. LogD7.4 was calculated using the following equation: LogD7.4 = log10[(counts in n-octanol phase)/(counts in buffer phase)].The LogD,18F-labeled 2 were incubated with 400 μL of balb/c mouse plasma  for upwards of 60 min at 37 °C. At the end of each incubation period, samples were quenched by addition of acetonitrile (0.5 mL), centrifuged to remove proteins, and finally passed through a 0.2 micron filter. The filtered samples were loaded onto the analytical radio-HPLC to check for metabolite formation, and analyses were conducted using Agilent ChemStation software.Stability in plasma was performed following published procedures47In vivo experiments were conducted in accordance with the guidelines established by the Canadian Council on Animal Care and approved by the Animal Ethics Committee of the University of British Columbia. Male immunodeficient NOD.Cg-scidIl2rgtm1WjlPrkdc/SzJ (NSG) mice were obtained from a breeding colony at the Animal Resource Centre of the BC Cancer Research Centre.6 HT-29 cells (in 100 μL PBS and Matrigel at 1:1 ratio) under the right dorsal flank. Biodistribution studies and PET/CT imaging were performed when tumors reached 7–9 mm in diameter.Animal model was established following previously published proceduresFor dynamic imaging study, tumor-bearing mice were sedated with 2% isoflurane inhalation and positioned prone onto the scanner bed. A 10 min baseline CT scan was obtained for localization and attenuation correction before radiotracer injection, using 60 kV X-rays at 500 mA, three sequential bed position with 33% overlap, and 220 degree continuous rotation. PET data were acquired in list mode acquisition, reconstructed using the 3d-OSEM-MAP algorithm with CT-based attenuation correction. The dynamic acquisition of 60 min was started at the time of intravenous injection with 6–8 MBq of the radiotracer. The list mode data were rebinned into time intervals  to obtain tissue time-activity curves. The mice were kept warm by a heating pad during acquisition. For static imaging study, the mice were briefly sedated for intravenous injection of the radiotracer (6–8 MBq), and allowed to recover and roam freely in their cages for 45 min. At that point, the mice were sedated with 2% isoflurane inhalation, placed on the scanner, and an attenuation correction CT scan was obtained as described above. A single static emission scan was subsequently acquired for 10 min.Biodistribution studies were performed to confirm the quantitative ROI uptake values observed from PET scans. At 1 h p.i., mice were euthanized. Blood was promptly withdrawn, and the organs/tissues of interest were harvested, rinsed with normal saline, blotted dry, and weighed. The radioactivity of the collected mouse tissues was counted and expressed as the percentage of the injected dose per gram of tissue (%ID/g).1–3 were designed using 4-dimethylaminobenzoic acid as the linker to connect a 4-fluorobenzyl group and a benzenesulfonamide motif for CA-IX targeting. This design also generated the needed cationic quaternary ammonium group that would prevent free diffusion of 1–3 into cells and binding to intracellular CA isoforms including CA-I and CA-II. In addition, 18F-labeled 1–3 could be prepared via copper-mediated aromatic radiofluorination reaction using an arylboronic pinacol ester precursor49,18F]fluorobenzyltriphenylphosphonium (18F-FBnTP), a myocardial perfusion PET tracerAs shown in 1–3 followed the procedures depicted in 4 was obtained in 99% yield by reacting 4-[(dimethylamino) methyl] benzoic acid with 2,3,5,6-tetrafluorophenol using N,N′-dicyclohexylcarbodiimide (DCC) as the coupling reagent. Reacting 4 with 4-fluorobenzyl bromide in acetonitrile afforded the quaternary ammonium bromide salt 5 as a precipitate which was isolated in 87% yield by filtration. The desired compounds 1–3 were obtained by coupling 5 in methanol with excess 3-(aminomethyl)benzenesulfonamide, 4-(2-aminoethyl)benzenesulfonamide, and 4-(aminomethyl)benzenesulfonamide, respectively. After evaporating methanol, the residue was triturated with tetrahydrofuran to obtain compounds 1–3 bromide salt in 93–99% yields.The preparation of compounds 1–3 were evaluated for four physiologically relevant CAs: CA-I, -II, -IV, and -IX (1–3 exhibited different inhibitory profiles. The derivatives have inhibition constants (Ki) in the ranges of 0.52–0.89, 0.07–0.72, 3.63–> 50, and 0.22–0.96 μM for CA-I, -II, -IV, and -IX, respectively. Relative to acetazolamide (Ki(CA-IX) = 0.03 μM), a pan CA inhibitor, the binding affinities of compounds 1–3 for CA-IX were substantially lower. With the best affinity towards CA-IX, compound 2 was selected for radiolabeling and in vivo experiments.After synthesis, the binding affinity of compounds  and -IX . CA-I an18F-fluorination precursor 8 is shown in 4 with 4-bromomethylphenylboronic acid pinacol ester afforded the quaternary ammonium bromide salt 6 in 14% yield. The ammonium bromide salt 6 was treated with silver triflate and converted quantitatively to the ammonium triflate salt 7. The desired precursor 8 was obtained in 64% by coupling 7 with 4-(2-aminoethyl)benzenesulfonamide.The preparation of the 18F-labeled 2 was performed by nucleophilic substitution of the precursor 8 with 18F-fluoride via copper-mediated aromatic radiofluorination reaction (18F-labeled 2 was performed by HPLC and 18F-labeled 2 was isolated in 10 ± 4% (n = 3) decay-corrected radiochemical yield with >98% radiochemical purity and 85.1 ± 70.3 GBq/μmol specific activity. An in vitro stability study was conducted by incubating 18F-labeled 2 at 37 °C in mouse plasma, and monitored by HPLC. As shown in 18F-labeled 2 was observed after 60 min incubation, suggesting high stability of 18F-labeled 2 in mouse plasma. Lipophilicity of 18F-labeled 2 was measured using traditional shake flask method7.4 (D7.4: distribution coefficient between n-octanol and pH 7.4 phosphate buffer) value was —0.79 ± 0.02, indicating that the tracer was hydrophilic.Synthesis of reaction 49–51. PuIn vivo imaging experiments were conducted in immunodeficient NSG mice bearing HT-29 human colorectal cancer xenografts. Biodistribution data and representative PET/CT images acquired at 1 h post-injection are shown in in vivo defluorination as uptake in bone was observed in negligible amount at 0.13 ± 0.02%ID/g. PET images are consistent with biodistribution data, as the gastrointestinal tract and kidneys showed the highest accumulation of activity. HT-29 xenografts were visualized in PET images with moderate tumor-to-background contrast. Analyzing the time activity curve for 18F-labeled 2 valeric acid and a succinyl acetazolamide group with 111In (via DOTA chelator) and 64Cu (via NOTA chelator) and acheived tumor uptakes values of 14.5–26.0%ID/g at 1 h p.i.29,99mTc-labeled acetazolamide derivative with excellent tumor targeting (22%ID/g at 3 h p.i.) and image contrast. Notably, both research groups used the human renal-cell carcinoma SK-RC-52 model for in vivo evaluations. Renal cell carcinomas commonly overexpress CA-IX due to perturbations of the von Hippel-Lindau (VHL) gene, which in turn regulates HIF-1αThe development of CA-IX targeting agents, particularly those derived from small molecule inhibitors has seen marked improvement in terms of tumor targeting and visualization. Initial efforts were hampered by the lack of target specificity, poor pharmacokinetics, and/or tracer instability24Ki values in the low nanomolar range. On the contrary, compound 2 selected for radiolabeling and evaluated in this study had only moderate binding affinity to CA-IX (Ki = 0.22 μM). A high binding affinity to the target of interest is one of many factors  that determine efficient tumor targeting and accumulationBeyond the use of radiometals, another major commonality shared by these successful tracers is the high affinity that they exhibit for CA-IX, typically with 1–3 to potentially target CA-IX for PET applications. Imaging and biodistribution data for 18F-labeled 2 showed clear visualization of tumor xenografts despite moderate uptake and tumor-to-background contrast. This is encouraging considering the relatively modest binding affinity of 2 to CA-IX. Therefore, our data demonstrate the use of cationic motif may be useful for designing future CA-IX tracers assuming high affinity binders can be obtained.We designed three cationic sulfonamide inhibitors