Angiostrongyliasis cantonensis. Although this nematode usually resides in the pulmonary arteries of rats, its incidental occurence in other hosts such as humans can cause optic neuritis and lead to serious vision sequelae. Nevertheless, there are currently no systematic studies conducted in this area.One of the most common causes of meningitis in South East Asia is angiostrongyliasis or infection by the parasitic nematode A. cantonensis on 7d, 14d and 21d, respectively. Electroretinogram (ERG), visual evoked potential (VEP), ophthalmoscopy and histology were examined on day 7d, 14d and 21d and tribendimidine (TBD) was later used to treat optic neuritis on day 14d for a week to evaluate its therapeutic effects.In order to study the pathogenesis of optic neuritis, mice were tried as a new animal model to study and challenge with A. cantonensis caused obvious inflammatory cell infiltration in the retina and optic nerve adventitia in day 14d and 21d followed by optic nerve fiber demyelination and retinal ganglion swelling at day 21d in the challenged mice. Prolonged VEP latency and decreased ERG amplitude were also observed on day 21. After treatment of TBD in the infected mice, retinal and optic nerve inflammation were alleviated, but VEP latency and ERG amplitude did not improve on day 21d and 28d.Infection of A. cantonensis can cause optic neuritis along with optic nerve demyelination and retinal ganglion cell damage in a mouse model. TBD alone treatment can improve the symptoms of optic neuritis, but does not aid in vision recovery, suggesting that both neuroprotective agents and Dexamethasone should be administered, along with treatment for the infection, to protect the optic nerve and ganglion cells. Furthermore, as the symptoms of optic neuritis caused by A. cantonensis in mice are similar to the optic neuritis in multiple sclerosis (MS) human patients, we suggest that the BALB/c mouse model provided in this study may be useful to explore therapies of optic neuritis in MS patients.The current study provides evidence that  Angiostrongylus (A). cantonensis is a parasitic nematode that causes Angiostrongyliasis . Fresh water snails harbor the third stage of the A. cantonensis larva (Larva), which is the infective stage. Consumption of raw or uncooked snails or fish is the most common manner, resultant in infection by A. cantonensis. In humans, 3rd stage larva mainly invade the central nervous system, causing meningitis, spinal meningitis, encephalitis and myelitis. The clinical manifestation of infection is eosinophilic meningitis[A. cantonensis[et al. conducted a clinical study of 3 optic neuritis cases caused by A. cantonensis. Larvae were found in the anterior chamber angle, vitreous body and retina and A. cantonensis antibody (29 KD) was detected in serum. Patients underwent visual acuity, random amplified polymorphic DNA (RAPD), VEP, and electroretinogram (ERG) testing as well as fundus examinations[A. cantonensis in Beijing, China, caused the attention of the Chinese government on the ponderance of A. cantonensis. In one survey about this outbreak, 25 severe cases were identified, 44% of which had different levels of visual disorders, such as photophobia, blurred vision, diplopia, visual field defects and/or muscae volitantes (‘floaters’). Even after treatment, a fairly substantial proportion of patients did not recover, seriously affecting quality of life[A cantonensis causes optic neuritis.ningitis-5, whichntonensis. The firntonensis. Since tntonensis. In 2008ntibody 2 KD was d of life,11. Up tA cantonensis to elucidate a pathogenetic mechanism of the disease and to develop therapeutic strategies for clinic treatment. In this study, mice that had been infected with A. cantonensis were used as a surrogate model to observe optic neuritis. Obvious VEP/ERG changes and pathological alterations of retina and optic nerve, such as retinal ganglion swelling and optic nerve demyelination, were observed in the infected mice. The effect of treatment with a new anthelmintic agent, Tribendimidine (TBD)[A. cantonensis is able to cause optic neuritis. It is necessary to develop new drugs, which can protect and nourish optic nerve and ganglion cells during the treatment of infection[The purpose of the present study was to establish a mouse model of optic neuritis caused by ne (TBD) was alsonfection.A. cantonensis, were collected from giant African snails (Achatina fulica) via homogenization and digestion of minced snail tissue that was placed in a pepsin–HCl solution  and incubated at 37°C for 2 h. The L3 in the sediment were washed with phosphate-buffered saline (PBS) and counted under an anatomical microscope. Four groups (n = 6 mice/group) were studied. In the 3 mice of each group, a total of 30 L3 were perfused into each BALB/c mouse; the other 3 mice in each group were not infected and served as controls. At the same time, infected animals were randomly assigned to either the treatment or non-treatment group . TBD was administered via gastric infusion to mice in the treatment groups once per day for 7 days. The dose of TBD was 100 mg/kg/d[BALB/c mice (20 – 40 g body weight) were purchased from the animal center laboratory at Sun Yat-sen University . The Institutional Animal Care and Use Committee approved all animal procedures. Larva III (L3) of  mg/kg/d. TBD wasMice were sacrificed by chloral hydrate asphyxiation and cervical dislocation at 7, 14, or 21 days post-infection. Paraffin embedded sections were then prepared from isolated optic nerves and whole eyes. Optic neuritis was detected by the presence of inflammatory cell infiltration on hematoxylin and eosin (H&E) staining and observed by microscopy . Retinal thickness was measured using Image Pro Plus 6.0 . Demyelination detection was done by using a 300 KV transmission electronic microscope  and axons were counted with Image Pro Plus 6.0.Mice in all four groups were anesthetized by intraperitoneal injection of chloral hydrate (0.42 mg/kg body weight) and secured to a moveable platform for ERG and VEP testing. The Flash ERG was recorded with chlorinated silver ball electrodes as described previously. For VEPTwo weeks before euthenasia, mice were anaesthetized with chloral hydrate, the skin was incised mediosagittally, holes were drilled into the skull above each superior colliculus  and 0.25 μl of Dextran biotin   was injected stereotactically into both superior colliculi.2) per retinal quadrant at eccentricities of 1/6, 3/6 and 5/6 of the retinal radius. Two independent investigators, who were blinded to the protocol, performed the cell counts.At the conclusion of the second recording session, the mice were euthanized by an overdose of chloral hydrate and were perfused via the aorta with 4% paraformaldehyde in PBS. The brain, optic nerves and both eyes were collected. The retinas were then dissected and soaked in horseradish peroxidase (HRP) at 37°C for 30 min. Retinas were then stained for 5 min with 3,3'-diaminobenzidine (DAB) and examined by light microscopy. RGC densities were determined by counting labelled cells in three areas  group to the control (infected untreated) group. Statistics were performed using IBM SPSS statistics 19 .ANOVA was used to compare retinal thickness, myelin sheath number, ganglion cell number, VEP P2 latency time and ERG amplitude at different times in infected versus control eyes. Student’s A. cantonensis though the cellular infiltrate appeared to have subsided at 28 d  symptoms in both. Therefore, mice have been commonly selected as an animal model to study the infection caused by A. cantonensis[A. cantonensis than other types of mice. Although there are clinical reports and experimental observations illustrating that A. cantonensis can cause optic neuritis in human beings and mice, there have been no systematic studies examining optic neuritis caused by A. cantonensis. Therefore, we carried out the systematic experiments by utilizing the mice in order to observe and treat the optic neuritis.Mice and human beings are both non-permissive hosts of ntonensis,17. In tA. cantonensis are funduscopic examination and ERG and VEP combined with immunologic diagnosis by ELISA and/or specific antibody detection in serum[Clinically, the most common techniques used to diagnose optic neuritis caused by in serum,19, thouin serum. Histoloin serum.2 of axons in the optic nerve decreased at day 14 and obvious demyelination appeared at day 21. This indicates that the A. cantonensis infection seriously affected the optic nerve, resulting in optic neuritis since demyelination is a common cause of optic neuritis. Shed myelin slows optic signal transduction causing an obvious visual disorder[In the present study, we utilized histological examination, such as H&E staining, employed transmission electronic microscopy and ERG/VEP testing to measure optic neuritis. Many inflammatory cell infiltrates were observed with H&E staining in the retinal ganglion cell layer and around the adventitia of the optic nerve at day 14 of infection. The retinal thickness increased in infected eyes, compared to normal controls, and peaked at day 21, which indicates that swelling may occur in the retina. Similarly, the result of electron microscopy examination showed swelling of the ganglion cells and internal nuclear layer. Interestingly, the retinal swelling subsided at day 28 after infection. The electron microscope examination of ganglion cells showed that the organelle vanished, which may be a reason for the thinner retina observed at day 28. On the other hand, the numbers/mmA. cantonensis infected mice.Ganglion cells are the most important neurons in the retina and their axons form the optic nerve. It is well known from the rat model of surgical axotomy of the optic nerve that 80 – 90% of retinal ganglion cells undergo apoptotic cell death following optic nerve transection,23. InflA. cantonensis infection can cause optic neuritis.It is known that RGC loss can occur before alterations in VEP potentials, which is detectable. However, when RGC death has reached a certain threshold, ERG potentials become affected and severe functional deficits in VEP responses occur. In the A. cantonensis can lead to optic neuritis. However, preventing and/or treating this disease is still a problem. In clinic, physical removal of the parasites via surgery, oral steroid therapy combined with an anthelmintic agent is the most effective therapy for the vision damage caused by parasites. It is because this treatment reduces intraocular inflammation and improves visual acuity to a certain extent[2 in the optic nerve increased in mice of the TBD-treated groups, compared to untreated groups. It may be that TBD killed A. cantonensis and lessened the eosinophil infiltration in the brain and optic nerve caused by this type of parasite, which in turn, decreased the demyelination of the optic nerve and reduced the death of ganglion cells. At day 14, the ERG in the TBD treatment group had recovered better compared with the untreated group, though they had still not reached a normal level. The better ERG recovery may be correlated with the increase in surviving ganglion cells. On the other hand, there was no obvious VEP recovery with TBD treatment. This is likely due to the fact that there were not enough surviving ganglion cells or optic nerve fibers to reach a certain threshold where they can function normally. This result is similar to what is observed clinically in cases of optic neuritis when other medicines are applied[A. cantonensis infection treatment and can not reverse the serious damage of ganglion cells and optic nerve effectively. As a result, other therapy methods for demyelination may be drawn, such as Multiple sclerosis (MS).The evidence so far points to the fact that the infection of n extent. In somen extent. Therefon extent. The inf applied. But it A. cantonensis results in pathological alterations and clinical manifestations, such as ganglion cell loss, demyelination and prolonged VEP latency, which are similar to MS[A. cantonensis or by MS may have some connecting links. It has been reported that helminthes may be a potential immunological therapy for MS. Helminthes invade a host and induce abnormal immunoregulation which could be used to treat MS[A. cantonensis is a type of helminth, but it invades the CNS directly causing symptoms similar to MS, thus it may aggravate MS instead of treating it. Although the nosogenesis of both diseases are very different, the methods to relieve symptoms used in MS therapy, such as combining neuroprotective agents with Dexamethasone treatment and interferon treatment, may provide new ways to treat optic neuritis caused by A. cantonensis[.MS is the most common non-traumatic cause of neurological disability in young adults in developed countries. It is an autoimmune CNS disease that has long been thought to be primarily characterized by inflammation and demyelination. The disar to MS. This imtreat MS. A. cantntonensis-32.A. cantonensis infection can cause optic neuritis and that TBD is an effective anthelmintic agent that could be used to treat this disease as it can effectively lessen the symptoms of optic neuritis. However, TBD is not an effective therapy for recovering vision. We suggest that TBD combined with a neuroprotective agent and Dexamethasone may provide better and more effective treatment for optic neuritis caused by A. cantonensis. Combining these treatments may help to prevent secondary injuries caused by A. cantonensis and may lessen other serious sequelae in the ocular region, such as visual impairment even vision loss. This will ultimately improve the quality of life for patients who suffer from this type of disease. Finally, due to the similarity of A. cantonensis and MS symptoms, therapies used for MS may also be helpful for treating optic neuritis caused by A. cantonensis.The above results indicate that cantinensis: Angiostrongylus cantonensis; VEP: Visual evoked potential; ERG: Electroretinogram; HRP: Horseradish peroxidase; BDA: Dextran biotin; CNS: Central nerve system; RGC: Retina ganglion cells; MS: Multiple sclerosis.TBD: Tribendimidine; ANOVA: Analysis of variance; A The authors declare that they have no competing interests.YF, XZ and LO carried out the experiments and performed the statistical analyses. YF and ZW drafted the manuscript. WL, WW helped to do the ophthalmologic examination section of the study. WC and FF provided help in animal experiment. ZW conceived the study and coordinated the project. XS helped to draft the manuscript. All authors read and approved the final manuscript.


Procedures that may optimize success in achieving live births from assisted reproductive technology (ART) continue to be examined. Not yet considered are the perspectives of fertility specialists regarding important developments in the fertility treatment field, current unmet needs, and anticipated future advances. In the current study, an 8-country survey of fertility specialists was conducted to provide a comprehensive, global depiction of fertility treatments across different regions.Fertility specialists from France, Germany, Italy, Spain, the United Kingdom (UK), the United States (US), China, and Japan were invited to participate in an online survey. Participants were eligible if they personally managed ≥25 patients/month who were experiencing difficulty conceiving, and if they had performed ART fertility treatment with ≥1 patient in the previous month. Quantitative questions addressed the number of patients seen, main infertility causes, number of cycles performed, ART procedure type, and ART outcomes. Qualitative questions covered diagnostic trends, unmet needs, important advances, and expected future developments.The number of fertility specialists who completed the survey included 29 in France, 33 in Germany, 23 in Italy, 38 in Spain, 34 in the UK, 91 in the US, 50 in China, and 65 in Japan. Patient volume increased over the prior 2 years according to 67 % (242/363) of the fertility specialists. As expected, ART outcomes all declined with age in all countries. ART outcomes varied by country, with the highest implantation, pregnancy, and live birth rates reported by fertility specialists in the US and China and the lowest rates reported in France and Italy. The most frequently reported unmet needs in fertility treatment were financial coverage, improved implantation rate, and egg donation. Most frequently named future advancements expected to change the fertility treatment field included improved embryo selection through imaging and/or metabolomics, improved embryo implantation rate, and use of preimplantation genetic diagnosis.This study, which follows a rigorous survey methodology, elucidates the current state of fertility specialists’ practices and perspectives on the global fertility treatment field, which highlights differences and similarities among countries. This research may inform further studies and procedural developments that might better improve and standardize ART. Considerable progress continues to be made in assisted reproductive technology (ART) fertility treatment since the birth of the first “test tube baby”, Louise Brown, in 1978 in the United Kingdom (UK), followed by other live births in Australia (1980), the United States (US) (1981), and in Sweden and France 1982) [ [2]. HowFertility specialists are continually examining ways to optimize the techniques and processes within the fertility treatment field to improve success in achieving live births. Recent targets of interest have included analyzing procedures for ovulation induction and triggering final oocyte maturation –6, improLarge ART fertility treatment registries exist through the European IVF Monitoring Consortium on behalf of the European Society of Human Reproduction and Embryology (ESHRE) in Europe  and the ART, and more specifically IVF, continues to change and further develop with new technologies. However, many questions remain, including: What have been the true innovations in the field of fertility treatment, and how have they affected the treatment of patients? What are the differences per country in fertility treatment? Where are fertility treatments headed in the future? In the current study, an 8-country survey of fertility specialists was conducted with the aim to provide a comprehensive depiction of fertility treatments across different regions. Using a standardized survey-based approach, the goal of the research was to provide both quantitative data and qualitative perceptions from fertility specialists regarding which developments have been most important in the field of fertility treatment within the past 30 years, the current unmet needs in fertility treatment, and which anticipated improvements will be “game changers” in fertility specialty practices in the coming years.Fertility specialists from the European Union Five , the US, China, and Japan were invited to participate in an online survey aimed at understanding current management of women experiencing difficulty conceiving. The criteria for the selection of these countries included having a high number of patients who undergo ART, the representation of a broad spectrum of fertility treatment regulations and practices across the included countries, and high access to the internet within each country. In the EU5 and US, a sample frame of 3975 fertility specialists was established by assembling physicians from public sources, such as the ESHRE annual ART reports and the CDC web site, that list fertility clinics . For ChiA survey was developed to assess current fertility treatment practices, with a focus on ART procedures. The survey was pre-tested through interviews and consultations with 33 fertility specialists throughout 21 countries. The online questionnaire used in the current study included both quantitative and qualitative questions. Quantitative questions covered the following topics: the number of patients having difficulty conceiving; the main causes for fertility issues; and for each age group split, the number of cycles performed, the type of ART procedure used, and the ART treatment outcomes. ART treatment outcomes were assessed using 4 outcome metrics: the proportion of cycles for which an embryo reached the developmental stage sufficient for the embryo to be transferred, the implantation rate, the proportion of cycles that resulted in pregnancy  and the proportion of cycles that resulted in a live birth . Qualitative questions covered trends in diagnosis, unmet needs in the field of fertility treatment, the perceived biggest improvement in fertility treatment in the past 30 years, and expected development(s) that could be the next “game changer” in fertility treatment. All respondents provided qualitative comments because these sections of the survey were mandatory.All survey data were analyzed in aggregate and the individual identities of the survey respondents were blinded to the study authors. The planned analyses for quantitative data were descriptive and included means and percentages. Qualitative data were analyzed thematically and coded according to the main themes of the survey questions. Any response that addressed multiple themes was counted as multiple comments.A total of 1435 physicians responded to the survey invitation and 363 met eligibility criteria and completed the survey. The number of respondents by country who completed the survey included 157 fertility specialists in the EU5 , 91 in the US, 50 in China, and 65 in Japan.The mean number of women with difficulty conceiving seen per month ranged from 63 in Italy to 236 in China Table . In termThe main causes for the difficulty conceiving were very similar across all countries, with male factor, both male and female factor, diminished ovarian reserve, and ovulatory dysfunction being the most frequently reported causes of infertility Table . The onlAmong the categories of fertility management, ART use was highest in the EU5, ranging from 47 % of fertility specialists in France to 75 % in Spain, compared with 37 % in the US, 38 % in China, and 27 % in Japan (Table The number of women who consulted for issues related to difficulty conceiving increased over the prior 2 years according to 67 % (242/363) of the fertility specialists. The percentage of survey respondents who reported an increase in patient volume ranged from 49 % in the US to 85 % in Germany and 94 % in China Fig. . FertiliThe mean number of ART procedures performed per year greatly varied across countries. The fertility specialists based in Japan, Italy, and the US had the lowest mean number of ART procedures at 170, 242, and 253 per year, respectively, followed by fertility specialists based in Spain (341 procedures), France (353), Germany (462) and the UK (492). Fertility specialists based in China had the highest mean number of ART procedures per year at 894. The proportion of ART procedures performed by age group is shown in Fig. Survey respondents reported the percentage of their patients who received ICSI, fresh embryo, and donor embryo ART cycles Table . ICSI waThe number of embryos, categorized as single embryo transfer (SET), dual embryo transfer (DET), and >2 embryos transferred (>DET) back to the patient at each ART cycle increased with increased patient age Table . The majAll 4 ART procedure outcome measures, including percentage of patients with implantable embryos, implantation rate, PR, and LBR, declined with age in all countries Figs.  and 6. TSurvey participants identified what they perceived to be the greatest improvements in the field of fertility treatment within the past 30 years. The relative rankings of the improvements varied by country. However, improvements that were most frequently cited included ICSI (ranging from 14 % of respondents in Japan to 83 % in France), vitrification and cryopreservation techniques that provide the ability to freeze gametes and embryos (ranging from 16 % of respondents in China to 66 % in Spain), improvement in hormonal drugs and treatment protocols (ranging from 18 % of respondents in China to 39 % in Germany), and the ability to culture embryos up to the blastocyst stage (ranging from 15 % of respondents in Japan to 41 % in the UK) Fig. . PGD wasFertility specialists reported current unmet needs for patients who have difficulty conceiving and choose to pursue fertility treatment. Among the most frequently reported unmet needs, better financial coverage was mentioned by survey respondents from all countries but France, ranging from 17 % of survey respondents in Italy to 67 % in the US Fig. . With thThe future advancements that the fertility specialists most frequently reported they expect to occur that could change the field of fertility treatment included improved embryo selection through imaging and/or metabolomics, greater use of PGD and PGS, and development of treatments that improve the implantation rate Fig. . Fertilith position in the other countries. As expected, ART outcomes decreased with increased patient age. There is a strong unmet need to develop techniques that will help preserve or restore fertility for older patients. The cost and/or absence of financial reimbursement for IVF fertility treatment appears to be an important barrier to patients’ access to fertility treatment.There are several key findings from the current survey of fertility specialists in 8 countries. There is consensus among the current survey results, the annual CDC ART registry report, and the annual ESHRE ART report that the number of infertility patients seeking fertility treatment is increasing and it is expected that the number of patients will continue to increase due to aging populations , 16. TheART outcome was reported to be the most successful in Spain, the US, and China. These countries also reported that PGD/PGS was one of the most important fertility treatment improvements in the past year, and this could potentially explain why these countries have better outcomes results. However, many other dimensions that have an effect on the outcomes of fertility treatment should be considered. Thus, the highest success rates in Spain, the US, and China cannot be driven only by the use of PGD/PGS. The number of embryos transferred, the use of fresh versus frozen embryos, as well as other environmental and psychological elements not measured in the current survey can also have an effect on clinical outcomes. The main unmet need reported by fertility specialists is better coverage for the cost of IVF. Fertility treatment is a highly technological field and is very costly, indicating it may be difficult to address this unmet need. Because ART is not fully reimbursed in the studied countries -- with the exception of France -- it is up to the leadership of the clinics to reduce the costs of the procedures.In terms of future development, the fertility specialists reported that improved embryo selection and implantation rates are expected to change the field of fertility treatment. There appears to be little room for improvement in the hormonal treatment used to produce eggs and several techniques are available to produce embryos, including IVF, ICSI, IMSI, and donor eggs. With the advent of time-lapse monitoring of embryo development and PGD/PGS procedures, embryo selection is one of the newest areas of development in fertility treatment. However, these procedures are approved in only a few countries and have raised some ethical discussions in most. The factors underlying the implantation rate of embryos are not well understood, specifically, there is a need to better understand why good embryos do not successfully implant. Finally, the survey did not attempt to find the root causes of infertility in order to move away from palliating the consequences of infertility and toward the prevention and best treatment of infertility.The current study survey results compare favorably with and further extend the findings within the most recently reported CDC ART National Summary Report . The CDCThe comparison of the current study survey results among European countries, China, and Japan with existing registry data is more challenging. The ESHRE registry report (2010) is based on 104 fertility clinics in France, 114 clinics in Germany, 202 clinics in Italy, 103 clinics in Spain, and 72 clinics in the UK . The curIt should be noted that this research has a number of limitations. As with any survey, our findings may be influenced by the recall and response bias of the surveyed individuals. Additionally, only a subset of fertility specialists participated in our survey, and as with all analyses, caution should be used when generalizing results to an entire population. However, the response rate obtained in our survey is in line with what would be expected for this type of survey . RegardiThe survey results provide a unique and comprehensive interim look at the fertility treatment field ahead of the next annual release of the ESHRE and CDC ART publications. The survey results identified a trend toward more frequent single embryo transfer, most likely due to improved identification of viable embryos resulting in an increased chance of pregnancy per embryo transfer. The survey also highlights differences and similarities among countries practicing fertility treatment. As reported by the fertility specialists who participated in the survey, the key important avenues that are expected to change in fertility treatment in the future are the improvement of embryo selection and the improvement of implantation rate.


Hyperuricemia is a common finding in chronic kidney disease due to decreased uric acid clearance. The role of uric acid as a risk factor for chronic kidney disease has been largely debated, and recent studies suggested a role of uric acid in the causation and progression of kidney fibrosis, a final common pathway in chronic kidney disease. Uric acid and xanthine oxidase may contribute to kidney fibrosis mainly by inducing inflammation, endothelial dysfunction, oxidative stress, and activation of the renin-angiotensin system. Besides, hyperuricemia induces alterations in renal hemodynamics via afferent arteriolopathy and contributes to the onset and progression of kidney fibrosis. Xanthine oxidase inhibitors may prevent kidney damage via lowering uric acid and/or inhibiting xanthine oxidase. However, there is still no sufficient evidence from interventional clinical researches supporting the causal relationship between uric acid and kidney fibrosis. The effect and role of xanthine oxidase inhibitors in preventing kidney fibrosis and chronic kidney disease progression must be further explored by performing future large scale clinical trials. Regardless of the underlying etiology, most forms of chronic kidney disease (CKD) are characterized by progressive fibrosis as a final common pathway, which eventually affects all substructures of the kidney leading to a final consequence of end-stage renal disease. Although there has been a great deal of research, a comprehensive understanding of the pathogenetic mechanisms of kidney fibrosis remains uncertain and this hampers the development of effective therapeutic strategies .Uric acid (UA) is the final breakdown product of purine degradation in humans, and elevated serum UA level, hyperuricemia, is causative in gout and urolithiasis due to the formation and deposition of monosodium urate crystals. Hyperuricemia is a common finding in CKD due to decreased UA clearance. Its role as a risk factor for CKD progression has been largely debated, and it was primarily considered as a marker or epiphenomenon of kidney damage , 3. HoweIn this review, after providing a brief background concerning UA physiopathology, we will focus on the mechanistic role of UA in kidney fibrosis. We will also review the role of XO and the effect of XO inhibitors in preventing kidney fibrosis and their associated mechanisms.Cell turnover leads to the production of adenosine, inosine, and guanosine. They degrade to hypoxanthine and xanthine, which are the substrates for the widely distributed XO in the formation of UA. XO catalyzes the oxidation of purine substrates, xanthine and hypoxanthine, producing both UA and reactive oxygen species (ROS). Thus, XO is one of the major enzymatic sources of ROS. Allopurinol and febuxostat are inhibitors of XO, and they reduce uric acid and ROS formation  10]..10].UA is the oxidation end-product of purine metabolism in humans and higher primates. Most other mammals, except for the Dalmatian dogs, can degrade UA further to water-soluble allantoin with the enzyme uricase, and as a result serum urate levels are about 10% of those in humans , 11. HowUrates are the ionized form of UA, and, at a physiologic pH of 7.4, over 95% of UA dissociates into urates, with 98% existing as monosodium urate. The serum urate level depends on dietary purines, the breakdown of endogenous purines, and the renal and intestinal excretion of urate. Hyperuricemia is defined as the accumulation of serum UA beyond its solubility point in water (6.8 mg/dL), and it develops due to UA overproduction, undersecretion, or both . The domAllopurinol, a purine inhibitor of XO, has been conventionally used for urate-lowering therapy to inhibit UA synthesis. A novel urate-lowering drug, febuxostat, is a potent nonpurine selective inhibitor of XO, and it inhibits both the reduced and oxidized forms of the enzyme in contrast to allopurinol that inhibits the reduced form of the enzyme only , 17. FebStudying the role of UA in kidney fibrosis, a process which eventually leads to CKD, is very difficult since uric acid is excreted primarily by the kidney, and hence a decrease in the glomerular filtration rate (GFR) is inevitably accompanied by a rise in the serum UA level. As such, studies in experimental animals in which serum UA can be modulated are critical to understand whether there is a role for UA in the causation or progression of CKD .In the past, hyperuricemia was thought to cause CKD, the so-called urate nephropathy, by the deposition of urate crystals in the renal interstitium. This results in a chronic inflammatory response and in progressive tubulointerstitial injury in a similar manner as seen with tophi in gouty arthritis . HoweverGenerating hyperuricemia in laboratory animals proved to be difficult due to the fact that most mammals have the uricase enzyme. Rodent models in which the uricase gene was knocked out showed renal failure due to extensive tubular crystal deposition and finally death . An alteAfter the year 2001, when the hyperuricemic rat model was developed by using OA  and untiα), fibrotic (TGF-β), and oxidative (HO-1) markers, along with pathologically confirmed kidney fibrosis. Lowering of UA levels with allopurinol reversed the kidney damage, suggesting that UA played a major role in the pathogenesis of kidney fibrosis [In another rat model with dietary intake of adenine, which may be the source of the UA as a purine base, adenine-fed rats showed hyperuricemia , 31. Adefibrosis . AnotherBased on the growing evidence that lowering UA levels with allopurinol prevented renal injury induced by hyperuricemia, the role of febuxostat in preventing kidney injury was investigated. In OA-induced hyperuricemic rats, febuxostat lowered UA levels and ameliorated systemic and glomerular hypertension as well as preglomerular arteriolopathy. In normal rats without hyperuricemia, febuxostat tended to lower UA levels and had no effect on blood pressure, glomerular pressure, and afferent arteriole morphology .There are some experimental studies that demonstrated the relationship between UA and renal hemodynamics. In one study, hyperuricemic rats fed OA not only developed systemic hypertension but also glomerular hypertension. Hyperuricemic rats showed increased glomerular capillary pressure with afferent arteriole thickening. Allopurinol prevented hyperuricemia, systemic and glomerular hypertension, and arteriolopathy. Glomerular capillary pressure and arteriolar thickening correlated with serum UA and systolic blood pressure. This study suggested that glomerular hypertension might be mediated by insufficient vasoconstriction of the afferent arteriole to systemic hypertension, allowing the transmission of systemic pressure to the glomerular capillary tuft . In anotHyperuricemia also contributed to renal progression in animal models of CKD. In 5/6 nephrectomy remnant kidney model, remnant kidney rats fed OA showed more severe renal failure, proteinuria, and histologic findings  compared to remnant kidney rats without hyperuricemia. Allopurinol reduced serum UA levels and prevented the functional and histologic changes in remnant kidney rats fed OA. This study also demonstrated that hyperuricemia accelerated renal progression by increasing renin expression in the renal cortex and cyclooxygenase-2 (COX-2) expression in the afferent arteriole. In particular, this study showed that increased COX-2 expression induced by hyperuricemia was associated with proliferation of vascular smooth muscle cells in preglomerular arteries . In anotβ1-induced profibrogenic progression in the mice, suggesting that lowering serum UA may be an effective therapeutic intervention to prevent the progression of diabetic nephropathy [Hyperuricemia has also been known to exacerbate renal injury in some animal disease models including cyclosporine (CsA) and diabetic nephropathy. Hyperuricemia frequently complicated CsA therapy. In one study using a model of CsA nephropathy, the rats developed hyperuricemia with arteriolar hyalinosis, tubular injury, and interstitial fibrosis. CsA nephropathy rats fed OA showed higher UA levels with more severe histologic findings compared to CsA nephropathy rats that were not treated with OA. This study also demonstrated that the mechanism did not involve intrarenal urate crystal deposition and appeared to involve activation of RAS and inhibition of intrarenal NO production . In anothropathy . Homo sapiens. This hypothesis was based on in vitro experiments which showed that UA is a powerful scavenger of singlet oxygen, peroxyl radicals, and hydroxyl radicals. UA circulating at an elevated level was proposed to be one of the major antioxidants of the plasma that protects cells from oxidative damage, thereby contributing to an increase in life span of human species and decreasing the risk of cancer [ Homo sapiens, a high level of UA is strongly associated with and in many cases predicts development of hypertension, visceral obesity, insulin resistance, dyslipidemia, diabetes, kidney disease, and cardiovascular and cerebrovascular events [While UA has been reported to be a potent antioxidant in the extracellular fluid , it has f cancer . On the r events . Antioxir events . Major sr events , 45, livr events , and hear events .2−/NO3−), systemic hypertension, renal vasoconstriction, and preglomerular arteriolopathy. Chronic administration of L-arginine, a substrate for endothelial NO synthase, increased the urinary excretion of NO2−/NO3− and preserved arteriolar structures probably mediated by the antiproliferative effect of NO on vascular smooth muscle cells, suggesting the role of endothelial dysfunction as a mediator of renal injury induced by hyperuricemia [In OA-induced hyperuricemic rat model, hyperuricemia caused intrarenal oxidative stress, increased expression of NOX-4 subunit of renal NADPH oxidase and angiotensin II, and decreased NO bioavailability. Hyperuricemic rats also showed systemic hypertension, renal vasoconstriction, and arteriolopathy. Tempol (superoxide scavenger) attenuated the adverse effect induced by hyperuricemia despite equivalent hyperuricemia, suggesting that UA might cause oxidative stress . In anoturicemia . A recenuricemia . An expeuricemia .κB and cytoplasmic phospholipase A2 [UA induces inflammatory responses. Hyperuricemia-induced inflammatory response mediates kidney injury via alteration of vascular and tubular cells in kidney. UA has the ability to induce monocyte chemoattractant protein- (MCP-) 1 in vascular smooth muscle cells, suggesting that it may have a role in the vascular changes associated with hypertension and vascular disease . UA alsoipase A2 . Hyperuripase A2 . UA contipase A2 . in vivo in mice [XO has been reported to be upregulated by various inflammatory stimuli such as lipopolysaccharide (LPS), hypoxia, and cytokines –60. Augm in mice .In the last decade, epithelial-mesenchymal transition (EMT), a process by which fully differentiated epithelial cells lose their epithelial characteristics and undergo phenotypic conversion to mesenchymal cells, has emerged as an important pathway leading to generation of matrix-producing fibroblasts and myofibroblasts in kidney fibrosis. In addition to kidney fibrosis, EMT has been known to play a pivotal role in embryonic development, wound healing, tissue regeneration, and cancer progression , 68.α-smooth muscle actin (α-SMA). Allopurinol significantly inhibited UA-induced changes in E-cadherin and α-SMA with an amelioration of kidney fibrosis at 6 weeks. In cultured rat renal tubular epithelial cells (NRK cells), UA induced EMT, which was blocked by the organic acid transport inhibitor, probenecid. UA increased expression of transcriptional factors associated with decreased synthesis of E-cadherin. UA also increased the degradation of E-cadherin via ubiquitination, which is of importance since downregulation of E-cadherin is considered to be a triggering mechanism for EMT. This study suggested that UA-induced EMT of renal tubular cells might be a novel mechanism explaining the association of hyperuricemia and renal progression after taking into account that EMT is an early phenomenon in kidney fibrosis [A recent study showed that UA exerted a direct effect on renal tubular cells by inducing EMT . OA-indufibrosis –71. FurtThe underlying mechanisms by which UA could cause kidney fibrosis have been reported in animal models since 2001, and there has been a growing interest in this topic and numerous retrospective and prospective observational studies have been performed to assess the relationship between UA and CKD. The majority of data support the role of UA as a cause or exacerbating factor for kidney fibrosis and progressive CKD. Although, over the last several years, some clinical intervention trials including randomized controlled trials (RCTs) have further supported this mechanistic role of UA –78, clinWith regard to the animal models in this research field, the uricase inhibitor OA-induced hyperuricemic rat model –29 was mFebuxostat, a chemically engineered nonpurine selective inhibitor of XO, received approval in February 2009 from the Food and Drug Administration for the chronic management of hyperuricemia in patients with gout . AlthougAt present, drug therapy for asymptomatic hyperuricemia is not actively recommended, and this negative approach is mainly due to the absence of evidence from adequately powered RCTs on the causality between hyperuricemia and the onset or progression of CKD. Considering the large sample size required for an adequately powered trial, an international collaboration is necessary.β, phosphatidylinositide-3-kinase, and chemokines to microRNAs, are undergoing clinical trials [Current research interests focus on developing new effective antifibrotic drugs to slow progression or even reverse chronic kidney injury, and several compounds which target various components of the fibrotic pathway, from signaling molecules that include TGF-l trials , 83. AltAfter taking into account the results of all the important experimental studies mentioned above, UA and XO may contribute to kidney fibrosis mainly by inducing inflammation, endothelial dysfunction, oxidative stress, and activation of RAS . Besides


Galvanic vestibular stimulation (GVS) is a low-cost and safe exam that tests the vestibulospinal pathway. HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a slowly progressive disease that precociously affects the vestibulospinal tract. This study compared electromyographic (EMG) responses triggered by GVS between asymptomatic individuals infected with HTLV-1 and those with HAM/TSP.Bipolar galvanic stimuli of 400 ms and 2 mA intensity were applied on the mastoid processes in 120 trials. The EMG response, and its short latency (SL) and middle latency (ML) components, was recorded from both soleus muscles in 13 healthy, HTLV-1-negative adults , and 26 individuals infected with HTLV-1, 13 asymptomatic (56 ± 8 years) and 13 with HAM/TSP (60 ± 6 years).The average value of EMG components in the group of healthy individuals was 55 ± 4ms (SL) and 112 ± 10ms (ML); in the group of asymptomatic HTLV-1 carriers, 61 ± 6ms (SL) and 112 ± 10ms (ML); and in the group with HAM/TSP, 67 ± 8ms (SL) and 130 ± 3ms (ML) (P=0.001). In the HTLV-1-asymptomatic group, the SL component was delayed in 4/13 (31%) exams and the ML component was normal in all. In the HAM/TSP group, the most common alteration was the absence of waves.A pattern of abnormal vestibular-evoked EMG response was found in the HTLV-1-neurological disease, which ranged from delayed latency among asymptomatic carriers to absence of response in HAM/TSP. GVS may contribute to early diagnosis and monitoring of non-traumatic myelopathies.


Drosophila melanogaster has been a popular model to study cAMP signaling and resultant behaviors due to its powerful genetic approaches. All molecular components  essential for cAMP signaling have been identified in the fly. Among them, adenylyl cyclase (AC) gene rutabaga and phosphodiesterase (PDE) gene dunce have been intensively studied to understand the role of cAMP signaling. Interestingly, these two mutant genes were originally identified on the basis of associative learning deficits. This commentary summarizes findings on the role of cAMP in Drosophila neuronal excitability, synaptic plasticity and memory. It mainly focuses on two distinct mechanisms  regulating excitatory and inhibitory synaptic plasticity related to cAMP homeostasis. This dual regulatory role of cAMP is to increase the strength of excitatory neural circuits on one hand, but to act locally on postsynaptic GABA receptors to decrease inhibitory synaptic plasticity on the other. Thus the action of cAMP could result in a global increase in the neural circuit excitability and memory. Implications of this cAMP signaling related to drug discovery for neural diseases are also described.The fruit fly  Since its discovery in 1958 , the cycMany isoforms of ACs and PDEs have been identified in the nervous system, indicating critical roles of cAMP in neural function . Indeed,Aplysia, it was demonstrated that cAMP signaling mediates short- and long-term facilitation (LTF) at sensorimotor synapses  at excitatory glutamatergic synapses  essential for the cAMP signaling pathway. Drosophila AC gene rutabaga and PDE gene dunce have been intensively studied to understand the role of cAMP signaling in the nervous system. These two mutant genes were originally identified on the basis of associative learning deficits (dunce and rutabaga) with defects in cAMP signaling  regulating excitatory and inhibitory synaptic transmission related to cAMP homeostasis. At the end, I comment on implications of this research on disease therapy.In this mini review, I summarize the findings on the role of cAMP in Aplysia sensory neuron, cAMP increased spike duration and excitability . In contrast to mammalian CNS, acetylcholine is the primary excitatory NT in Drosophila CNS  in the mossy fibers and CA3 pyramidal cells in hippocampal slices . The cAMchannels . Studieslitation , which ccurrents  or by accurrents . We have neurons . Presyna neurons . Further neurons . All thes Figure .Drosophila nervous system. This increase was observed not only at excitatory but also inhibitory synapses. This can be contradictory because an increase in inhibitory synaptic transmission by cAMP is expected to reduce overall strength of excitatory synaptic transmission in certain neural circuits. Therefore, we further examined the role of cAMP signaling in inhibitory GABAergic synaptic transmission in Drosophila  receptor subunit is widely expressed in several regions of the Drosophila brain  increases neuronal excitability and presynaptic release of NTs in osophila . When poosophila . This cola brain  and its la brain . Based oosophila , the actosophila .Drosophila brain has shown the presence of a large number of inhibitory GABAergic interneurons, some of which innervate into the MB  are involved in the transduction of US signals   . Differebe, etc) . The synbe, etc) . Since rrutabaga can be easily explained as its basal cAMP level is too low so that no neural input signal can induce proper cAMP-dependent synaptic plasticity mediating learning memory. Interestingly, dunce mutants with high levels of cAMP in MB neurons also show defects in short-term memory  and thus reduces the effects of cAMP. However, this remains to be explored in Drosophila nervous system. Taken together, all these findings strongly suggest that disruption of cellular cAMP homeostasis can alter excitatory cholinergic and inhibitory GABAergic synaptic plasticity and hence cause defects in associative learning, although the underlying mechanisms leading to this effect can be different .Learning and memory defects in m memory . Furthersynapses . Severalm memory . Althoug defects . TherefoDrosophila can be directly implicated in mammalian systems including human.Cyclic AMP is the most abundant and important second messenger in the nervous system. Therefore, it makes sense that its signaling involves a variety of physiological and pathological processes such as learning and memory, pain, drug addiction and neurodegeneration. Molecular components in cAMP-PKA signaling pathway should be excellent targets for the development of new therapeutic strategies. These components in cAMP signaling are very well conserved throughout animal groups and, therefore, findings from www.dartneuroscience.com). Since an increase of cAMP-specific PDE mRNAs was observed in early stages of AD  and mental retardation. Given the importance of cAMP signaling in neural plasticity and cognition, any molecular component in this signaling pathway can be a potential target for drug development to enhance cognition. Particularly two enzymes have drawn more attention. The first target is a PDE which down-regulates cAMP-PKA signaling. Therefore, PDE inhibitors will prolong cAMP signaling and produce higher levels potentially enhancing cognition. Indeed, a PDE4 inhibitor HT-0712 has been shown to improve hippocampus-mediated memory in mice . HT-0712es of AD , PDE inhrutabaga fly mutants are consistent as these flies showed defects in synaptic plasticity and short-term memory. Therefore, drugs that stimulate AC activities can be useful to treat memory deficits although no drug is on a clinical test yet. AC is an attractive drug target to be further explored.The second promising drug target for cAMP signaling is AC. AC1/AC8 double knockout mice showed loss of LTP as well as memory . FurtherDrosophila has been used as a favorable model animal to study actions of addictive drugs such as cocaine and alcohol . Rubinstein-Taybi syndrome (RTS), a genetic disorder showing mental retardation and physical abnormalities  is known alcohol . A recenstration  demonstrGiven the suitability of multiple drug targets in cAMP signaling, one important question is the therapeutic safety in addition to drug potency. In other words, how can we deliver a drug to a specific target in order to minimize toxic side effects? Chemogenetic tools have been developed to enhance specific drug delivery spatially and temporally . One sucDrosophila, dual regulatory roles  of cAMP signaling are to increase the strength of excitatory neural circuits on one hand, but to act locally on postsynaptic GABA receptors to decrease inhibitory synaptic plasticity on the other. Thus the action of cAMP could result in a global increase in the neural circuit excitability and memory. The cAMP signaling is also implicated in pain processing, AD and drug addiction. All these normal and disease-related behaviors are mediated through interaction between specific neural circuits comprised of excitatory and inhibitory synapses. Therefore, knowledge gained from the studies of cAMP signaling can contribute to the development of new or more effective drugs. Since molecular components and functions of cAMP signaling pathway have been well conserved in Drosophila, it could be a useful animal model to study mechanisms underlying behaviors mediated by cAMP signaling at the molecular, physiological and circuit levels. The fly can also be an excellent drug discovery platform for diseases with defects in cAMP signaling.In this commentary, I focused on the role of cAMP signaling in neural excitability, synaptic plasticity, learning and memory. On the basis of work from The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


HTLV-3 and HTLV-4 are two recently identified viruses closely related to HTLV-1, which have not yet been linked to any diseases. Similarly to HTLV-1 and HTLV-2, which both express antisense transcript-derived HBZ and APH-2, respectively, HTLV-3 and HTLV-4 harbor antisense genes termed APH-3 and APH-4. These two encoded proteins contain an atypical bZIP domain but are still able to down-regulate Tax-mediated LTR activation, alike HBZ and APH-2. Since HBZ and APH-2 affect Jun-dependent transcription differently, our goal was to assess the impact of APH-3 and APH-4 on the transactivation potential of Jun family members. Co-IP experiments first showed that both APH-3 and APH-4 interacted with all tested Jun members. Jun/APH complexes significantly enhanced Jun-dependent transactivation of the human collagenase promoter containing a single AP-1 binding site as well as a construct bearing a minimal promoter and AP-1-binding sites. APH-3 and APH-4 deletion mutants and point mutations of specific leucine residues demonstrated that this functional interaction was mediated by the bZIP-like domain. Using a construct with a minimal promoter bearing GAL4-binding sites and APH-3/APH-4 expression vectors fused to the GAL4 DNA binding domain, we further showed that, in contrast to HBZ, APH-3 and APH-4 did not contain an activation domain. These results highlight the varying capacity of different HTLV-encoded antisense proteins to act upon transactivation mediated by Jun transcription factors. Taking into account the previously reported association between HBZ and ATL development, determining the functional differences of these HTLV antisense proteins could contribute in better understanding this association.


S-glutathionylation for ROS, and S-nitrosylation for NO. These changes may alter the activity, stability, and interaction with other molecules or subcellular location of proteins, changing the entire cell dynamics and contributing to the maintenance of homeostasis. However, despite the recent advances about the roles of ROS and NO in signaling cascades, many challenges remain, and future studies focusing on the signaling of these molecules in planta are still necessary.The natural environment of plants is composed of a complex set of abiotic stresses and their ability to respond to these stresses is highly flexible and finely balanced through the interaction between signaling molecules. In this review, we highlight the integrated action between reactive oxygen species (ROS) and reactive nitrogen species (RNS), particularly nitric oxide (NO), involved in the acclimation to different abiotic stresses. Under stressful conditions, the biosynthesis transport and the metabolism of ROS and NO influence plant response mechanisms. The enzymes involved in ROS and NO synthesis and scavenging can be found in different cells compartments and their temporal and spatial locations are determinant for signaling mechanisms. Both ROS and NO are involved in long distances signaling (ROS wave and GSNO transport), promoting an acquired systemic acclimation to abiotic stresses. The mechanisms of abiotic stresses response triggered by ROS and NO involve some general steps, as the enhancement of antioxidant systems, but also stress-specific mechanisms, according to the stress type , and demand the interaction with other signaling molecules, such as MAPK, plant hormones, and calcium. The transduction of ROS and NO bioactivity involves post-translational modifications of proteins, particularly  A typical plant cell has more than 30,000 genes and a large number of proteins, many of which are still unknown, and these proteins may have their activity and/or function altered by several types of post-translational modifications . Therefo2O2) and the hydroxyl radical . ROS have distinct biological properties, a short half-life and high chemical reactivity   . NO is aTable 1) . Due to Table 1) . CurrentTable 1) .Nitric oxide and ROS are involved in and interact with each other in a wide range of cellular processes, which include response to abiotic stresses , defenseFigure 1), and their temporal and spatial localization is critical for signaling . NADPH oxidases are integral membrane proteins that promote the transfer of cytoplasmic NADPH electrons to extracellular oxygen, forming apoplast . Severalapoplast . Other oapoplast . In addiion SAA; . SAA enaion SAA; .2O2 as a result of their metabolic activity, which involves processes such as photorespiration, the glyoxylate cycle, and β-oxidation  or signal detection by apoplastic proteins and membrane receptors  is especially important because it catalyzes the removal of to water . In combto water , once thto water .Figure 1), which can be divided into reductive pathways, including the action of xanthine oxidoreductase in the peroxisomes , relatively stable molecules in solution that may act as reservoirs of NO (S-nitrosylation of glutathione (GSH) by NO and can be transported in the phloem, thus contributing to the transport of this signaling molecule over long distances, which plays an important role in SAA  and maintains in its active form through a mechanism that removes electrons from NR and alters its activity. Another class of hemoglobins, the non-symbiotic hemoglobins (nsHb), particularly those belonging to the GLB1 class, have also been reported to have NO dioxygenase activity and to promote the degradation of NO in certain circumstances, such as hypoxia , is also regarded as a mechanism of NO elimination and involves the modulation of mitochondrial activity , the product of the reaction between NO and S-nitrosylation, which consists of NO binding to cysteine residues in target proteins, is apparently the principal mechanism for the transduction of the NO bioactivity. S-nitrosylation can also occur via trans-nitrosylation, that is, by the transfer of NO from an S-nitrosylated residue to another thiol group through the action of low-molecular weight nitrosothiols, such as GSNO , which may be important for the inactivation of this regulatory protein after the initial response of plants to stress , reversible or irreversible, including S-glutathionylation, disulfide bond formation, and sulfhydration   . The exaigure 2) . Heavy migure 2) , while iigure 2) . Interesigure 2) . In addiigure 2) .According to their concentrations, ROS and NO can cause oxidative/nitrosative stress in cells or may act as signaling molecules. At low concentrations, NO contributes to increased tolerance of the plants to metals in various ways, for example, by promoting metal binding to the cell wall, preventing their entry into the cell  or promoFigure 2)  . Droughtigure 2) . The metigure 2)  and xantigure 2) . The genigure 2) , which cn of ROS . In paran of ROS .Arabidopsis thaliana   .Arabidopsis (Atnoa1) which show deficiency in NO synthesis and hypersensitivity to salt stress   . Similarigure 2) . Howeverigure 2) . In factl stress .2O2, has been shown to be involved in signal transduction that culminates in increased expression of heat shock genes, which encode proteins that play critical roles in the maintenance of cellular homeostasis in stressful conditions  showed lower concentrations of NO and increased susceptibility to cold stress  and non-enzymatic antioxidant mechanisms , cyclic nucleotides, plant hormones, and mitogen-activated protein kinases . The MAPK cascades, for example, are activated by ROS, NO, and hormones, representing a convergence point for these signaling molecules. MAPK signal transduction involves a phosphorylation cascade comprising MAPK kinase kinase (MAPKKK), MAPK kinase (MAPKK), and finally, MAPK, which is activated after phosphorylations of threonine and tyrosine residues in the conserved motif T-X-Y . In these organisms, NO binding to a soluble guanylate cyclase increases the activity of this enzyme up to 200 times, with a consequent increase in the formation of cGMP . In planactivity . In Chlapathways . Two difproteome . In addiproteome , ROS andproteome .2+ is a highly versatile signaling molecule that plays a central role in the response to environmental stressors , phosphatases regulated by Ca2+ and by changes in Ca2+ channels and pumps . GG wrote the introduction and the topic “Interaction among ROS, NO, and Other Signaling Molecules.” JO outlined the manuscript together with FF, guided and led the discussions and made a critical review of all the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Drosophila, the vertebrate and mammalian equivalent of the pathway has been studied extensively for its role in cancer development and progression. As this pathway regulates key target genes involved in development, its action also allows for the modulation of the microenvironment to prepare a tumor-suitable niche by manipulating tumor cell growth, differentiation, and immune regulation, thus creating an enabling environment for progression and metastasis. In this review, we will summarize recent scientific discoveries reporting the impact of the Hedgehog signaling pathway on the tumor initiation process and metastatic cascade, shedding light on the ability of the tumor to take over a mechanism crucially intended for development and normal function.Cancer poses a serious health problem in society and is increasingly surpassing cardiovascular disease as the leading cause of mortality in the United States. Current therapeutic strategies for cancer are extreme and harsh to patients and often have limited success; the danger of cancer is intensified as it metastasizes to secondary locations such as lung, bone, and liver, posing a dire threat to patient treatment and survival. Hedgehog signaling is an important pathway for normal development. Initially identified in  In order for tumor initiation and metastasis to occur, tumors usurp several signaling pathways that contribute to normal growth and tissue repair. The normal development of mammalian embryos involves multiple signaling pathways that regulate cell proliferation, survival, and differentiation such as the Hedgehog (Hh) , Wnt 2]2], TGF-βHh signaling involves a complex network of molecules. The canonical Hh signaling pathway can be initiated by three ligands: Desert hedgehog (DHH), Indian hedgehog (IHH) or Sonic hedgehog (SHH). These bind to the 12-pass transmembrane protein receptors Patched1 (PTCH1) and Patched 2 (PTCH2), which were initially found to form a complex with single pass co-receptor proteins: Cell adhesion molecule-related/downregulated by oncogenes (CDON) and Brother of Cdo (BOC) , 12. TheIn the nucleus, the GLI transcription factors bind DNA and modulate the transcription of a multitude of genes including Fox, Myc, and cyclin D among others that are involved in tissue development, differentiation, epithelial-mesenchymal transition (EMT), and stem cell maintenance. Recently, GLI1 protein transcript was found to undergo alternative splicing establishing two isoforms with different functions: an N-terminal deletion and a truncated GLI1 variant . The trunon-classically as a result of crosstalk between diverse intracellular signaling networks.Although the Hh signaling pathway is normally activated upon ligand binding to the receptor, multiple mechanisms have been involved in enhancing GLI protein activity in the absence of Hh ligands. In breast cancer, TGF-β signaling was shown to activate GLI2 target genes culminating in enhanced bone metastasis . In basaThe Hh signaling pathway must be strictly controlled in order to prevent aberrant activation. The Hh signaling pathway can be activated by four different mechanisms. And though the downstream effects of Hh activation are ultimately proliferation and differentiation, it is notable that its mode of activation differs depending on the cancer type , 27.The Hh signaling pathway can be activated classically through ligand binding or non-classically in the absence of the ligand , both ofThe non-classical mode of activation is, as mentioned above, independent of Hh ligand presence, which occurs irrespective of PTCH receptors loss of function or gain of function of SMO; this mode is found in BCC , and canThe first identified naturally occurring Hh blocker is cyclopamine, which causes developmental abnormalities in animals. The Beachy group first reported the inhibitory effect of cyclopamine on Hh signaling to occur by directly binding and inhibiting SMO . HoweverThe first FDA approved Hh inhibitor for clinical use is vismodegib (Genentech) marketed as Erivedge . ApproveCurrently, there are several Hh inhibitors employed in clinical trials for multiple types of cancers. For example, sonidegib is being tested on patients with advanced hepatocellular carcinoma  and in cIn the ensuing paragraphs, we will summarize recent scientific discoveries reporting the impact of the Hh signaling pathway on the tumor initiation process and metastatic cascade, shedding light on the ability of tumors to takeover a mechanism crucially intended for development and normal function. We have specifically focused on the recent advances and knowledge garnered to address novel tumor properties that pertain to creating a favorable microenvironment known as the updated hallmarks of cancer as described by Hanahan and Weinberg Fig. .Fig. 2ThThe most pronounced characteristic of cancer is the unrestricted proliferation of cells. As the main function of Hh signaling is to promote fetal development and patterning, it is expected that signaling through the pathway would upregulate genes involved in cellular proliferation and survival, a function that tumors manipulate. Not only does Hh signaling promote the over-proliferation of cancer cells themselves, but it also modulates cells found in the local microenvironment. Wilkinson and colleagues found that human prostate cancer associated fibroblasts (CAFs) have the capability of forming primary cilia; this was associated with upregulation of canonical Hh signaling . AdditioCancer by definition is the uncontrolled replication of cells. Cancers successfully employ telomeric activation to maintain maximum replication; this is supported by the Hh pathway’s ability to upregulate telomerase activity. As the normal function of Hh signaling pathway is to promote embryonic development, it essentially controls and influences cellular division allowing its implication in various cancers as a driver for telomerase activity. The telomerase subunit is made up of telomerase reverse transcriptase (TERT) and telomerase RNA component, both of which make up the complex that drives cell replication . RecentlIt is widely accepted that tumors adapt to hypoxic environments and survive by upregulating genes that promote anaerobic metabolism such as hypoxia-inducible factor-1α (HIF-1α) ; this isHelicobacter pylori-driven gastritis that progressed to gastric metaplasia, Gli1 was found to drive myeloid cell differentiation and upregulation of IL-6, IL-1β, and TNFα, three important cytokines for activating STAT3 signaling, the main promoter of the inflammatory response [Cancer has long been associated with inflammatory responses , 83. Infresponse . These eresponse . In gastThe formation of new limbs as development progresses requires the creation of blood vessels through the induction of pro-angiogenic factors such as vascular endothelial growth factors (VEGF) and fibroblast growth factors (FGF). Both VEGF and FGF are direct transcriptional targets of the Hh signaling pathway . In an iAlthough the majority of published literature largely associates Hh signaling with increased angiogenesis, several reports have emerged in pancreatic cancer models showing increased vascularization enhancing drug delivery upon the inhibition of paracrine Hh signaling in pancreatic stroma using SMO inhibitors –96. ThusAlthough the human immune system is fully capable of eliminating foreign matter and abnormal cells, cancer cells evade the immune system through various mechanisms . Cancer One of the most important mechanisms by which cancers support their growth and immune system evasion is through avoiding apoptosis , 108. ApThe ability of tumor cells to metastasize is permitted through the flexibility of the cells to alter their shape and function, break through the basement membrane, extravasate into lymph and blood vessels, and travel to distant locations where they occupy a distinct microenvironment , 115. SeHh signaling and cancer stem cells: Given the role of Hh signaling during development when there is a dynamic shift between cellular phenotypes from epithelial and mesenchymal type, it comes as no surprise that this is recapitulated in cancer progression [gression . Stainingression . A more gression . Likewisgression . p63, a gression . Also ingression . Also ingression . The invThe progression of cells through the cell cycle in order to undergo replication requires the successful advancement through many checkpoints which would suppress growth if cells are experiencing chemical and physical stress or DNA damage. Cancer cells though defective in structure and function, employ different signaling pathways, including the Hh pathway, to bypass those regulatory checkpoints . AberranA possible mechanism by which tumors can evade cell growth suppression other than cell cycle checkpoints is their ability to regulate autophagy. Autophagy is the process by which cells get degraded and their components recycled as a result of cellular stress or infections . In streIn addition to the impact of Hh signaling on profoundly influencing several cancer processes, it also desensitizes cancer to therapies. In breast cancer, the OPN oncoprotein was shown to activate non-classical Hh signaling and GLI transcription, resulting in the upregulation of multidrug resistance genes viz. ATP-binding cassette transporters (ABCB1) and ATP-binding cassette sub-family G member 2 (ABCG2). Treatment of cells with GLI inhibitor GANT-61 or silencing GLI1 expression abrogated this effect . In prosHh signaling also desensitizes tumor cells to radiotherapy. In hepatocellular carcinoma, SHH-driven signaling induced PTCH1 and Gli1 expression; this protected cells against ionizing radiation (IR) . The useIt is evident from the presented compilation of literature that the Hh signaling pathway is extensively studied and significantly linked to several cancer types. Aberrations in almost all molecular members of the pathway have been reported across multiple malignancies. The pathway’s ability to promote replicative immortality, evade genomic instability and growth suppression, sustain proliferative signaling, and switch cellular energetics are crucial for the tumor cells’ ability to grow uncontrollably, avoiding multiple levels of homeostatic cellular regulation that prevent damaged cells from surviving. As the tumors establish in their niche, they employ Hh signaling to induce angiogenesis, take advantage of tumor promoting inflammation and escape immune system surveillance to facilitate tissue invasion and metastasis.Derris glabrescens plant, Glabrescione B is among the newly identified, naturally occurring small molecule inhibitor, functioning in a similar mechanism to GLI inhibitors GANT-58 and 61 [In closing, Hh signaling is activated across multiple cancer types in many different ways: through mutations or through non-classical ligand-independent or through non-canonical modes. Hh signaling influences every aspect of a tumor cell: initiation, establishment and its metastatic journey. With the promising success of inhibitors of this pathway in the clinic, the pathway continues to offer the scope to direct and design inhibitors to function at multiple levels. At present, a large cadre of chemically modified pharmacological inhibitors has been developed to target SMO. In order to pursue the elimination of the rapidly-evolving tumor and its associated milieu, it is necessary to continue efforts to target this pathway at all levels. Avenues to target and block the activity of ligands, block co-receptors Boc, Cdo, and Gas1 and interfere with the activity of GLI transcription factors are currently being explored by many laboratories. Discovered in the 8 and 61 . The rec8 and 61 . With th


Di-(2-ethylhexyl) phthalate (DEHP), a commonly used plasticizer, has an adjuvant effect in combination with ovalbumin (OVA). The adjuvant effect of DEHP has already been verified in our previous studies. In this study, to further investigate whether thymic stromal lymphopoietin (TSLP) was involved in the DEHP-adjuvant effect, DEHP was administered through a daily gavage exposure route. Mice were sensitized with ovalbumin (OVA) to trigger allergic responses, and an anti-TSLP monoclonal antibody was used to neutralize the effect of TSLP. Biomarkers including cytokines in bronchoalveolar lavage fluid (BALF), serum total IgE and TSLP content in the lung were detected. In addition, airway hyperreactivity and lung sections were examined. Collectively, these data indicated a salient Th2 response which was characterized by the upregulation of Th2-type cytokines, such as interleukin 4 (IL-4), IL-5 and IL-13. Moreover, the eosinophil number in BALF and the eosinophil cationic protein (ECP) in the lung were seen to have increased significantly. However, neutralization of TSLP with an anti-TSLP mAb reversed the adjuvant effect of DEHP on airway inflammation, structural alterations in the airway wall and increased airway hyperresponsiveness (AHR) to methacholine induced by the OVA allergen, suggesting that TSLP was an effective target site for suppressing the adjuvant effect of DEHP co-exposure. In recent years, reports of about the illegal use of the phthalate plasticizer, di-(2-ethylhexyl) phthalate (DEHP), have raised concerns among medical institutions, regulatory agencies and the public. DEHP is widely used as a plasticizer in polyvinyl chloride from which it can leach and thence be absorbed by the human body. DEHP exposure is associated with the development of wheezing and allergic airway diseases, and has been shown to contribute to asthma occurrence in Sweden –2. Addit+ OVA T cells [Thymic stromal lymphopoietin (TSLP) is first isolated from the supernatant of a murine thymic stromal cell line, initially identified as a pre-B-cell . As an a T cells . Neutral T cells .Diisononyl phthalate (DINP) has similar physical and chemical properties as DEHP, and can aggravate AD-like skin lesions via a TSLP-mediated pathway . DibutylAllergic asthma is a common chronic disease that occurs in the bronchial epithelium, and its representative symptoms include lung inflammation, airway hyperresponsiveness (AHR) and mucus overproduction . EvidencIn this study, a chronic OVA-induced mouse model of asthma was used to investigate the role of TSLP in the DEHP-adjuvant effect on the coallergen (OVA). Local treatment by anti-TSLP mAb was used to block the effect of TSLP, which was the link between DEHP co-exposure and the on-going adjuvant effect on allergic inflammation.All experimental procedures were approved by the Office of Scientific Research Management of Central China Normal University, and the Application for the Use of Animals was certified on Nov. 8, 2011  . AccordiSPF male Balb/c mice (5–6 weeks) were purchased from the Hubei experimental animal centre  and housed in an environment with 12-hour light-dark cycle, 20–25°C and 50–70% humidity. OVA-free food and tap water were offered ad libitum. Prior to administration, the mice were quarantined for at least a week.2A by intraperitoneal (i.p.) injection (20 μg per mouse) (IgG control group). Briefly, mice were sensitized with saline or 50 μg OVA plus 1.75 mg Al(OH)3 in 300 μl saline on day 20 (by i.p. injection), day 34, day 41 and day 48 (by subcutaneous injection). For the TSLP blocking experiment, the mice received 20 μg of anti-TSLP monoclonal antibody (mAb)  or the isotype control IgG2A via i.p. injection  60 minutes prior to OVA sensitization, the dose of anti-TSLP mAb being the same as that in a previous study [The schematic diagram of the treatment schedule is shown in us study . All micBlood samples were collected by heart puncture. After centrifugation  the serum was collected and stored at -70°C before analysis. The lowest detection limit of total IgE was 0.156 ng/ml, and the sensitivities of OVA-specific IgE and OVA-specific IgG1 assays were both 0.1 ng/ml. All operations were conducted according to the manufacturer’s instructions.Cytokine levels were detected using ELISA kits  according to the kits’ instructions. The lowest detection limit of IL-4 and IFN-γ were both 15 pg/ml, and the sensitivities of IL-5 and IL-13 were 5 pg/ml and 4 pg/ml respectively.Lungs were lavaged three times with 0.5 ml, 0.6 ml and 0.7 ml ice-cold saline using a tracheal cannula and a 1 ml polyethylene syringe. The volumes of qualified samples were 1.3 to 1.5 ml. The supernatant of BALF samples was collected after centrifugation  and stored  for analysis. The pellets of BALF samples were then resuspended with cold sterile saline, and counted using a haematology analyser . The number of eosinophils (EOS) and the ratio of neutrophils (NEUT) in BALF were recorded.i.p. injection. When the mice showed no response to a pin prick acupuncture on their feet, the skin of the neck was carefully incised, the trachea was intubated, and quickly connected to a small computer-controlled ventilator via the tracheal cannula; According to the manufacturers’ instructions of the AniRes2005 lung function system , the ratio of expiration/inspiration was preset at 1.5:1 and the actual respiratory rate was preset at 90 breaths/min. While the volume of air used in the mechanical ventilator was 8 ml/kg, and for 20 g mouse the volume air was 0.16 ml per breath. The AniRes2005 software was then run. When all the parameters were stable, methacholine  was injected into the jugular vein at doses of 0.025, 0.05, 0.1 and 0.2 mg/kg successively every 5 minutes using the injector needle through the fixed tracheal cannula. The software recorded the inspiratory and expiratory resistance R-areas (Ri and Re) and the lung dynamic compliance (Cldyn). The R-area (100 Pa·ml-1) represents the area between the peak curve of Ri or Re. The baseline level was recorded during the 250s time period after MCH entered the vein, while the lowest value of Cldyn (ml·1000 Pa-1) was analysed to quantitatively assess lung compliance [The airway hyperresponsiveness (AHR) measurement was conducted 24 h after the final aerosol challenge using an AniRes2005 lung function system  according to the manufacturer’s instructions. Mice were anesthetised with pentobarbital sodium (Sigma) at a dose of 100 mg/kg by mpliance .Lungs were excised from the thoracic cavity, inflated, fixed with 4% neutral buffered polyformaldehyde and then cut into slices. Tissues were embedded in paraffin and stained with hematoxylin&eosin (H&E), Periodic acid-Schiff (PAS), or Masson’s trichrome (MT). Pictures were taken using a DM 4000B Microscope (Leica), and analyzed with Image-Pro Plus 6.0 to determine the average optical density (AOI) of the PAS and MT stained sections.TSLP levels in lung and BALF were measured with the mouse TSLP ELISA kit . According to the manufacturers’ instructions of eBioscience mouse TSLP ELISA kit, the functional minimum sensitivity is 16 pg/ml and the standard curve range is 16–2000 pg/ml.p-value of less than 0.05 was considered to be a significant difference.Analysis of variance (ANOVA) was used for statistical analysis. The data were presented as the mean ± standard error of the mean (SEM). One-way analysis of variance (one-way ANOVA) was used to determine the levels of difference between groups. A p<0.01) (p<0.01). Compared with the DEHP+OVA group, we observed that neutralization of anti-TSLP mAb significantly inhibited the elevation of total IgE (p<0.01), OVA-sIgE (p<0.01) and OVA-sIgG1 (p<0.01)  . It was (p<0.01) .p<0.05 or p<0.01), the DEHP+OVA group (p<0.01), the anti-TSLP group (p<0.05 or p<0.01) and the IgG control group (p<0.01) (p<0.01) in the DEHP+OVA group when compared to the OVA group. Furthermore, pre-treatment with anti-TSLP mAb diminished the increase of IL-4, IL-5 and IL-13 concentrations in BALF (p<0.01), as well as there being an apparent increase in the ratios of IFN-γ and IL-4 in BALF (p<0.01) in the anti-TSLP group when compared to the DEHP+OVA group  and the ratio of Th1 cytokine (IFN-γ) and IL-4 in BALF were detected using ELISA kits to assess the nature of the lymphocyte cytokine response to DEHP and/or OVA . IL-4, I(p<0.01) . In addiVA group .+ cells into the airway, we counted the number of inflammatory cells in BALF. The data on cell count demonstrated that blocking TSLP with anti-TSLP mAb effectively eliminated the DEHP-adjuvant effect on elevated eosinophil counts and on the percentage of neutrophils in BALF . ECP levels rose significantly in the DEHP+OVA group (p<0.05) in comparsion to the OVA group  between the DEHP+OVA group and OVA group. This case indicated that 10 mg/kg bw/day DEHP co-administration promoted AHR in OVA-sensitized mice, but pre-administration of anti-TSLP mAb effectively nullified this adjuvant effect, so values of Ri, Re and Cldyn in anti-TSLP group all changed significantly (p<0.05 or p<0.01), compared with that in DEHP+OVA group.To assess the change in AHR, we compared airway responses to MCH in different groups . In all As shown in p<0.01) in comparison to that in the saline group (p<0.05). Neutralization by anti-TSLP mAb significantly decreased PAS-positive cell count in the airway, when compared with that seen in the DEHP+OVA group (p<0.01)  .p<0.01) (p<0.05). Anti-TSLP mAb pre-administration decreased the degree of the collagen layer under the lamina propria in comparison to the DEHP+OVA group (p<0.05)  stained lung tissues . The colp<0.01) . The mor(p<0.05) .Several key findings were illustrated in the histological analysis of lung tissue slices stained with H&E, PAS or MT : (1) Thep<0.01 or p<0.05). The significant elevated TSLP levels in the DEHP+OVA group compared to the OVA group was one of the effects formed by DEHP co-exposure with OVA sensitization, which was so-called DEHP-adjuvant effect. Moreover, the TSLP levels in lung and BALF were both lower in the anti-TSLP group than in the DEHP+OVA group (p<0.05 or p<0.01).As the data in In this study, we concentrated on the possible association between TSLP and the hypersensitive reaction in a DEHP oral exposure mouse asthma model, and tried to discover the mechanisms relating to these DEHP immunoadjuvant effects.Recent animal studies have demonstrated that DEHP may promote and aggravate allergic asthma by an adjuvant effect –30. PrevTSLP anchors at the epithelial cells and acts as a key switch to trigger Th2 responses in allergic asthma, usually primarily produced by the damaged epithelial cells . Diverseet al [The typical markers of allergic asthma are significantly elevated IgE and enhanced airway hyperresponsiveness –43. In tet al . In addiet al . AHR is et al . When AHet al . DEHP, aet al , 48. HowThe adaptive immunity for allergic asthma is initiated when the allergen interacts with airway epithelia, so the bronchial epithelial cells play a vital role in the early stages of allergic asthma. In the present mouse asthma model, the allergen OVA induced the epithelial cells to secrete TSLP, when there was DEHP co-exposure with OVA, the levels of TSLP in pulmonary tissues and BALF were significantly increased. Excessive TSLP activated more dendritic cells and thus facilitated the priming and recruitment of more Th2 cells to the lung, and more Th2 cytokines and more allergen specific IgE antibody and IgG1 were produced Figs  and 8. TNotably, there were several special designs in the present study. First, the OVA group was set as the positive control facilitating the detection of potential DEHP-adjuvant effects, and the DEHP+OVA group was used as another positive control for detecting the effect of the anti-TSLP mAb neutralization in comparison to the anti-TSLP group. Second, subcutaneous administration, intraperitoneal injection, and inhalation exposure are the more effective methods for inducing the adjuvant effect of DEHP than oral exposure . EspeciaTaken together, our study demonstrates that DEHP at a dose of 10 mg/kg bw/day has immunoadjuvant effects on airway inflammation induced by OVA allergen, and TSLP is an effective target site for suppressing the adjuvant effect of DEHP co-exposure.S1 Fig(JPG)Click here for additional data file.


HTLV-1 is associated with two immunologically distinct diseases: HTLV-1-associated myelopathy/tropical spastic paraparesis and adult T cell leukemia. The genesis of these diseases is believed to be associated with the route (mucosa versus blood) and mode (cell-free versus cell-associated) of primary infection as well as the modulation of dendritic cell (DC) functions. To explore the role of DCs during early HTLV-1 infection in vivo, we used a chimeric HTLV-1 with a replaced envelope gene from Moloney murine leukemia virus to allow HTLV-1 to fuse with murine cells, which are generally not susceptible to infection with human retroviruses. We also used a CD11c-diphtheria toxin receptor transgenic mouse model system that permits conditional transient depletion of CD11c(+) DCs. We infected these transgenic mice with HTLV-1 using both cell-free and cell-associated infection routes in the absence and presence of DCs. The ablation of DCs led to an enhanced susceptibility to infection with cell-free but not cell-associated HTLV-1 in both CD4 and non-CD4 fractions, as measured by the proviral load. Infection with cell-free virus in the absence of DCs was also found to have increased levels of Tax mRNA in the non-CD4 fraction. Moreover, depletion of DCs significantly dampened the cellular immune response against both cell-free and cell-associated virus. These results uniquely differentiate the involvement of DCs in early cell-free versus late cell-associated infection of HTLV-1 and highlight a significant aspect of viral immunopathogenesis related to the progression of adult T cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis after the initial infection.


To know the options for fertility preservation in women with cervix, ovarian or endometrial cancer.To understand the role and limitations of imaging in selection of patients for fertility preserving options.To recognise the appearances of early stage disease that fulfil the criteria for eligibility for fertility preservation.A significant number of women with gynaecological cancer are of childbearing age and have not completed their families. In these younger women, consideration of fertility-sparing options is very important. This poster will describe the fertility-sparing treatment options that are currently available and delineate the role of imaging in the selection of suitable patients. A review of the current literature will be presented to provide an evidence base. In cervix cancer, MRI is used to delineate the size, position and stage of the tumour for the selection of patients suitable for radical trachelectomy. In complex adnexal masses, MRI with diffusion and perfusion is used to categorise the likelihood of invasive or borderline malignancy in order to allow potential fertility preservation where possible. In endometrial cancer, MRI is used to rule out imaging signs of invasive disease prior to consideration of hormonal treatment. Imaging is also used in patient follow-up for the detection of recurrent disease. Each of these scenarios will be illustrated.Imaging plays a major role in the management of patients who are considering fertility-preserving treatment options for gynaecologic malignancy.


Ursolic acid (UA), a triterpenoid compound, is reported to have a glucose-lowering effect. However, the mechanisms are not fully understood. Adipose tissue is one of peripheral tissues that collectively control the circulating glucose levels.The objective of the present study was to determine the effect and further the mechanism of action of UA in adipocytes.The 3T3-L1 preadipocytes were induced to differentiate and treated with different concentrations of UA. NBD-fluorescent glucose was used as the tracer to measure glucose uptake and Western blotting used to determine the expression and activity of proteins involved in glucose transport. It was found that 2.5, 5 and 10 µM of UA promoted glucose uptake in a dose-dependent manner . 10 µM UA-induced glucose uptake with insulin stimulation was completely blocked by the phosphatidylinositol (PI) 3-kinase (PI3K) inhibitor wortmannin (1 µM), but not by SB203580 (10 µM), the inhibitor of mitogen-activated protein kinase (MAPK), or compound C (2.5 µM), the inhibitor of AMP-activated kinase (AMPK) inhibitor. Furthmore, the downstream protein activities of the PI3K pathway, phosphoinositide-dependent kinase (PDK) and phosphoinositide-dependent serine/threoninekinase (AKT) were increased by 10 µM of UA in the presence of insulin. Interestingly, the activity of AS160 and protein kinase C (PKC) and the expression of glucose transporter 4 (GLUT4) were stimulated by 10 µM of UA under either the basal or insulin-stimulated status. Moreover, the translocation of GLUT4 from cytoplasm to cell membrane was increased by UA but decreased when the PI3K inhibitor was applied.Our results suggest that UA stimulates glucose uptake in 3T3-L1 adipocytes through the PI3K pathway, providing important information regarding the mechanism of action of UA for its anti-diabetic effect. The prevalence of diabetes has dramatically increased and results in a considerably higher rate of mortality worldwide. Insulin resistance has been considered as a well-known metabolic disorder of diabetes, which is closely related with serious complications such as cardiovascular and kidney diseases Although some effective therapeutic drugs have been developed and used for many years to treat diabetes, most of these drugs produce undesirable or severe side effects, such as induction of fat accumulation Ursolic acid (UA) is a natural pentacyclic triterpenoid and present in many different plants, fruits and herbs. Accumulating evidence has shown that UA possesses multiple nutritional and pharmacological functions Ursolic acid, cytochalasin B, insulin, 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, wortmannin, SB203580, compound C, protease inhibitor and bovine serum albumin (BSA) were purchased from Sigma . High glucose Dulbecco’s modified Eagle’s medium (DMEM) was from Mediatech, Inc. . Fetal bovine serum (FBS) was bought from PAA Laboratories . Bovine calf serum (BCS) was purchased from Cayman Chemical Company . The BCA protein assay kit was obtained from Thermo Scientific . RIPA lysis buffer was from Millpore . Protein loading buffer was from Bio-Rad . Antibodies against phospho-phosphoinositide-dependent kinase (pPDK), phosphoinositide-dependent kinase (PDK), phospho-protein kinase C (PKC), protein kinase C (PKC), phospho-AS160 (pAS160), AS160, GLUT4, glucose transporter 1 (GLUT1), phospho-phosphoinositide-dependent serine/threonine kinase (pAKT), phosphoinositide-dependent serine/threonine kinase (AKT) and clathrin were from Cell Signaling Technology, Inc. . 2-NBD-glucose was purchased from Invitrogen Life Technologies . BM chemiluminescence blotting substrate kit was from Roche Diagnosis .3T3-L1 mouse embryo fibroblasts were obtained from American Type Culture Collection  and cultured in DMEM containing 10% BCS until confluent, and were then maintained in the same medium for additional 2 d. The cells were then induced to differentiate according to the method reported previously After differentiation, 3T3-L1 adipocytes were cultured in the presence of 2.5 to 50 µM of UA for 24, 48 and 72 h, respectively. At each time point, the cells were treated with MTT assay reagents (1 mg/mL) for 4 h and the resulting formazan was solubilized in 150 µL dimethyl sulfoxide (DMSO) and further diluted 10 times with DMSO. The absorbance was read at 570 nm on a Varioskan Flash spectral scanning multimode plate reader .2PO4, 1.2 mM MgCl2, 2.3 mM CaCl2, 4.8 mM NaHCO3, 10 mM HEPES and 0.3% BSA) containing 100 µM NBD-glucose, 2.8 mM glucose and different concentrations of UA for 2 h at 37°C in the presence or absence of 1 µg/mL insulin. After 2 h incubation, the reaction was terminated by 20 µM of cytochalasin B. The cells were washed twice with cold Krebs Ringer bicarbonate buffer (4°C), and then 100 uL cold PBS (4°C) were added to each well. The fluorescence intensity was immediately measured at 466/550 nm on a Varioskan Flash spectral scanning multimode plate reader . For the inhibition experiments, the cells were pretreated with PI3K inhibitor wortmannin (1 µM), MAPK inhibitor SB203580 (10 µM), and AMPK inhibitor compound C (2.5 µM), respectively, for 30 min.3T3-L1 preadipocytes were cultured in 96-well plates and induced to differentiate for 8 d. The cells were then starved overnight in DMEM with 0.1% BSA and further incubated in Krebs Ringer bicarbonate buffer .The statistical analyses were performed using SPSS 13.0 statistical program . The treatment effect was determined using one-way ANOVA and followed by a post-hoc Dunnett's or Bonferroni's multiple comparisons test, where a P value less than 0.05 was considered significant. Data are presented as means ± SD.The MTT assay results revealed that UA at concentrations up to 35 µM did not affect cell viability after 24 or 48 h of incubation . *P<0.05 and **P<0.01 vs. the control of 0 µM UA; CC indicates 2.5 µM of coumpound C and SB indicates 10 µM of SB203580.(TIF)Click here for additional data file.