The Hippo pathway has emerged as a key signaling pathway that regulates a broad range of biological functions, and dysregulation of the Hippo pathway is a feature of a variety of cancers. Given this, some have suggested that disrupting the interaction of the Hippo core component YAP and its paralog TAZ with transcriptional factor TEAD may be an effective strategy for cancer therapy. However, there are currently no clinically available drugs targeting the YAP/TAZ–TEAD interaction for cancer treatment. To facilitate screens for small molecule compounds that disrupt the YAP–TEAD interaction, we have developed the first ultra-bright NanoLuc biosensor to quantify YAP/TAZ–TEAD protein–protein interaction (PPI) both in living cells and also in vitro using biosensor fusion proteins purified from bacteria. Using this biosensor, we have performed an in vitro high throughput screen (HTS) of small molecule compounds and have identified and validated the drug Celastrol as a novel inhibitor of YAP/TAZ–TEAD interaction. We have also demonstrated that Celastrol can inhibit cancer cell proliferation, transformation, and cell migration. In this study, we describe a new inhibitor of the YAP/TAZ–TEAD interaction warranting further investigation and offer a novel biosensor tool for the discovery of other new Hippo-targeting drugs in future work. The Hippo signaling pathway was originally identified as the key regulator of organ size control ,2,3,4. MSince TEAD is the major transcription factor mediating YAP/TAZ oncogenic functions, it has been proposed that direct disruption of the YAP/TAZ–TEAD interaction would be an attractive strategy to restrain the transcriptional outputs of YAP/TAZ and the Hippo pathway for cancer therapy ,15,16,17Oplophorus Gracilirostris  in a 96-well plate was incubated with increasing concentrations of Celastrol for overnight at 4 °C. The next day, 100 ng of SmBiT–YAP or SmBiT–TAZ fusion protein was added into the 96-well plate and incubated for 5 min. Then, 10 µL NanoGlo Live Substrate (1:25 dilution from stock) was added into each well, mixed, and luciferase activity was immediately measured using the GloMax Navigator Microplate Luminometer.In the case of fusion proteins, a defined amount of each pair of proteins was mixed and, after adding the substrate, NanoLuc luciferase assay was performed using Turner Biosystems 20/20 luminometer  or GloMax Navigator Microplate Luminometer . To evaluate the dose-dependent effects of Celastrol on the TAZ–TEAD biosensor activity in vitro, 100 ng of LgBiT-TEAD-His in 40 µL/well buffer [30 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl4 cells (H1299 and A549) were seeded in triplicate in 24-well plates and after 24 h were treated with 1 µM of each small molecule. After four days, cells were counted. To investigate dose dependent effect of Celastrol on cell proliferation, 4 × 104 H1299 and MDA-MB-231 cells were seeded in triplicate into each well of 12-well plates. After 24 h, cells were treated with different concentrations  of Celastrol and counted three days after treatment. Data are shown as mean ± S.D.To evaluate the effects of 10 “hit” compounds on cell proliferation, 2 × 10® 2.0 Assay kit (Cat #G9242) according to the manufacturer’s protocol from Promega.Cell viability determination was performed using CellTiter-Glo2 H1299 cells were seeded in six-well plate. The next day, cells were treated with different concentrations of Celastrol. After 10 days, cells were washed with cold PBS, fixed with pure cold methanol for 2 min, and then stained for 5 min with staining solution containing 0.05% crystal violet in 25% methanol.Here, 5 × 105 H1299 and MDA-MB-231 cells were seeded in 24-well plates  and grown until 100% confluent. A wound was generated by removing the insert according to company protocol and cells were treated with 0.5, 1, and 2.5 µM Celastrol. Images were captured using a Nikon TE-2000U  inverted microscope after 16 and 18 h of treatment for H1299 and MDA-MB-231 cells, respectively.Here, 2 × 10Extracted samples were separated by 12% SDS-polyacrylamide gel electrophoresis (PAGE) (Bio-Rad) and transferred to nitrocellulose membranes. Antibodies for western blot analysis were as follows: mouse monoclonal anti-Myc (9E10) from Roche ; mouse monoclonal anti-Flag (F1804) and mouse monoclonal anti-β-actin (AC-15) from Sigma; mouse monoclonal YAP antibody (63.7) from Santa Cruz Biotechnology  and mouse monoclonal TEAD4 (ab58310) from Abcam . First antibody incubation was followed by probing with the corresponding secondary antibody and developing using Amersham Imager . Uncropped images of western blottings are available in 2, and 3 mM DTT) overnight at 4 °C with or without small molecule compound (10 µM). The next day, the buffer was aspirated and cell lysate containing TEAD1-Myc (20 µg in 200 µL), pre-incubated with small molecules, was added. After 2 h incubation, the membrane was washed three times (5 min) with 1% NP40 lysis buffer followed by western blotting using anti-myc antibody.For semi HTP dot blot analysis, HEK293T cells were transiently transfected with TEAD1-Myc. After 48 h, cells were harvested and lysed in 1% NP40 lysis buffer. GST-YAP fusion protein (500 ng in 2 µL) was placed on nitrocellulose membrane as a dot and allowed to dry for 5 min at room temperature. The membrane was then incubated in buffer  overnight at 20 °C. Bacterial cells were lysed by sonication, bacterial lysates were centrifuged to collect soluble fractions, and tagged proteins were isolated from the supernatant via Ni-NTA or GST affinity purification. Proteins were subjected to dialysis over night at 4 °C using a standard buffer  and, after concentrating, they were analyzed by SDS-PAGE, and stored at −80 °C.CodonPlusRIPL or BL21DE3 escribed . After i2, and 3 mM DTT)) were distributed into 384-well plates. Then, a spectrum library (2688 compounds) was added into a 384 well plate (10 µM) and incubated for overnight at 4 °C. The next day, 2 µL/well (0.5 µL SmBiT-YAP-His (100 ng) + 1.5 µL buffer) was added into the 384-well plate and incubated for 5 min. Biosensor activity was measured after adding 5 µL of NanoGlo substrate proceed to luciferase activity measurement (emission 460 nm). Screening was performed a second time with incubation of drug with SmBiT-YAP-His first, followed by addition of LgBiT-TEAD-His. Compounds that reduced luciferase bioluminescence of the YAP–TEAD biosensor by ≥2 fold were considered primary hits. Secondary screens were performed on these primary hits using the biosensor and five different concentrations  of each compound.HTS for 2688 small molecules were performed in the Department of Cancer Genetics and Genomics, Small Molecule core, at the Roswell Park Cancer Institute. Experimental conditions were as follows. 40 µL/well [0.5 µL (100 ng) LgBiT-TEAD-His + 39.5 μL buffer  using Polyjet transfection reagent (SignaGen SL100688) according to the manufacturer’s instructions. The next day, cells were trypsinized, and triplicates of cells were seeded in 24-well plates (2 × 105 cells per well). After 10 h, when cells were completely attached and expanded, the cells were treated with different concentrations of Celastrol . Twelve hours after treatment, the cells were lysed using passive lysis buffer (200 µL) and luciferase assays were performed using a Luciferase Assay Kit (Promega) and GloMax Navigator Microplate Luminometer. Luciferase fold-change was calculated based on the ratio of luciferase activity of the reporters in treated cells, to that of non-treated control cells.In order to have the same cell source and transfection efficiency for all samples used for each reporter assay, 3 µg of CTGF-Luc, CYR61-Luc, or STBS-Luc reporter were first transfected into HEK293T cells in 100 mm dishes   according to the manufacturer’s instruction. Fifty ng RNA/reaction was used to perform qRTPCR using the SuperScript III Platinum SYBR Green One-Step qRT- PCR Kit (Invitrogen). Then, 18S rRNA was used as an internal control.t-test (two-tailed) was used for statistical analysis. A value of p ≤ 0.05 was accepted as statistically significant.Data shown in the graphs are the average of at least triplicate experiments except for qRT-PCR, which was performed in duplicate. Unless otherwise specified, data are expressed as the mean ± S.D. Student’s In conclusion, we have successfully developed the first YAP/TAZ–TEAD NanoBiT biosensors that can easily be used to study the YAP–TEAD PPI and screen small molecules that target this complex. Such compounds disrupting YAP–TEAD interaction may be useful for the treatment of Hippo pathway–related diseases .


Four h after exposure to 10 mg L−1 ZnO NPs, we noticed a disturbance of PSII functioning that became more severe after 12 h. However, after a 24 h exposure to 10 mg L−1 ZnO NPs, we observed a hormetic response, with both time and dose as the basal stress levels needed for induction of the adaptive response. This was achieved through the reduced plastoquinone (PQ) pool, at a 12 h exposure, which mediated the generation of chloroplastic H2O2; acting as a fast acclimation signaling molecule. Nevertheless, longer treatment (48 h and 72 h) resulted in decreasing the photoprotective mechanism to dissipate excess energy as heat (NPQ) and increasing the quantum yield of non-regulated energy loss (ΦNO). This increased the formation of singlet oxygen (1O2), and decreased the fraction of open reaction centers, mostly after a 72-h exposure at 10 mg L−1 ZnO NPs due to increased Zn uptake compared to 5 mg L−1. We characterized zinc oxide nanoparticles (ZnO NPs) by dynamic light scattering (DLS) measurements, and transmission electron microscopy (TEM), while we evaluated photosystem II (PSII) responses, Zn uptake kinetics, and hydrogen peroxide (H The small size of nanoparticles (NPs) provides them with special physical and chemical properties that are not found in bulk materials allowing their utilization, among others, in agricultural products, catalysis, cosmetics, electronics, energy production, engineering, food industry, pharmaceutics and textiles ,2,3,4,5.Among the variety of metal NPs that are often used for marketable purposes, zinc oxide (ZnO) NPs are the most commonly used ones ,7,8. ZnO−1 soil and 76–760 μg L−1 water , and a higher mean rate (Vc) in comparison to the 10 mg L−1 solution  than in the higher solution (42 h) (max) and the equilibrium concentration (Ceq) displayed their higher values at the higher ZnO NPs concentration (10 mg L−1) (−1 dry wt at 5 mg L−1 and 1086.0 ± 33.7 μg g−1 dry wt at 10 mg L−1)  , Table 2 mg L−1) . Moreovemax × t)/(mK + t); C, in μg g−1 dry wt; mK, in hours; t, in hours; Ceq, in μg g−1 dry wt; Teq, in hours; Vc, concentration/hours. In parentheses, standard errors are given.The fits correspond to a Michaelis-Menten equation: C =  compared to control values, with the exception of the 24 h treatment that ΦPSΙΙ increased at 10 mg L−1 ZnO NPs and did not differ compared to control values at 5 mg L−1 ZnO NPs  increased significantly at 5 mg L−1 ZnO NPs compared to control, while at 10 mg, L−1 increased after 24 h of exposure, but decreased at further exposure time (48 h and 72 h)  b. ΦNPQ antration b. NO) did not differ compared to control values after exposure to 5 mg L−1 ZnO NPs  of PSII decreased significantly at both ZnO NPs concentrations, with the exception of the 24 h exposure, where the ETR increased at the high concentration and did not differ compared to control values at the low concentration a. With tntration a. C. nodosa leaf blades exposed to 5 mg L−1 was significantly higher than 10 mg L−1  increased at the low concentration compared to control, while at the high concentration it increased after 24 h of exposure, but decreased significantly at further exposure time (48 h and 72 h) b. After 0 mg L−1 b. qp), did not differ compared to control values after 4 h, 24 h, and 48 h exposure to 5 mg L−1 ZnO NPs but reduced after 72 h  pool (a measure of PSII open reaction centers)  and the plant species ,12,22,25C. nodosa cellular, physiological and biochemical measurable responses (biomarkers) to metallic elements  have been proposed as early warning signals of alterations in seawater quality [C. nodosa, as the test material in evaluating the effects of metal oxide nanoparticles [2 NPs, discharged by industry [In  quality ,33,38. Harticles , despiteindustry . NPs areindustry ,48,49.Photosynthetic organisms via the process of photosynthesis, transform the light energy into chemical energy with the collaboration of PSII and PSI, while the most susceptible constituent of the photosynthetic apparatus to environmental stresses is believed to be PSII ,17,18,50−1 ZnO NPs with a more than twice initial rate and higher mean rate (Vc) than 10 mg L−1 (PSΙΙ) compared to 10 mg L−1, after a 24 h exposure  than at 10 mg L−1 (Teq = 42 h), resulting in a significantly higher regulated non-photochemical energy loss as heat (ΦNPQ) after 48 h and 72 h exposure, compared to 10 mg L−1  of C. nodosa leaf blades exposed to 10 mg L−1 for 48 h and 72 h (PSΙΙ (NO (1O2) production. ΦNO consists of chlorophyll fluorescence internal conversions and intersystem crossing, indicative of 1O2 formation via the triplet state of chlorophyll (3chl*) [1O2 formation, mostly after 72 h exposure to 10mg L−1 ZnO NPs, was due to an increased Zn uptake compared to 5 mg L−1  ,53,54. T5 mg L−1 .Fv/Fm), and the redox state of plastoquinone (PQ) pool (qp), was also observed previously due to increased Zn accumulation [2O2 [qp) and increased H2O2 generation was noticed  indicate excess photon supply and associated ROS production [2O2 is moving throughout leaf veins, serving as a signaling molecule and triggering a stress-defense response [qp)   and a sip) (PSΙΙ a and ETRp) (PSΙΙ a observel levels e. HoweveC. nodosa leaf blades exposed to 10 mg L−1 ZnO NPs depicts a hormetic response. A hormetic response suggests that a basal stress level is needed for adaptive responses [−1 ZnO NPs and the time required for the induction of this mechanism was 24 h exposure. Hormesis has been extensively documented in plants, revealing that biphasic dose-responses occur commonly ,66. Thusesponses ,69. This


Introduction: Additional or better markers are needed to guide the clinical monitoring of patients with non-muscle-invasive bladder cancer (NMIBC). Aim: To investigate the influence of occupational exposures and genetic polymorphisms on recurrence and progression of NMIBC. Methods: The study includes 160 NMIBC patients. We collected on questionnaire information on demographic variables, lifetime smoking history, lifetime history of occupational exposure to aromatic amines and polycyclic aromatic hydrocarbons. Genetic polymorphism ; N-acetyltransferase 1; 2 ; cytochrome P450 1B1 (CYP1B1); sulfotransferase 1A1 (SULT1A1); myeloperoxidase (MPO); catechol-O-methyltransferase (COMT); manganese superoxide dismutase (MnSOD); NAD(P)H:quinone oxidoreductase (NQO1); X-ray repair cross-complementing group 1; 3  and xeroderma pigmentosum complementation group (XPD)) was assessed in peripheral blood lymphocytes. DNA adducts were evaluated by 32P-postlabeling. Predictors of recurrence  and progression (transition of tumor from low-grade to high-grade and/or increase in TNM stage) were identified by multivariate Cox proportional hazard regression with stepwise backward selection of independent variables. Hazard ratios (HR) with 95% confidence interval (95%CI) and two-tail probability of error  were estimated. Results: The risk of BC progression decreased with the homozygous genotype “ValVal” of both COMT and MnSOD . The results on BC recurrence were of borderline significance. No occupational exposure influenced recurrence or progression. Conclusion: Our results are supported by experimental evidence of a plausible mechanism between cause  and effect (decreased progression of tumor in NMIBC patients). The genetic polymorphisms associated with better prognosis may be used in clinic to guide selection of treatment for patients initially diagnosed with NMIBC. However, external validation studies are required. Approximately 75% of incident cases of bladder cancer (BC) present as non-muscle-invasive BC (NMIBC) and 25% as muscle-invasive BC (MIBC) . RecurreThe standard conservative therapy—transurethral resection (TUR) of the bladder tumor combined with intravesical instillations with Bacillus Calmette-Guérin and/or intravesical chemotherapy (BCG)—can hinder recurrence, but the results in view to progression are inconsistent ,7,8.Whether intravesical instillations increase cancer-specific survival (CSS) is yet debated ,9. A tooIt has been reported that the clinicopathological variables, used in the risk tables of European Organization for Research and Treatment of Cancer (EORTC), are not able to accurately predict the risk of disease recurrence and progression in individual patients . LikewisThere is an increasing evidence that genetic polymorphisms could have a role in estimating the prognosis of disease and response of treatment ,19,20,21The influence of occupational exposure on BC prognosis was investigated in 794 bladder cancer patients from Germany. A shorter relapse-free survival, though not significant, was observed in some occupations including those entailing exposure to aromatic amines . LikewisGenome wide association studies show that genetic variants have relevance on both etiology and prognosis for a number of tumors, including colorectal, pancreatic, breast, lung, and prostate cancer ,31,32,33The aim of the present study is to investigate the extent to which the occupational exposures and genetic polymorphisms influence the recurrence and progression of the tumor in a group of NMIBC patients of a retrospective hospital based case-control study ,38,39,40The present study includes the “cases” arm stemming from an earlier hospital-based case-control study fully described in previous publications ,38,39,40During hospital admission (July 1997 to December 2000), information on demographic variables, lifetime smoking history, dietary habits, lifetime occupation history was collected by a trained interviewer through standardized questionnaire; blood samples were collected and on the same day processed by centrifugation for obtaining peripheral blood lymphocytes (PBLs). The Brescia ethical committee  approved the study .N-acetyltransferase 1 (NAT1) fast, NAT2 slow, cytochrome P450 1B1 (CYP1B1) V432 L, sulfotransferase 1A1 (SULT1A1) R213H, myeloperoxidase (MPO) G-463A, catechol-O-methyltransferase (COMT) V108 M, manganese superoxide dismutase (MnSOD) A-9 V, NAD(P)H:quinone oxidoreductase (NQO1) P187S, X-ray repair crosscomplementing group 1 (XRCC1) R399Q, XRCC3 T241 M, and xeroderma pigmentosum complementation group (XPD) K751Q polymorphisms was assessed using Amplification Refractory Mutation System assay and using the GeneAmp PCR System 9700 . PCR were followed by enzymatic digestion and PCR-RFLP analysis, as previously described [p-value > 0.05. The polymorphisms were dichotomized according to additive genetic models on the basis of a priori evidence and on previous studies conducted on this study population. Genetic biomarkers were coded as 0/1 variables as follows: GSTM1 ; GSTP1 ; GSTT1 (“NULL” = 1); NAT1 (“S” = 1); NAT2 (“S” = 1); SULT (“1A1/1A1 = 1); MPO (“A/A” = 1); COMT (“WW” = 0); MnSOD (“WW” = 1); NQO1 (“MM” = 1); CYP1B1 (“WW” = 0); XRCC1 (“G/G” = 0); XRCC3 (“C/C” = 0); XPD (“A/A” = 0). The genotype frequency of MPO (“A/A” = 1); SULT (“1A1/1A1 = 1); GSTM1 ; GSTP1 ; GSTT1 (“NULL” = 1); MnSOD (“WW” = 1); COMT (“WW” = 0); XRCC1 (“G/G” = 0); XPD (“A/A” = 0); XRCC3 (“C/C” = 0); NQO1 (“MM” = 1); NAT1 (“S” = 1); NAT2 (“S” = 1) were 0,04; 0.04; 0.64; 0.52; 0.22; 0.22; 0.20; 0.11; 0.19; 0.12; 0.06; 0.69; 0.60, respectively. Automated DNA extraction was performed following the manufacturer’s instructions as previously described . Genotypescribed ,38,39,40Occupational exposures to polycyclic aromatic hydrocarbons (PAHs) and aromatic amines (AAs) were estimated as previously described . Two dic32P-postlabeling after enrichment with Nuclease P1 as previously described [p = 0.028) were found to be positively associated with occupational AAs exposure but not with BC risk. Therefore DNA adducts were likely biomarkers of exposure. However, the responsible electrophilic chemical could not be identified because adducts detected by the nuclease P1 method of 32P–post-labeling are not specific.Aliquots of 5 μg DNA were assayed for the presence of bulky-DNA adducts by escribed ,42. Resoescribed . A 0/1 vescribed , DNA addAge at the first TUR biopsy was broken down in three classes: <60; 60–69; and ≥70 years. Smoking was categorized in three levels: current smokers; former smokers; nonsmokers.Time to first recurrence of bladder cancer was the time elapsed between date of diagnosis and date of histological confirmation of a newly found bladder or prostatic urethra tumor following at least one tumor-negative follow-up cystoscopy or two surgical resection sessions for the primary tumor. Furthermore, a dichotomous variable was coded for presence/absence of recurrence.transition from low-grade  to high-grade ;increase in T stage ;increase in N stage ;increase in M stage ;cystectomy for therapy-resistant or “uncontrollable” disease (no case).Time to first progression of bladder cancer was the time elapsed between data of diagnosis and date of a recurrence at which one or more of the following events occurred: Additionally, a dichotomous variable was coded for presence/absence of progression. We coded a dichotomous variable which was 0  or 1 . The variable did not have a significant effect in two models of Cox Proportional Hazard for BC progression and for BC recurrence (data not shown).The distribution of patient characteristics  by recurrence or progression status was separately analyzed with the chi-square test, setting at 0.05 the threshold of significance. The presence of an interaction between occupational exposure and adducts/polymorphisms associated to bladder cancer prognosis was never significant. The likelihood ratio tests were:p = 0.8446 − Adducts × exposure to PAH, bladder cancer recurrenceChi-Square (1d.f.) = 0.04, p = 0.3177 − Adducts × exposure to aromatic amines, Chi-Square (1d.f.) = 1.00, p = 0.8033 − COMT × exposure to PAH, progressionChi-Square (1d.f.) = 0.06, p = 0.1681 − COMT × exposure to aromatic amines, progressionChi-Square (1d.f.) = 1.90, p = 0.6895 − MnSOD × exposure to PAH, progressionChi-Square (1d.f.) = 0.16, p = 0.3967 − MnSOD × exposure to aromatic amines, progression.Chi-Square (1d.f.) = 0.72, p-value). The statistical power of one regression coefficient in a Cox Proportional Hazard model, holding constant the coefficients of the other covariates, was computed through a STATA command by specifying the sample size (=160 patients), the effect size (the hazard ratio supplied by the model) and the overall probability of failure (=42/160 = 0.26 for BC progression).Kaplan–Meier curves were plotted for the favorable genotypes, and log rank test was applied to compare the difference between the event free survival times of each genotype. To identify significant predictors for recurrence and progression we used the multivariate Cox proportional hazard regression analysis with stepwise backward selection of independent variables. The program estimates the hazard ratio (HR) with 95% confidence interval (95%CI) and two-tail probability of error  were included in the analysis and followed up; the follow-up time was 4.63 ± 2.65 years. Using the HR provided by the model, the estimated power was 0.9996 for the combined homozygous polymorphisms of MnSOD and COMT.p-value of the chi-square test. A significant result was obtained for NQO1 (p = 0.005) and DNA Adducts (p = 0.019). The disease prognosis was better  for the homozygous genotype “MM” of NQO1. p-value of the chi-square test. Borderline significant results were obtained for MnSOD (p = 0.049), COMT (p = 0.084), NQO1 (p = 0.065) and DNA Adducts (p = 0.068). The disease prognosis was better  for the homozygous genotypes: “WW” for COMT; “WW” for MnSOD; “MM” for NQO1. MnSOD and COMT were combined in a three-class variable. With none, one single and both homozygous polymorphisms, the cases of progression were, respectively, 12 (39%), 26 (27%) and 4 (11%); the chi-square for trend was 6.69 (p = 0.0097). The different risk of progression was graphically represented by the Kaplan-Meier survival curves. As shown in 2 = 9.73; degrees of freedom = 2; p-value = 0.0077). p = 0.036) and level of DNA adducts . Time to first recurrence of bladder cancer was in average 1131 days (about three years) with a standard deviation of 819 days. p-value) including only one predictor: exposure to PAHs (Model 1) or AAs (Model 2). Although no result was statistically significant, exposure to AAs and to a lesser extent to PAHs seem to increase the risk of recurrence. p = 0.022) and the homozygous genotype “WW” of MnSOD . Time to progression of bladder cancer was in average 1690 days (about 4.6 years) with a standard deviation of 966 days. After stepwise backward selection of predictor variables, p = 0.006). A backward stepwise selection was applied to identify polymorphisms possibly associated to the risk of recurrence/progression. Thereafter, for bladder cancer progression, an interaction could be observed between the two selected polymorphisms; the p-value = 0.006 observed for carriers of both homozygous polymorphisms , 1 (one or another) and 2 (both homozygous polymorphisms). orphisms  correspoHomozygous genotype “ValVal” of both MnSOD and COMT decreased the risk of progression of NMIBC. Increasing age and level of DNA adducts increased the risk of recurrence.Strengths of this research pertain to the comprehensive and specific assessment of environmental and occupational bladder cancer risk factors, with a thorough assessment of major genetic polymorphisms related to bladder cancer as well as determination of some biomarkers of biological effect.An obvious limitation of the present study is a sample size that might not be large enough to detect weaker relationships or associations based on few cases of recurrence and progression  that are used in the EORTC Genito-Urinary Group tables to predict the probabilities of recurrence or progression of NMIBC to MBIC . The EurThe above missing information could be a possible limitation because it might affect the final analysis in terms of adjustment for potential covariates. It is worthy to note that, generally, tumor focality and size are poorly documented in medical charts . AlthougA statistical model seeks only to best describe the data. Below, we specify the relationships between the variables in terms of the biological processes that are thought to have given rise to the data. 2−) [2O2) is a more stable ROS compared to O2− and can easily diffuse throughout the cell, where it may be further processed by catalase (CAT) or glutathione peroxidase (GPx) to O2 and H2O [In all mammalian cells the aerobic respiration inevitably produces reactive oxygen species (ROS); the latter can be induced also by ionizing radiation, polycyclic aromatic hydrocarbons and smoking . MnSOD i2−) , which p2−) . Followi and H2O .A polymorphism encoding for either valine  or alanine (Ala) at codon 16 has been described in the human MnSOD gene . Ala is The role of MnSOD and its changes in gene expression during tumorigenicity in several cancer cell lines have been areas of intense investigation in the last decades; these studies have been reviewed in recent years ,49,51. IAccording to Hempel , publicl2O2 production. Expression of pro-metastatic and pro–angiogenic factors  were upregulated in the metastatic line and shown to be H2O2-dependent [A highly metastatic line 253JB-V) has been obtained from human transitional BC cells JB-V has . Given tependent . On the other hand, a study was performed on paraffin-embedded tissues  that were treated with immunohistochemical staining for catalase, superoxide dismutase and glutathione peroxidase. With respect to superficial transitional cell carcinomas, catalase and MnSOD expression was significantly lower in invasive transitional cell carcinomas, thus appearing to be associated with progression of BC .Worthy to notice, in several types of cancer, the ratios MnSOD/catalase and MnSOD/glutathione peroxidase have been suggested as biomarkers for tumor progression and metastasis .While the above-cited literature took into account the level of gene expression, our work considers the polymorphism, which causes replacement of alanine with valine at amino acid 16  . The pol2−) into H2O2, any decrease in activity in MnSOD tends to increase O2− and decrease H2O2 (Equation (1) below), with varying and contrasting effects depending on cancer type and stage of the disease.As reported by Hung  and conf2− damages DNA (resulting in bladder cancer initiation) while H2O2 promotes tumor proliferation and metastasis via pro-metastatic and pro–angiogenic factors , leading to bladder cancer progression. In our investigation, we postulate that OCOMT is an enzyme that catalyzes the transfer of a methyl-group from S-adenosylmethionine to a catechol-containing substrate molecule. By catalyzing the methylation of various endobiotic and xenobiotic substances, COMT might protect DNA from damage . In 1995Data about the relationship between COMT polymorphism and bladder cancer risk are limited. No statistically significant effects of the COMT genotypes on bladder tumorigenesis were observed among men from Italy ,39 or FrHomozygous genotype “ValVal” of both MnSOD and COMT decreased the risk of progression of NMIBC. The results are supported by experimental evidence of a plausible mechanism. The genetic polymorphisms associated with better prognosis may be used in clinic to guide selection of treatment for patients initially diagnosed with NMIBC. Before, however, external validation studies are required.


However, its application is impeded by poor rate capability and rapid capacity fading. In this work, a MnO/carbon hybrid material, in which small-sized MnO nanoparticles are tightly anchored on carbon fibers (denoted as MnO@CFs), was prepared by annealing the electrospun precursor fibers at 650 °C. When applied as the anode material of the Li-ion battery, the small size of MnO shortens the Li-ion diffusion path, and the carbon fibers not only greatly improve the conductivity but also efficiently buffer the MnO structure strain during the charge–discharge process, endowing the MnO@CFs electrode with a good rate capability (185 mAh g In the past 10 years, rational electrode design with nanomaterials has been proved to overcome well the problems associated with new battery chemistries . For exa process ,18,19.x/C) composite materials, such as FeOx/C, CoOx/C, NiO/C, and SnO2/C, due to their versatility and facility [x nanoparticles. Detailed electrochemical studies confirm that electrospun MOx/C hybrid materials exhibit an enhanced electrochemical Li-storage performance including rate capability and cyclic stability, when they are compared with the monomeric MOx electrodes [Recently, spinning techniques such as electrospinning, centrifugal jet spinning, and force-spinning have had a great application in the fabrication of metal oxide/carbon  are tightly anchored on carbon fibers (denoted as MnO@CFs). The small size of MnO shortens the Li-ion diffusion path, and carbon fibers not only greatly improve the conductivity but also efficiently buffer MnO structure strain during the charge–discharge process. When applied as the anode materials of the Li-ion battery, the hybrid architecture endows the MnO@CFs electrode with a good rate capability and cyclic stability even at a high current density.3) was obtained from Alddin Industrial Co. . N,N-dimethylformamide (DMF) was purchased from Tanjin Fuyu Fine Chemical Co., Ltd. . Polyacrylonitrile (PAN) was supplied by Sigma-Aldrich Co. . Manganese(III) acetylacetonate (Mn(acac)3 was vigorously stirred for 10 h to form a transparent solution. Then it was loaded into a plastic syringe equipped with a 23 gauge spinning nozzle. The applied voltage was 10.8 kV, and the distance between nozzle and collector was 15.0 cm. The flow rate was controlled by a syringe pump at 6.0 μL min–1. The fibers were first stabilized at 230 °C for 3.0 h in air and then further annealed at 650 °C for 1.0 h under N2 atmosphere to obtain MnO@CFs. For comparison, carbon fibers (CFs) were also prepared by a similar electrospinning route in the absence of Mn(acac)3. Besides, MnO NPs were obtained by directly pyrolyzing Mn(acac)3 at 650 °C for 1.0 h under N2 atmosphere mosphere .Powder X-ray diffraction (XRD) was characterized by using a Philips X’pert X-ray diffractometer with Cu Kα radiation (λ = 1.5418 Å). The scanning electron microscopy (SEM) was performed on a Supra-40 scanning electron microscope . For SEM characterization, the sample powder was directly fixed by conductive tape. The transmission electron microscopy (TEM) was performed on a JEM-2100 . To characterize TEM, the sample was first dispersed in ethanol by ultrasonic treatment for 15 min, then transferred to a copper mesh by adding dropwise a suspension solution of the sample. Element distribution mapping characterization was carried out by an Oxford INCA energy dispersive X-ray detector equipped on JEM-2100. The X-ray photoelectron spectroscopy (XPS) study was accomplished by an ESCLAB MKII X-ray photoelectron spectrometer with a monochromatic Mg Kα X-ray source. The Raman spectrum was recorded on a Monovista-CRS500 confocal Raman spectrometer . Thermogravimetric analysis (TGA) was performed on a DTG-60H analyzer .N-methyl-2-pyrrolidone with weight ratio of 80:10:10 were mixed into a slurry. Then the slurry was pasted to a copper foil current collector (12 mm in diameter) at an active material loading of ~1.0 mg cm−2 (thickness ~6−8 µm), and vacuum-dried at 120 °C overnight. The mass capacities were calculated based on the composite. The electrolyte consisted of 1.0 M LiPF6 in ethylene carbonate/diethyl carbonate (1:1 by volume) solution. The galvanostatic charge–discharge test was performed using a LAND CT2001A system  in the voltage range of 0.01–3.0 V. Electrochemical measurements were performed using CR2025 coin cells . Active materials, carbon black, and poly(vinylidene fluoride) in θ = 35.0°, 40.6°, 58.8°, and 70.4° were well indexed to (111), (200), (220) and (311) reflections of cubic-phase MnO (JCPDS: No. 78-0424). Due to a low graphitization degree, the broad peak at 2θ = ~26°, corresponding to the (002) diffraction of carbon fibers (JCPDS: No. 41-1487), was not obvious for MnO@CFs. However, it could be further confirmed by Raman spectra. As shown in −1, respectively. The peak at 1560 cm‒1, named the G-band, corresponds to an E2g vibration mode of sp2-bonded carbon atoms in graphene sheets. The peak at 1332 cm−1, named the D-band, is associated with carbon atoms with structural defects and disorder in the graphitic structure [D/IG) is calculated to be 1.49, also implying a low graphitization degree of carbon fibers. The low graphitization degree can be mainly attributed to the low carbonization temperature (650 °C), although the incorporation of nitrogen element in the carbon fibers also induces structure deformation. What is more, there is a Raman peak at 626 cm−1, which corresponds to the characteristic vibration of Mn–O in MnO [2O evaporation, carbon combustion) and the weight gain (MnO oxidation to Mn2O3) [2O3 [tructure . The into Mn2O3) . Based oO3) [2O3 , the MnO3/2 (641.4 eV) and Mn 2p1/2 (653.3 eV), respectively, and the result is very consistent with previous reports [XPS technique was used to study the surface element composition and electronic state of MnO@CFs. As shown in n 2p1/2 63.3 eV, r reports .TEM and SEM were applied to investigate the microstructures of products. The SEM images in 2+ to Mn0 (MnO + 2Li+ + 2e‒ → Mn + Li2O) [0 to Mn2+ and Li2O decomposition (Mn + Li2O →MnO + 2Li+ + 2e‒) [We first investigated the electrochemical performance by cyclic voltammetry (CV). As shown in  + Li2O) . This pe + Li2O) . The ano+ + 2e‒) ,40. It i−1. Two voltage plateaus at ~0.5 and ~1.2 V during the discharge/charge process are very consistent with the CV curves. The first discharge/charge capacities are 1050 and 640 mAh g−1, respectively, and the initial coulombic efficiency is 61%, being higher than the 56% of pristine MnO . What is more, MnO@CFs still delivers a discharge capacity of 185 mAh g−1 at 5.0 A g−1, however, the value for MnO dramatically declines to 60 mAh g−1. It can be concluded that MnO@CFs exhibit a better rate capability than MnO, especially at high current density, and it also confirms the key role of carbon fibers in improving the rate capability of MnO. tine MnO c. It is ectrodes ,42. The odes [−1 d. The st−1 for 500 cycles  is a powerful technique to analyze the electrode kinetics. The EIS for MnO@CFs and MnO electrode was conducted at 1.2 V after five galvanostatic discharge–charge cycles, and the impedance patterns were recorded in the frequency range from 10 mHz to 100 kHz. As shown in In summary, MnO@CFs were prepared by a convenient electrospinning approach, and their lithium-storage performance investigated. Due to the shortened Li-ion diffusion pathway, enhanced electronic conductivity and improved strain buffer for MnO volume change, the unique architecture endows MnO@CFs with a good rate capability and cyclic stability, making it an alternative anode material for the Li-ion battery.


With increasing multidrug resistance and contraindication for corticosteroid use, the goal of this study was to develop thymosin beta-4 (Tβ4) as an adjunctive therapy to antibiotics for the treatment of bacterial keratitis that effectively promotes enhanced wound healing, host defense, and inflammation resolution. Disease outcome was assessed by clinical score, slit lamp photography, and histopathology. Cytokine profile, bacterial load, PMN infiltration, and Griess and reactive oxygen species (ROS) levels were determined. Adjunct Tβ4 treatment resulted in a significant improvement compared to PBS, Tβ4, and most remarkably, ciprofloxacin, correlating with changes in mediators of inflammation and wound healing. Collectively, these data provide evidence that wound healing is an essential aspect in the development of new therapies to treat corneal infection. Use of adjunctive Tβ4 provides a more efficacious approach for bacterial keratitis by addressing both the infectious pathogen and deleterious host response. Pseudomonas aeruginosa and Staphylococcus aureus are the two bacteria most commonly associated with this type of infection [Microbial keratitis is a rapidly progressing, visually debilitating infection of the cornea that can lead to corneal scarring, endophthalmitis, and perforation. Corneal opacification, a complication of keratitis, is among the leading causes of legal blindness worldwide, second to cataracts. nfection ,2,3,4. Infection . Risk fanfection . Currentnfection ,8. As renfection ,2. Clininfection ,10. To tTβ4 is a small, naturally occurring 43-amino-acid protein that promotes wound healing and reduces corneal inflammation . It is h6 CFU/mL of the cytotoxic Pseudomonas aeruginosa strain ATCC 19660 was applied topically to the wounded corneal surface. Mice were randomized into four different treatment groups consisting of either PBS as controls, Tβ4 (0.1%), ciprofloxacin (0.3%), or Tβ4 + ciprofloxacin, administered topically (5 μL) 3× per day beginning 24 h after infection. Uninfected, normal corneas were appropriately used as an additional control where noted. All animals were treated in a manner authorized by Wayne State University Institutional Animal Care and Use committee (protocol 16-090) and conformed to the Association for Research in Vision and Ophthalmology’s statement on the Use of Animals in Ophthalmic and Vision Research.The left central cornea of 8-week-old C57BL6 (B6) female mice  were scarified as previously described . A 5 µL Infected eyes were observed daily in a blinded fashion and disease response was graded using an established grading scale  for statWhole eyes were enucleated from each treatment group at 5 days p.i. and processed for histopathological analysis. Briefly, eyes were immersed in PBS, rinsed, and fixed in 1% osmium tetroxide, 2.5% glutaraldehyde, and 0.2 M Sorenson’s phosphate buffer (pH 7.4) at a 1:1:1 ratio for 3 h at 4 °C. Eyes were rinsed (0.1 M phosphate buffer), dehydrated in graded ethanols and propylene oxide, and next infiltrated and embedded in Epon-araldite. Sections (1.5 μm thick) were stained with Richardson’s stain, observed, and photographed .Pseudomonas isolation agar in triplicate and plates were incubated overnight at 37 °C. Results are reported as CFU/cornea ± SEM.Corneas from B6 animals treated with PBS, Tβ4, ciprofloxacin, or Tβ4 + ciprofloxacin were collected at 5 days p.i. and the number of viable bacteria was quantitated. Individual corneas were homogenized in sterile 0.9% saline containing 0.25% BSA. Serial 10-fold dilutions of each sample were plated on 2, 20 mM NaH2PO4, 20 mM Tris-HCL, pH 7.4, 30 mM D-glucose, 7.5 μM DCFH-DA; ThermoFisher Scientific, Waltham, MA, USA) for 1 h at 37 °C [At 5 days p.i., corneal lysates from each treatment group were incubated in reaction buffer  containing 0.5% hexadecyltrimethyl-ammonium (HTAB). Samples were processed as previously described . The cha5 PMN  with a sNO levels were determined (as previously described ) by measTotal RNA was isolated from individual whole corneas at 3 and 5 days p.i. for gene expression analysis using RNA-STAT 60  according to the manufacturer’s recommendations and quantified by spectrophotometric determination (260 nm). 100 ng of total RNA was reverse transcribed and used to produce a cDNA template and amplified as previously described . All priNormal (uninfected) and infected corneas were removed at 3 and 5 days p.i. from B6 mice treated with PBS, Tβ4, ciprofloxacin, or Tβ4 + ciprofloxacin. Individual corneas were homogenized in 1 mL RIPA buffer  with a protease inhibitor cocktail . An aliquot of each supernatant was assayed in duplicate for IL-1β and MIP-2 per the manufacturer’s instruction . The reported sensitivity of these assays was 4.8 pg/mL for IL-1β and 1.5 pg/mL for MIP-2. Results are expressed as average ng of each cytokine/mL ± SEM.U test . For all other experiments, a one-way ANOVA followed by Bonferroni’s multiple comparison test (GraphPad Prism) was used for analysis of 3+ groups. An unpaired Student’s t test was used for comparison between 2 groups. Data were considered significant at P < 0.05. A minimum of N = 5/group/time point were used unless otherwise stated. All experiments and/or measurements were carried out in triplicate and representative data from a typical experiment is shown.Sample sizes were determined statistically prior to experimentation based on previous work ,28,29,31Clinical scores, accompanied by representative photographs, were recorded  to initiHistopathological sections from each treatment group  further Viable bacteria were determined by direct plate counts from infected corneas at 5 days p.i., as shown in Select proinflammatory mediators, known to play a role in bacterial keratitis , were inmRNA levels of laminin α3 (LAMA3), laminin β3 (LAMB3), laminin γ2 (LAMC2), fibronectin, integrin α5 (ITGα5), ITGβ1, urokinase (uPA), urokinase receptor (uPAR), and TGF-β1 were measured at 3 and 5 days following infection in the four treatment groups, as shown in Protein levels of fibronectin A, ITGα5 To begin elucidating potential mechanisms by which Tβ4 exerts its proresolving effects on the inflammatory response, we assessed expression of lipid mediator biosynthetic cyclooxygenase (COX) and lipoxygenase (LOX) enzymes, as shown in We next examined select lipid mediator end products, proresolving RvD1 and proinflammatory LTB4, along with their respective receptors, FPR2 and BLT1. As shown in Microbial keratitis is one of the most common and destructive of corneal diseases in humans, ultimately culminating in blindness. However, even less severe cases of keratitis develop some level of corneal opacity and compromised visual acuity. Development of corneal opacity is not limited to infection, though, and can be caused by injury and other eye diseases as well. Edema, inflammatory cell infiltration, fibrosis, and vascularization can all contribute to corneal opacity. Development of a therapeutic agent that clears or limits corneal scarring and promotes corneal wound healing without adverse side effects continues to be a major hurdle in the clinical setting and is the focus of the current study. Herein, we examined the therapeutic potential of Tβ4 as a combination therapy with antibiotics, revealing strong clinical implications relevant to human treatment of bacterial keratitis and potentially other conditions that result in corneal opacity.Using an established murine model of bacterial keratitis, we examined the therapeutic potential of Tβ4 as an adjunct therapy to ciprofloxacin. Although it is best known for its wound-healing capacity, Tβ4 has anti-inflammatory properties ,34 and iP. aeruginosa [We also provide evidence that proper bacterial clearance is crucial to the anti-inflammatory and wound-healing effects carried out by Tβ4. Previous in vitro work has shown that Tβ4 exhibits moderate antimicrobial activity against ruginosa ; howeverTo develop the most effective treatment possible for corneal infectious diseases, we must focus on two major aspects of pathogenesis—clearance of the invading pathogen and tissue restoration/homeostasis. As mentioned, previous studies have shown that Tβ4 promotes corneal wound healing. Sosne et al. have shown improved wound healing with Tβ4 treatment using dry eye and alkali burn models ,34. It hAdditionally, our findings indicate that Tβ4 adjunct therapy holds promise in overcoming limitations of current treatment modalities, namely the immunosuppressive effects associated with steroids. More importantly, we show that Tβ4 works synergistically with ciprofloxacin to enhance therapeutic action—a highly relevant aspect in light of increasing multidrug resistance in the major pathogens known to cause keratitis ,2. Taken


Solanum tuberosum L. This group comprises 40% of the total soluble proteins in potato tubers. Here, patatin (40 kDa) was extracted from potato fruit juice using ammonium sulfate precipitation (ASP) and exposed to high hydrostatic pressure (HHP) treatment . We investigated the effect of HHP treatment on the structure, composition, heat profile, and antioxidant potential, observing prominent changes in HHP-induced patatin secondary structure as compared with native patatin (NP). Additionally, significant (p < 0.05) increases in β-sheet content along with decreases in α-helix content were observed following HHP treatment. Thermal changes observed by differential scanning calorimetry (DSC) also showed a similar trend following HHP treatment; however, the enthalpy of patatin was also negatively affected by pressurization, and free sulfhydryl content and surface hydrophobicity significantly increased with pressurization up to 450 MPa, although both interactions progressively decreased at 550 MPa. The observed physicochemical changes suggested conformational modifications in patatin induced by HHP treatment. Moreover, our results indicated marked enhancement of antioxidant potential, as well as iron chelation activities, in HHP-treated patatin as compared with NP. These results suggested that HHP treatment offers an effective and green process for inducing structural modifications and improving patatin functionality.Patatin represents a group of homologous primary storage proteins (with molecular weights ranging from 40 kDa to 45 kDa) found in  Solanum tuberosum L.) is the most important non-grain food crop in the world and is central to global food security. The potato is a member of the Solanaceae family, which includes tomatoes, peppers, aubergines (eggplants), petunias, and tobacco. According to recent Food and Agriculture Organization estimates, China is the biggest global potato producer, with 87.26 million metric tons which constitutes almost one-third of the total worldwide production [Potato (oduction . The rapSoluble proteins present in potato fruit juice (PFJ) have been divided into three groups: patatins 30%–40%), protease inhibitors 40%–50%), and other proteins 10%–15%)  [0%–40%, p0%, and o%–15% (ma4)2SO4 precipitation is one of the most stable methods used to isolate high-yield patatin exhibiting better physicochemical and structural characteristics [Existing methods for extracting potato-protein isolates include ultrafiltration, ion exchange, gel permeation, affinity and mixed-mode chromatography, carboxymethyl cellulose complexation, acid and heat coagulation, and ethanol and various salt-precipitation methods ,13.  is required to improve their biological activities. Recently, several studies focused on eco-friendly techniques, such as the use of high hydrostatic pressure (HHP), which is relatively more economical and effective . HHP is S. tuberosum L. and evaluated the effect of HHP on its secondary structure and thermal stability in order to compare the functional properties of NP and HHP-treated patatin regarding their antioxidant potential and iron-chelating activities.Here, we isolated and purified patatin to a high purity from −1 and remained constant. Dialysis through a semipermeable membrane is among the most convenient methods for removing salts or other small molecules following purification. Dialyzed samples were removed from the membrane and stored at −20 °C following lyophilization.Patatin is slightly acidic, although at neutral pH and ambient temperature it exists as a dimer held together by non-covalent hydrophobic forces . Two pea2 = 0.998. Results indicated total protein content 96.218 µg·mL−1.The protein content of lyophilized patatin as determined using the Kjeldahl method  was 89.2As shown in Due to the glycoprotein composition of patatin, studies revealed that ~6% of patatin consists of neutral sugar and hexosamine . UsuallyThis might be explained by the interconversion of glucose into galactose, whereas a lower effect of HHP treatment was observed on xylose and mannose content. This was likely due to application of high pressure unfolding the protein secondary structure, thereby detaching some of the bound neutral sugar groups from the protein.FTIR spectra depicted pressure-induced changes in the patatin secondary structure according to characteristic shifts in several band frequencies . The res−1 and 1538 cm−1 associated with amide I and amide II bands, respectively. The spectral shapes of amide I (1600–1700 cm−1) and amide II (1500–1550 cm−1) did not change considerably when treated at a pressure level of 250 MPa. The absorption associated with the amide I band was due to stretching vibrations of the C=O amide bond and reflects the secondary structure of the protein. However, HHP-induced modifications led to a pronounced decrease in intensity of this characteristic peak. Based on the pressure effects on the widths of the depolarized and weakly polarized bands, the reorientation rate of the chains appeared dampened along with increasing pressure. The bandwidth of the strongly polarized bands (symmetric CH2-stretching) was an indication of interchain interactions [−1 and 1538 cm−1 . We observed a band in NP at 933 cm−1, which reduced abruptly in all HHP-treated samples. Our findings agreed with those of Hoppe [Both the NP and HHP-induced patatin samples returned a single major band at 1658 cmractions . Changes538 cm−1 . These fi et al. . For the330 cm−1 . An impoof Hoppe , who rep2 of 66.62 °C and a ΔH of 24.03 J·g−1. As shown in d2 from 65.88 °C to 65.64 °C was observed in patatin samples treated with 250 MPa to 550 MPa, respectively, indicating low-affinity protein-protein interactions as a result of aggregation [p < 0.05) reduction from 24.03 J·g−1 to 3.056 J·g−1 with increased pressurization from 250 MPa to 550 MPa. These findings agreed with those previously reported for rapeseed protein and soy protein, with decreases in ΔH from 10.25 J·g−1 to 3.72 J·g−1 and 7.8 J·g−1 to 0.6 J·g−1, respectively [Patatin thermal stability as measured by DSC and influenced by HHP is shown in regation . The ΔH ectively ,38. ThesResults presented in Decreases in α-helix conformation and random-coil structures along with increased β-sheet conformations induced by HHP treatment from 250 MPa to 550 MPa might result from the formation of intermolecular hydrogen bonding, which ultimately results in insoluble protein aggregates that modify secondary structure .o has greater significance in elucidating protein function associated with biological phenomena [o were induced by HHP probed by ANS as indicated following ANS binding to proteins via electrostatic interactions, resulting in fluorescence signals upon binding to hydrophobic domains. Fluorescence spectra are mainly attributed to tryptophan, tyrosine, and phenylalanine residues, with tryptophan specifically contributing to the fluorescence quantum yield, which decreases as their exposure to solvent increases [p < 0.05), with increases in Ho  at 250, 350 and 450 MPa respectively, as compared with those observed in NP (Ho = 180.4 ± 1.5).Hhenomena . ANS wasncreases . As showo = 587.5 ± 1.8) was observed when treated at 550 MPa, the gradual increase in Ho in HHP-treated samples might be attributed to the increased exposure of buried nonpolar (hydrophobic sites), which were ultimately bound to the ANS probe and resulted in increased fluorescence. The lower S0 values in NP might be attributed to the unexposed hydrophobic groups becoming buried and less accessible to ANS. The highest Ho value occurred at 450 MPa (Ho = 2434.7 ± 2.4), which might be explained by protein dissociation, leading to the exposure of additional hydrophobic regions. These results are in agreement with previous findings reporting HHP-induced hydrophobicity in different proteins [o values determined by the same ANS probe were slightly higher than those derived from lentil legumin-like protein [While a dramatic drop  in free-SH content  along with increased HHP treatment at 250, 350 and 450 MPa respectively.Positive and negative charges of side chains are randomly distributed over the patatin sequence, and it contains one cysteine residue . Figure −1 of energy is required to disrupt disulfide bonds above the normal HHP-treatment ranges (~600 MPa). Therefore, covalent peptide linkages usually remain unaffected by HHP treatment [−1), which might be due to re-arrangement of free-SH in other peptide regions to form S-S linkages leading to protein aggregation [Increases in free-SH content in the presence of HHP at up to 450 MPa might be attributed to the pressure-induced exposure of embedded SH groups inside the structure or cleavage of S-S linkage. Usually, 213.1 kJ·molreatment . The marreatment . Previoureatment ,31. We oregation ,38,54. Pregation . SDS-PAGregation . Decreasregation . However−1. Liu et al. [50 value for patatin (45 kDa) associated with DPPH-radical-scavenging activity at 0.582 mg·mL−1 [The results of DPPH-radical-scavenging assays are shown in u et al.  reported mg·mL−1 , was sig mg·mL−1 . This di mg·mL−1 .HHP treatment at 250 MPa resulted in relatively fewer conformational changes and less functional activity as compared with those observed at higher pressure ranges. This could be attributed to the reversible behavior of the protein due to its elasticity and weak interactions between intermolecular and intramolecular forces . These r−1 and 60 µg·mL−1, with a NetAUC standard equation of Y = 0.899x + 2.581 (R2 = 0.993). We observed a strong correlation between patatin concentration and antioxidant potential for the NP protein and patatin treated with 250 MPa, 350 MPa, 450 MPa, and 550 MPa. Patatin pressurized at 550 MPa showed maximum activity at 4 mg·mL−1 and yielded 110,700 ± 106 µM TE 100 g−1, followed by results from pressurization at 450 MPa , which was relatively higher than results for NP patatin . Treatment at 250 MPa resulted in higher TE values in a dose-dependent manner, which might be attributed to the increased availability of the patatin active site following pressurization. Comparison of patatin results with those of other Solanaceae family members revealed that NP exhibited a higher ORAC value as compared with tomato and eggplant, but lower than that of pepper, thereby indicating that it might be a more effective antioxidant relative to some family members [The ORACs of NP and HHP-treated patatin are presented in  members .2+ complex formation in the presence of chelating agents is indicated by reduced coloration of the reaction mixture. Several proteins from plants, as well as their hydrolysates, were studied to explore their metal chelating abilities [p < 0.05) iron-chelating activity (2+-chelation ability as compared with that observed by NP patatin. HHP treatment at 550 MPa resulted in the highest activity (87.7% at 3 mg·mL−1), followed by 450 MPa (84.8%), 350 MPa (82.4%), and 250 MPa (76.2%). However, at the same concentration, the NP showed relatively lower chelation ability (71.8%). Although both NP and HHP-treated patatin showed chelation activity >50% at all concentrations, dose-dependent effects were not significant from 1 mg·mL−1 to 3 mg·mL−1. Our findings indicated improved Fe2+-chelating activity in patatin as compared with that observed in other plant proteins, such as cannabis protein hydrolysates (50% at 2.2 mg·mL−1), sweet potato protein hydrolysate (34.74% at 2 mg·mL−1), and whey protein hydrolysate (<60% at 8% protein) [2+-chelating activity at ~89% at 1 mg·mL−1 [Inhibition of ferrozine-Febilities . Both NPactivity . All HHPprotein) ,62. Howe1,1-Diphenyl-2-picrylhydrazyl free radical (DPPH) free radical (DPPH), trolox , 2,4,6-tris(2-pyridyl)-s-triazine) (TPTZ) and 2,2′-azobis (2-aminopropane) dihydrochloride (AAPH) were obtained from Sigma-Aldrich . DEAE-sepharose fast flow and superdex 200 10/300 were obtained from . All other chemicals used in this study were of analytical grade unless otherwise stated.S. tuberosum L.) were purchased from the local market and stored at 4 °C. Potatoes were carefully sorted, washed, and peeled, followed by chopping (1 × 1 × 5 cm). Chopped potatoes were kept in 50 mM NaHSO3 to prevent enzymatic browning. PFJ was extracted using a domestic juice extractor , filtered through a 120-mm mesh sieve, and the starch slurry was allowed to sediment. The PFJ was centrifuged twice at 3000× g for 10 min  to remove all traces of starch from the juice.Patatin was isolated from potatoes according to the procedure of Racusen and Foote  with sli4)2SO4 to 60% saturation as described by Seppala et al. [2SO4. After 1 h at 4 °C, the suspension was centrifuged , and the resulting precipitate was washed twice with half of the starting volume of 50 mM sodium phosphate buffer (pH 7.0) containing (NH4)2SO4 up to 60% saturation. Subsequently, the protein isolate was filtered using an ultrafiltration (UF) column with a MW cut-off of 20 kDa and a reverse osmosis cellulose acetate membrane  at 4 °C to remove low MW compounds and salts. The retentate was subsequently freeze-dried and stored at −20 °C. Additional patatin purification was performed using an ÄKTA Protein Pure M1 system with unicorn software and HiTrap DEAE-sepharose FF , followed by transfer to a Superdex 200 10/300 column  to obtain high-purity patatin.Ammonium sulfate precipitate (ASP) was prepared from PFJ by adding  was referred to as NP.HHP treatment was performed using a high-pressure device  and a hydraulic type cell with an inner capacity of 1 L and a water jacket for temperature control. Patatin solution (2%) was prepared in mili-Q water, and 10 mL of each patatin solution was packed in polyethylene bags under vacuum to remove air bubbles. All solutions were then pressure-treated at 25 °C for 15 min at 250 MPa, 350 MPa, 450 MPa, and 550 MPa. The target pressure was reached at a rate of ~250 MPa·minv/v) β-mercaptoethanol (a thiol-reducing agent), 2% (w/v) SDS, 10% glycerol, 150 mM EDTA-Na, sucrose (60% (w/v)), and 0.01% bromophenol blue and denatured at 97 °C in a water bath for 5 min. Each denatured sample (20 μL) along with 8 μL of prestained protein-standard solution  was loaded into each well of a dual-plate electrode assembly with running buffer  SDS (pH 8.3), and 0.01% (v/v) β-mercaptoethanol) at 30 mA constant power for ~30 min at room temperature. The electrophoresed gel was transferred to staining solution (0.25% Coomassie Brilliant Blue R-250 in 10% (v/v) CH3COOH and 40% (v/v) methanol) on a shaker with gentle agitation for 2 h. The gel was destained with 40% (v/v) methanol and 10% (v/v) CH3COOH, and gel imaging was performed upon appearance of clear electrophoretic band patterns.Patatin MW determination was performed under reducing conditions using one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis  according the method described by Laemmli et al. , with slThe monosaccharide composition of NP and HHP-treated patatin was determined using the method of Ogutu and Mu , with so−1 at 25 °C), eluent B contained 1 M sodium acetate (NaOAc) in 250 mM NaOH, and eluent C consisted of only 250 mM NaOH. All eluents were filtered through 0.2-μm filter paper  prior to use. Standard solutions containing neutral sugars  with concentration ranges of 0.001 ppm, 0.05 ppm, 0.1 ppm, 0.5 ppm, 1 ppm, and 5 ppm in a single run (35 °C) at a flow rate of 0.5 mL·min−1 were used to confirm the linearity of the detector response and to determine the relative response factors. Each sample (10 μL) was injected using a 0.45-µm Ministart filter membrane at the same flow rate as that of the standard.Eluent A consisted of Mili-Q water (0.055 μS∙cmw/w)) and pressed under hydraulic pressure at ~100 bars to obtain an even, clear pallet disk of ~13-mm diameter. Infrared spectra were recorded on an IR spectrophotometer  equipped with an attenuated total reflection ATR system  and a deuterated triglycine sulfate detector . Spectra in the range of 4000 cm−1 to 400 cm−1 were obtained with an average of 64 scans and a resolution of 4 cm−1 at room temperature. Each spectrum was baseline-corrected against a blank (KBr only). The absorption peaks were fixed using spectrometer-coupled software OPUS version 6.5 (Bruker).Primary structural changes between NP and HHP-treated patatin were determined using FTIR spectroscopy. Lyophilized samples (NP and HHP-treated) were pulverized with potassium bromide , peak, denaturation (Td2), and off-set (Toff) temperatures, as well as the enthalpies of denaturation (∆H), were recorded from the thermogram using Universal Analysis 2000 software (TA Instruments).The thermal denaturation and stability of NP and HHP-treated patatin were analyzed using a differential scanning calorimeter  . NP and −1) were prepared in 10 mM phosphate buffer (pH 7.0). Far-UV CD spectra were recorded as the average of triplicate scans (190–240 nm) in a 0.1 cm path-length quartz cuvette using a scan speed of 1000 nm·min−1, a wavelength step of 1 nm, and a response time of 0.5 s. Input unit was Ɵ in deg·cm2·dmol−1. All spectra were baseline corrected by subtraction of the spectrum of a protein-free phosphate buffer .CD allows the study of protein stability, folding, and interactions. CD measurements were performed using a MS-450 spectropolarimeter . All samples , and interpretation of CD spectra was performed using the Online Circular Dichroism Analysis program DICHROWEB . The algo of NP and HHP-treated patatin was measured using 1-anilino-8-naphthalene sulfonate (ANS) as the hydrophobic fluorescence probe [w/v) in 10 mM phosphate buffer (pH 7.0) were thoroughly mixed with 20 mL of freshly prepared ANS. The mixtures were shaken vigorously and stored for 10 min in the dark. The fluorescence intensity (FI) of each sample was measured at 390 nm (excitation), and emission within the range 300 nm to 800 nm was recorded using a fluorometer . Net FI of each solution (FINet) was calculated as:The Hce probe . ANS solw/v) was calculated using linear regression analysis and used as an index of Ho.The initial slope of FI versus protein concentration  were prepared in 0.086 M tris-glycine buffer . An aliquot (3 mL) of the sample was then mixed with 40 µL Ellman’s reagent  in tris-glycine buffer. The mixture was incubated at room temperature for 30 min, followed by centrifugation at 1000× g for 5 min. The absorbance of the mixture was measured at 412 nm using a UV-Vis spectrophotometer, with tris-glycine buffer used as a blank. Results consisted of the absorbance value divided by the molar extinction coefficient of 13,600 mol·L−1·cm, and free-sulfhydryl (SH) content was expressed as µmol·g−1 of protein. All measurements were conducted in triplicate.NP and HHP-treated samples were determined using Ellman’s reagent according to the method of Beveridge et al. , with sl−1) was added to 2 mL of freshly prepared DPPH solution (0.1 mM in 95% ethanol). The mixture was vortexed using a mixer  and incubated in the dark at 27 °C for 30 min. The absorbance of each solution was measured at 517 nm using a UV-vis spectrophotometer  at room temperature. The lower absorbance of the reaction mixture indicated higher radical-scavenging activity. Radical-scavenging activity was calculated as the percentage of DPPH discoloration using the following equation:−1 DPPH solution after incubation (30 min) at room temperature, and “AD” represents the absorbance of 2 mL of 0.1 mmol·L−1 DPPH solution with 1 mL Milli-Q H2O.The DPPH-radical-scavenging activity of patatin was measured according to the method described by Zhang et al. , with mo−1) were added to 20 μL phosphate buffer in 96-well microtiter plates. Subsequently, 20 μL sodium fluorescein solution (63 nmol·L−1) was added to each well, followed by incubation at 37 °C for 15 min. After incubation, 140 μL of freshly prepared AAPH (18.28 mmol·L−1) solution was pipetted into the duplicate wells, whereas other duplicate wells were diluted with 140 μL of phosphate buffer  alone. After 10 min of vigorous shaking, fluorescence intensity was measured using a ChameleonTM V multi-label microplate reader . The system was set to fluorescence mode, and the fluorescence intensity of each well was recorded 60 times at 2-min intervals. The excitation and emission filter wavelengths were set at 485 nm and 535 nm, and the detection temperature was kept constant at 37 °C.An ORAC assay was performed according to the method described by Prior et al. . Phospha−1) was prepared. The linear regression equation was y = 0.8898x + 2.5805 (R2 = 0.9929). ORAC values of the samples were expressed as μg Trolox equivalent per 100 g of sample (μg TE 100 g−1). The results were interpreted according to the ORAC equation as follows:i represents the relative fluorescence intensity of the reaction solution. The AUC corresponds to the relative fluorescence decay, and (Δt) represents the interval time, with Δt in this study at 2 h. AUCsample and AUCblank represent the AUCs of the sample and the blank, respectively, and net AUC represents the net AUC between the sample and the blank.The fluorescence intensity of each sample was determined without the effect of AAPH  in order to calculate the relative fluorescence intensity using Equation (1). The relative fluorescence intensity was used to calculate the area under the curve (AUC) using the approximate integration method shown in formula (2). ORAC values were expressed as the net area under the curve (netAUC) between the samples and the blank as shown in Equation (3). A calibration curve for the trolox standards . Each sample (600 µL) was mixed with freshly prepared FeSO4 (60 µL) and 2.42 mL Milli-Q H2O and incubated for 30 min at room temperature with continuous shaking (80 rpm). After incubation, 120 µL of 5 mM ferrozine solution was added to the mixture, and decolorization due to Fe2+ dissociation was monitored by determining the absorbance at 532 nm. Milli-Q H2O and EDTA were used as negative and positive controls, respectively. The percentage of inhibition of ferrozine–Fe2+ complex formation was determined using the following formula:Determination of ferrous ion-chelation activity was performed according to the method of Miao et al.  and at dp < 0.05.Results were expressed as the mean ± standard deviation (SD) of triplicate experiments unless otherwise stated. GraphPad Prism software version 5.0  and SAS 8.1 software  were used for all statistical analyses and to generate data plots. OriginPro 8.5.1  was used to plot FTIR spectra. Statistical significance was determined at p < 0.05) along with increases in HHP treatment. Furthermore, we observed significant enhancement of antioxidant potential in HHP-treated patatin as compared with that observed in the NP. Our findings suggested that pressurization might significantly contribute to patatin stabilization. The structural, thermal, and bio-functional properties were dependent upon the intensity of HHP treatment. Therefore, this suggested that patatin from S. tuberosum L. might constitute a potential antioxidant for use in enhancing human health. Moreover, our findings revealed the potential application of HHP treatment as an economical and effective means of modification that might contribute to the conversion of certain proteins into value-added ingredients. Despite this progress, there remain many unanswered questions and unexplained avenues of research to be explored.Patatin was effectively isolated and purified from PFJ and modified by HHP treatment (250–550 MPa). The effects of HHP treatment on the structure, composition, heat profile, and antioxidant potential indicated significant changes in patatin structure and physical properties, which enhanced its physicochemical behavior. HHP treatment also enhanced the antioxidant potential of patatin according to results measuring DPPH-radical-scavenging ability and ORAC. Iron-chelation activity was also improved significantly (


Dirofilaria immitis strains at 3 µg/kg after single and multiple oral dosages. Three studies were conducted to evaluate higher oral moxidectin doses for efficacy against confirmed ML-resistant D. immitis strains.Moxidectin has previously shown limited efficacy (≤ 44.4%) against confirmed macrocyclic lactone (ML)-resistant D. immitis L3 and randomly allocated to treatments. Study 1: 6 groups of dogs (n = 8) were inoculated with JYD-34 (Day − 30) and treated as follows: T01, negative control; T02–T05, moxidectin at 3, 6, 12 or 24 µg/kg, respectively, on Day 0 only; T06, moxidectin at 3 µg/kg on Days 0, 30 and 60. Study 2: 10 groups of dogs (n = 5) were inoculated (Day − 30) with either JYD-34  or ZoeLA  and treated as follows: T01 and T02, negative controls; T03–T05, moxidectin at 24, 40 or 60 µg/kg, respectively, on Days 0, 28 and 56; T06 and T09, moxidectin at 3 or 60 µg/kg on Day 0 only; T07, T08 and T10, moxidectin at 24, 40 or 60 µg/kg, respectively, on Days 0, 28 and 56. Study 3: 5 groups of dogs (n = 5) were inoculated with ZoeMO (Day − 28) and treated as follows: T01, negative control; T02, moxidectin at 3 µg/kg moxidectin on Day 0 only; T03–T05, moxidectin at 24, 40 or 60 µg/kg, respectively, on Days 0, 28 and 56. All dogs were necropsied for adult heartworm recovery ~ 4–5 months post-inoculation.Dogs were inoculated with 50 All moxidectin-treated dogs showed significantly lower worm counts than controls. The efficacy of moxidectin administered once at 3 µg/kg was 19% (JYD-34), 44.4% (ZoeLA) and 82.1% (ZoeMO). Increasing both the dose and the number of dosages of moxidectin improved efficacy, with 100% protection obtained using three dosages of moxidectin at either 40 µg/kg  or 60 µg/kg (ZoeLA). Three dosages of 24 µg/kg were also highly effective, providing ≥ 98.8% efficacy for all three strains.Increasing both the dose and number of consecutive monthly dosages of moxidectin improved the efficacy against ML-resistant heartworms. Based on these data and other technical considerations, the 24 µg/kg dose was considered the optimal dose for further commercial development. However, canine HW disease incidence continues to increase in many countries around the world including the USA  (T01) or HPMC capsules filled with the appropriate amount of hand-pulverized ProHeart® tablets to deliver a dose of 3, 6, 12 or 24 µg/kg moxidectin on Day 0 only (T02–T05) or on Days 0, 30 and 60 (Groups T01 and T06)  were inoculated with 50 psule] T0 or HPMC n = 5) of dogs were inoculated with 50 D. immitis L3 on Day − 30; four groups  were given the JYD-34 strain and six groups  were given the ZoeLA strain. On Day − 3, dogs were moved to their allocated treatment pens. Treatment consisted of either a placebo (a tablet containing inert ingredients) or oral moxidectin (ProHeart® tablets shaved to deliver the correct dose). Placebo tablets were administered on Days 0, 28 and 56 . Moxidectin was administered at 3 (T06), 24 , 40  or 60 µg/kg , and treatments were given either on Day 0 only (T06 and T09) or on Days 0, 28 and 56   were allocated to treatment following a randomized block design within room, with block based on pen location within room and Day − 35 body weight. Ten groups (n = 5) and pens according to a randomized block design. Block was based on pen location and Day − 34 body weights. All animals were inoculated with 50 D. immitis L3 on Day − 28 (ZoeMO strain) and then treated with either a placebo (tablet containing inert ingredients) or oral moxidectin (ProHeart® tablets shaved to deliver the correct dose). Placebo tablets were given on Days 0, 28 and 56. Moxidectin treatments were administered on Day 0 only at 3 µg/kg (T02) or on Days 0, 28 and 56 at 24 (T03), 40 (T04) or 60 µg/kg (T05)  were allocated to five treatment groups  consisted of approximately equal numbers of male and female dogs. No dog had ever received ProHeart® 6 or any ML-containing product within 90 days prior to the start of the study.All dogs were housed individually within a mosquito-proof facility in indoor cages that complied with accepted animal welfare legislation and guidance. They were fed an appropriate maintenance diet of a commercial dry canine ration and had access to water ® or WITNESS® Heartworm Antigen Test Kit, Zoetis, Parsippany; SNAP Heartworm RT Test, Idexx, Westbrook, ME, USA; Solo Step® Canine Heartworm Test, Heska, Loveland, CO, USA) at least 1 week prior to L3 inoculation. Heat treatment of serum prior to HW antigen testing was not performed. To identify any infections not previously detectable at study start, additional blood samples were collected on Day 59 (Studies 1 and 3) or Day 60 (Study 2) and examined for D. immitis microfilariae and tested for adult D. immitis antigen. In Study 1, blood samples were also collected on Days 104 and 122 and examined for adult D. immitis antigen to detect infection due to experimental inoculation.Dogs were determined to be free of HW infection by modified Knott’s test, and commercially available adult HW antigen tests  collected from naturally infected dogs from the middle to southeastern USA. JYD-34 (Studies 1 and 2) was originally collected from a naturally occurring field case of canine HW in Illinois in July 2010. ZoeMO-2012 (ZoeMO) (Study 3) was collected in December 2012 from the same dog from which the original JYD-34 strain had been collected 2.5 years earlier . The dog had been maintained in mosquito-proof quarters with no additional administration of macrocyclic lactones during the intervening time. ZoeLA (Study 2) was collected from a naturally occurring field case of canine HW in Louisiana in June 2013. All three isolates were validated as infective strains through the diagnosis of circulating microfilariae, positive heartworm antigen test results and adult worm recovery in recipient animals. Additional details on these strains are provided by McTier et al. .Aedes aegypti mosquitoes reared and maintained at Zoetis  as previously described  transformation was applied to all counts prior to analysis, and the geometric means (back-transformed least squares means) were calculated.The experimental unit for treatment was the individual dog. Within each study, the numbers of adult Percent efficacy, relative to the control group and based on geometric means, was calculated as follows:Treatment differences were assessed between control and treated groups using contrasts in a general linear mixed model analysis of natural logarithm transformed worm counts and a significance level of α = 0.05. The models included the fixed effect of treatment and random effects of room (study 2 only), block within room, and error. SAS PROC MIXED was used to fit the statistical models and the Kenward–Roger option was used to calculate degrees of freedom for the test-statistics used for pairwise treatment comparisons. All analyses were carried out using SAS/STAT Release 9.3 .D. immitis strain were positive for adult worms at necropsy, and worm counts confirmed the adequacy of the infections  ≤ 13.21, P < 0.0001 for all treatments except for a single dose of moxidectin at 3 µg/kg (T02), t(34) = 2.33, P = 0.0259; Study 3, 7.34 ≤ t(16) ≤ 16.0, P < 0.0001)  showed significantly reduced adult worm counts compared to control dogs  and approaching the efficacy of one dose at 24 µg/kg (53.2%)  showed similar results to those obtained using the JYD-34 strain when treated with three consecutive monthly dosages of moxidectin; only one dog treated with 24 µg/kg moxidectin had a single worm at necropsy, and a dose of either 40 (T04) or 60 µg/kg (T05) was 100% effective in preventing the development of D. immitis  provided > 99% efficacy in dogs inoculated with the ZoeLA strain, with 1 of 5 dogs infected. A similar pattern was observed at 60 µg/kg, with a single dose (T09) providing 88.2% efficacy (5 of 5 dogs infected) and three dosages  providing complete prevention in all dogs.There were no mortalities among the dogs involved in any of the three studies. Various abnormal health observations were made sporadically during these studies across all treatment groups . Most signs resolved with either no or minimal treatment . All clinical signs recorded during the studies were deemed to be unrelated to treatment with moxidectin.D. immitis strains to MLs is real and has the potential to become more widespread [D. immitis populations with reduced susceptibility to MLs.The increasing number of HW LOE reports in dog populations across the USA is well documented. Between 2000 and 2002, the number of LOE cases reported to the US Food and Drug Administration, Center for Veterinary Medicine rose from 405 to 951, and the following 12 months (2002–2003) saw another increase to 1503 . Twenty despread , 33, 43.D. immitis strains  well characterized in terms of their response to MLs, Bourguinat et al. [The three strains used in the present evaluations, JYD-34, ZoeMO and ZoeLA, have been well characterized as ML-resistant , 36, 37.t et al. . These dD. immitis in dogs when compared across various effectiveness studies [D. immitis provided by moxidectin likely rests in its inherent potency and its physicochemical and pharmacokinetic properties [Historically, moxidectin has been shown to be the most potent of the MLs in preventing the development of  studies , 46–48. operties , 49. Moxoperties –52. Thesoperties . Moxidecoperties , 39, 54.operties . This reD. immitis strains provided by repeated dosages has not been reported for any of the present commercially available oral preventives  of oral moxidectin increased the efficacy against all three ML-resistant HW strains  evaluated, with three consecutive monthly dosages of ≥ 40 µg/kg moxidectin providing complete preventive efficacy. Additionally, three dosages of moxidectin at 24 µg/kg was highly effective (≥ 98.8%) in preventing the development of all three HW strains, with only a single dog in each group (1 of 5) having just one or two worms. This high level of protection against ML-resistant D. immitis populations with reduced susceptibility to MLs, with an adequate margin of safety in the most sensitive patient population (collies). For these reasons, 24 µg/kg moxidectin was selected as the optimal dose for further commercial development in a new oral HW preventive.Furthermore, based on the existing very high efficacy  obtained after three months of dosing with 24 µg/kg moxidectin against all three resistant strains and the unique attributes of the moxidectin molecule, it is likely that four or more consecutive monthly dosages of 24 µg/kg moxidectin may provide additional protection. This dose should provide robust HW preventive efficacy against the strains to which most dogs in the USA will be exposed, including D. immitis with complete protection obtained using three dosages of moxidectin at either 40 µg/kg  or 60 µg/kg (ZoeLA). Three consecutive monthly dosages of 24 µg/kg moxidectin provided very high efficacy (≥ 98.8% efficacy with 4 out of 5 dogs heartworm-free) against all three ML-resistant strains. Moxidectin administered at 24 µg/kg was selected as the optimal oral dose to develop as a safe and effective means of protecting dogs against HW disease in the face of increasing ML resistance.Increasing both the dose and the number of consecutive monthly oral dosages of moxidectin resulted in increased preventive efficacy against ML-resistant


Apis mellifera) during their lifetime is to feed the larval offspring. In brief, young workers (nurse bees) secrete a special food jelly that contains a large amount of unique major royal jelly proteins (MRJPs). The regulation of mrjp gene expression is not well understood, but the large upregulation in well-fed nurse bees suggests a tight repression until, or a massive induction upon, hatching of the adult worker bees. The lipoprotein vitellogenin, the synthesis of which is regulated by the two systemic hormones 20-hydroxyecdysone and juvenile hormone, is thought to be a precursor for the production of MRJPs. Thus, the regulation of mrjp expression by the said systemic hormones is likely. This study focusses on the role of 20-hydroxyecdysone by elucidating its effect on mrjp gene expression dynamics. Specifically, we tested whether 20-hydroxyecdysone displayed differential effects on various mrjps. We found that the expression of the mrjps (mrjp1–3) that were finally secreted in large amounts into the food jelly, in particular, were down regulated by 20-hydroxyecdysone treatment, with mrjp3 showing the highest repression value.One of the first tasks of worker honey bees ( Molting or ecdysis (from ancient Greek: ἐκδύω (ekduo)) in insects is induced by ecdysteroids, a group of steroid hormones named after the first isolated molting hormone ecdysone ,2,3. At Apis mellifera), where hemolymph ecdysteroid titers decline prior to the pupal molt, increase again in young unpigmented pupae with intense eye pigmentation (~600–800 pg/µL or ~320 pmol/µL of ecdysteroids), and drop to a minimum during cuticle melanization before ecdysis (~50–150 pg/µL and ~60 pmol/µL of ecdysteroids) . T. T17]. TMRJP1–9) ,25 that MRJP1–9) . HoweverMRJP1–9) ) [17]. Tted diet . This cimarkedly ,24,26. Bmarkedly , the regecdysone . Furtheron stage . This is5 pg/µL) , we expeA. mellifera), sealed brood frames containing pupae with dark eyes were taken from one colony belonging to the university’s apiary , Germany) and placed into an incubator at 34 °C with 60% relative humidity. Immediately after hatching, honey bee workers were assigned to six cages of 60 bees each. The cages were kept at 30 °C and a humidity level of 60%. The bees received water ad libitum and 7 g food dough  per day, which either did not contain 20-hydroxyecdysone (20E) (control group—3 cages) or was supplemented with 0.1 mg/g 20E  (20E group—3 cages). This concentration was previously shown to be effective. In brief, when employing larval feeding experiments that resulted in pupation and adult emergence, there was only a slight increase in mortality observed  [mrjps compared to well-fed controls [For the collection of newly emerged honey bees (v/v) diethylpyrocarbonate). RNA quantity and quality were photometrically determined  and the total RNA was stored at −80 °C. From 1 µg total RNA, cDNA was synthesized according to Buttstedt et al. [Before RNA extraction, the eyes were removed from the bees’ heads, as compound eyes are known to contain PCR inhibitors . Every ht et al. , and purTM SYBR® No-Rox Kit . Nine bees per cage were analyzed, which were pooled to three cohorts consisting of three bees each. Initially, ribosomal protein S5α (rpS5α), actin related protein 1 (arp1), proteasome subunit beta type-1 (pros26), and peptidyl-prolyl cis-trans isomerase-like 2 (ppil2) were chosen as reference genes, but only ppil2 showed a Ct (threshold cycle) value standard deviation (SD) below 1 [Quantitative real-time PCR (qPCR) was performed with 1 µL cDNA (adjusted to a concentration of 15 ng/µL DNA) using the SensiMix  below 1  within tt values of all investigated genes were determined with the CFX Connect TM RealTime System  and the associated software . All samples were measured in technical duplicates and subjected to a repeat test cycle when the Ct value deviation value exceeded 0.5. The amplification efficiency was determined by serial dilution qPCR  4.9.0 . The denp > 0.20) were analyzed with one-way ANOVA with subsequent post-hoc Scheffe test. In addition, partial correlations between consumed food amount and the day after hatching were performed. The raw relative gene expression dataset was not normally distributed . However, normal distribution was obtained by Box-Cox transformation . The influences of variables such as cage identity and 20E administration on gene expression of all nine mrjps were analyzed using a general linear model (GLM), regarding the cage as a random factor, and using a post-hoc Bonferroni test. The expressions of the significantly down-regulated genes (mrjp1–3) were correlated to consumed food amount with a Spearman rank correlation. Acini volumes were normally distributed  after log transformation, and thus analyzed by one-way ANOVA with post-hoc Scheffe test.All statistical analyses were performed with STATISTICA 8.0 . The significance level for all tests was set to α < 0.05. Differences in the control and 20E group regarding consumed food  . In addition, ten forager and ten nurse bees were collected from a hive and also measured. Acini volumes were calculated according to Omar et al. .p < 0.001) , but neither group showed any significant differences regarding consumed food on a specific day  (p = 0.83).During the incubation time in the cages, bees in the control group and the 20E group consumed an increasing amount of food dough over time  . In regap = 0.18 and 0.48, respectively). mrjps in relation to the reference gene. In the control group, the expression of mrjp1 was highest , followed by mrjp2 (491 ± 239), whereas expressions of mrjp3, 7, 4 , and 5 and 6 , were moderate. Finally, expressions of mrjp8 and 9 were considerably lower  than for all other mrjps , downregulation was moderately high for mrjp1 and 2 , and substantially higher for mrjp3   compared to the control group (19.8 ± 1.9 mg) at day 3 . As the expression of all three genes did not correlate with food consumption, the reduced gene expression in the 20E group can directly be ascribed to the effect of 20E.A significant effect of cage identity in general, or an interaction between gene expression patterns and cages, was not detected  . To exclat day 3 , food comrjps was moderate and not significant , MS = 142.87, F = 17.30, p = 0.053). However, there was a significant interaction between genes and treatment . Indeed, 20E had an effect on some but not all mrjps. This dynamic pattern of 20E effecting mrjp gene expression regulation became most prominent for mrjp3 transcriptional control, as illustrated by the hierarchical clustering, where mrjp3 formed its own cluster within the group of mrjp1–7  [The overall effect of 20E on the expression of all  mrjp1–7 . Interesmrjp expression in the caged workers, but had also an effect on acini volumes (p < 0.001). Workers in the 20E group had significantly decreased acini volumes (1.53 ± 0.76 mm2 × 10−3) compared to workers in the control group (2.64 ± 0.89 mm3 × 10−3)  (3 × 10−3) . Additionally, though control bees did not significantly differ from forager bees (3.00 ± 1.64 mm3 × 10−3) , bees fed with 20E had smaller acini volumes than foragers   . However< 0.001) .mrjps and significantly repressed the expression of the most prominent food jelly mrjps (mrjp1–3). Whether or not this is a direct hormonal effect cannot be unequivocally verified by analyzing the achieved data set, but three scenarios are possible:(1)mrjp promotor regions [mrjp gene expression, mediated by sole agonist activity at the nuclear ecdysone receptor complex, seems an unlikely explanation for the observed mrjp gene expression effects. However, the EcR/Usp dimer might also activate the expression of unknown target genes that in turn repress mrjps.One way in which steroids act in target tissues is by binding to nuclear receptors that recognize thereupon specific DNA sequences, arranged as direct or inverted repeats (for review see Henrich & Brown ). Hither regions . However regions ). Theref(2)mrjp regulation.Besides the classical nuclear receptor action of ecdysone described above, a nongenomic action involving membrane receptors and second messengers  has repeatedly been suggested ,42. A fi(3)mrjps might be reduced simply because of a reduced availability of the precursor” building block” protein.The production of vitellogenin, being the precursor protein for food jelly proteins ,15, can 20-Hydroxyecdysone (20E) slightly decreased overall expression of all mrjp1–3, especially, are massively upregulated in brood rearing nurse bees, compared to caged bees of the same age, given a protein-poor diet  [mrjp expression more similar to nurse bees in the hive, compared to the caged bees without protein in their diet  compared to nurse bees from the hive (4.35 ± 1.82 mm3 × 10−3), despite comparable mrjp expression. Thus, efficient food jelly protein production is probably dependent on two steps: (1) basic upregulation of mrjp expression, in comparison to the reference gene, after pollen consumption, which is most likely dependent on vitellogenin; and (2) increased acini volumes, to enable, in general, the much higher protein production in nurse bee hypopharyngeal glands [In a previous study, we showed that 00-fold) . Caged ceir diet . Thus, mn levels ,44. Althn levels , caged wl glands . This hyl glands , a factol glands , again il glands . This timrjps during this phase. Subsequently, ecdysteroids decrease in the hemolymph, probably leading to cessation of mrjp gene repression, accompanied by an induction of expression due to pollen consumption after hatching. This circumstance is most important for MRJP1–3, as these proteins are the main proteins in RJ [As the concentrations of ecdysteroids are high during the pupal phase of the bees ,7,9, it ns in RJ , and themrjps (mrjp1-3), with mrjp3 showing the highest downregulation (18-fold), while having no effect on mrjp4-9.We here show that food supplementation with 20E has indeed an inhibitory role on the expression of the most abundant food jelly