Cystic echinococcosis is a global public health problem; however, the drugs  currently recommended by WHO for its treatment, have limited efficacy. Therefore, novel drugs are required to provide more choices for the treatment of this disease.Echinococcus granulosus protoscoleces, germinal cells and metacestodes in vitro. The in vivo efficacy of UA was investigated in mice following secondary infection with E. granulosus. Furthermore, the corresponding ultrastructural damage induced by UA was evaluated by electron microscopy.The anthelmintic effects of ursolic acid (UA) were tested on In vitro, 45.95 ± 5.30% of protoscoleces were killed by UA at 40 μg/ml, while the growth of more than 90% of germinal cells was inhibited by UA at 10 to 40 μg/ml. The same effect was observed in metacestodes 7 days after treatment with UA at 10, 20 and 40 μg/ml, and more than 50% of metacestodes showed loss of integrity at the end of the experiment. In vivo, metacestode weight was significantly reduced following oral administration of UA at 200 and 100 mg/kg . Additionally, ultrastructural damage, such as alternations in germinal cell morphology and formation of vacuoles and lipid granules were observed in parasites treated with UA in vitro, while detachment of the germinal layer from the laminated layer was also seen in metacestodes in vivo.E. granulosus in vitro and in vivo, thus implicating UA as a potential anti-echinococcosis agent.UA was demonstrated to exert parasiticidal activity against  Echinococcus granulosus (sensu lato) in the larval stage, represents an important public health problem due to its wide geographical distribution as well as its medical and economic impact [E. granulosus involves dogs and other canids as definitive hosts, and numerous mammalian species, including domestic livestock and humans as intermediate hosts. Once the eggs are ingested by humans, hydatid cysts may develop, mostly in the liver and lungs. Despite its importance with regard to health, there are only two drugs  available for the treatment of echinococcosis, both exhibiting some adverse effects and limited efficacy in the chronic stage of the disease [Cystic echinococcosis (CE), also known as hydatid disease, which is caused by taeniid cestodes c impact . China ic impact . The lif disease . TherefoEchinococcus spp. based on the similarities between cancer cells and parasites. Tamoxifen, which was widely used for treating primary breast cancer, inhibited the survival of E. granulosus protoscoleces and metacestodes at 10 to 50 μM, and a reduction in cyst weight was observed following the administration of 20 mg/kg tamoxifen [E. multilocularis metacestodes in vitro although the activity of this drug was lower than that of albendazole and induced adverse effects in vivo [E. multilocularis. These results suggest that novel candidate drugs for the treatment of CE might be found among the chemicals with antitumor effects.To date, several drugs that inhibit cancer cell proliferation have been evaluated for their effects on amoxifen . The pro in vivo , as well in vivo , exhibitE. granulosus. As an ursane-type pentacyclic triterpenic acid, UA is ubiquitous in the leaves and berries of natural medicinal plants [In the present study, we investigated the efficacy of ursolic acid (UA) against l plants , 9. UA il plants –12. UA hl plants . UA was l plants  and antil plants  agent. Fl plants  and helml plants –19.Echinococcus granulosus. Moreover, the in vivo efficacy and cytotoxicity of UA were also investigated in experimentally infected mice.In this study, we evaluated the in vitro anthelmintic effects of UA on protoscoleces, germinal cells and metacestodes of UA (purity > 98%) and mebendazole (purity > 98%) were purchased from Aladdin  and Sigma-Aldrich , respectively. These powders were prepared as a 10 mg/ml stock solution in DMSO for in vitro experiments. On the day of use, the stock solutions were diluted in DMSO to the desired concentration. For in vivo experiments, these two chemicals were suspended in 1% tragacanth solution and stored at 4 °C. Unless stated otherwise, all culture media and reagents were purchased from Gibco-BRL .E. granulosus G1 strain [Liver hydatid cysts were obtained from sheep suffering from echinococcosis immediately after slaughter at abattoirs in Qinghai, China. The protoscoleces from the cysts were rinsed 5–8 times with PBS containing penicillin G (500 U/ml) and streptomycin (500 U/ml) for the collection of live parasites. Female Kunming (KM) mice  were purchased from the SLAC Laboratory Animal Center . Each mouse was inoculated intraperitoneally with 2000 protoscoleces. The genotype of protoscoleces from sheep and germinal cells from secondary infected mice was identified as −5 M L-dithiothreitol and 100 μM L-cysteine), 2 mM glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin G, and 100 μg/ml streptomycin, at 37 °C under 5% CO2. The culture medium was changed every 4–5 days. Metacestodes were collected aseptically from the mice more than 10 months after secondary infection with E. granulosus protoscoleces. Only the cysts distributed in the abdominal cavity and not adhering to the tissues were selected for experiments. After being washed 4–5 times with PBS, metacestodes (diameter < 1 cm) were maintained using the same culture conditions as those used to maintain protoscoleces. The larger metacestodes were maintained in PBS for 24 h to exclude contamination with host cells. Then germinal cells were collected and cultured as previously reported [Protoscoleces were maintained as previously described  with minreported . The culE. granulosus germinal cells, E. granulosus protoscoleces (as positive control) and host tissues  using RNeasy Mini Kit  according to the manufacturer’s instructions. The first-strand cDNA was then synthesized from total RNA treated with DNase I  using the ReverTra Ace qPCR Kit . PCR was performed using primers for the specific amplification of E. granulosus EF-1α [Germinal cells (without host cell contamination) were characterized by qPCR as follows: RNA was extracted from us EF-1α  and mousus EF-1α .2O. Amplification was performed using the following conditions: 95 °C for 30 s and 40 cycles of denaturation at 95 °C for 5 s, annealing at 60 °C for 10 s and polymerization at 72 °C for 30 s. The PCR products were then heated to 95 °C for 10 s. A melting curve was created by cooling the products to 65 °C and then heating to 95 °C at a rate of 0.1 °C/s while measuring of the fluorescence simultaneously. PCR products were separated by 2% agarose gel electrophoresis and stained with Goodview  before visualization under UV light.The qPCR reaction was performed using the C1000 Touch Thermal Cycler  in a final volume of 20 μl: 10 μl SYBR Green PCR Master Mix (Thermo Fisher Scientific), 0.8 μl of each primer (10 pmol/μl), 2 μl cDNA product, 6.4 μl ddHn = 50/well) or germinal cells (approximately 5 × 104/well) were seeded in 96-well microtiter plates  in 200 μl medium. Metacestodes (n = 6/well) were plated in 6-well microtiter plates (Costar) in 6 ml medium. UA was dissolved in DMSO and added to the medium at a final concentration of 1 to 40 μg/ml; 0.5% DMSO served as the control.Protoscoleces  that had been infected with E. granulosus protoscoleces for 8 months were randomly divided into four groups . These animals received UA (suspended in 1% tragacanth solution) via the oral route at 200 and 100 mg/kg/day (UA-200 and UA-100), and mebendazole (MBZ) at 25 mg/kg/day (MBZ-25)  for 28 consecutive days. The remaining group was treated orally with 1% tragacanth solution (1% TRA) as a control. During the treatment, mice were monitored daily for changes in behavior and body weight changes as well as survival. Two weeks after treatment, all mice were sacrificed by cervical dislocation, and the cysts in the peritoneal cavity were isolated and weighed. The efficacy of the treatment was assessed based on mean cyst weight [KM mice , respectively, as previously described .4 cells/well). UA was added, and plates were incubated for 72 h at 37 °C under 5% CO2. Cell viability was determined with CCK-8 kits using the same method used to evaluate the viability of germinal cells. The effects of UA on non-carcinogenic cell lines were analyzed in the range 1.56–50.00 μg/ml, and on cancer cell lines in the range 0.39–12.5 μg/ml.Cytotoxicity was determined using three non-carcinogenic cell lines  and three cancer cell lines (A2058 cells cultured in RPMI1640 and A172 cells and HCT-8 cells cultured in DMEM) ; all media were supplemented with 10% FBS. The six cell types were seeded in 96-well microtiter plates  and Tox50 (concentration that induced 50% cell toxicity) values were calculated using the probability unit method with SPSS version 17.0; P < 0.05 was considered to indicate statistical significance.The differences in mean cyst weights among groups were assessed by Kruskal-Wallis H-test and paired comparisons. ICAfter UA treatment at 40 μg/ml for 3 days, 45.95 ± 5.30% of protoscoleces were killed, while there was no significant effect on their viability at 20 μg/ml Fig. . Dead prUltrastructural damage was revealed by TEM in the protoscoleces treated with UA at 40 μg/ml for 3 days compared with the control protoscoleces cultured in 0.5% DMSO medium (which resulted in a death-rate of 6.43 ± 1.00%). No changes in ultrastructure were observed throughout the experimental period in the control group Fig. . In contE. granulosus germinal cell culture (Ct = 24.03 ± 0.23), thus confirming the absence of host cells  and protoscoleces (Ct = 24.13 ± 0.62).In the qPCR analysis, there was no amplification of mouse GADPH in lls Fig. , Lane 6,E. granulosus protoscoleces, UA had a considerably stronger, and also dose-dependent, effect on germinal cells over 3 days. At 10, 20 and 40 μg/ml, UA resulted in a dramatic reduction of viable cells, with the inhibition rate higher than 90% . Furthermore, daily oral administration of MBZ at 25 mg/kg significantly decreased the cyst weight . Oral administration of UA at 200 and 100 mg/kg also reduced the growth of the metacestodes in vivo . Furthermore, the cyst weight was significantly reduced in the MBZ-25 group compared with that in the UA-200 (P = 0.044) and UA-100 (P = 0.045) groups, although there was no significant difference between the UA-200 and UA-100 groups (P = 0.940).Intraperitoneally infected KM strain mice were exposed to different treatment regimens involving MBZ and UA for 4 weeks as indicated in Table TEM and SEM studies of metacestodes isolate from mice following treatment with UA revealed extensive parasite tissue damage Fig. . Hydatid50 values for the three non-carcinogenic cell lines were 13.69 ± 1.39, 11.41 ± 2.24 and 13.97 ± 1.67 μg/ml, respectively. In contrast, the cancer cells were more sensitive to UA, with Tox50 values of 2.54 ± 0.20, 2.68 ± 0.04 and 2.80 ± 0.02 μg/ml, respectively.The cytotoxicity of UA was assessed in mammalian non-carcinogenic cell lines  and cancer cell lines  using the CCK-8 method. The ToxE. granulosus and the in vivo efficacy in experimentally infected mice. Although treatment with 40 μg/ml UA for 3 days only resulted in the death of 45.95 ± 5.30% protoscoleces, the survival of germinal cells was found to be inhibited in a dose-dependent manner, with > 90% inhibition at 10, 20 and 40 μg/ml. These results indicated that germinal cells were more sensitive to UA than protoscoleces. This information has important implication for the potential treatment of echinococcosis, because germinal cells are the only proliferative part of the parasite and are usually considered as the main target for chemotherapy [Since drug research and development is time-consuming and expensive, a drug repurposing strategy offers an alternative route to significantly shorten the traditional drug development process . In the otherapy . In addiotherapy . Our res50 was calculated to be 11–14 μg/ml, which was 2- to 3-fold in excess of the measured IC50 values observed for germinal cells. These results proved that UA was more effective against parasites than normal host cells, but also indicated a potential safety concern. Hence, during the 28 days of treatment, we paid close attention to the conditions of the animals. However, no mice died during the experiments, while two mice and one mouse died in the control and MBZ-25 groups, respectively, which preliminarily indicated that the use of UA for the treatment of murine-echinococcosis was safe in our conditions. In addition, the regimen used in this study has been proved to be safe in the treatment of parasites and cancer in animals and humans [To test the in vivo efficacy, UA was administered at 200 and 100 mg/kg for the treatment of echinococcosis according to the regimen adopted for the treatment of cancers with UA , 28. Aftd humans . Still, Echinococcus spp. For example, UA was reported to induce apoptosis through suppression of the components of the ERK/MAPK signaling pathway [Echinococcus spp. [E. multilocularis was reported to play a functional role in the E. multilocularis metacestode [E. granulosus hydatid cyst fluid, cyst membranes and protoscolices, and are involved in pathology-related tissue remodeling [The efficacy of UA against echinococcosis in the murine model might be attributed to its direct effect on the parasite, as well as its impact on host according to the mechanisms of UA against cancers. It was reported that UA can inhibit cell growth, migration and invasion in many cancer cell models and exert antiproliferative effects  via seve pathway , 30, whicus spp. , 32. Humacestode . In addiacestode , 34 relamodeling , 36.Poor solubility of UA in aqueous solutions greatly limits its application . In the E. granulosus protoscoleces, germinal cells and metacestodes in vitro and in vivo. Thus, UA is implicated as a potential anti-echinococcosis agent. However, its mechanism and the application prospects with proper formulation remain to be investigated.The findings of the present study demonstrate the efficacy and parasiticidal activity of UA against


There is a direct relationship between fructose intake and serum levels of uric acid (UA), which is the final product of purine metabolism. Recent preclinical and clinical evidence suggests that chronic hyperuricemia is an independent risk factor for hypertension, metabolic syndrome, and cardiovascular disease. It is probably also an independent risk factor for chronic kidney disease, Type 2 diabetes, and cognitive decline. These relationships have been observed for high serum UA levels (>5.5 mg/dL in women and >6 mg/dL in men), but also for normal to high serum UA levels (5–6 mg/dL). In this regard, blood UA levels are much higher in industrialized countries than in the rest of the world. Xanthine-oxidase inhibitors can reduce UA and seem to minimize its negative effects on vascular health. Other dietary and pathophysiological factors are also related to UA production. However, the role of fructose-derived UA in the pathogenesis of cardiometabolic disorders has not yet been fully clarified. Here, we critically review recent research on the biochemistry of UA production, the relationship between fructose intake and UA production, and how this relationship is linked to cardiometabolic disorders.  Uric acid (UA) is the final product of purine metabolism. It is a well-known risk factor for gout . MoreoveThe increased incidence of gout among rich people in the 18th and 19th centuries was principally due to high consumption of purine-containing meat. However, UA levels are rising in the 21st century too, with mean levels of >5.5 mg/dL in women and >6.0 mg/dL in men . This caRecent research suggests that hyperuricemia may be caused by elevated activity of the enzyme xanthine oxidase (XO) . XanthinHigh UA levels favor adipose tissue formation, which was originally an evolutionary advantage for humans . NowadayHere, we review the most relevant discoveries in the field, focusing on (i) the role of UA in cardiometabolic disorders; and (ii) the link between fructose consumption, high blood UA levels, and associated disorders, particularly CVD. We conducted a literature search of different scientific databases  for peer-reviewed studies focusing on XO, hyperuricemia, fructose, and CVD. The search strategy was designed to retrieve studies published in English from journal inception to 2016. We used an assessment framework to appraise the quality of basic research studies, prognostic studies, and methodological considerations in the analysis and publication of observational studies. The screening, study selection, and data extraction was undertaken by three independent authors. Disagreement was resolved by discussion and, if required, by a fourth independent author. We have assessed the clinical and methodological heterogeneity across the studies and, where available, included meta-analyses whenever these have been performed. +, whereas XO-FAD has higher reactivity for molecular oxygen, producing higher levels of hydrogen peroxide (H2O2) and the superoxide anion (O2−). O2− is then converted into oxygen and H2O2, either spontaneously or catalyzed by the superoxide dismutase (SOD) enzyme, . Second, there is the purine salvage pathway, which economizes the intracellular energy expenditure and is regulated by hypoxanthine-guanine phosphoribosyltransferase (HG-PRTase) . Catabol enzyme, A,B 14]..+, where enzyme, A 17]. A. A+, wheoduction ,18 , and as oligo- and polysaccharides (fructans) in many plants. It is also used as an added sweetener for food and drink, and as an excipient in pharmaceutical preparations, syrups, and solutions [Fructose is present as a monosaccharide in fruits and vegetables , as a diolutions . Followiolutions . It is nolutions . Severalolutions ,53,54. Hd-glyceraldehyde. When there is a high intake of fructose, phosphorylation into fructose 1-phosphate is fast, but the reaction with aldolase is slow , and particularly peroxynitrite (OONO−) [UA has been described as a “paradox molecule” with opposing roles. At physiological concentrations (much higher than ascorbate concentrations in plasma), it is a powerful oxygen radical scavenger in extracellular hydrophilic environments such as human plasma. It may protect the erythrocyte membrane from lipid peroxidation . UA can  (OONO−) ,62,63. H (OONO−) ,64. Rece (OONO−) ,66. It d (OONO−) ,68 and b (OONO−) . Fructos (OONO−) ,70. 2− and H2O2. XO levels are substantially elevated in patients with coronary disease or carotid stenosis, and there is an inverse relationship between XO levels and endothelium-dependent vasodilation [Oxidative stress significantly contributes to the development of insulin resistance and imbalance in vascular homeostasis, including endothelial cell dysfunction, atherosclerosis, vascular calcification, and impaired myocardial energetics, stimulating the production of interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) ,72. Hemedilation . Accordidilation ,75. Extrdilation ,77 activdilation . As a redilation . In thisdilation .− is a potent non-radical oxidant species formed by the reaction between NO and O2−, which commonly occurs in the vascular endothelium [−, producing a nitrosated derivative [OONOothelium . NO is uothelium ,83. Sincothelium ,85. Severivative ,86,87. TPhysiologically, circulating UA levels increase with age. UA levels are lower in women of childbearing age, rising after menopause to levels similar to those in men . There iSeveral in vitro and in vivo studies have shown that high fructose consumption increases blood UA levels ,55,56,91A relatively old meta-analysis of 21 controlled feeding trials of at least seven days showed that isocaloric exchange of fructose for other carbohydrates did not affect serum UA. However, in nondiabetic participants, hypercaloric supplementation of control diets with fructose (+35% excess energy) at extreme doses (213–219 g/day) significantly increased serum UA compared with the control diets alone ) . This meNotably, the fructose-induced increase in serum UA may cause acute damage (post-assumption). By excluding shorter studies, the meta-analysis may have excluded insights into this issue . FinallyLecoultre and colleagues performed a study on healthy subjects with two approaches: four to six days on an isoenergetic low-fructose diet (LFrD) and then either 6 days on a hyperenergetic HFrD with 34% excess energy as fructose or four days on a weight-maintenance HFrD in which 30% starch was substituted with fructose . There wMany experiments have shown that hyperuricemia may have a potential role in endothelial dysfunction and reduced NO bioavailability. However, several other studies have failed to demonstrate that an increase in blood UA levels can contribute to the onset of coronary and cardiovascular diseases. These studies also found no association between raised serum UA levels and the incidence of cardiovascular events ,102,103.The well-characterized Mediterranean cohort of the Brisighella Heart Study has a high intake of fruit . Here, tIn general, epidemiologists and other scientists have attempted to prove a causal link between dietary fructose intake and metabolic disorders, such as obesity, diabetes, and metabolic syndrome. However, while some studies have found a link, other studies have not .Recent relevant clinical research has sought to verify if XO inhibition could benefit patients with high circulating UA levels, focusing on the XO inhibitor allopurinol and its active metabolite oxypurinol. Allopurinol has displayed beneficial effects on blood pressure in adolescents with newly diagnosed essential hypertension , and in XO inhibition has also been shown to improve a range of surrogate markers in hyperuricemic patients with CVD . Wu and In summary, a specific causal link between fructose consumption, hyperuricemia, and CVD has not yet been established. There is an association between UA and established cardiovascular risk factors, and there is a limit to how much population-based studies can adjust for confounding variables. As such, it is not yet possible to conclude that fructose intake is the main contributor to an increase in blood UA, and that this detrimentally affects vascular health. Further studies are required to prove or exclude a causal correlation between dietary fructose intake, UA production, and metabolic disorders. This would allow researchers to better understand which patients would obtain the greatest preventative benefit from reducing their UA levels, with diet and/or with XO inhibitors. An important issue to consider is the bioavailability of XO inhibitors, especially the ability to cross cell plasma membranes. To address this, members of our group recently developed a cell-based biosensor that only measures intracellular XO activity and its inhibition by drugs that cross cell membranes . The sci


Recent reports have shown a rapid rise in the incidence of renal cell carcinoma (RCC), and Wnt (Wingless-related integration site) signaling pathway is important in RCC. Frizzled 8 (FZD8) is a member of Frizzled (FZD) receptor family which could activate canonical or non-canonical Wnt/β-catenin pathways. Nevertheless, the role of FZD8 in RCC is poorly investigated. The immunohistochemical analysis showed high expression of FZD8 in RCC tissues compared with peri-tumor tissues. FZD8 knockdown decreased the ability of proliferation and metastasis of RCC cells. Research revealed that the FZD8 regulated the transcription of Cyclin D1, c-Myc, and could promote the epithelial to mesenchymal transition (EMT) by mediating Vimentin and Snail through the Wnt/β-catenin signaling pathway. In addition, the results of our experiment revealed that FZD8 is involved in the regulation of non-canonical Wnt signaling pathway. These data suggested that the expression of FZD8 may play an important role in the proliferation and metastasis of RCC, and serve as a putative promising drug target for human RCC therapy. RCC is the common type of malignant tumor in adult urologic neoplasms . The incWnt/β-catenin signaling is one of the major signaling pathways in cell fate regulation and pathological process. Overexpression of Wnts and FZDs or the expression of a constitutively active FZD regulate various processes which are important for cancer progression, including tumor initiation, growth, and metastasis . The funRecently, the results of multiple studies have shown that the expression levels of several members of the FZD family, acting in both canonical and non-canonical Wnt signaling pathways, were up-regulated in various cancers. The FZD family includes ten genes in humans and their down regulation inhibits the Wnt signaling pathway leading to the suppression of cell proliferation, migration, invasion, motility and metastasis of tumor cells . For exaP<0.01), compared with those in the matching adjacent non-tumor tissues  were used to silence FZD8 mRNA expression. In order to confirm the target effects produced by shRNA, we used qRT-PCR that both stably transfected FZD8 shRNA and knockdown the FZD8 expression in 786O and A498 cell lines expressing the gene, whereas the scramble control shRNA had no effect Figure  and 2B. Then, we examined the effects of FZD8 expression in knocking-down, the canonical Wnt signaling pathway in RCC cells. FZD8 shRNA-1 and FZD8 shRNA-2 substantially decreased FZD8 protein levels compared to control group Figure . The cytIn the study of FZD8 co-receptors, we were focused on the lipoprotein (LDL) receptor-related protein-5 (LRP5), LRP6, RYK, and receptor tyrosine kinase-like orphan receptor (ROR) 2. In the previous studies reports, the low-density LRP5 regulates the osteoblastic metastases and the tumor caused new bone formation in prostate cancer . LRP6 fa2+ pathway . *P<0.05 was considered statistically significant, and extremely significant difference **P<0.01 and ***P<0.001.Results were presented as the mean±SD for at least three experiments for each group. Statistical software SPSS 19.0 was used for all analysis. All qRT-PCR reactions were run in triplicate, and the mean threshold cycles were figured up. The average expression levels of FZD8 were normalized using β-actin as a reference gene, and 2


Radiolabeling of maxadilan-DTPA (680 MBq/nmol) was more efficient than of DTPA-maxadilan (55 MBq/nmol). Conjugation with DTPA slightly reduced receptor binding affinity in vitro: IC50 values were 3.2, 21.0 and 21.0 nM for maxadilan, natIn-DTPA-maxadilan and maxadilan-DTPA-natIn respectively. Upon i.v. injection maxadilan-DTPA-111In accumulated specifically in INS-1 tumors (7.30 ± 1.87%ID/g) and in the pancreas (3.82 ± 0.22%ID/g). INS-1 tumors were clearly visualized by small animal SPECT/CT. In conclusion, this study showed that the high affinity of maxadilan to the PAC1 receptor was maintained after DTPA conjugation. Furthermore, radiolabeled maxadilan-DTPA accumulated specifically in INS-1 tumors and, therefore, may qualify as a useful tracer to image insulinomas.There is an ongoing search for new tracers to optimize imaging of beta cell-derived tumors (insulinomas). The PAC1 receptor, expressed by insulinomas, can be used for targeting of these tumors. Here, we investigated whether radiolabeled maxadilan could be used for insulinoma imaging. Maxadilan was C- or N-terminally conjugated with DTPA (termed maxadilan-DPTA or DTPA-maxadilan respectively). BALB/c nude mice bearing subcutaneous INS-1 tumors were injected with either In-111-labeled maxadilan-DTPA or In-111-DTPA-maxadilan. Biodistribution studies were carried out at 1, 2 and 4 hours after injection and SPECT/CT imaging 1 and 4 hours after injection of maxadilan-DTPA- Biochemical diagnosis is based on low blood glucose levels or elevated proinsulin, insulin and C-peptide levels in plasma and these tests can be complemented with fasting tests (which result in hypoglycemia if insulinoma is present)3. The most widely used nuclear medicine imaging strategy for detecting insulinomas is somatostatin receptor scintigraphy (SRS) using 111In-labeled octreotide. SRS is based on targeting somatostatin receptors (SSTR), which are over-expressed in sixty percent of insulinomas4. Because the remaining forty percent has either low or no expression of this receptor, many insulinomas are not being detected by scintigraphy based on somatostatin receptor targeting4. More preferably, PET (Positron Emission Tomography) imaging with octreotide analogues can be used, since PET is more sensitive than SPECT imaging, due to the higher spatial resolution of clinical PET scanners and higher sensitivity in detection of emitted gamma photons. A few promising studies were conducted using SSTR PET/CT, although these studies showed that a small percentage of insulinomas remain undetected8.The most common pancreatic neuroendocrine tumors (NETs) are insulinomas. Insulinomas are tumors derived from the insulin-producing beta cells located in the islets of Langerhans in the pancreas. Clinical symptoms include symptomatic hypoglycemias as well as weight gain (due to increased food intake to compensate for hypoglycemia) and neurological symptoms2 (cholecystokinin), VPAC1  and GLP-1 (glucagon-like peptide 1) receptors4. Several preclinical SPECT and PET studies in RipTag2 mouse models using the GLP-1 analogues exendin-3 and exendin-4, showed specific tracer accumulation in insulinomas via the GLP-1 receptor11. In the first pioneering clinical PET/CT and SPECT/CT studies with exendin-4 in insulinoma patients, small insulinomas, which could not be detected by conventional imaging methods , CT (Computed Tomography), SRS)17, were accurately diagnosed. The success of visualizing neuroendocrine tumors is partially dependent on the type of receptors expressed in the tumors, as was shown previously by Baumann et al.5. For example, malignant insulinomas have a low incidence and expression density of the GLP-1 receptor, but show high expression of the somatostatin receptor, where in benign insulinomas the expression profile is more favorable for the GLP-1R19. Therefore, to improve the diagnostic tool box, the search for new tracers for detection of insulinomas is ongoing.More than ten years ago, it was demonstrated that almost all insulinomas express several neuropeptide receptors, including CCK22.Vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase activating polypeptide (PACAP), and their receptors PAC1, VPAC1 and PAC2 have emerged as important factors in islet cell function (insulin secretion) and growth and differentiation of neuroendocrine tumors including insulinomas. As these receptors are highly expressed in insulinomas and beta cells, they are potential targets for beta cell and insulinoma imagingin vitro receptor autoradiography with VPAC1 and VPAC2 subtype-selective ligands, respectively, insulinomas have been demonstrated to bind high levels of radiolabeled selective VPAC1 receptor ligands, although substantially less than GLP1 selective ligands4. As PAC1, VPAC1 and VPAC2 mRNAs were detected in a rat insulinoma cell line with PAC1 binding prevailing over that of VPAC1 and VPAC223, the PAC1 receptor seemed to be a promising alternative for targeting insulinomas. The natural mammalian ligands at the PAC1 receptor are PACAP38 and the truncated form PACAP27. They bind to PAC1, VPAC1 and VPAC2, whereas VIP only binds to VPAC1 and VPAC2. Since PACAP38 and PACAP27 are non-selective PAC1/VPAC1/VPAC2 ligands and unstable in plasma24, they are not suited for in vivo targeting of PAC1 receptors. More than two decades ago it was demonstrated that the stable analogue maxadilan shared features with ligands of the PACAP family26. Maxadilan is a 61-amino acid vasoactive peptide, derived from sand flies28, and has been demonstrated to bind specifically and with high affinity to the PAC1 receptor but not to either VPAC1 or VPAC228.Using 111In-labeled maxadilan to image rat insulinoma xenografts (INS-1) in a nude mouse model. For this purpose maxadilan was either C- or N-terminally conjugated with DTPA to allow labeling with 111In. These compounds were evaluated for their radiolabeling properties and in vitro and in vivo binding characteristics.In the present study we have examined the ability of 111In-DTPA-maxadilan and maxadilan-DTPA-111In could be labeled with a specific activity of 55 and 680 MBq/nmol, respectively. The radiochemical purity after purification exceeded 95%.111In was analyzed in human serum. The radiochemical purity directly after labeling was >95%  for the receptor. DTPA conjugation resulted in a somewhat lower affinity as indicated by slightly higher IC50 values: 18.3 and 13.2 nM (p < 0.001 and p < 0.001) for DTPA-maxadilan and maxadilan-DTPA, respectively. Labeling of DTPA-maxadilan or maxadilan-DTPA with 115In did not significantly reduce the affinity (p = 0.07 and p = 0.12 respectively) compared to the unlabeled compounds.The results of the ICin vivo PAC1 receptor targeting study with 111In-labeled maxadilan in BALB/c nude mice bearing subcutaneous INS-1 tumors are summarized in Fig. 111In in the tumor was 7.76 ± 1.37%ID/g, which was significantly higher than that of 111In-DTPA-maxadilan (4.82 ± 0.87%ID/g) (p = 0.0037). Also, pancreatic uptake was significantly higher for maxadilan-DTPA-111In compared to 111In-DTPA-maxadilan, . Accumulation in both tumor and pancreas could be blocked by an excess of unlabeled maxadilan, demonstrating specific uptake of the peptides via the PAC1 receptor (1.54 ± 0.49%ID/g and 1.24 ± 0.48%ID/g for maxadilan-DTPA-111In and 111In-DTPA-maxadilan respectively in tumor tissue and 0.59 ± 0.07%ID/g and 0.43 ± 0.04%ID/g respectively for pancreatic uptake). Renal uptake was very high and similar for both peptides , which could not be blocked with an excess of unlabeled maxadilan. Furthermore, in all other dissected organs, except for blood and spleen, receptor-mediated uptake of maxadilan was observed. Since these results showed that maxadilan-DTPA-111In has higher accumulation in tumor and pancreatic tissue than 111In-DTPA-maxadilan, this peptide was used to study the pharmacokinetics. Figure 111In-labeled maxadilan-DTPA.The results of the SPECT/CT images of BALB/c nude mice bearing subcutaneous INS-1 tumors are shown in Fig. 111In-labeled maxadilan-DTPA and INS-1 tumors in the right shoulder were clearly visualized. In addition to tumor and kidney, accumulation of the tracer in the liver was observed. Figure Images were acquired one and four hours after injection of We demonstrate here for the first time that indeed radiolabeled maxadilan is a promising new tool to image subcutaneously transplanted insulinomas in mice.111In with high specific activity while preserving the affinity for the PAC1 receptor. High and specific uptake of this tracer in both subcutaneous INS-1 tumors and pancreas was found. SPECT imaging clearly visualized the s.c. INS-1 xenografts. Both, pancreatic as well as tumor uptake were receptor mediated, as was demonstrated by blocking experiments with unlabeled maxadilan.We were able to radiolabel maxadilan with 111In was at least twelve times higher than that of 111In-DTPA-maxadilan. This could possibly be explained by hindrance of the radiometal incorporation into the DTPA molecule, due to either the secondary or tertiary structure of maxadilan or a conformational change in the peptide as a result of the conjugation.Since the conjugation of a DTPA moiety can affect the affinity of the peptide for the receptor, the peptide was conjugated with DPTA at either the C- or the N-terminus and the molecules were compared. The maximum specific activity of maxadilan-DTPA-29. Therefore, it was thought likely that conjugation of DTPA to the N-terminus could influence the binding affinity. However, it has also been shown in previous studies that when the disulfide bridge at the N-terminal part of maxadilan is removed or changed, the receptor binding capacity of maxadilan is preserved30. Nevertheless, as was determined in our in vivo biodistribution studies, the C-terminally modified maxadilan-DTPA-111In analogue showed higher uptake in the tumor, therefore this compound was used for further characterization.Conjugation of DTPA to either the C-or N-terminus of maxadilan influenced the receptor binding affinity significantly. Maxadilan contains four cysteine residues, which form two disulfide bridges, one of which is located close to the N-terminus. Furthermore, it is known that the N-terminal residues of maxadilan are important for receptor binding32. As demonstrated in the SPECT images, maxadilan-DTPA-111In clearly visualized the subcutaneous INS-1 tumors and they were easily distinguished from the specific accumulation of radiolabeled maxadilan in various other tissues.The biodistribution shows specific accumulation of maxadilan in most organs. This is in line with the widespread PAC1 receptor expression and function in the pituitary, adrenal medulla, pancreas, stomach, colon, lung, and heart111In, 68Ga or 18F, show a more favorable biodistribution compared to this maxadilan tracer. Although those previously-studied tracers show high tumor and kidney uptake similar to maxadilan, the tumor-to-normal-organ ratios are more optimal, which can be correlated with expression levels of the different receptors that are targeted34. Future studies need to be conducted to compare radiolabeled maxadilan with existing relevant PET agents in animals, before moving to clinical trials.Other preclinical studies using INS-1 tumour-bearing mice have shown that GLP-1 receptor targeting ligands, labeled either with 68Ga-labeled somatostatin receptor imaging and radiolabeled GLP-1 receptor imaging were introduced for diagnosis of insulinomas. These tracers would be useful for differentiating benign from malignant tumors or in diagnosis of metastatic NETS. However, the sensitivity is not optimal and false positive or false negative cases continue to be described using somatostatin receptor targeting35. False negative-SSTR PET/CT could be explained by small lesions (missed because the resolution of the scanner is not sufficient to detect them) or low somatostatin receptor expression . A solution to the latter problem would be the use of a combination of different radiotracers, thus targeting alternative receptors. This was also suggested by Reubi et al., who conducted an elegant in vitro study proving that a cocktail of three different ligands was able to detect all tested NETs36. Tumors with no or low receptor density, which would be missed in vivo when using only one radiotracer, have a higher probability of being detected when multiple receptors are targeted. In addition, if one of the tracers had a very high uptake in the tumor, this high uptake could help to overcome the limited spatial resolution and also visualization of small lesions.Recently, clinical imaging techniques such as 37. Due to the high accumulation of radiolabeled maxadilan in the kidneys, and to a lesser extent in other organs, the feasibility of peptide receptor radionuclide therapy with maxadilan would be questionable. Insulinomas may not be the only NETs in which radiolabeled maxadilan could play a diagnostic role. In a study carried out by Pisegna et al. high expression levels of the PAC1 receptor in rat gastric ECL (enterochromaffin-like) cells were observed39. Currently, 111In-DTPA-octreotide or 68Ga-DOTATOC/68Ga-DOTA-octreotide are used for the detection of gastric neuroendocrine tumors, which originate from ECL cells40. Furthermore, PAC1 receptor expression has been demonstrated in Lewis lung tumour transplants41. Radiolabeled maxadilan could therefore be a possible candidate for detecting both ECL derived tumors and a subtype of small cell lung cancer, in addition to somatostatin receptor tomography. Moreover, there is evidence that there is high expression of PAC1 in human tumors such as paragangliomas, neuroblastomas, pituitary adenomas and endometrial cancers45 with expression found also in human lung cancers46. Importantly, most of these studies were conducted in vitro, so in vivo studies are needed to explore the ratio between peptide uptake in PAC1 receptor positive tumors and that in normal tissues where PAC1 is widely expressed.In addition to diagnostic imaging, some somatostatin analogues, when radiolabeled with a beta emitter, are used as therapeutic agentsin vivo PAC1 targeting in patients with insulinoma. Radiolabeled maxadilan, therefore, represents a new tracer to image insulinomas using SPECT in addition to or in combination with octreotide and exendin for the identification of benign or malignant insulinoma. Furthermore, the expression of PAC1 in other human tumors indicates a broader application for the usage of radiolabeled maxadilan. Thus, the newly generated PAC1 selective high affinity radiopeptide maxadilan can be employed for multireceptor tumour targeting in vivo.In conclusion, radiolabeled maxadilan accumulates efficiently and specifically in INS-1 tumors and could potentially be used for p-NH2-Bn-DTPA via an extra C-terminal glutamic acid. The quality and purity of the peptides was determined using RP-HPLC (reversed-phase high performance liquid chromatography) and mass spectrometry (MS) by the manufacturer. RP-HPLC showed a purity of 95.35%, 93.9% and 100% and a retention time of 15.18 min, 15.81 min and 14.46 for maxadilan, DTPA-maxadilan and maxadilan-DTPA respectively. With MS the expected mass of the peptides was confirmed . The amino acid sequences of the peptides are shown in Table DTPA-conjugated and native maxadilan were purchased from Think Peptides . N-terminal conjugation of DTPA (DTPA-Maxadilan) was performed with isothiocyanate-DTPA  via a beta-alanine spacer and C-terminal conjugation (Maxadilan-DTPA) with 111In by adding 111InCl3 to 1 µg peptide in 0.1 M MES buffer, pH 5.5. After incubation at RT for 20 min, 50 mM EDTA (ethylenediaminetetraacetic acid)  was added to a final concentration of 5 mM. Quality control was performed by Instant Thin Layer Chromatography (ITLC) on silica gel ITLC strips . As a mobile phase 0.1 M EDTA in 0.1 M NH4Ac, pH 5.5 was used . The reaction mixture was purified on a disposable PD-10 desalting column , which was eluted with 10 ml PBS, 0.5% (v/w) bovine serum albumin (BSA) and fractions containing radiolabeled maxadilan were pooled.DTPA-maxadilan and maxadilan-DTPA were labeled with 111InCl3 as previously described and incubated with human serum (1:10) at 37 °C. Before addition of human serum and 1, 2, 4 and 24 hrs after incubation with human serum, samples were taken, mixed with acetonitrile (1:1) and centrifuged for 5 min at 5000 g to precipitate serum proteins. The supernatant was analyzed using RP-HPLC on a C18 reversed-phase column  and ITLC. The column was eluted with a linear gradient of 0.1% TFA  in acetonitrile (3% to 100% over 10 min) with a flow rate of 1 ml/min. ITLC was performed as described above.Maxadilan-DTPA was labeled with 5 MBq 47 was maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 mM HEPES, 50 µM β-mercaptoethanol, 1 mM sodium pyruvate, 100 units/ml penicillin and 100 µg/ml streptomycin, in a humidified 5% CO2 atmosphere at 37 °C. The cells were harvested by trypsinization with trypsin/EDTA.The rat insulinoma cell line INS-150) of maxadilan, DTPA-maxadilan, maxadilan-DTPA, 115In-DTPA-maxadilan and maxadilan-DTPA-115In was determined using suspensions of INS-1 cells. Labeling of DTPA-maxadilan and maxadilan-DTPA with natIn was performed as previously described48. Unlabeled and 115In-labeled peptides were added to the cells  in eppendorf tubes to final concentrations ranging from 0.1 to 300 nmol (n = 3) together with 1,000 Bq maxadilan-DTPA-111In. After 4 h incubation on ice, the cells were centrifuged at 3,000 × g, the supernatant was removed and the cells were washed with 1 ml ice-cold PBS and the radioactivity in the cell pellet was determined in a well-type gamma counter . The IC50 value was calculated by one-site competition analysis with Graphpad Prism .The 50% inhibitory concentration . When the tumors had grown to approximately 5 mm in diameter, groups of five mice were injected with either 370 kBq 111In-DTPA-maxadilan or maxadilan-DTPA-111In (peptide dose: 13 pmol). For both peptides, an additional group of 5 mice was co-injected with an excess  of unlabelled maxadilan to determine the nonspecific binding of the peptides to the cells. Mice were euthanized 2 h p.i. and blood, muscle, tumor, heart, lung, spleen, pancreas, stomach, intestine, adrenals, kidney and liver were dissected, weighed and the radioactivity concentration was determined.In order to assess the feasibility of targeting insulinomas with radiolabeled maxadilan, female BALB/c nude mice (6–8 weeks old) were injected subcutaneously with INS-1 cells  were injected with 370 kBq maxadilan-DTPA-111In and mice were euthanized at 1, 2 or 4 h p.i. The radioactivity concentration in the organs was measured in a gamma counter.To examine the pharmacokinetics of maxadilan-DTPA-111In (13 pmol). A separate group of tumor-bearing mice was co-injected with an excess of unlabelled maxadilan . One and four h p.i. SPECT/CT images were acquired using a dedicated small animal SPECT scanner . SPECT images were acquired with a 0.6 mm pinhole mouse collimator with an acquisition time of 50 min. The images were reconstructed with OSEM  using the U-SPECT-Rec software . The settings for the CT were as follows: spatial resolution, 160 μm; 40 kV; 612 μA.BALB/c nude mice bearing subcutaneous INS-1 tumors were injected intravenously with 9 MBq maxadilan-DTPA-t test was used for determination of significance. A p-value below 0.05 was considered as significant. For the competitive binding assay the F-test was used to manually calculate significance.Statistical analysis was done using GraphPad Prism version 5.03 for Windows. The unpaired


This study evaluated current fertility care for CKD patients by assessing the perspectives of nephrologists and nurses in the dialysis department.N = 312) and dialysis nurses (N = 1211).Two different surveys were distributed for this cross-sectional study among Dutch nephrologists (p < 0.01).Response rates were 50.9% (nephrologists) and 45.4% (nurses). Guidelines on fertility care were present in the departments of 9.0% of the nephrologists and 15.6% of the nurses. 61.7% of the nephrologists and 23.6% of the nurses informed ≥50% of their patients on potential changes in fertility due to a decline in renal function. Fertility subjects discussed by nephrologists included “wish to have children” (91.2%), “risk of pregnancy for patients’ health” (85.8%), and “inheritance of the disease” (81.4%). Barriers withholding nurses from discussing FD were based on “the age of the patient” (62.6%), “insufficient training” (55.2%), and “language and ethnicity” (51.6%). 29.2% of the nurses felt competent in discussing fertility, 8.3% had sufficient knowledge about fertility, and 75.7% needed to expand their knowledge. More knowledge and competence were associated with providing fertility health care (In most nephrology departments, the guidelines to appoint which care provider should provide fertility care to CKD patients are absent. Fertility counseling is routinely provided by most nephrologists, nurses often skip this part of care mainly due to insufficiencies in self-imposed competence and knowledge and barriers based on cultural diversity. The outcomes identified a need for fertility guidelines in the nephrology department and training and education for nurses on providing fertility care. Chronic kidney disease (CKD) is associated with a decrease in reproductive function –4. AlthoWhere little is documented on fertility management and counseling in male CKD patients, several studies exist on the management of fertility in female CKD patients , 8. CounAdequate fertility care, including counseling, management, and referral to a physician specialized in fertility, determines whether the pregnancy and maternal and fetal outcomes are successful . A multiThe aim of this multidisciplinary study was to examine current fertility care for CKD patients from the perspectives of the nephrologists and nurses working in the dialysis unit. Guidelines appointing who should provide fertility care within nephrology departments were focused on, as were practice patterns of nephrologists and nurses regarding informing, discussing, counseling on fertility, and referral to a physician specialized in fertility. In addition, the evaluation among nurses addressed possible barriers toward discussing fertility care and tools needed to provide it. Nurses’ competence, knowledge, and education regarding fertility in CKD patients were evaluated, as well as how these factors influence fertility care currently provided by them.n = 318) who were member of the Dutch Federation of Nephrology (Nefrovisie) were requested to participate in the survey. A total of 312 questionnaires were sent to nephrologists’ home addresses; six obtained addresses were out of date. Non-respondents received a reminder letter two and/or four months after the initial mailing.Data for this cross-sectional study were collected among nephrologists and nurses using two separate surveys. All practicing Dutch nephrologists (n = 63). Thirty-four centers (54.0%) agreed on participation, and all their employed dialysis nurses and nurses specialized in nephrology received a questionnaire (n = 1171) at their work address. An e-mail was sent to non-responding centers received 2 and/or 4 months after the initial invitation as a reminder. Participating centers received a motivational e-mail after 4 months asking them to motivate their staff on returning the questionnaire. In addition, 40 questionnaires were handed out during an informative meeting for nurses specialized in nephrology resulting in a total of 1211 distributed questionnaires.For the survey among nurses, all Dutch dialysis centers  were approached for participation (n = 7). No modifications were made as no remarks were made. The first sheet of the questionnaire contained demographic questions and an opt-out possibility. The (remaining) ten items assessed:Guidelines on fertility care within the nephrology department.Nephrologists’ practice patterns regarding informing, discussing, and counseling on fertility, including referral to physicians specialized in fertility.Fertility subjects addressed by the nephrologist.The authors developed both questionnaires as validated instruments for assessing the aims of the studies were not available. The questionnaires were derived from instruments used in previous studies evaluating healthcare in medical departments –13. ContGuidelines on fertility care within the dialysis or nephrology department.Nurses’ practice patterns regarding informing, discussing, and counseling on fertility with dialysis patients.Competence of the nurse in providing fertility care to dialysis patients.Possible barriers to discussing fertility with dialysis patients.Current knowledge and education on fertility in dialysis patients, including influence on fertility care currently provided by the nurse.Tools that could assist in providing fertility care to dialysis patients.The nurses’ questionnaire was pilot-tested at the Leiden University Medical Centre as well. Twenty-three nurses working in the dialysis department inspected layout, linguistics, and comprehensiveness of items. No comments were made, so the final questionnaire was identical. Demographic questions and an opt-out possibility were placed on the first sheet; the other ten questions assessed:p values of <0.05 were considered statistically significant.Data were analyzed using IBM SPSS statistics 23 . Descriptive statistics were used to describe demographic information and the answers given by the respondents. Bivariate associations were calculated using the Cochran–Armitage trend test. Two-sided Since no patients or interventions were involved during both surveys, no formal ethical approval was needed in the Netherlands.n = 28), “not practicing” (n = 5), and “not interested” (n = 2). Fifteen respondents were excluded since they were specialized in pediatric nephrology. A total of 113 (36.2%) questionnaires were analyzed.In the nephrologist survey, a total of 159 of the 312 questionnaires sent were returned (response rate 50.9%). Thirty-eight respondents were not willing to participate, and reasons for not participating were, for instance, “lack of time” (n = 7), “insufficient time” (n = 4), and “not enough experience” (n = 2). Including the 23 questionnaires from the pilot study, a total of 546 surveys were used for analyses. Demographic information of the respondents of both surveys is displayed in Table Of the 1211 questionnaires sent to the nurses, 550 were returned (response rate 45.4%). Twenty-three nurses declined participation, three questionnaires were excluded because less than 50% was filled in, and one respondent was excluded for not working as a nurse. Reasons not to participate were, for example, “no interest” , “nephrologist and nurse together” , and “nephrologist, nurse and nephrology social worker” .Both surveys assessed if clear guidelines were present within nephrology departments regarding which care provider is accountable for discussing patients’ fertility. Ten nephrologists (9.0%) answered positively on the question if clear guidelines were present. Seven (6.3%) noted that their guidelines indicated the nephrologist was accountable for advising CKD patients on fertility matters, and one nephrologist (0.9%) noted the gynecologist was responsible. Of the nurses, 84 (15.6%) were aware of guidelines within their department regarding the accountability for discussing patients’ fertility. Thirty-one nurses (6.2%) stated these guidelines pointed out their own group of professionals as accountable. Other answers given concerning accountability were the “nephrologist” (p = 0.004 resp. p < 0.001). No association was found between experience or awareness of guidelines and providing information among nephrologists.Nephrologists and nurses were asked how often they informed their dialysis patients about the association between a decline in renal function, dialysis, and reduced fertility. Their answers on this question are listed in Table n = 96, 88.1%) advised their female patients with renal failure not to become pregnant. Fifty-eight (62.4%) did so when their patients reached CKD stage 4 and 12.9% (n = 12) discouraged pregnancy in stage 5. Other nephrologists advised their patients to avoid pregnancy during stage 1 , stage 2 , and stage 3  of CKD. Besides advising their female patients to prevent pregnancy before renal transplantation, nephrologists discussed several other subjects regarding fertility with their female patients. These included: wish to have children , health risks of pregnancy , and inheritance of the disease . With their male patients, they also discussed their wish to have children  and inheritance of the disease . On top of that, almost three-quarter of nephrologists  inquired after erectile dysfunction. With regards to referring CKD patients to a physician specialized in fertility, on average the nephrologists referred 14.5% (SD 27.0) of female patients and 9.7% (SD 22.5) of male patients.A majority of the nephrologists  and female patients . On the question “What could be helpful for the discussion of fertility”, 86.0% (n = 467) answered informative brochures, 55.6% (n = 302) thought training to discuss fertility would be helpful, and 15.1% (n = 82) recommended posters about fertility in the waiting room. Almost 45%  felt that the nephrologist should initiate the conversation on fertility so nurses could then refer to that conversation. Self-reported answers to this question were, for example, “more privacy or private appointments to discuss fertility” , “more knowledge” , and “referral to a physician” .Nurses with more years of experience discussed fertility more often with male  answered affirmatively. Next, the nurses were asked to rate their own level of knowledge regarding fertility in CKD patients. Forty-seven nurses (8.7%) stated to have no knowledge at all, 254 (47.0%) answered “a little” and 194 (35.9%) had some knowledge. Forty-five nurses (8.3%) rated their own knowledge as sufficient and one nurse (0.2%) answered in having a lot of knowledge. Nearly 30% of the nurses  felt competent to discuss fertility with their patients, three-quarters  stated to be in need of expanding their knowledge. Nurses with less knowledge, who did not felt competent to discuss fertility or who were in need of expanding their knowledge, informed their patients less often on fertility. Furthermore, these nurses discussed fertility issues less often with their patients and noted “insufficient training” as a barrier to discuss fertility more often than other nurses. The p values ranged from <0.001 up to 0.01.On the question “Is the subject fertility in CKD patients addressed during your training to become a nurse?” two-thirds of nurses (This analysis was the first to evaluate the extent of fertility care and information provided by nephrologists and nurses working in the dialysis unit for CKD patients. The study identified the absence of clear guidelines on this important part of renal health care in the majority of the Dutch nephrology and dialysis departments. Despite the lack of guidelines that appoint a health care professional to provide fertility care, the study shows that most nephrologists routinely discuss fertility during consultation. Unfortunately, this trend is not present throughout the whole nephrology department; nurses working in the dialysis unit who were unaware of guidelines or who had less work experience often skipped discussing fertility. However, the nurses are willing to provide this part of renal care in cooperation with the nephrologist. A suggestion was made that the nephrologists could initiate the subject of fertility enabling the nurses to continue the conversation during their own consultation with their patients. Still, multiple reasons exist that restrain nurses from assessing this subject. Besides the age of the patient, major barriers that withhold nurses from discussing fertility with their CKD patients derive from diversity in language, ethnicity, culture, and religion. Dealing with these factors in a diverse patient population requires certain skills, knowledge, and attitudes defined as cultural competence . CulturaThis study revealed the importance of training and nurses’ self-imposed knowledge and feelings of competence in providing fertility care. Unfortunately, study outcomes also indicated that these components were not self-evident among nurses of the dialysis department. This lack of training, self-imposed knowledge, and competence might be explained when focusing on the current educational system. Even though almost 70% of the nurses received education on fertility in CKD during their nurse training, a pressing need for additional training in discussing fertility exists. These lacuna in competence, knowledge and training regarding fertility issues is a problem not confined to the nurses working in nephrology; similar challenges arise among nurses in other medical departments as well , 18.This study was a pioneer in assessing fertility care provided by renal care providers for CKD patients. In this light, a limitation of the study is the lack of formal comparison as no previous study of this nature has been conducted. Response bias may have occurred due to low responses in both surveys. However, this may also be interpreted as a lack of interest or knowledge regarding the subject. In addition, no validated questionnaires exist that assess the study aims. For this reason, two questionnaires were developed, which have not been formally validated. Validation of these two surveys was not performed as they will not be reused. Nevertheless, the authors attempted to develop reliable, literature-based and pilot-tested instruments. Finally, response bias may have occurred as socially desirable answers may have been given. As a result, answers might have been over or underestimated.In order to achieve a cooperative fertility care system in CKD for both nephrologists and nurses, study results suggest several adjustments need to be made which involve both care providers. First, clear guidelines should be formulated within all nephrology departments to ensure multidisciplinary fertility care is provided. These guidelines should highlight the roles in providing fertility care of both nephrologists and nurses of the dialysis department, so any uncertainties regarding for instance accountability will be diminished. At the request of nurses themselves, informative brochures on fertility should be made available within nephrology departments and dialysis units to hand out to patients. In order to establish a functional system, nurses’ competence and knowledge regarding discussing fertility, especially in culturally diverse populations, should be improved. Renal care providers’ competence and skills regarding discussing fertility are of great importance as it is their ethical responsibility to explain the complexity of fertility in CKD to their patients . LiteratGuidelines that appoint who should provide fertility care to CKD patients are absent in the majority of Dutch nephrology departments. Current fertility care is not provided in a cooperative manner: most of the nephrologists assessed fertility routinely during consultation; less experienced nurses working in dialysis units often skipped this part of renal care. Besides a patients’ age, a lack of competence, insufficient knowledge and training, unawareness of guidelines, and barriers based on cultural diversity are reasons for nurses to omit providing fertility care. To achieve a cooperative fertility care system in CKD for both nephrologists and nurses, results emphasize the need for guidelines on this part of renal care in Dutch nephrology and dialysis departments and the provision of training to improve of nurses’ competence regarding cultural diversity and fertility. Finally, more research should be performed to determine patients’ needs and preferences regarding this important part of renal healthcare, especially among male patients.


Bone metastases often occur in prostate cancers, lung cancers and breast cancers. Bone alkaline phosphatase (BALP) is one of the most commonly used serological markers for clinical evaluation of bone metabolism. Here, we reported that high concentrations of uric acid (UA) caused decrease of BALP levels and revealed that the effect of high concentrations of UA on the BALP expression was through inhibition of its promoter activity. Our results suggested physicians to think about serum UA status of patients with advanced cancer to avoid misdiagnosis when BALP was used to diagnose or assess the extent of bone metastases. Tumor metastasis is a leading cause of death in patients with advanced cancer –3. Bone The serological markers of bone metabolism mainly consist of bone formation and resorption markers, such as the bone formation markers tALP, BALP, OC and P1NP and the bone resorption markers BSP, NTX, CTX and TRACP 5b –37. Bonet-test, p value < 0.0001) than the healthy subjects (16.13 ± 4.87 μg/L)  Table . The resL) Table .To confirm whether high UA concentrations lead to decrease of BALP levels, we tested the BALP levels of 3 dialysis patients 10 minutes before and 10 minutes after renal dialysis. UA concentrations of these 3 patients after renal dialysis decreased from 342 to 53 μM, 493 to 208 μM, and 498 to 202 μM, respectively Figure . In contBased on the above data, we demonstrated that there was a negative correlation between UA concentrations and BALP levels in general population.Since high UA concentrations can falsely decrease TRACP 5b levels due to a method-related systematic error, we examined whether high UA concentrations interfered with BALP immunoassay. We performed interference experiments according to the procedure we have described in “Materials and Methods”. We spiked serial dilutions of UA into BALP standard samples and a serum sample from one patient with confirmed bone metastasis, respectively. As a result, we did not observe that BALP levels were affected by gradually increasing doses of UA in BALP standard samples or the patient’s serum sample Figure . TherefoTo examine whether high UA concentrations affected the expression of BALP gene, the mRNA levels of BALP were first determined in human osteosarcoma cell line (Saos-2) after treatment with a variety of doses of UA. The results showed that UA treatment decreased the mRNA levels of BALP in dose- and time-dependent manners Figure . Then, wUric acid, a metabolic end-product of purine nucleotides, is considered closely related to many kinds of diseases, such as metabolic syndrome, kidney disease, hypertension and cardiovascular disorders –43. As aIn this study, we first evaluated the differences between a total of 91 patients with high concentrations of UA and 91 healthy subjects. The results showed the average BALP level of the high-UA individuals was significantly lower than the healthy subjects. Meanwhile, we showed that lower UA concentration resulted in increase of the BALP in patients undergoing hemodialysis through renal dialysis. These data suggested that BALP was negatively affected by high UA concentrations, just as TRACP 5b. However, we confirmed that high UA concentrations did not interfere with BALP immunoassay, which was different from TRACP 5b. To disclose how high UA concentrations negatively affected the BALP levels, we detected the mRNA and protein levels of BALP in Saos-2 treated with UA or not. The results showed that high UA concentrations suppressed the mRNA and protein expression of the BALP. Recent researches have shown UA can regulate target genes expression by some different ways. UA induces ET-1 gene expression by the activation of ERK pathway via ROS generation . UA stimAs we know, BALP was widely used serological marker in the detection of bone metabolism. Many studies used BALP to assess bone growth and the extent of bone metastasis –56. HoweAlthough we have made clear that high UA concentrations negatively affected TRACP and BALP levels, two widely used bone metabolic markers, we do not know whether other bone metabolism markers such as OC, NTX and CTX are affected by high UA concentrations. We will further disclose whether serum UA status have effects on the level of other bone metabolic markers in our future work to decrease the misdiagnosis rate of bone metabolism.This study was approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University. All samples were collected from August 2014 to February 2016. Patients with cancer, hepatitis, renal dysfunction and inflammatory disease were excluded from serum collection. A total of 91 patients, including 25 women and 66 men  formed the study group. Additionally, 91 healthy subjects were sampled as the control group, including 25 women and 66 men .The ALT, AST, Glu, UREA, CREA and UA quantitation were analyzed using an Olympus AU5400 automatic chemical analyzer and commercial kits  according to the instruction manual. WBC and NEU % were counted by the Sysmex XE-2100 hematology analyzer . The levels of CEA and AFP were measured by electrochemiluminescence immunoassay (ECLIA) on an Elecsys E 602 . Serum BALP was detected by using Unicel DxI 800  device via the chemiluminescence enzyme immunometric method.(1) A known concentration of BALP standard sample was divided into 5 aliquots. Serial dilutions of UA  were prepared from UA standard subject. The 4 aliquots were then spiked at 3:2 with each UA solution. This generated 4 distinct test samples with the same BALP concentration, final UA concentrations ranging from 100 to 800 μM. An aliquot containing double distilled water (DDW) instead of UA served as a blank. Then measured the BALP concentrations of each aliquot. (2) A UA standard sample was spiked with a serum sample from one patient with confirmed bone metastasis  at 0:10, 1:9, 2:8 and 3:7, respectively. This generated 4 pools whose final UA concentrations were 229.6, 606.6, 983.7 and 1360.7 µM. Then measured and corrected (multiplied by the dilution) BALP concentrations of each aliquot.2, harvested by trypsinization, and subcultured twice weekly.The human osteoblast-like cells (Saos-2) and human embryonic kidney 293 cells (HEK293) were obtained from ATCC and maintained in DMEM  supplemented with 10% fetal bovine serum  with 1% penicillin streptomycin, at 37°C in 5% COSigma-Aldrich, USA) for 0, 12, 24 or 48 h. Total RNA of cells was extracted with Trizol reagent  according to manufacturer’s instructions. cDNA was transcribed from total RNA using PrimeScript™ RT Master Mix  . The mRNA level of BALP and GAPDH were quantified by real time-PCR using SYBR® Premix EX Taq™ (Tli RNaseH Plus)  in ABI 7300 real-time PCR system (Applied Biosystems Inc.). The forward and reverse primer sequences were designed using Olige 7 software and listed in Table Saos-2 cells were seeded at a density of 200,000 cells/well in 6-well dishes. Cells were grown in DMEM with 10% FBS, and then 24 hour later incubated with 0, 0.2, 0.4 mmol/L UA  according to manufacturer’s protocol. The forward and reverse primers were designed using Olige 7 software and listed in Table Kpn I and Bgl II  and then subcloned to a promoter-less vector pGL3-Basic restrictively digested with Kpn I and Bgl II. The recombinant plasmids were tested and sequenced, and the positive cloned plasmid was named as pGL3-BALP.A 5′ flanking upstream (including promoter region) of BALP (NM_000478.5) (from -473 to +154 nt relative to transcription start site) was amplified from genomic DNA by PrimeSTARTransient transfections were carried out in HEK293 cells using Lipofectamine2000  according to the manufacturer’s suggestion. Cells were vaccinated in a 48-well culture plates and when the cells had grown to 70–80% before transfection. For luciferase assays, 200 ng of pGL3-BALP plasmid was cotransfected into HEK293 cells with 2 ng of control pRL-TK plasmids as an internal control. After 6 hours, cells were incubated with different doses  of UA, respectively. After 24 hours, cells were harvested and luciferase assays were measured with the Dual Reporter assay system  using FB12 luminometer . The relative luciferase activities (RLA) were calculated by normalizing the fluorescence luciferase with internal standard Renilla luciferase.t-tests were used for significance testing between groups of continuous data. For all statistical comparisons, a p value < 0.05 was considered statistically significant.The statistical software package SPSS17.0  was used for data analysis. Data were presented as means ± SD and represented at least three independent experiments. Two-tailed Ethical clearance for this study was obtained from the Ethics Committee at the First Affiliated Hospital of Nanjing Medical University. Because all the samples used in this study were collected from clinical residual specimen, written informed content from each patient was waived. This study was conducted in accordance with the Declaration Helsinki.


Rct) compared to a reduced graphene oxide (rGO) electrode or a bare glassy carbon electrode (GCE). The limit of detection and sensitivity of PrGO for the detection of UA are 0.19 μM (S/N = 3) and 0.01 μA/μM, respectively, in the range of 1–300 μM of UA.An attractive electrochemical sensor of poly/reduced graphene oxide electrode (PrGO) was developed for an electrochemical technique for uric acid (UA) detection in the presence of ascorbic acid (AA). PrGO composite film showed an improved electrocatalytic activity towards UA oxidation in pH 6.0 (0.1 M PBS). The PrGO composite exhibited a high current signal and low charge transfer resistance ( Uric acid (UA) is present in human biofluids like urine and blood. It is a protein metabolism byproduct which is also found in large amounts in certain foods that may cause some harm because humans do not have any enzyme to break down uric acid, thus UA will accumulate and contribute to diseases like gout or kidney stones. Gout is a disease where uric acid crystals are formed in the joints which cause a painful inflammatory response . TherefoPoly (PEDOT), polyaniline (PANI) and polypyrrole (Ppy) are examples of conducting polymers (CPs) that have been reported as useful materials for making biosensors . In sensAs a two-dimensional (2D) carbon material, graphene exhibits excellent properties which provide low-cost manufacture, high mechanical strength, good conductivity and high surface area ,15. GrapIn this work, PrGO composite was prepared by using a cyclic voltammetry technique. The surface electrochemical properties of PrGO composite were compared with rGO, PEDOT and bare GCE. The electrocatalytic activity of UA oxidation was studied and discussed. The results indicated that PrGO composite was well-suited and well-defined for the fabrication of a UA biosensor in the presence of ascorbic acid (AA) due to its excellent electrochemical performance. PrGO revealed a low detection limit towards oxidation of UA and it also possesses an excellent analytical performance, with good stability, high sensitivity and selectivity for UA. Based on our best knowledge, the use of PrGO composite for the electrocatalytic determination of UA in the presence of AA has not been studied. Thus, this work will focus on the fabrication of a PrGO composite on a GCE substrate for UA detection in the presence of AA by using a differential pulse voltammetry (DPV) technique. The PrGO composites were characterized using Fourier transform infrared (FTIR) spectroscopy, cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and field emission scanning electron microscopy (FESEM).2PO4) and dipotassium hydrogen phosphate (K2HPO4) were supplied by Merck . Uric acid (UA), ascorbic acid (AA) and 3,4–ethylene-dioxythiophene (EDOT) were provided by Sigma-Aldrich . Phosphate buffer solution (PBS) was prepared by adjusting the pH value using an appropriate quantity of standard stock solutions of 0.1 M KH2PO4 and 0.1 M K2HPO4. Deionized water (18.2 MΩ·cm) from a Milli-Q system  was used to prepare all solutions.Potassium chloride (KCl) and graphene oxide (GO) were obtained from Graphenea  and Fisher , respectively. Potassium dihydrogen phosphate (KH3)−distilled water followed by deionized water for 10 minutes. PrGO composite was deposited electrochemically onto the GCE in a solution containing 0.01 M EDOT and 0.01 mg/mL GO solution by using the CV technique between 1.2 V and 1.5 V for three cycles (scan rate: 0.1 V/s).Electrodeposition of PrGO was accomplished by using a potentiostat . The GCE was cleaned with alumina slurry (0.5 μm) and sonicated in 1:1 nitric acid (HNO3Fe(CN)6/K4Fe(CN)6 containing 0.1 M KCl with frequency from 10 kHz to 0.1 Hz. The amperometric was performed at 0.5 V. Field emission scanning electron microscopy  was applied to examine the morphology of the composites. The presence of functional groups in the PrGO composite was identified by Fourier transform infrared spectroscopy . The pH of the solutions was measured with a pH meter , which was calibrated with standard buffer solutions.The electrochemical measurements were performed using a three-electrode system consisting of a counter electrode (Pt wire), reference electrode (Ag/AgCl) and the working electrode . The measurements were performed at room temperature by using a potentiostat (Autolab M101). Cyclic voltammetric scans were applied from −0.2 to 0.6 V (vs. Ag/AgCl). The EIS analyses were carried out at open circuit potentials (OCP) in 5 mM KThe electrodeposition of PrGO was performed by applying 1.2 to −1.5 V in 0.01 mg/mL GO and 0.01 M EDOT for three cycles. The cyclic voltammogram in SEM images and FTIR spectra of PEDOT, GO, rGO and PrGO composites are shown in −1 which is attributed to the C-S stretching mode, whereas, the bands at 1318, 1658 and 2796 cm−1 result from the C-O-C, C=O and sp3 C-H stretching modes, respectively. The GO spectrum shows peak absorptions assigned to C=O and O-H groups at 1317 cm−1 and 3310 cm−1, respectively. After the reduction of GO, the intensities of the oxygenated functional group peaks decrease compared to GO. As for PrGO composite, a series of peaks was observed at around 1300, 1600, 2000 and 3000 cm−1 which are associated with the functional groups present in PEDOT and rGO film. Hence, it revealed that PEDOT was successfully incorporated with rGO as shown in the SEM image.The FTIR spectrum in 3[Fe(CN)6] as a redox probe in 0.1 M KCl solution. A redox peak was seen with a separation peak (ΔpE) of 129 mV for bare GCE, while the ΔpE values for rGO and PrGO are 178 and 68 mV, respectively. The rGO film possesses larger ΔEp than bare GCE and the redox peak currents decreased significantly due to the aggregation of rGO [Ep value for PrGO is smaller than those of bare GCE and rGO, indicating the rate of electron transfer and redox peak current for PrGO is significantly increased which is a result of the excellent electrical conductivity of the combination of PEDOT and rGO films [Ip) according to the Randles-Sevcik equation [n is the number of electrons taking part in the redox reaction (n = 1), A is the electroactive surface area (in cm2), D is the diffusion coefficient (D = 6.5 × 10−6 cm2·s−1), C is the bulk concentration (in mol·cm−3), v is the scan rate (in V·s−1). The values of the electrochemically active surface area for bare GCE, rGO and PrGO are calculated as 0.065, 0.046 and 0.100 cm2, respectively. The results reveal that the PrGO electrode has larger reaction surface area than the rGO and bare GCE.n of rGO  that cauGO films . The eleequation :Ip=(2.63Fe(CN)6/K4Fe(CN)6 (1:1) and 0.1 M KCl. At low frequency, an inclined straight line indicates the diffusion limiting step characteristic for the electrochemical processes [Rct) that lead to the limitation of the electron transfer rate between the redox probe and electrode interface. As seen in Rct value of rGO (648 Ω) is lower compared to the Rct of bare GCE (1202 Ω) due to the conductive properties of the rGO that can accelerate the electron transfer rate. After the modification of the GCE with PrGO, the Rct is significantly reduced to 8.23 Ω due to the excellent combination of the conductivity of PEDOT and rGO. The changes in Rct indicate that the PrGO composite possesses superior conductivity that enhances the electrocatalytic activity of the electron surface of the PrGO electrode.EIS is a practical analysis to identify the properties of electron transfer rate occurred on the surface of the electrodes. rocesses . While tThe important factor that needs to be optimized in the determination of the analyte is the pH of the analyte-containing solution. The pH effect on the determination of 20 μM of UA in 0.1 M PBS at PrGO electrode was observed within the range of 5.0–9.0 by using the DPV method as shown in Ep (V) = −0.058 pH + 0.692 (R2 = 0.999) (inset of +) and two electrons (2e−) [Hence, as the pH of the PBS solution increased, the peak potential is negatively shifted which demonstrates that protons are involved in the processes of UA oxidation at the PrGO electrode surface ,38. The inset of A. This rns (2e−)  which ocμM UA for various scan rates. The results reveal that below than 100 mV/s, the anodic peak current (Ipa) is directly proportional to the scan rate (V)  . Thus, the presence of high concentrations of AA has no significant influence on the detection of UA.In order to establish the applicability of PrGO for the selective determination of UA in the presence of AA, simultaneous changes of the AA concentrations were investigated at a fixed concentration of UA (450 μM). As shown in Ipa (μA) = 0.2 + 0.01 CUA(μM) (R2 = 0.992) and the LOD (S/N = 3) was measured to be 0.19 μM, which was calculated from typical formula of LOD = 3σ S−1 [Ipa(μA) = −1.421 + 1.35 CUA (μM) (R2 = 0.9814) and the LOD measured is 0.32 μM (S/N = 3). This result indicates that DPV has better detection than the amperometric technique. A comparison between the proposed electrode and other modified electrodes towards detection of UA is tabulated in DPV and amperometry techniques were employed for the determination of the limit of detection (LOD). Based on the DPV technique, the peak current increases linearly with increasing UA concentration in the range of 1–300 μM with a linear regression equation of = 3σ S−1 . Figure The accuracy and effectiveness of the proposed sensor in a real application were tested using the standard addition method in four samples of human urine without any pretreatment. The human urine samples underwent a dilution process using 0.1 M PBS (pH 6.0). After the samples were appropriately diluted and spiked with a certain amount of UA, the analysis was performed using DPV . The spiIn order to measure the PrGO electrode stability, repeatability and reproducibility tests were performed. The PrGO electrode offered a highly reliable current signal during 10 consecutive measurements using the same electrode in 1 mM UA with a relative standard deviation (RSD) of 3.86%. This indicates that this electrode does not experience any clogging on its surface throughout the measurements. The sensor reproducibility was measured by determining 1 mM UA with four different modified electrodes. This modified sensor depicted an excellent reproducibility with RSD value of 4.27%.A PEDOT-doped reduced GO (PrGO) composite for the detection of UA was successfully prepared using the cyclic voltammetry technique. The PrGO composite provided a high peak current and low charge transfer resistance compared with bare GCE and rGO. A facile technique for the detection of UA was investigated and PrGO revealed good stability and a low detection limit with a broad linear range. PrGO composite film is not only able to serve as a sensitive and selective sensor towards UA, but it also resolved the difficulty of observing discrete UA and AA oxidation peaks, indicating that PrGO composite film is a promising candidate applicable for real applications.


Giardia duodenalis is a worldwide protozoa known causing diarrhea in all vertebrates, humans among these. Homologous recombination is a mechanism that provides genomic stability. Two putative recombinases were identified in G. duodenalis genome: GdDMC1A and GdDMC1B. In this article, we describe the identification of conserved domains in GdDMC1A and GdDMC1B, such as: DNA binding domains  and an ATPcap and Walker A and B motifs associated with ATP binding and hydrolysis, phylogenetic analyses among assemblages and three-dimensional structure modeling of these recombinases using bioinformatics tools. Also, experimental data is described about LD50 determination for ionizing radiation in trophozoites of G. duodenalis. Additionally, as recombinases, GdDMC1A and GdDMC1B were used to rescue a defective Saccharomyces cerevisiae Δ rad51 strain under genotoxic conditions and data is described.Giardia duodenalis trophozoites”  The data described here are related to the research article entitled “Characterization of recombinase DMC1B and its functional role as Rad51 in DNA damage repair in  Specifications TableValue of the data•Bioinformatics tools allow to identify conserved domains and/or function in recombinases GdDMC1A and GdDMC1B.•Structure-based protein alignment allows to identify conserved domains/functions in recombinases GdDMC1A and GdDMC1B with respect to other recombinases.•Phylogenetic analyses allow to cluster these recombinases with other organisms to determine whether they are closely related.•Comparative modeling analysis between predicted structure of GdDMC1A and GdDMC1B can identify differences in protein structure and make some predictions.1Giardia duodenalis were identified  and phylogenetic reconstructions and 3D modeling were made to identify similarity , Fig. 3.entified  and compentified , Fig. 5.entified  and LD50hozoites .2Saccharomyces cerevisiae strain with either GdDMC1A or GdDMC1B containing plasmids looking for a rescue phenotype under DNA damaging conditions and G. duodenalis. This experimental design was used only for this work.Bioinformatic tools  were used to create


The Hedgehog (Hh) signaling pathway plays an essential role in the growth, development, and homeostatis of many tissues in vertebrates and invertebrates. Much of what is known about Hh signaling is in the context of embryonic development and tumor formation. However, a growing body of evidence is emerging indicating that Hh signaling is also involved in postnatal processes such as tissue repair and adult immune responses. To that extent, Hh signaling has also been shown to be a target for some pathogens that presumably utilize the pathway to control the local infected environment. In this review, we discuss what is currently known regarding pathogenic interactions with Hh signaling and speculate on the reasons for this pathway being a target. We also hope to shed light on the possibility of using small molecule modulators of Hh signaling as effective therapies for a wider range of human diseases beyond their current use in a limited number of cancers. Drosophila embryo, and Hh pathway components have subsequently been found to be remarkably conserved between invertebrates and vertebrates where the pathway also plays key roles in several types of cancers, some of which are being treated with drugs that inhibit signaling ..decapentDrosophila, a forward genetic screen was performed to identify point mutations that might disrupt the Hh-dependent activity of NS1. A point mutation was recovered resulting in an alanine to valine substitution at position 122 that reduced this activity in flies and in transfected cells [Using ed cells . InteresThe precise mechanism by which Hepatitis viruses, HIV, EBV, and H.p. interact with Hh signaling has not been completely elucidated, however, all interactions and often the deleterious effects could be blocked by eliminating Gli ,39,66 anDrosophila as a model organism to study disease genes. For example, a series of genetic epistasis experiments and quantitative imaging experiments using Förster resonance energy transfer-Fluorecence lifetime imaging (FRET-FLIM) assays revealed that NS1 interacts directly with the transcriptional mediator, Ci/Gli1, parroting the HBV X/Gli1 interaction [The mechanism by which NS1 modulates Hh signaling was analyzed using the extensive array of genetic and molecular tools available when using eraction ,55. FurtTo ensure efficient use of their genomes, pathogenic interactions are usually established with more centrally connected host proteins so that several processes may be targeted concomitantly. These virulence factors constantly evolve their interface residues, either to evade or to optimize their binding capabilities to host proteins . Thus, sAs Hh signaling has significant roles in tissue homeostasis and remodeling, we speculated that Hh signaling may be activated as a part of a defense mechanism during infection to promote tissue repair . Indeed,In the lung, acute activation of Shh signaling in epithelial cells was observed upon naphthalene injury, which was strikingly similar to the activation observed during prenatal development . Shh is in vitro stimulation of cultured skin cells with Hh ligand promoted production of angiogenic signals, increased proliferation of fibroblasts, and increased migration, adhesion, and tube formation of endothelial progenitor cells to aid in wound closure [In the skin, Asai et al. found that Ptch1 expression is upregulated in wounds in mice and that  closure . Further closure ,72,73,74Similarly, Shh signaling in pancreatic fibroblasts is required for tissue repair during the onset of pancreatic cancer and pancreatitis due to a mutation in the proto-oncogene, K-ras, as a deletion of one allele of Gli1 or mice lacking expression of Shh in the pancreatic epithelium significantly impaired tissue remodeling and cytokine expression in this model . This stWhere repair processes fall short, however, tissue fibrosis can occur, marked by the formation of detrimental scar tissue. Perhaps not surprisingly, there is also a strong link between tissue fibrosis and Hh signaling in several tissues inflicted with chronic ailments, primarily driven by the Hh-dependent activation of epithelial to mesenchymal transition of cells and the resultant excessive extracellular matrix deposition . PulmonaHh ligand secreted by liver cells in response to damage causes cellular differentiation of fibroblasts and myofibroblasts which are critical for normal liver regeneration ,85,86. HHh ligand and target genes, including those involved in extracellular matrix (ECM) production, have also been shown to be upregulated in a mouse model of renal fibrosis following injury where protection was provided by cyclopamine treatment or by deficiency in Gli1 ,91. SimiThese data clearly establish the role of Hh signaling in tissue repair processes where unstrained signaling can cause harmful fibrotic outcomes. This aligns with a model in which pathogen-dependent tissue damage causes a Hh-mediated repair response . Some viDrosophila by Lee et al., Hedgehog signaling was found to be required as a first line of defense against harmful uracil-secreting pathogens in the gut by triggering the production of microbicidal reactive oxygen species (ROS) [Cadherin 99C (Cad99C), however, could rescue this phenotype, and subsequent experiments determined that Cad99C-dependent formation of signaling endosomes stimulated the enzyme, dual oxidase (DUOX), to produce ROS in response to uracil detection.The Hh pathway also has postnatal roles in defining the immune response which is conserved from flies to humans, although the relevant signaling output and how it is applied as a defense mechanism differs between vertebrates and invertebrates. In a study conducted in es (ROS) ,95,96. FIn mammals, Hh signaling takes a multipronged approach in regulating the immune system by controlling several T-cell features such as differentiation, proliferation, and activity ,98. Shh Analysis of thymocyte phenotypes in mouse mutants of Hh pathway components show that Hh signaling is important for regulating T-cell proliferation rates, determining the final differentiation step between CD4 vs. CD8, controlling various steps throughout the conversion process of double negative cells to single positive cell, as well as modulating the strength of T-cell receptor (TCR) signaling to influence TCR repertoire selection ,102,103.Direct induction of cytokine expression—secreted factors of immune cells that have an effect on other cells—is another capability of Hh signaling. Peripheral CD4 T-cells retain expression of Hh pathway components and can respond to Hh signaling following TCR-activation ,81. Hh shelper 2 (Th2) cells [Consistent with Hh controlling immune cell proliferation and cytokine induction, some auto-immune disorders ,112,113 2) cells . This oc2) cells ,110. Thu2) cells . Similarly, influenza infection strongly induced expression of cytokines CXCL-10 and IL6, the latter at least partially by a direct, cell-autonomous interaction between Hh signaling and NS1 . InteresCollectively, these studies indicate that select populations of immune cells are primed to respond to the Hh signal, often resulting in proliferation and an upregulation of a subset of cytokines. Increasing Hh signaling, as occurs in certain tissues during HBV, HCV, EBV, and HIV infection, may help the virus evade the immune response by disrupting the balance of immune components available to extract the infection . In contSmall molecule modulators of Hh signaling have been used in basic research for several years now to detect links between signaling and specific phenotypes of interest. Currently, CDC-0449 (Vismodegib) and LDE225 (Sonidegib), both Smoothened inhibitors, and arsenic trioxide (ATO), a Gli1/2 inhibitor, have been approved by the Food and Drug Administration (FDA) to treat basal cell carcinoma (BCC) and certain leukemias, respectively, whereas many others are still in clinical trials B 3,119,,119,120.Treatment both in vivo and in vitro with Vismodegib or the Gli inhibitors, Gant-58 and Gant-61, has successfully reversed the detrimental phenotypes caused by several of the ailments mentioned in this review. For example, Vismodegib reduced liver fibrosis and tumor formation in a mouse model of fibrosis-associated HCC  and amelThus, it stands to reason that therapeutic uses of FDA-approved molecules that inhibit Hh signaling may be expanded for use as potent inhibitors to treat several pathogenic infections. In contrast to the currently available therapies, such as vaccines and antivirals, which target strain-specific and rapidly-mutating viral proteins, treatments that target highly-conserved host targets may ultimately provide superior and continual protection across a broader spectrum of strains.Interestingly, HIV-infected cells and tissues appear to be responsive to treatment with both Hh antagonists and agonists—the former directed to infected cells, the latter directed to the local uninfected environment. In the former case, Gli inhibitors were shown to decrease HIV-dependent proliferation and migration of mouse kidney podocytes in vitro . In the These HIV studies reinforce the importance of carefully selecting a relevant treatment since the cellular processes regulated by this signaling pathway are expansive and unforeseeably complex. This may require a combinatorial approach in which several drugs are targeted to different Hh-dependent processes, which may further strengthen or reduce signaling in a context-dependent fashion. This may be possible by using a combination of drugs that target pathway components with both positive and negative roles in signaling. Additionally, defining the precise mechanism in which viral factors interact with the pathway would, no doubt, restrict the search for useful drugs. The Hh signaling pathway has emerged as a target of several pathogens in recent years, where cases of both pathogen-dependent increases and decreases in pathway activity have been observed. These changes in signaling correlate to an exacerbated detriment to the host and sometimes also as protection, as in the case of influenza. We speculate that the key postnatal roles that Hh signaling plays in wound repair and the immune response are critical features that would be appealing for pathogenic control. Potential new therapies involving Hh inhibitory and stimulatory compounds that disrupt or reverse these interactions could derive from these findings and suggest an important new avenue for further investigation.