To evaluate the survival benefit of radiotherapy (RT) in esophageal cancer (EC) patients aged ≥ 80.Records for all EC patients aged ≥ 65 years were extracted from the Surveillance, Epidemiology, and End Results database. Chi-square test compared the characteristic and treatment between patients aged ≥ 80 with those aged 65–79. Focusing on patients aged ≥ 80, we employed multivariable logistic regression to identify the association between selection of RT and patients’ characteristics. Survival curve was employed to visualize the survival rate and multivariable Cox proportional hazard model was established to quantify the effect of RT on overall survival (OS) and cancer special survival (CSS).P < 0.001). Selection of RT in patients aged ≥ 80 were associated with cancer histology (P < 0.001), grade (P = 0.024) and stage (P < 0.001). RT significantly improved the OS (hazard ratio(HR) = 0.717) and CSS (HR = 0.722) . Further stratified analysis found the improvement were only significant in the localized  and regional stage patients  .Patients aged ≥ 80 were more likely to be white male and have localized EC (all Our study suggested EC patients aged ≥ 80 benefit from RT only if the cancer is in localized/regional stage. Esophageal cancer (EC) is the one of the most common and fatal malignant tumor worldwide . It is eStudying the prognosis factors and treatment benefit of EC patients aged ≥ 80 may help patients and clinicians make decisions that are more appropriate. For example, radiotherapy (RT) constitutes one of the most important treatment for EC patients. However, the related adverse reactions of RT, such as pneumonia, cardiac toxicity, electrolyte imbalance, bone marrow suppression, pulmonary and even systemic infection, often staggers the choice of using it . A clearThe Surveillance, Epidemiology, and End Results (SEER) Program of the National Cancer Institute (NCI) provides an authoritative source of information about cancer incidence and survival in the United States. SEER currently collects and publishes cancer incidence and survival data from population-based cancer registries covering approximately 28% of the U.S. population. The SEER program is the only comprehensive source providing information about stage of the cancer at the time of diagnosis and patient survival data .In the present study, we conducted a retrospective population-based study for EC patients aged ≥ 80 using SEER database. We explored clinical characteristics, prognosis factors and efficacy of RT for EC patients aged ≥ 80. This study represents the first large sample size based descriptive analysis of EC patients aged ≥ 80.P < 0.001). Surgery treatment rate declined dramatically with age. Among EC patients aged ≥ 80 years, up to 83.9% patients had not been treated with surgery, while over two thirds of patient ultimately received RT. The median survival time (MST) of patients aged over 80 significantly shorter than those aged 65–79 . Same trend was also shown in 1, 3, 5-year overall survival rate.A total of 12,407 EC patients were enrolled in the present study, including 9,946 patients aged 65–79, and 2,461 patients aged ≥ 80 (19.8%). Characteristics of each age category were displayed in We investigated the survival rate, clinicopathologic characteristics including stage distribution and treatment status of EC patients aged ≥ 80 years at the different periods of time. Among EC patients aged ≥ 80 years diagnosed between 2004–2013, only 14.4% patients had been treated with surgery, while 64.0% patient ultimately received RT. The RT served as the main treatment for the patients all over the period. The MST and 1, 3, 5-year overall survival rate increased over the years. Detailed characteristics were presented in P < 0.001), grade (P = 0.024) and stage (P < 0.001). Patients with regional stage cancer were more likely to receive RT as compared to patients with localized , but a reverse trend was seen in distant stage (OR = 0.757).We further investigated possible characteristics associated with RT in patients aged ≥ 80. We removed the patients treated by surgery to exclude its interference. A total of 2,066 EC patients aged ≥ 80 were eligible for further analysis by multivariable logistic regression analysis and models were adjusted for potential confounders listed in Table P < 0.001). No significant association was found for histology and grade. Patients who received RT showed better prognosis compared to patients without RT .As shown in Figure P = 0.527) and CSS .To further specify the benefit in patient subgroup, stratified Cox regression analysis was conducted to evaluate the impact of RT on OS and CSS by stage, adjusting for year of diagnosis. As presented in Table Finally, we delineated the effect of RT on survival in EC patients aged ≥ 80 using stratified survival curves. As shown in Figure RT is the most important therapy for EC patients aged ≥ 80. However, the survival benefit of RT for this group has yet not been well evaluated. Using large national cohort data from SEER database, we compared the clinical and pathological characteristics of patients aged ≥ 80 to those aged 65–79. In addition, we also analyzed the prognostic factor as well as survival benefit of RT in EC patients aged ≥ 80. To the best of our knowledge, our study constitutes the first large database based observational study focusing on RT benefit of EC patients aged ≥ 80.Comparison of the clinical and pathological characteristics revealed several different characteristics for patients aged ≥ 80. As anticipated, the number and proportion of EC patients aged ≥ 80 increased significant across period. An interesting finding was, although most of EC patients aged ≥ 80 were still male, the female ratio had increased comparing with that of 65–79. This result was consistent with the previous report  that theRT and surgery are two main treatments strategies for EC, especially localized EC . AlthougOur survival analysis demonstrated the importance of stage information when choosing RT for EC patients aged ≥ 80. RT served as the standard treatment plan for localized EC , 17. PreOnly a very few number of EC patients aged ≥ 80 received surgery. To focus on evaluating the benefit of RT, we ruled out this group of patients. It should be noted that, due to the lack of medical information in the SEER database, we were unable to obtain details of chemotherapy. Potential chemotherapy may influence survival, which lead to biased results. However, patients aged ≥ 80 are mostly unlikely to tolerate chemotherapy , 20. We In conclusion, our study provided creditable evidence for characteristics and outcome for EC patients aged ≥ 80 based on large, population-based registry. Our study suggested that EC patients aged ≥ 80 benefit from RT only if the cancer is in localized/regional stage.Public SEER data from 1973 to 2013  was extracted for the study (reference number: 12420-Nov2015). The latest release of SEER*Stat Software (Version 8.3.2) was used to clean the raw SEER data [Patients diagnosed with primary EC and aged ≥ 65 years were eligible for analysis. EC cases were confirmed using the World Health Organization International Classification of Disease for Oncology 3rd Edition . Disease stages were defined by SEER historic stage criteria, including localized (invasive but confined to organ of origin), regional (extension beyond organ of origin but no distant metastasis), or distant (distant metastasis) . HistoloInclusion criteria contained: (1) diagnostic confirmation by positive histology; (2) active follow-up; (3) only one malignant primary indicator. Individuals were excluded with: (1) unknown age, sex, grade, race, SEER historic stage; (2) surgery status of “Recommended, unknown if performed”, “unknown”, “death certificate” or “autopsy only”; (3) RT of “Recommended, unknown if performed” or “Unknown”.Survival time was evaluated using evaluated OS and CSS. Survival data was extracted at 1-month intervals for a minimum follow-up of 4 months and a maximal follow-up of 60 months to minimize the effect of chemotherapy and exclude patients not survive long enough to receive cancer-directed therapy. OS was determined from the SEER records of survival time  and vital status. CSS was determined from the SEER cause-specific death classification variable and was evaluated from the date of diagnosis to the date of EC specific death.Enrolled patients were first divided into 2 subgroups by age: 65–79 and over 80. Patient’s characteristics and treatment were compared by Chi-square test. Subsequently, focusing on survival evaluation of RT, patients aged between 65–79 and patients who received surgery were ruled out from further analysis Figure . Multiva


The emergence and female numbers produced per P. elaeisis female were higher in A. gemmatalis pupae from caterpillars fed an artificial diet treated with glyphosate. However, emergence was lower than 50% when the caterpillars were fed on soybean leaves treated with glyphosate offered ad libitum (3–5 times). Palmistichus elaeisis died before parasiting A. gemmatalis pupae treated with malathion. Diflubenzuron reduced the P. elaesis sex ratio in the second generation. Tebuconazole and triflumuron did not cause side-effects on this parasitoid. A continuous exposure to glyphosate by the host may lead to side-effects on P. elaeisis emergence, but its moderate use is acceptable for this parasitoid. Diflubenzuron had severe transgenerational side-effects. Tebuconazole fungicide and triflumuron insecticide are compatible with P. elaeisis in sustainable integrated pest management (IPM) programs, while malathion can not be included in them.New plant protection strategies focus on minimizing chemical pesticide use and increasing their compatibility with biological control agents. The objective was to evaluate the side-effects of glyphosate, diflubenzuron, malathion, tebuconazole and triflumuron (at 720, 45, 400, 150 and 20 g ai ha However, pesticide applications may have direct or indirect negative effects on these non-target organisms3, reducing the strength of their ecological services4.Beneficial arthropods provide ecological services such as dung burial, pest control and pollination5, but generalist ones can contribute to the control of arthropod pests. In Brazil, management of soybean (Glycine max (L.) Merrill) pests includes biological control6. Palmistichus elaeisis Delvare and LaSalle, 1993 (Hymenoptera: Eulophidae) is a generalist and gregarious endoparasitoid of Coleoptera and Lepidoptera pupae7. This parasitoid is released to control Anticarsia gemmatalis Hubner (Lepidoptera: Noctuidae) in soybean crops8, however, pesticides are still widely used in this crop6.The significant contribution of parasitoids to pest control is due to their high host specificity9, which may disrupt their efficiency for biological control11. Organophosphates are broad-spectrum insecticides with high toxicity for non-target arthropods12; some herbicides have shown side-effects on different non-target organisms15. Fungicides are generally non-toxic for insects, but may have synergistic effects with other pesticides16; while growth regulator insecticides (GRI) are safer for biological control agents5, but can have lethal and sublethal effects on immature stages or on reproduction17.Natural enemies of insects are susceptible to pesticides18. The low toxicity of pesticides for non-target arthropods is an important component for sustainable integrated pest management (IPM)19. Parasitoids and predators can be exposed directly to pesticides during pesticide applications or, more commonly, indirectly by host hemolymph and contaminated prey tissues11. In this way, these insects remain exposed to sublethal doses for longer time periods than lethal doses19. The exposure to pesticides does not necessarily result in the death of natural enemies20, but their effects may comprise the development, longevity and fecundity of these insects, as well as behaviors involved in mobility, foraging for hosts (or prey) and mates17.New plant protection strategies focus on minimizing chemical pesticide use, and seek its compatibility with biological control agents21. Soybean rust (Phakopsora pachyrhizi Sid.) is a devastating foliar disease of this crop22, and the fungicide tebuconazole is widely used to control this disease. IPM programs established in soybean crops in Brazil, have allowed an over 50% reduction in insecticide use23. However, A. gemmatalis infestations sometimes require the application of insecticides. Among the most used are the GRI’s diflubenzuron and triflumuron, but when infestations are high density, contact insecticides such as malathion are applied23. Knowledge of the side-effects of these pesticides on P. elaeisis is scarce, and requires further research.In Brazil, glyphosate-resistant (GR) soybean varieties represent 86% of the cultivated area, making the use of this herbicide for weed management inevitableP. elaeisis fed with A. gemmatalis pupae from caterpillars exposed to these pesticides.The objective of this study was to evaluate the side-effects of diflubenzuron, glyphosate, malathion, tebuconazole and triflumuron, pesticides used for soybean crops, on the performance of Palmistichus elaeisis was exposed indirectly to pesticides, authorized for soybean cultivation in Brazil, through treated hosts. Anticarsia gemmatalis caterpillars were fed an artificial diet or soybean leaves treated with glyphosate and/or tebuconazole to obtain the pupae. Additionally, A. gemmatalis pupae from caterpillars fed only an artificial diet (non-treated) were immersed in different pesticide solutions . Glyphosate increased the number of parasitoids emerged and females produced per female with 164 and 25 individuals, respectively. This herbicide and tebuconazole did not affect the other P. elaeisis reproductive parameters (Table P. elaeisis (>80%) on A. gemmatalis pupae, from caterpillars fed with soybean leaves treated with glyphosate and tebuconazole, was similar to that of the control. However, the emergence of this parasitoid was lower than 50% from pupae in the treatment with soybean leaves treated with glyphosate. Palmistichus elaeisis emergence was lower from A. gemmatalis pupae whose caterpillars were fed with leaves from the GR soybean cultivar CD214-RR with or without glyphosate treatment. Anticarsia gemmatalis pupae fed with this cultivar also had the lowest weight, and the cephalic capsule width and longevity of P. elaeisis females was smaller than other treatments, possibly due to poor nutrition of this parasitoid with these smaller size pupae  with only six females per female being produced. This treatment presented the lowest sex ratio in the second P. eleasis generation , was a good food source for A. gemmatalis.Exposure to extreme environmental conditionsP. elaeisis from A. gemmatalis pupae, whose caterpillars were fed an artificial diet immersed in glyphosate and tebuconazole solutions, shows the safety of these pesticides for this parasitoid. The greater number of progeny of this parasitoid with glyphosate may be due to this herbicide stimulating oviposition. This side-effect of glyphosate was observed in Polyphagotarsonemus latus Banks (Acari: Tarsonemidae), and Tetranychus bastosi (Tuttle) Baker and Sales (Acari: Tetranychidae) when exposed at 360 g ae/ha14. Effects of this herbicide vary between species31, and had contrasting effects on foraging behavior of the predator spiders Tigrosa helluo Walckenaer and Pardosa milvina Hentz (Araneae: Lycosidae)13; caused early activation of anantioxidant defense in Drosophila melanogaster Meigen (Diptera: Drosophilidae)31; and severely reduced the reproduction and fecundity of Chrysoperla externa Hagen (Neuroptera: Chysopidae)15, but improved this parameter for P. latus and T. bastosi14. Effects of glyphosate-based herbicides could be mainly associated with chemicals  not specified on the label. Different glyphosate formulations decreased the parasitism and egg viability rates of Telenomus remus Nixon (Hymenoptera: Platygastridae) between 20 to 75%32. In addition, detrimental effects of glyphosate are more likely to occur over the long term with continuous use of this herbicide15. This explains the low elaeisis emergence from A. gemmatalis pupae, whose caterpillars were exposed to glyphosate each time that soybean leaves were offered ad libitum (3–5 times). Glyphosate use in the field is acceptable for the performance of this parasitoid, because applications of this herbicide for weed control are not frequent over short time periods.High parasitism and emergence rates of P. eleais agrees with findings from the “International Organization for Biological Control–West Palaearctic Regional Section (IOBC/WPRS)”-Working Group “Pesticides and Beneficial Organisms”, indicating that this fungicide is not harmful to parasitoids and predators33. In addition, this compound is an antifungal agent for rearing lepidopteran larvae on an artificial diet34. However, a synergistic effect of this fungicide with other pesticides16, can not be discounted.The lack of tebuconazole side-effects on the development, reproduction and longevity of P. elaeisis mortality by malathion contact or inhalation before parasitism showed the lethal impact of this broad-spectrum insecticide. Mortality by malathion is attributed to its rapid transformation to oxygenases enzymes, to malaoxon and isomalathion inhibiting the acetylcholinesterase and other enzymatic systems functioning in its detoxification12. Palmistichus elaeisis can not detoxify the malathion but this insecticide presents low specificity and may attract natural enemies35. Malathion did not cause mortality of A. gemmatalis pupae, possibly due to its rapid degradation36, and to the cuticular protection reducing insecticide uptake.Total P. elaeisis generation, and the parasitism and emergence of the second. However, diflubenzuron reduced the sex ratio of individuals from the second generation. This could be due to reduced sperm production or male sterility, since some hymenopterans have arrenotocal parthenogenesis where males develop from unfertilized eggs and females from fertilized ones37. In addition, this GRI can have ovicidal activity leading to a reduction in the reproduction rates of this parasitoid38. The possible sterility of P. elaeisis males from diflubenzuron exposure from the first generation onwards requires further investigation, because IGR side-effects are different depending on species39. The sex ratio and longevity of the parasitoid Tamarixia radiata Waterston (Hymenoptera: Eulophidae) was not affected with diflubenzuron40, but this insecticide had an indirect impact on the reproduction and population dynamics of the predator Podisus nigripinus Dallas (Hemiptera: Pentatomidae)38. Diflubenzuron and pyriproxyfen delayed the development from egg to pupa, emergence of adults and reduced the lifespan of Hyposoter didymator Thunberg (Hymenoptera: Ichneumonidae) females of the F2 generations, demonstrating the transgenerational effects of these GRI’s41. Chlorfluazuron inhibited testicular development and spermatogenesis of Spodoptera litura F. (Lepidoptera: Noctuidae) males42. Anagrus nilaparvatae Pang et Wang (Hymenoptera: Mymaridae) adults exposed to chlorfluazuron had no mortality, but its fertility and the longevity of females was reduced43. Evaluating the pesticide effects in more than one generation, provides an understanding of the unidentified effects in a first analysis4. Transgenerational effects caused by diflubenzuron on reproductive P. elaeisis parameters, even without exposing the second generation to the insecticide, strongly suggests that direct contact in the field could have lethal effects on this parasitoid, since first generation individuals were exposed only through indirect contact from a treated host.The GRI’s, diflubenzuron and triflumuron, did not affect the population dynamics of the first P. elaeisis development may be related to its action mechanism as a chitin synthesis inhibitor, but with little impact on beneficial insects, with low absorption through ingestion by these insects44. Triflumuron did not affect the sex ratio of the second generation of Trichogramma galloi Zucchi (Hymenoptera: Trichogrammatidae)5, and others GRI’s are also harmless for non-target arthropods such as spirotetramat for Chrysoperla carnea Stephens (Neuroptera: Chrysopidae) and Adalia bipunctata L. (Coleoptera: Coccinellidae) larvae and adults45; methoxyfenozide for several non-target arthropods2; and tebufenozide, hexaflumuron, and tebufenozide for Trichogramma species adults47.The low toxicity of triflumuron for P. elaeisis. Continuous exposure of the host to glyphosate may represent long-term risks, but a responsible use of this herbicide is acceptable. Diflubenzuron had transgenerational side-effects in the reproductive performance of the second P. elaeisis generation, even without exposing this parasitoid to the insecticide. Direct contact of diflubenzuron could have lethal effects on P. elaeisis. The fungicide tebuconazole and the insecticide triflumuron are compatible with P. elaeisis in IPM programs, and malathion can not be included in these.The herbicide glyphosate and the insecticide diflubenzuron reduced the performance of 48, with 125 g bean, 62.4 g beer yeast, 100 g wheat germ, 100 g soy protein, 50 g casein, 35 g agar, 5 g nipagin, 6 g ascorbic acid, 3 g sorbic acid, 6 mL formol at 40% in water, and 10 mL vitaminic solution. Second instar caterpillars were reared in 500 mL plastic containers, receiving soybean leaves ad libitum (3–5 times) or an artificial diet according to the treatments until pupae stage. Plastic pots were kept in an acclimatized chamber at 25 ± 2 °C, 70 ± 10% relative humidity and 14:10 h (light:dark) photoperiod.Caterpillars of the pest were obtained from the Laboratory of Biological Control of Insects (LCBI-BIOAGRO) at the Universidade Federal de Viçosa (UFV), where this insect is reared on an artificial dietAnticarsia gemmatalis pupae with 48–72 h-old  were exposed to P. elaeisis females for 24 h at a density of 6:1 females:pupae48, in the same rearing conditions as for A. gemmatalis. These new offspring were used in the experiments.Adults were kept in glass tubes (14.0 × 2.2 cm) with honey droplets as food source. The tubes were closed with a cotton plug. −1). These pots were kept in the greenhouse of the Soybean Breeding Program at the UFV in Viçosa, Minas Gerais, Brazil, and the plants were watered daily. Three plants with 3–4 true leaves were kept after germination and used from V6 to R6 phenological stages49.Two GR cultivars (CD212-RR and CD214-RR) and two conventional ones (CD201 and OC14) were used. The four cultivars were obtained from Coodetec . Five soybean seeds were planted per pot (3 L) with substrate (soil: organic matter) fertilized with ammonium sulphate  for 5 s, and offered to ad libitum (3–5 times) to A. gemmatalis caterpillars from the second to the fifth instar. Control leaves were immersed in water. Because this lepidopteran can have different preference levels for soybean cultivars as a source of food50, two GR cultivars (CD212-RR and CD214-RR) were used for glyphosate, and two conventional ones (CD201 and OC14) for tebuconazole, so that this factor does not alter the interpretation of our results.Soybean leaves were immersed in the corresponding pesticide solution for 5 s, and offered Anticarsia gemmatalis pupae from caterpillars fed an artificial diet were immersed quickly (5 s) in the different pesticide  solutions or water (control).17, therefore, performance of second P. elaeisis generation with GRI’s, diflubezuron and triflumuron, was studied. Anticarsia gemmatalis pupae from caterpillars fed only on an artificial diet were used, and were not submerged in insecticide solutions.Growth regulator insecticides are related to transgenerational side-effects on reproduction of non-target arthropodsA. gemmatalis pupae, obtained from caterpillars fed an artificial diet or soybean leaves treated with pesticides according to the respective treatments described previously, were exposed to 48–72 h-old P. elaeisis females for 48 h. Each experimental unit consisted of one pupa for every six P. elaeisis females  placed into glass tubes (14.0 × 2.2 cm). The experiments were conducted in a completely random design with 16 replications, and glass tubes were kept in an acclimatized chamber at 25 ± 2 °C, 70 ± 10% relative humidity with a 14:10 h (light:dark) photoperiod.In all experiments, 48–72 h-old P. elaeisis (egg to adult), parasitism percentage not considering natural host mortality51, emergence percentage of progeny, and the cephalic capsule width (mm) of the parasitoids emerged from each A. gemmatalis pupae were evaluated. The cephalic capsule was measured with a micrometric ocular in a stereoscopic microcopy. Sex ratio was calculated as Rs = female number/total parasitoid number. Individuals were sexed according to the antenna and abdomen morphological characteristics of this parasitoid52.Life cycle duration of Anticarsia gemmatalis pupae weight and P. elaeisis reproductive parameters were submitted to ANOVA. Statistical analysis was performed with Statistix software version 9.0 . The means were compared using Tukey’s test at 95% probability level when necessary. Palmistichus elaeisis parasitism and emergence (%) were submitted to non-parametric analysis at 95% probability.


Aim. The usefulness of photodynamic therapy (PDT) for treating sentinel lymph node (SLN) metastasis was evaluated. Materials and Methods. Verteporfin, a hydrophobic photosensitizer, forms a soluble aggregate with poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB). The concentrations of verteporfin were determined by measuring the fluorescence emitted at 700 nm. Seven days after the inoculation of A431 cells at the forearm of BALB/c nude mice, PMB-verteporfin was injected at dorsum manus and 75 J of light energy was delivered for 1 minute. Fifty-three mice were randomly assigned to the combination of PMB-verteporfin injection and light exposure, light exposure alone, PMB-verteporfin injection alone, and no treatment groups. Ten days after PDT, brachial lymph nodes, which were considered as SLNs, were harvested and evaluated. Results. The concentration of verteporfin in SLN was significantly higher than other organs. The combination of PMB-verteporfin injection and light exposure group significantly reduced the SLN metastasis (13%) comparing with no treatment group (52%), light exposure alone group (57%), and PMB-verteporfin injection alone group (46%). Conclusions. These data suggested that PDT using PMB as a nanotransporter of verteporfin could be a minimally invasive treatment of SLN metastasis in breast cancer and represent a potential alternative procedure to SLNB. Axillary lymph node dissection (ALND) has been integral part of breast cancer surgery since the description of the radical mastectomy . The ALNThe SLNB has become a gold standard procedure for axillary lymph node evaluation in clinically node-negative patients, and emerging data show that the survival benefits of the ALND may not be greater than the SLNB alone in patients with up to 2 positive SLNs –7. In otAlthough the SLNB is much less invasive comparing with the ALND, it is still associated with complications such as lymphedema, numbness, and pain –10. MoreA photodynamic therapy (PDT) involves the systemic or local administration of photosensitizer followed by its subsequent activation by broadband red light. In the presence of oxygen, the activated photosensitizer can generate reactive oxygen species that cause cell damage and ultimately cell death . Vertepoco-n-butyl methacrylate) (PMB) with 30 unit% of MPC units and molecular-weight below 5.0 × 104 can be dissolved in an aqueous medium and form stable polymer aggregates [A 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer has the same polar group (phosphorylcholine group) of phospholipids constructed as cell membranes and possesses excellent biocompatibility, that is, reduction of protein absorption and inhibition of platelet adhesion at the surface of the MPC polymer , 20. Thugregates   were obtained from the American Type Culture Collection . The A431 cell line with stable expression of green fluorescence protein (GFP) (A431-GFP cells) was obtained by transfection of pEGFP-N1  followed by G418 selection. The cells were maintained in DMEM supplemented with 10% heat-inactivated foetal bovine serum  in a humidified atmosphere of air containing 5.0% COAll animal experiments were conducted according to Keio University's institutional guidelines for the care and use of laboratory animals in research. BALB/c nude mice were purchased from Oriental Yeast Co., Ltd. . They were maintained under specific pathogen-free conditions in the Keio University Experimental Animal Center on a standard laboratory chow diet and had access to tap water ad libitum. Six-week-old female mice weighing 15 to 20 g were used in experiments.5 A431-GFP cells/50 μL at forearm of BALB/c nude mice. The brachial lymph nodes, which were considered as SLNs, were harvested after 7 days and examined by stereoscopic fluorescence microscope , on ice and stirred on a magnetic stirrer for 1.0 h at room temperature in order to evaporate dichloromethane. Finally, aqueous solution of verteporfin in PMB aggregate (PMB-verteporfin) was obtained.PMB was synthesized and purified as previously described. The composition of the MPC units and BMA units was 30 mol% and 70 mol%, respectively. Verteporfin was dissolved in dichloromethane at concentration of 100 mg/mL. In the meanwhile, the PMB was dissolved at concentration of 50 mg/mL in PBS. Then, 200 The diameter of each component was measured using a particle size analyser, Zetasizer nano .PMB-verteporfin (4 mg/mL) was administered as single bolus injections at each dorsum manus of 12 mice, to give a dose of 0.2 mg/body. One hour later, organ samples including SLN, lung, liver, kidney, and brachial skin were harvested from each mice, weighed, and lyophilized. N,N-dimethylformamide was added to each freeze-dried samples, which were then homogenized using a MagNA Lyser  at 6,500 rpm for 30 sec and centrifuged to extract verteporfin. The concentration of verteporfin was calculated from the fluorescence emitted at 700 nm (excitation at 430 nm) using the microplate reader, Synergy 4 Multimode .2 for a total treatment time of 1 minute and irradiance ranged from 0.18 to 0.76 W/cm2. During irradiation the temperature was kept at 20°C. After 10 days from PDT, the SLNs were harvested and evaluated by stereoscopic fluorescence microscope. The microscopic image of SLN treated with PDT revealed a small amount of nuclear disruption  using an Optical Fuel laser . Q-band excitation was established at this wavelength. The light dose was 75 J/cmsruption .t-test. The inhibitory effect on SLN metastasis was analysed in Mann-Whitney U test using SPSS PASW Statistics 18. The p value of <0.05 was defined as being statistically significant.The concentration of verteporfin was given as means ± standard deviation. The SPSS PASW Statistics 18  was used for analyzing the difference of concentration using unpaired Student's μg/g tissue in SLNs. Concentrations of verteporfin in lung, liver, kidney, and brachial skin of mice were 0.38 ± 0.38 μg/g, 1.38 ± 0.93 μg/g, 0.26 ± 0.06 μg/g, and not detected, respectively . The injection site of PMB-verteporfin did not show any damage including inflammation or ulceration.The concentration of verteporfin in SLNs was significantly higher than other organs (p < 0.05) (p < 0.05).The combination of the PMB-verteporfin injection and light exposure group significantly reduced the SLN metastasis comparing with no treatment group  . InhibitIn this study, the PMB efficiently delivered verteporfin to the SLN via subcutaneous injection and the PDT using the PMB as a nanotransporter of verteporfin revealed inhibitory effect against SLN metastasis.The PDT is still not widely used for cancer treatment because of the limited specificity of the photosensitizers for cancerous tissue. In other words, healthy noncancerous tissue may also be damaged by photosensitizers following irradiation. One of the major complications of the PDT was cutaneous phototoxicity because photosensitizers might accumulate in normal tissue such as skin. After the infusion of the photosensitizers, patients are required to avoid direct sunlight for a period of 7–10 days on average , 23. TheSeveral studies have been conducted on the correlation between the identification rate of the SLN and the size of radioisotope colloids and found that small-sized (200–400 nm) colloids were superior to regular-sized (400–1000 nm) colloids both in the detection rate by lymphoscintigram and the intraoperative identification rate of the SLNs , 24. The3 to 150 mm3 [Among several photosensitizers, the verteporfin was picked up in our study because its activation wavelength (689 nm) is longer than other systemic PSs. Light with long wavelength can penetrate tissue deeply. Light with longer wavelength is necessary to reach SLN and activate PSs . The tre 150 mm3 . Moreove 150 mm3 .Another advantage of the PDT might be repeated treatment. Although radiotherapy (RT) also could be useful to eradicate microscopic disease in axillary lymph nodes, RT usually could be given to patients only once. However, the PDT could be given to patients several times because of favourable toxicity profile.Axillary lymph node status has traditionally been a guide to decide adjuvant treatment. However, current guidelines support differences in adjuvant systemic treatment based on the intrinsic subtype and multigene assay rather than the number of positive lymph nodes . AccordiThe results of this study should be interpreted with caution. First, only one cancer cell line was used. Further studies using other cell lines would be valuable to confirm these findings. Second, fixed doses of verteporfin and light exposure were used. Although the doses used achieved good antitumor effects, dose-response studies would help to determine the optimum doses of both verteporfin and light exposure. Third, the lymph node metastasis rate of the murine SLN metastasis model in this study was 60%. Stable metastasis model with higher rate was necessary to validate the usefulness of PDT against SLN metastasis models.Taken together, these results indicate that PDT using PMB as a nanotransporter of hydrophobic verteporfin might be noninvasive treatment against the SLN metastasis. As the roll of the ALND for primary breast cancer has been considered to diminish over recent years, patients with up to 2 SLNs involvement could be treated with the SLNB alone. Although the SLNB is much less invasive procedure comparing with the ALND, it is still associated with complications such as lymph edema, numbness, and pain.The PDT using PMB-verteporfin could avoid the SLNB in most of clinically node-negative breast cancer patients.


To examine the association between lifetime alcohol consumption and significant liver disease in type 2 diabetic patients with NAFLD. A cross-sectional study assessing 151 patients with NAFLD at risk of clinically significant liver disease. NAFLD fibrosis severity was classified by transient elastography; liver stiffness measurements ≥8.2 kPa defined significant fibrosis. Lifetime drinking history classified patients into nondrinkers, light drinkers , and moderate drinkers (any period with intake >20 g/day).p = 0.008) and to be Caucasian (p = 0.007) and to have a history of cigarette smoking (p = 0.000), obstructive sleep apnea (p = 0.003), and self-reported depression (p = 0.003). Moderate drinkers required ≥3 hypoglycemic agents to maintain diabetic control (p = 0.041) and fibrate medication to lower blood triglyceride levels (p = 0.044). Compared to lifetime nondrinkers, light drinkers had 1.79  and moderate drinkers had 0.91  times the odds of having liver stiffness measurements ≥8.2 kPa . Compared with lifetime nondrinkers, light and moderate drinkers were more likely to be male ( In diabetic patients with NAFLD, light or moderate lifetime alcohol consumption was not significantly associated with liver fibrosis. The impact of lifetime alcohol intake on fibrosis progression and diabetic comorbidities, in particular obstructive sleep apnea and hypertriglyceridemia, requires further investigation. Analysis of a large T2DM cohort , are more likely to develop cirrhosis, and have a 2-3-fold increased risk of mortality from chronic liver disease . It is tIn diabetic patients with NAFLD who have an increased risk of nonalcoholic steatohepatitis (NASH) and fibrosis, the effect of modest alcohol consumption on fibrosis progression and patient outcomes remains unclear. In order to investigate this, we examined the association between lifetime alcohol consumption and liver stiffness, a noninvasive marker of liver fibrosis, in a cohort of diabetic patients at risk of clinically significant NAFLD. Additionally, we examined the association between alcohol consumption and other comorbidities and medication use.n = 96), or from primary care practices within the Metro South Hospital and Health Services District (n = 55), between October 2015 and November 2016.Consecutive patients with T2DM were offered participation in a cross-sectional study when they attended the Endocrine Clinic at the Princess Alexandra Hospital, Brisbane .Data were collected prospectively by the study clinician (PP). Firstly, medical history was obtained during a consultation in the liver clinic. Patients underwent a clinical assessment and were subsequently informed about their diagnosis.Medical history was obtained using a structured questionnaire. Interview items included self-reported sociodemographic characteristics, history of tobacco or recreational drug use, previously diagnosed liver disease, other medical conditions, and use of medications. Alcohol intake was assessed using a standardized questionnaire and the Alcohol Use Disorders Identification Test  (AUDIT).Clinical assessment included anthropometric measurements , laboratory tests , transient elastography, and liver ultrasound. Metabolic syndrome was defined as central obesity , plus any two of the following four factors: raised fasting plasma glucose or previously diagnosed type 2 diabetes, raised blood pressure or treatment of previously diagnosed hypertension, raised triglycerides or reduced HDL cholesterol, or specific treatment for these lipid abnormalities . ruling out disease with at least 90% sensitivity, as described by Cassinotto et al. [Transient elastography with simultaneous Controlled Attenuation Parameter (CAP) was performed after a 3-hour fast using FibroScan® technology  using the standard M or XL probes. Examinations were performed by a trained clinical nurse (with experience in performing more than 250 liver stiffness measurements (LSM)) and reviewed by a hepatologist (KAS) with extensive FibroScan experience (more than 2000 LSM performed). Recommended standard FibroScan operating procedures were followed along with adherence to criteria for definition of reliable LSM: minimum of 10 valid measurements with a success rate of ≥60% and IQR ≤30% of the final (median) result. The XL probe was used when the skin-capsule depth was ≥2.5 cm. Although optimal liver stiffness cut-off values in NAFLD remain under discussion, for the purposes of this study, we used the following cut-off values: 8.2 kPa for severe fibrosis and 10.5 kPa for the diagnosis of cirrhosis, respectively, foro et al. .Liver ultrasound was performed by one of the sonographers at the Department of Diagnostic Radiology, Princess Alexandra Hospital, Brisbane, trained in advanced liver imaging. The Princess Alexandra Hospital is the referral center for the state-wide liver transplant service, and over 1650 liver ultrasounds are performed in the department each year. Steatosis was determined by increased hepatic echogenicity and beam attenuation, resulting in the renal cortex appearing relatively hypoechoic to the liver parenchyma, absence of the normal echogenic walls of the portal vein, and poor visualization of the diaphragm and deep portions of the liver . Evidencn = 28) for clinical indications, based on increased likelihood of advanced disease or patient interest in participating in clinical therapy trials. Histological changes of NAFLD  were assessed and scored according to accepted criteria [NAFLD severity was broadly classified by transient elastography and liver ultrasound. Participants were categorized as “no advanced disease”  and “clinically significant liver disease” (LSM ≥ 8.2 kPa). Liver biopsy was performed in a subset of the patients  version 24.0 . Participants' sociodemographic and clinical characteristics were described using frequency distribution and percentage and mean and standard deviation . Pearson's chi-square tests were used to compare proportions (Fisher's exact test was used when cell counts were less than 5) and one-way ANOVA was used to compare means between groups. AllBivariable logistic regression analysis was used to determine the odds of being categorized as having “clinically significant liver disease” compared to “no advanced disease.” Obesity or girth, age, and gender, as variables of clinical relevance, were included in the model. Odds ratios (OR) and 95% confidence interval (CI) were reported.2 and mean girth of 120.8 ± 18.6 cm. The cohort included 46 lifetime nondrinkers, 70 light drinkers , and 35 moderate drinkers . Moderate drinkers had significantly higher median lifetime alcohol consumption of 66.7 g/week compared with light drinkers (4.8 g/week) and nondrinkers (0 g/week) (p = 0.000) and had a mean of 23.7 (±10.9) years from their period of regular drinking >20 g/day up to the time of enrolment in the study. In validation of the alcohol history-based subgrouping, moderate drinkers had a significantly higher alcohol consumption score and alcohol-related harm score (4) when compared with light drinkers (2) and nondrinkers (0)  . A post hoc analysis demonstrated a significant difference between moderate and light drinkers' alcohol consumption (p = 0.035), further corroborating the alcohol history taken at the time of interview.One hundred and fifty-one “at-risk” patients with T2DM and NAFLD were reviewed in the NAFLD clinic over a 12-month period. Overall, the mean age of subjects was 61 ± 10.3 years and 63.6% were males. The majority of subjects were Caucasian, with a mean body mass index (BMI) of 35.9 ± 8.3 kg/mp = 0.008) and Caucasian (p = 0.007) and to have a history of cigarette smoking (p = 0.000). Girth and BMI were not significantly different between the three cohorts (p > 0.05). Although there was no significant difference in glycated hemoglobin (HbA1c) or insulin therapy between the three cohorts, significantly more moderate drinkers required ≥3 hypoglycemic agents to maintain diabetic control (p = 0.041), suggesting that moderate drinkers may have worse diabetic control. In particular, a higher proportion of moderate drinkers (49%) were using a glucagon-like peptide type 1 (GLP-1) receptor agonist or a dipeptidyl peptidase-4 (DPP-4) inhibitor, compared to light drinkers (26%) and nondrinkers (20%) (p = 0.027).The demographic and clinical characteristics of the 151 subjects according to lifetime drinking history are summarized in p = 0.033) but this was not statistically significant following adjustment for age and BMI (p = 0.086). Importantly, a higher proportion of moderate drinkers (20%) were taking a fibrate medication to lower blood triglyceride levels, compared to light drinkers (4%) and nondrinkers (11%) (p = 0.044). Serum low-density lipoprotein (LDL) levels were significantly lower in females with a history of moderate alcohol intake (p = 0.045); however, this should be interpreted with caution as the number of females in this subgroup was very small (n = 7) (data not presented). There was no significant difference in HDL levels or use of statin therapy between the three cohorts (p > 0.05).Serum triglyceride levels were higher in moderate drinkers (p = 0.003). Seventy-one percent of moderate drinkers had a history of obstructive sleep apnea compared to 36% of light drinkers and 17% of nondrinkers. Self-reported depression was also significantly more common in moderate drinkers (71%), compared to light and nondrinkers .Interestingly, the prevalence of obstructive sleep apnea (OSA) differed significantly between patients with and without alcohol consumption (n = 28), and an additional 8 patients had liver imaging diagnostic of cirrhosis. Twenty-nine of these 36 patients had liver stiffness measurements ≥8.2 kPa and 23 of these 29 patients (79%) had bridging fibrosis or cirrhosis.LSM met quality criteria in 134 patients. Median LSM was 6.3 kPa with a range from 2.6 to 63.9 kPa and required the use of the XL probe in 83%. Eighty-eight of 134 patients (65.7% of the cohort) had LSM <8.2 kPa, suggesting the absence of severe fibrosis. Liver biopsy was performed in a subset of the patients (p > 0.05). In females, the proportion of patients with LSM ≥ 8.2 kPa was 35% in nondrinkers and 50% in light drinkers. However, none of the 7 female patients with a history of moderate alcohol intake had LSM ≥ 8.2 kPa. Compared to lifetime nondrinkers, light drinkers had 1.79  and moderate drinkers had 0.91  times the odds of having LSM ≥ 8.2 .There was no significant difference between those patients with LSM <8.2 kPa or ≥8.2 kPa and their alcohol consumption, dependence, alcohol-related harm, and overall AUDIT score. Compared to male lifetime nondrinkers, the proportion of male light and moderate drinkers with LSM ≥8.2 kPa  was not significantly different  and other comorbid conditions remains controversial. Due to the high prevalence of NAFLD and increased risk of advanced fibrosis in people with T2DM, determining the effect of light or moderate alcohol intake on liver disease progression in these patients is crucial. The present study indicated that, in diabetic patients at risk of clinically significant NAFLD, light or moderate lifetime alcohol consumption was not associated with a protective effect on liver fibrosis. Of concern, alcohol consumption was associated with an increased prevalence of metabolic comorbidities, specifically hypertriglyceridemia and OSA, and a history of smoking and self-reported depression.Increasing evidence suggests that moderate alcohol consumption (≤30 g/day) reduces the risk of coronary artery disease and other vascular conditions associated with the metabolic syndrome –27. Howe2) presenting for weight loss surgery, modest alcohol consumption (<200 g/week) was associated with a reduction in NASH, but the effect was not significant after controlling for diabetes or insulin resistance index [Prior studies examining the relationship between alcohol, obesity, and liver injury have produced conflicting results. In comparison with obesity alone, an earlier cross-sectional study showed that the combination of obesity and heavy alcohol intake (>60 g/day) was associated with a 1.3-fold increased risk for steatosis detected by liver ultrasound . The effce index . In compce index .Few studies have examined the effect of modest alcohol consumption in the presence of preexisting NAFLD. Heavy episodic drinking  was associated with fibrosis progression on repeat liver biopsy or development of end-stage liver disease after a mean of 13.8 years . In contIn our study, fasting serum triglyceride levels were higher in moderate drinkers, and a higher proportion of moderate drinkers (20%) were taking a fibrate medication to lower blood triglyceride levels, compared to light drinkers (4%) and nondrinkers (11%). Although high alcohol intake is known to increase blood triglyceride levels, prior studies of the effect of low-to-moderate alcohol intake on triglycerides have provided variable results . As alcoWe have previously shown a high burden of multimorbidity in diabetic NAFLD patients, with the most common coexisting chronic conditions including ischemic heart disease, obstructive sleep apnea, self-reported depression, respiratory diseases, and osteoarthritis . Our datStudy strengths include the prospective collection of history of alcohol consumption by the study clinician before a diagnosis was established and face-to-face patient interviews to overcome literacy concerns and patient understanding of questions. The downside of using face-to-face interviews is that they may promote social-desirability bias, where patients answer questions in a certain way or feel self-conscious in the presence of an interviewer, thereby inaccurately or incompletely disclosing alcohol consumption. This was a small single-center study and therefore our findings may not be truly representative of the wider population with T2DM and NAFLD. In order to ensure we were assessing the impact of alcohol intake in patients with NAFLD, participants with alcohol intake >20 g/day within 5 years prior to study entry were excluded. However, these exclusion criteria may have led to a selection bias excluding patients in whom moderate ongoing alcohol intake may have been beneficial, resulting in resolution of NAFLD. Although liver biopsy remains the gold standard for the diagnosis and assessment of liver fibrosis, the method of assessing advanced fibrosis used in this study (LSM) is an accepted noninvasive procedure for identification of advanced fibrosis .Lifetime alcohol consumption was not significantly associated with liver fibrosis in T2DM patients when assessed by LSM. Of concern, alcohol consumption was associated with an increased prevalence of metabolic comorbidities, specifically hypertriglyceridemia and OSA, and a history of smoking and self-reported depression. This study highlights the importance of taking a detailed alcohol history from patients regarded as “without a history of significant alcohol consumption,” as the impact of alcohol on diabetes comorbidities cannot be dismissed and requires further investigation.Supplementary table 1: AUDIT questionnaire breakdown according to lifetime drinking history.


HTRA2 p.G399S, and ANO3 p. W490C, p. R484 W and p. S685G mutations.Essential tremor (ET) is characterized by a frequent family history. No monogenic form of ET has been identified. We aimed at exploring ET patients to identify distinct subgroups and facilitate the identification of ET genes. We tested for the presence of HTRA2 and ANO3 mutations screening.Between June 2011 and November 2013, all consecutive patients suspected with ET were prospectively included in a prospective, monocentric study. Family history, age at onset (AAO), features of tremor, benefit of alcohol and drugs, electrophysiological recording findings were collected. Sanger sequencing was performed for p < 0.01) and ET family history (p < 0.01) were more frequent and the disease progression less severe (p < 0.0001). Neither HTRA2 nor ANO3 mutation were identified in our patients.Sixty eight patients were investigated. Fourteen diagnosed with psychogenic (5) or dystonic tremor (9) were excluded. Regarding the 54 ET patients, mean AAO was 48 years (6–77), and mean disease duration 15 years (1–55). Bimodal distribution of AAO was consistent with phenotypic subgroups. In patients with AAO before 30 years, marked benefit of alcohol  and marked benefit of alcohol, to facilitate the identification of ET genes. Electromyographic recording remains a support to distinguish ET from differential diagnosis.  According to the Consensus Statement of the Movement Disorder Society (CSMDS) , essentiClinical practice emphasizes ET heterogeneity, and the limits of the above definition: family history is frequent but may be lacking, histogram of age at onset (AAO) demonstrates bimodal distribution with a first peak in childhood or adolescence  and a seFUS gene [LINGO1 and SLC1A2 polymorphisms have been only identified in genome-wide association studies [HTRA2 variant has very recently been associated in one family with ET and optional parkinsonism [ANO3 mutations were reported in a spectrum of dystonic patients initially presumed to have familial essential tremor [Such heterogeneity added to the lack of stringent diagnostic criteria or of biomarkers and to the evidence for non mendelian inheritance could explain the weak profitability of genetic studies and their difficulty to find reproducible data . Indeed,FUS gene  was foun studies –11. An Hinsonism . Howeverl tremor .HTRA2 and ANO3 mutations recently identified as causes of family ET with extrapyramidal features.We aimed at analysing a cohort of patients with ET to identify distinct phenotypic subgroups in order to facilitate the identification of genes responsible for ET, and searched for From June 2011 to September 2013, all the patients referred in our tertiary movement disorders center with suspected ET, according to the CSMDS [Following items were collected: family history, AAO, current age, clinical features of tremor, severity scales  performance subscale ), markedUsing (1) surface electrodes on extensor and flexor muscles of both forearms, and on cervical muscles, and (2) an unidirectional piezoelectric accelerometer placed on the index finger of dominant hand, the following items were analysed: the conditions of occurrence of the tremor , frequency, regularity, agonist-antagonist synchronicity, distribution, and distractibility. Accelerometer signal was bandpass filtered at 20–500 Hz. Postural and intention tremor were analyzed during postural maintenance and action . The effect of mental calculation was tested at rest. Patients with atypical tremor including distractible electrophysiological patterns  suggestive of psychogenic tremor and patients with suspected dystonic tremor  were excluded from the study.FMR1 premutation was investigated in all patients with onset after 50 years in order to rule out fragile X-associated tremor ataxia syndrome (FXTAS) [HTRA2 p.G399S, and ANO3 p.W490C, p.R484 W and p.S685G variants recently reported as causes of ET, DNA was extracted from whole blood using illustra™ DNA Extraction kit BACC3 .. The coding exons of HTRA2 and ANO3, reported recently [www.gatc-biotech.com/). Sequenced were analysed using the SEQSCAPE v2.5 software .Cupper, ceruloplasmin and thyroid stimulating hormone (TSH) serum levels were analysed in each patient.  (FXTAS) . For scrrecently , 13 wereCerebral MRI was performed in 28 cases and, dopamine transporter single-photon emission tomography (DAT-SPECT) was performed in 8 of the 10 patients who also had rest tremor.Statistical analyses were performed using parametric and non-parametric tests, according to the data distributions. Association between categorical variables was assessed using Fisher-exact tests, because of the small populations in each level of the qualitative variables.Correlation between quantitative variables has been tested using Pearson correlation tests and Spearman correlation tests, in order to check for linear and non-linear correlation assumption.Link between a qualitative and a quantitative variable was checked using the non-parametric Kruskal-Wallis test, because of the nature of continuous variables distributions. The Gaussian assumption of these formers was not always checked (Shapiro-Wilk test), implying the use of those non-parametric tests.Alpha risk was fixed to 5%. Analyses were performed using the R software (version 3.0.2).Sixty-eight patients were investigated. Taken together, clinical and neurophysiological findings were finally consistent with psychogenic tremor in 5 patients and dystonic tremor in 9 patients who were excluded.The main clinical and electrophysiological data of the remaining 54 patients are summarized in Tables p = 0.008 and p = 0.0007, respectively). TETRAS score was higher when head tremor was present (p < 0.001) but head tremor was positively correlated with neither age of onset nor tremor duration.The 54 studied patients had a mean age of 62 years range, 18 to 80) and had a mean history of tremor of 14.5 years . The mean AAO was 48 years . Tremor age of onset showed a bimodal distribution  an associated lower limbs and 14 (26%) an associated head tremor. Mean frequency of tremor was 6.3 Hz (3.8–11) in upper extremities. All patients had postural tremor, 46 patients (85%) an associated intentional tremor and 10 (18%) an associated rest tremor.FMR1 permutation were negative. No patients had HTRA2 p.G399S, or ANO3 p.W490C, p.R484 W and p.S685G variant.Biological analysis  was normal in all patients. All the 18 patients investigated for Brain MRI was normal in 13/28 cases, but demonstrated vascular leucopathy in 9, cortical frontal atrophy in 2, incidental meningioma in 2  and a frontal right meningomia (2 cm in diameter)), and asymptomatic aneurysm of a cerebral artery in 2 patients.DAT-SPECT demonstrated the loss of dopaminergic neurons in 4/8 patients with rest tremor.p = 0.28, p = 1.0, p = 1.0, respectively); marked benefit of alcohol ; associated rest tremor ; symmetrical tremor ; associated head tremor ; associated lower limbs tremor ; frequency ; TETRAS performance subscale ; evolution speed  .Treatments and efficacy of treatments are summarized in Table We aimed at identifying extreme phenotype of ET that could be distinguished from other ET variants in order to find genes responsible for ET which appears to be heterogeneous regarding both clinical and genetic basis. Indeed, the identification of gene causing ET remains an important genetic challenge in the field of movement disorders.Our study confirmed the bimodal distribution of AAO and the frequent positive family history of ET , 17 thatWe found that early onset of tremor, marked benefit of alcohol consumption and positive family history of ET should be considered as such peculiar phenotype also characterized by a less severe progression of the disease. Thus, in order to identify genes causing ET, we recommend to perform genetic analysis in patients with such extreme phenotype. However, these new findings need to be confirmed in larger series. Benefit of alcohol could be quantitatively assessed  and coulFor the identification of such peculiar phenotype, electrophysiological recording remains a strong support. Indeed, we confirmed the difficulty in some patients to distinguish ET from differential diagnosis such as dystonic tremor or to some extent psychogenic tremor. Therefore, we also recommend to perform electrophysiological recordings for the selection of patients with genuine ET for future genetic studies.p < 0.01) and TETRAS subscale performance (p < 0.01). Likewise, head tremor which was present in only 11 patients  [p < 0.001). In the same way, the higher frequency of lower limb tremor in patients with earlier age at onset could be due to the natural history of the disease rather than to a peculiar phenotype. However, this statement remains to be confirmed by further studies.We found that tremor frequency and head tremor should not be considered as determinant factors for the identification of a peculiar phenotype. In our series, some findings could be explained by the natural history of ET rather than by different phenotypes of ET, since patients with earlier age at onset had longer disease duration. Indeed, an age-associated decrease in tremor frequency (approximately 0.06 to 0.08 Hz/year ) has bee series) , was onlp = 0.31) nor TETRAS subscale performance (p = 0.2). Some authors suggested that in patients with severe, old and disseminated ET, rest tremor could be explained by diffusion of the pathologic process to the dopaminergic system. However recent post-mortem studies of a few ET patients with rest tremor did not reveal Lewy body pathology [Rest tremor was present in 18% patients, but in opposite to previous data , no corrathology , and in HTRA2 p.G399S variant has recently been identified to be a risk factor with overlapping family ET and PD but we did not find this variant in our cohort [ANO3 mutations in any of our patient using Sanger sequencing. These data have to be replicated in a larger sample of ET patients.Nevertheless, epidemiological studies have shown that ET could be a risk factor for Parkinson’s disease (PD) . Additior cohort . In the r cohort , 25. We Finally, our series confirmed the therapeutic efficacy of propranolol and primidone , and to Larger cohorts are probably needed to confirm that the factors identified in the present study  correspond to an extreme phenotype of ET. Our results suggest to study separately patients with such peculiar phenotype in order to finally find gene responsible for ET.


To investigate the lncRNA profiling during tilapia peritoneal macrophages (TPMs) activation and discuss the relationship between lncRNA and mRNA.Streptococcus agalactiae (Sa) antigen, heat shock protein 70 (HSP70) and HSP70+Sa. The expressions of lncRNA and mRNA were confirmed by qPCR. 356 lncRNA, 10173 mRNA and 1782 transcripts of uncertain coding potential (TUCP) were differentially expressed by pairwise comparison. These lncRNAs were shorter in length, fewer in exon number and higher in expression levels as compared with mRNAs. 683 lncRNAs and 4320 mRNAs were co-located, while 316 lncRNAs and 9997 mRNAs were in co-expression networks. Seven mRNAs  were involved in both networks of LNC_00035 and LNC_000466. Differentially expressed genes were involved in signaling pathways, such as “phosphorylation”, “cytokine-cytokine receptor interaction”, “endocytosis” and “MHC protein complex”. LNC_000792, LNC_000215, LNC_000035 and LNC_000310, with cis and/or trans relationships with mRNAs, were also involved in ceRNA network.RNA sequencing was used to investigate the lncRNA and mRNA profiles of TPMs activation following stimulation with These results might represent the first identified expression profile of lncRNAs and mRNAs in tilapia macrophages activated by HSP70 and Sa. Streptococcus agalactiae (Sa) is one of the most important pathogens causing severe streptococcicosis for tilapia industry of China. Currently, there are no effective chemicals or vaccines available against this disease for commercial use yet. Identification of the immunological responses between Sa and host immune system is of utmost importance given the economic burden of the disease to the aquatic industry. The activation of mammal macrophage is crucial for its immunological functions, and similar functions have been reported in fish macrophages [+T cells [et al. reported that the TNFα and IL1β mRNA in liver, head kidney, and spleen increased after intraperitoneal injection of HSP70 in Wuchang Bream [rophages –4. Previrophages . In mammrophages , 7, and rophages . These o+T cells , and sev+T cells –12. As s+T cells –15, inding Bream , and levng Bream . This meng Bream , 19, andng Bream . However+T cells [+T cells and CD11C+ dendritic cells [via adsorption of miR-199a-5p to increase expression of PU.1 and ACVR1B [Long non-coding RNA (lncRNA) is known to play a key role in gene regulation network in mouse and human. For example, lncRNA participated in the activation of CD8+T cells , CD4+T cic cells , 23. Impic cells , and PU.d ACVR1B . HoweverBefore RNA sequencing, the interaction of HSP70, Sa and HSP70+Sa with TPMs was confirmed by using fluorescein labelling and qPCR examination. As shown in Figure By pairwise comparison, we found lncRNA, mRNA and TUCP were differentially distributed among groups. Totally, 356 lncRNAs, 10173 mRNAs and 1782 TUCPs were differentially expressed genes from pairwise comparisons Figure . The moscis (trans (cis and trans) were demonstrated, which was consistent with the FPKM predictions  in TPMs treated with Sa and/or HSP70 , while 316 lncRNAs and 9997 mRNAs were in co-expression  network. Next we randomly selected five lncRNAs, LNC_00035, LNC_000215, LNC_000310, LNC_000358 and LNC_000466, to demonstrate the relationships between lncRNAs and corresponding mRNAs. As shown in Figure cis and trans relationships with their corresponding mRNAs. For example, 371 mRNAs were involved in the trans network with LNC_00035, which also had 11 co-located mRNAs. Specifically, we found seven mRNAs  were involved in both networks of LNC_00035 and LNC_000466. However, some lncRNAs had only cis or trans relationships with mRNAs (data not shown).To investigate the relationship between lncRNAs and their neighboring coding genes, we randomly selected 400000 mRNAs, 100000 lncRNAs, and 800000 random gene pairs formed by lncRNAs and their neighboring genes. We observed a more correlated expression pattern of lncRNAs with their neighboring gene pairs (mean correlation: 0.01899) than random coding gene pairs (mean correlation: 0.01687), and the expression pattern of lncRNAs showed a relatively higher correlation than coding gene pairs (mean correlation: 0.01752) Figure . We alsotrans with lncRNAs but not the mRNAs cis with lncRNAs. The highest number of differentially expressed genes  in biological process were related to “regulation of biological process” in HSP70+Sa vs. HSP70 group, “nucleic acid metabolic process” in HSP70+Sa vs. Sa and Sa vs. Control groups, and “biological regulation” in HSP70 vs. Control group  analysis with the selected mRNAs which neighbor lncRNAs or have high co-expression with lncRNAs in four comparison groups. GO terms enrichment was found mostly in mRNAs ol group . Kyoto Ep Figure . While tp Figure . As for p Figure . We nextp Figure . Comparep Figure .cis and/or trans relationships with mRNAs in lncRNA targets data, were also involved in ceRNA network. Subsequent investigation showed that the expressions of these lncRNAs were divergent. For instance, LNC_000792 was significantly increased in Sa- and HSP70-stimulated groups compared to HSP70+Sa group, while LNC_000035 had an opposite expression profile in these groups  within a ceRNA network, we scanned all differentially expressed lncRNAs and mRNAs during TPMs activation against fish miRNA database . We founs Figure .Tilapia HSP70 could activate TPMs , which wThe aim of this study was to identify lncRNA during TPMs activation. We found that 797 transcripts were predicted as novel lncRNAs in tilapia. Previous studies showed that lncRNAs could be classified into six types: sense, anti-sense, bidirectional, intronic, intergenic and small RNA (sRNA) host lncRNA . In our cis [trans [cis, while 316 lncRNAs with 9997 mRNAs were in trans, indicating both cis and trans regulatory models dominate and may play essential roles on TPMs activation. Take LNC_000310 as an example, 14 mRNAs were involved in trans and 9 mRNAs were in cis, indicating LNC_000310 was involved in both trans and cis regulations. Evidence suggests that lncRNAs may regulate and associate with expression of neighboring mRNAs [cis relationship of LNC_000310, IL13Ra1 chain is crucial for induction of the alternative macrophage activation by IL13 [cis with LNC_000358, was reported to be essential in activation of TLR2/4 shared signaling [trans with lncRNAs may also be involved in the activation of TPMs. For instance, we found that 250 mRNAs were involved in trans network of LNC_000215. Among them, at least GFRA3 [cis and/or trans with mRNAs, may exert their functions through their associated mRNAs, which play crucial roles in HSP70-induced TPMs. However, these predicted functions of lncRNAs need experimental verification in our future studies.Recent studies reported that some lncRNAs regulate gene expression of their neighborhood in cis , and nons [trans . In the ng mRNAs . Further by IL13 . Additioignaling , which iignaling . Similarst GFRA3 , NFATC1 st GFRA3  and ADCYst GFRA3 , 39 mighOur GO analysis revealed that “metabolic process” and “biological regulation” were important processes during TPMs activation, and the KEGG pathway analysis supported these findings. In mammals, metabolic pathways have been documented to be involved in macrophage activation –42. Genecis and/or trans network, were involved in the ceRNA network as well. In the mRNAs that were targeted by the four lncRNAs, ENSONIT034160R126779 and ENSONIT034160R106484 were the mRNAs that were also the targets for both LNC_000215 and LNC_000035 in the ceRNA network. This highlights the crosstalk of genes in regulatory networks during TPMs activation.The ceRNA hypothesis was proposed as a novel regulatory mechanism between non-coding RNA and coding RNA , 49. TheAll experiments were conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and Welfare. Sample collection protocol was approved by the Committee on the Ethics of Animal Experiments of the Guangxi Institute of Fisheries (Permit Number: 200099). Animals were humanely sacrificed as necessary to ameliorate suffering.Oreochromis niloticus brood stocks were obtained from the National Tilapia Seed Farm . Streptococcus agalactiae bacteria cultivation was performed as previously reported [6 cells per well in 24-well plates for 24 h. A final volume of 200 μl macrophages in triplicate were stimulated with 100 μg/ml HSP70, 1.0×105 cells/well Sa antigen, 100 μg/ml HSP70 plus 1.0×105 cells/well Sa antigen and media control at 27°C in 5% CO2 for 24 h. Interaction of HSP70 and Sa antigen with macrophages were verified by fluorescence technique as previously reported [reported . HSP70 preported . Peritonreported . The obtTotal RNA used for library construction and PCR was isolated from each individual sample using TRIzol reagent . DNase I was added to remove contaminating genomic DNA. Integrity of RNA was evaluated by using the RNA Nano6000 Assay Kit of the Bionalyzer 2100 system . Purity and quantity of total RNA were tested by using a Nanodrop spectrophotometer .We used 3 μg RNA per sample as input material for the RNA preparations. Ribosomal RNA was removed by Epicentre Ribo-zero™ rRNA Removal Kit  followed by rRNA free residue cleaning by ethanol precipitation. Libraries were constructed using the rRNA-removed RNA by NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina®  according to the manufacturer's instructions. The library fragments were purified with AMPure XP system  to select cDNA fragments between 150 to 200 bp in length. Library quality was evaluated on the Agilent Bioanalyzer 2100 system. The libraries were sequenced on an Illumina Hiseq 2500 platform at the Novogene Bioinformatics corporation .Oreochromis niloticus genome sequence assembly (Bowtie v2.0.6 and Tophat (v2.0.9)). Finally, the mapped clean reads of each sample were assembled by both programs of Cufflinks [Firstly, all raw reads with fastq format were processed through perl scripts to obtain the clean data (clean reads) by removing those reads containing adapter or ploy-N and low quality reads. Then, Q20, Q30, and GC content of the clean data were calculated and accessed. Subsequently, the high quality clean reads were mapped with the ufflinks .transcripts to evaluate the extent and quality of the ORF by searching the sequences with known protein sequence database [transcripts. CNCI was used to distinguish coding and non-coding sequences according to the spectrum of the nearby trinucleotide and effectively predict the incomplete and anti-sense transcripts. Pfam Scan (v1.3) was used to identify the occurrence of the known protein family domains in the PFAM database [transcripts without coding potential were considered as lncRNA candidates. The coding genes 10k/100k upstream and downstream of lncRNA were classified as the cis target gene, and the expression level of each transcript was used to identify trans target gene of lncRNAs. Depending on the relationships between lncRNAs and their neighboring protein-coding genes, lncRNAs could be classified into six types: (1) sense, (2) anti-sense, (3) bidirectional, (4) intronic, (5) intergenic, and (6) small RNA (sRNA) host lncRNA [The CPC, CNCI, and PFAM were used to distinguish mRNAs from lncRNAs. CPC program was used to clarify the coding and non-coding database . CPC valdatabase . The trat lncRNA . In this2O. The cycling was initiated by a single denaturing cycle of 95°C for 3 min followed by 40 cycles at 95°C for 20 s, 56-60°C for 20 s, and 72°C for 30 s. Specific primers used in this study were listed in −ΔΔCt value method [Purified total RNA was synthesized by using the PrimeScript RT reagent . QPCR was performed on an ABI 7500 PCR instrument using SYBR green reagents  following the manufacturer's protocol. Each qPCR reaction (25 μ L) was comprised of 12.5 μ L 2 × SYBR Green qPCR Master Mix , 1 μ L primer, 2 μ L cDNA, and 8.5 μ L HTranscripts with change folds >2.0 and P < 0.05 were considered as differentially expressed genes. To explore the roles of these genes, KEGG (http://www.kegg.jp/) was used to confirm the pathway enrichment and GO analysis (http://www.geneontology.org) was performed to identify the roles of differentially expressed lncRNAs and mRNAs. The GO terms included biological process, cellular component, and molecular function. The corresponding target genes of the differentially expressed lncRNAs were mapped to GO terms and integrated discovery DAVID program (http://david.abcc.ncifcrf.gov/). The p-value indicated the significance of the pathway, and lower p-value indicated higher significance of the pathway.The quantification of lncRNAs and coding genes was calculated by Cuffdiff (v2.1.1) . Transcrhttp://www.mircode.org/), and then lncRNAs and/or mRNAs targeted by miRNAs were analyzed by using TarBase program (http://www.microrna.gr/tarbase). The ceRNA network was built and illustrated by using Cytoscape (v3.4.0). Analyses were performed by Novogene Bioinformatics corporation .LncRNAs or mRNAs can interact with miRNAs through their MicroRNA Recognition Elements (MREs) within a ceRNA network. Firstly, the differentially expressed lncRNAs and/or mRNAs were selected in each sample. To build the ceRNA network, the interactions between differentially expressed lncRNAs and miRNAs were predicted by miRcode program . An absolute fold change ≥2, Streptococcus agalactiae; TUCP: transcripts of uncertain coding potential; HSP70: heat shock protein 70; ORF: open reading frame; qPCR: quantitative polymerase chain reaction; lncRNA: long non-coding RNA; GO: gene ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; FPKM: fragments per kilobase of exon per million fragments mapped; MREs: microRNA recognition elements; CeRNA: competing endogenous RNAs.TPMs: tilapia peritoneal macrophages; Sa:


Iron disorder and abnormal expression of hepcidin play important roles in many diseases, but it is still unclear in chronic periodontitis (CP) and type 2 diabetes mellitus (T2DM). We aimed to assess ferritin and hepcidin levels in serum and saliva of CP patients with or without T2DM.Serum and unstimulated whole saliva samples were collected from 88 participants, who were categorized into 4 groups based on the presence or absence of CP or T2DM. Demographics and general health parameters were recorded. Full-mouth clinical periodontal parameters including probing pocket depth, clinical attachment loss, bleeding index, and plaque index were recorded. Chemiluminescence microparticle immunoassay and enzyme-linked immunosorbent assay were used to detect ferritin and hepcidin concentrations, respectively, in serum and saliva.P < 0.05). Serum hepcidin and serum ferritin are linear correlated (P < 0.001). Serum hepcidin/ferritin values in the CP with T2DM group were significantly lower than those in the T2DM and control groups. Moreover, salivary ferritin levels in the CP and T2DM groups were higher than those in the control group (P < 0.05). There was positively correlation between salivary ferritin and serum ferritin (P = 0.017). Hepcidin concentrations were relatively low in saliva.Serum ferritin and hepcidin levels in the CP and CP with T2DM groups were higher than in the control group (These results suggest that iron overload and hepcidin inadequacy existed in CP with T2DM patients. Salivary ferritin might provide a reference for body iron load.ChiCTR-ROC-17012780 Chronic periodontitis (CP) is a bacteria-induced chronic inflammatory disease of tooth-supporting tissues . Type 2 Iron is an indispensable nutrient for the human body; it is essential to maintain systemic iron homeostasis because iron disorder in the body can lead to various clinical diseases . Recent Iron stores can be detected using several techniques including liver biopsy , iron stAfter receiving approval from the Ethical Committee of the Second Xiangya Hospital of Central South University, we recruited 22 T2DM patients, 22 CP patients, 22 CP patients with T2DM, and 22 healthy individuals; the age of the participants ranged 40–83 years. All participants provided a written informed consent, and all steps of the clinical examination and sampling procedures were explained to each participant. Participants were diagnosed with T2DM by specialist physicians at the hospital according to the American Diabetes Association criteria . A singlhttp://apps.who.int/trialsearch/Trial2.aspx?TrialID=ChiCTR-ROC-17012780).Data regarding demographic and clinical characteristics, including age, body mass index (BMI), fasting blood glucose (FBG), triglyceride (TG), cholesterol (CHOL), high-density lipoprotein (HDL), low-density lipoprotein (LDL), alanine transaminase (ALT), and aspartate transaminase (AST), were recorded in physical examination reports of all participants. Clinical registration information of this study could be found on the International Clinical Trials Registry Platform  and enzyme-linked immunosorbent assay (ELISA), respectively. Ferritin assay was performed according to the manufacturer’s instructions described in the assay procedure. A two-step immunoassay was required. Ferritin present in the sample binds to antiferritin-coated microparticles. After washing, antiferritin acridinium conjungate was added. Subsequently, Pre-Trigger and Trigger Solutions were added to the reaction mixture; the resulting chemiluminescent reaction was measured as relative light units (RLUs). A direct relationship exists between ferritin concentrations in the sample and RLUs detected by the ARCHITECT i optical system. Hepcidin concentrations in saliva and serum were determined using an ELISA kit  according to the manufacturer’s protocol. Ferritin and hepcidin levels in serum and saliva were presented as ng/mL.P values < 0.05 represented statistical significance.Data analysis was performed using the SPSS statistical program . We compared characteristics of participants in each group, including age, FBG, BMI, TG, CHOL, LDL, and AST by using analysis of variance (ANOVA), while HDL, ALT were compared using the nonparametric Kruskal–Wallis test. Constituent ratio of gender was analyzed by Chi-square test. As a result of the skewed distributions of periodontal parameters, non-parametric tests kruskal-Wallis were used, when we analyzed periodontal condition. The constituent ratio of gender was analyzed using chi-square test. Test of normality showed that log serum ferritin, serum hepcidin, 1/(serum hepcidin/ferritin), log salivary ferritin, 1/salivary hepcidin, and log  were in accordance with the normal distribution. Thus, analysis of covariance (ANCOVA) was used both for comparing levels of ferritin, hepcidin, and hepcidin/ferritin in serum and saliva among each group and for controlling the effect of age. We analyzed the relationship between serum hepcidin and serum ferritin; serum ferritin and salivary ferritin by curve fitting. In addition, stepwise linear regression analysis was used to explore risk factors of serum ferritin, hepcidin, and hepcidin/ferritin that could be entered into the model. P < 0.05). However, no significant intergroup differences were noted in terms of sex, BMI, TG, CHOL, HDL, LDL, ALT, and AST (P > 0.05).Table P < 0.05).Periodontal parameters including PD, CAL, BI, and PI of each group are enlisted in Table P1 = 0.008) and CP with T2DM (P2 = 0.015) groups were higher than those in the control group. The CP group exhibited higher serum ferritin compared with the T2DM group (P3 = 0.036). The change trend of serum hepcidin was similar to that of serum ferritin. Serum hepcidin levels were higher in the CP (P1 = 0.003) and CP with T2DM (P2 = 0.036) groups than in the control group. Moreover, serum hepcidin levels were higher in the CP group than in the T2DM group (P3 = 0.04). The serum hepcidin/ferritin value was significantly lower in the CP with T2DM group (mean = 0.88) than in the T2DM and control groups (mean = 1.00 for both). Serum hepcidin levels were positively correlated with levels of serum ferritin .Concentrations of ferritin, hepcidin, and hepcidin/ferritin were represented as median and interquartile range Table , Fig. 1.1–X13, Table 11–4.991X2–100.828X1). Regarding serum hepcidin, the results showed that gender, age, HDL, and absence or presence of CP could be entered into the model (Y = 359.086 + 47.604X11–30.911X7–1.946X2–54.76X1). Regarding the serum hepcidin/ferritin value, results revealed that age and absence or presence of CP could be entered into the model (Y = 0.717–0.143X11 + 0.008X2).We selected serum concentrations of ferritin and hepcidin and value of hepcidin/ferritin as dependent variables and considered gender, age, BMI, FBG, TG, CHOL, HDL, LDL, ALT, AST, absence or presence of CP, absence or presence of T2DM, and duration of T2DM as independent variables X1–X1, Table 32 = 0.064, P = 0.017, Fig. We performed curve fitting between serum ferritin concentration and salivary ferritin level. The results showed that there was correlation between them (RTo our knowledge, no previous study has explored concentrations of ferritin and hepcidin and the hepcidin/ferritin ratio in serum and saliva of CP patients with or without T2DM. The key findings of the present study were that patients with CP and T2DM exhibited significantly higher serum ferritin and hepcidin levels and lower serum hepcidin/ferritin ratio compared with controls. Absence or presence of CP was a significant predictor of serum ferritin concentration and the hepcidin/ferritin ratio. In addition, there was correlation between serum ferritin concentration and salivary ferritin level. Thus, these results suggested that iron overload and hepcidin inadequacy existed in CP with T2DM patients. CP might be a potential risk factor for iron overload and hepcidin inadequacy. Besides, salivary ferritin might provide a reference for body iron load.Iron is required in the body for several biological processes, but it is also harmful when in excess . Iron ovHepcidin is a key regulator of systemic iron homeostasis, and its unbalanced production is responsible for the pathogenesis of various iron disorders . A previSince hepcidin is regulated by iron levels, the concentration of hepcidin alone could not effectively reflect the ability of the body to regulate iron load. The hepcidin/ferritin ratio has been reported to reflect the adequacy of hepcidin production for a given iron load; when this ratio decreases, the hepcidin produced is insufficient for the iron overload in the body , 44, whiCP is an inflammatory disease of the periodontal supporting tissues caused by microorganisms in the dental biofilm . The majWhole saliva is composed of secretions from major and minor salivary glands as well as the gingival crevicular fluid . Human sOur results imply that Iron overload and hepcidin inadequacy existed in CP with T2DM patients. Furthermore, salivary ferritin level might provide a reference for body iron load. Additional studies are needed to explore other potential influential factors of salivary hepcidin concentrations.


Androgen receptor (AR) is widely expressed in breast cancer (BC) but its role in estrogen receptor (ER)-positive tumors is still controversial. The AR/ER ratio has been reported to impact prognosis and response to antiestrogen endocrine therapy (ET).We assessed whether AR in primary tumors and/or matched metastases is a predictor of efficacy of first-line ET in advanced BC. Patients who had received first-line ET (2002–2011) were recruited, while those given concomitant chemotherapy or trastuzumab or pretreated with > 2 lines of chemotherapy were excluded. ER, progesterone receptor (PgR), Ki67 and AR expression were assessed by immunohistochemistry, and HER2 mainly by fluorescent in-situ hybridization. Cut-offs of 1 and 10% immunostained cells were used to categorize AR expression.p = 0.028). AR status in primary tumors or metastases was not associated with progressive disease (PD) as best response. In contrast, Ki67 ≥ 20% and PgR < 10% showed a statistically significant association with PD as best response.Among 102 evaluable patients, biomarkers were assessed in primary tumors in 70 cases and in metastases in 49, with 17 patients having both determinations. The overall concordance rate between primary tumors and metastases was 64.7% (95% CI 42%-87.4%) for AR status. AR status did not affect TTP significantly, whereas PgR and Ki67 status did. AR/PgR ≥0.96 was associated with a significantly shorter TTP (HR = 1.65, 95% CI 1.05-2.61, AR expression does not appear to be useful to predict the efficacy of ET in advanced BC, whereas Ki67 and PgR exert a greater impact on its efficacy. Androgen receptor (AR) is emerging as an important mediator in the biology and therapy of breast cancer (BC). It has a role in breast carcinogenesis, varying on the basis of the expression of other biomarkers, such as estrogen receptor (ER), progesterone receptor (PgR), human epidermal growth factor receptor 2 (HER2/neu), and a prognostic effect has also been observed .AR is a nuclear transcription factor, which is activated by the binding of steroid hormone androgens, similarly to other members of this family of receptors including ER and PgR .AR protein is expressed in about 70–90% of BC. Its levels may however vary depending on the location considered (cytoplasmic and/or nuclear), the cutoff of expression , and the antibody used for immunohistochemistry (IHC) , 4.AR expression in different BC subtypes is also variable. It is found in about 75% of ER-positive, 50–60% of HER2-enriched, and 20-40% of triple negative BC, respectively [Depending on the expression of other hormone receptor proteins and their ligands, the AR pathway may promote or inhibit cell proliferation. Moreover, this interplay between AR, ER and their ligands is complicated by the possible conversion of androgens to estrogens. A cross talk between AR pathway and HER2/neu pathway is also known , 8.AR and ER can compete for the binding to estrogen response elements (EREs) on specific genes . So the Moreover a high AR expression can activate the epidermal growth factor receptor (EGFR), promoting an agonist effect of tamoxifen on ER pathway, and this aberrant mechanism could be blocked by enzalutamide +/− gefitinib .Currently AR is under investigation in the clinical setting as a therapeutic target in BC. Enzalutamide and other anti-androgens appear suitable treatment options for AR-expressing BC , 14.A favorable prognostic role of AR in BC was identified in various studies. This effect was confirmed, in terms of both disease-free survival (DFS) and overall survival (OS), in a large meta-analysis including almost 8000 patients with stage I-III BC. In other studies AR expression independently predicted longer BC specific survival and lower risk of recurrence , 15, 16.We previously showed the value of PgR and Ki67 to predict benefit from first-line endocrine therapy in a case series of patients with advanced luminal breast cancer . In thisA retrospective analysis was performed for this study. Patients with locally advanced or metastatic BC histologically confirmed treated with first-line ET from 2002 to 2011 at Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori, IRST , were included. Patients who received concomitant chemotherapy or trastuzumab or pretreated with more than 2 lines of chemotherapy were excluded. All patients were aged ≥18 years and were followed up for a period ranging from 5 to 13 years. We collected data from medical records of these patients. Subsequently we gathered tumor tissue samples of these patients for AR assessment.The original hematoxylin-eosin stained sections were reviewed by two pathologists responsible for selecting pathological inclusions representative of tumor tissue and for the analysis of clinical pathological features, such as histologic grade.The predictive value of AR expression, alone or in relation to the other conventional biomarkers, to select the patients responsive to first-line endocrine therapy was the primary endpoint.IRST and AVR (Area Vasta Romagna) Ethics Committee , reviewed and approved the study and informed consent was obtained from all patients.Data were collected from electronic databases. The following characteristics were evaluated for each single patient.Patient’s data: age at diagnosis. Tumor’s data: 1) histotype ; 2) tumor classification at diagnosis  according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual 7th Edition; 3) nodal classification ; 4) presence of metastases at diagnosis (M0-1); 5) ER, PgR, Ki67, HER2 status in primary tumor and metastasis. Treatments and outcomes: 1) adjuvant chemotherapy; 2) adjuvant ET; 3) type of first-line ET; 4) best objective response (complete response (CR), partial response (PR), stable disease (SD), PD, according to RECIST criteria); 5) time-to-progression (TTP), meant as the time in months from the beginning of first-line ET until progression or last tumor response evaluation available.All datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.Hormone receptors, Ki67, HER2 and AR status of the primary tumor, metastasis or both were assessed for all patients enrolled. The major part (79%) of the biomarkers detection was performed in the Pathology Unit of Morgagni Pierantoni Hospital in Forlì .Tumor samples obtained during surgery were fixed in neutral buffered formalin and embedded in paraffin. Four-micron sections were mounted on positive-charged slides for each patient . REporting recommendations for tumour MARKer prognostic studies (REMARK) and the European Quality Assurance guidelines were used. Immunostaining for conventional biomarkers and AR expression was performed using the Ventana Benchmark XT staining system  with the Optiview DAB Detection Kit . ER, PgR, Ki67 , HER2  and AR  antibodies were used. For ER, PgR, Ki67 and HER2 detection, tissue sections were incubated for 60 min with antibodies diluted 1:80, 1:40, 1:100 and 1:350, respectively, in antibody diluent . The AR antibody was pre-diluted by the supplier. Sections were incubated for 16 min and automatically counterstained with hematoxylin II . Biomarker positivity was detected and semiquantitatively quantified as the percentage between immunopositive tumor cells and the total number of tumor cells. Two independent observers evaluated all the samples and any disagreement was resolved by consensus after joint review using a multihead microscope. Hormone receptors, Ki67 and HER2 biomarkers were classified on the basis of the St. Gallen and ASCO-CAP guidelines –20. In pPatients and tumor features were described according to descriptive statistics.Positive or negative AR status in both tumor and metastasis was used to define the concordance between tumor and metastasis, while discordance was defined as positivity at one site and negativity at the other one. The concordance rate was calculated as the proportion of concordant cases with respect to the total number of patients. The two-sided exact binomial 95% confidence interval (95% CI) was estimated.AR expression was analyzed in relation to the other conventional biomarkers , best response to therapy , and time to progression (TTP) (months). Kaplan-Meier method was used to estimate TTP and compared with the logrank test. Hazard ratios (HR) and their 95% CI were calculated using the Cox regression model.The Chi Square test was used to evaluate the association between categorical variables and best response and the non-parametric ranking test (Wilcoxon test) was used to evaluate the relationship between median value of AR expression and Ki67 classes .To determine the optimal cutoff values of AR/ER and AR/PgR ratios at a median TTP time Receiver operating characteristic (ROC) curves were used.p values < 0.05.Analyses were performed using SAS Statistical software . Statistical tests were significant for One hundred and two patients were evaluable according to the selection criteria and the availability of tissue samples. Median age was 60 years (range 33-85). Seventy eight percent of them were diagnosed as ductal and 14% as lobular histotype. Metastatic disease at diagnosis was found in 26 out of 102 (25.5%). Ninety two percent were treated with an aromatase inhibitor as first-line endocrine therapy, and letrozole was the most frequent aromatase inhibitor administered (45%). For detailed description of patients’ characteristics see Table Biomarker’s expression  was assessed in primary tumors in 70 cases and in metastases in 49, with 17 patients having both assessments Fig.  (Table 2ER was negative (< 1%) in 3 samples (4.3%) of primary tumors and in none of the metastatic samples. PgR was negative (< 1%) in 10 (14.3%) primary tumors and in 12 (24.5%) metastases. Ki67 was low (< 20%) in 59.1% of primary tumors and in 61.2% of metastases. HER2 status was positive in about 15% of cases both in primary tumors and metastases. In Fig. p = 0.017).AR status Fig. , consideAR status in primary tumors or metastases was not associated with PD as best response Table .Table 3Rp = 0.62); clinical benefit was 90.7% and PD 9.3% for AR-positive tumors defined by a cutoff ≥10%, versus 79% and 21%, respectively, for AR-negative tumors (p = 0.18). Conversely Ki67 showed a significant association with PD as best response, with 26.9% PD with high Ki67 versus 4.3% PD with low Ki67 (p = 0.009). PgR-negative status defined by a cutoff < 1% showed just a trend in terms of association with PD as best response (p = 0.079), while significant results (p = 0.031) were obtained by using ≤10% PgR as cut off value.Clinical benefit rate, defined as CR or PR or SD, was 88.5% and PD 11.5% for AR-positive tumors defined by a cutoff ≥1%, versus 83.3% and 16.7%, respectively, for AR-negative tumors (p = 0.884); similarly for AR ≥10% median TTP was 16 months (95% CI 13.0–19.0) vs 13.8 months (95% CI 11.0–42.1) for AR < 10% (p = 0.935). AR/PgR ≥0.96 was associated with a significantly shorter TTP  . Differences in TTP according to AR status were not statistically significant (Table  95% CI 1.0–19.0 vMost of ER-positive BCs are AR-positive (about 80-90% of them) and the coexpression of AR, ER and PgR is associated with a better prognosis and well-differentiated phenotype –23.Some studies suggest that the levels of expression of AR and its relation to ER expression levels in primary tumors predict benefit from adjuvant endocrine therapy with tamoxifen .This study was not powered to determine whether AR expression could predict response to tamoxifen or fulvestrant, as the majority of patients received aromatase inhibitors and very few patients received those therapies.Nevertheless our findings suggest that AR expression does not predict the efficacy of first-line endocrine treatment (mainly based on aromatase inhibitors in our study) in ER- or PgR-positive advanced BC, both in term of TTP and PD as best response.These results might be influenced by the limited amount of AR negative cases in this subtype of BC. Moreover, the stronger predictive effect of PgR and Ki67 could make the role of AR expression less evident, as suggested by the predictive significance of the AR/PgR ratio.Cochrane et al. . evaluatMoreover we evaluated AR/PgR ratio and we found a potential predictive value of this parameter at the cut-off of 0.96. This finding could be explained by the stronger predictive value of PgR alone in comparison with AR alone, as PgR < 10% and Ki67 ≥ 20% showed a significant association with PD as best response, and PgR ≤1% and Ki67 ≥ 20% were significantly associated with shorter TTP.PgR and Ki67 seem to be useful to select patients with a higher probability of being responsive to first-line endocrine therapy for metastatic BC, whereas AR expression does not appear to be useful. The AR expression could acquire more relevance when anti-androgen therapy will be available for BC patients.