Compared to PRE-concentrations, elevated (p < 0.05) T was observed at IP on WK2–WK5 (mean difference: 135–511 pg·mL−1), at 30P on WK3 (mean difference: 81.0 ± 30.1 pg·mL−1) and WK5 (mean difference: 56.6 ± 22.7 pg·mL−1), and at 60P on WK3 (mean difference: 73.5 ± 29.7 pg·mL−1) and WK5 (mean difference: 74.3 ± 28.4 pg·mL−1). Compared to PRE-concentrations, elevated (p < 0.05) C was noted on all weeks at IP (mean difference: 9.3–15.9 ng·mL−1) and 30P (mean difference: 6.0–19.9 ng·mL−1); significant (p < 0.006) elevations were noted at 60P on WK1 (mean difference: 9.1 ± 3.0 ng·mL−1) and WK5 (mean difference: 12.8 ± 2.9 ng·mL−1). Additionally, TC was significantly reduced from PRE-values by 61% on WK1 at 60P (p = 0.040) and by 80% on WK5 at 30P (p = 0.023). Differences in T, C, and TC were also observed between weeks at specific time points. Although each workout affected concentrations in T, C, and/or the TC ratio, changes appeared to be modulated by the presence of overload and workout duration. During periods of elevated training or competition, athletes and coaches may consider monitoring these hormones for consistency and as a means of assessing workout difficulty.To determine the salivary steroid response to high-intensity functional training (HIFT) competition workouts, saliva samples were collected from ten recreationally trained male and female competitors during a 5-week (WK1–WK5) international competition. Competitors arrived at their local affiliate and provided samples prior to (PRE) their warm-up, immediately (IP), 30-min (30P), and 60-min (60P) post-exercise. Samples were analyzed for concentrations of testosterone (T), cortisol (C), and their ratio (TC). Generalized linear mixed models with repeated measures revealed significant main effects for time ( Since 2005, the number of CrossFit® affiliates has increased ~1000 fold  and participation in the CrossFit® Open (CFO) has increased from 26,000 competitors in 2011 to over 380,000 competitors in 2017 [High-intensity functional training (HIFT) is a group-based fitness strategy that utilizes aerobic, gymnastic, and weightlifting exercises performed at a high-intensity and in a constantly varied fashion to promote general physical preparedness . Trainin−1) [Limited data suggests that HIFT is indeed a high-intensity (or vigorous) exercise strategy. Although the severity differs between workout designs , they ha−1) ,6, marke−1) , and inf−1) . Despite−1) ,10,11 an−1) ,10,11 maTestosterone (T) is an anabolic hormone that leads to the activation of many important anabolic processes including increases in transcription, translation, signaling-enzymes, and structural proteins . While t® GamesTM. Although the format and composition for each CFO workout is unique, they typically resemble HIFT workouts [Evidence suggests that several programming variable combinations will influence the acute hormonal response to resistance exercise ,17,21. T® for a minimum of 2 years, and they had never progressed beyond the preliminary stage of the Reebok CrossFit GamesTM . Of the five females included in this study, four were naturally cycling while the fifth was using a hormone-based contraceptive (Medroxyprogesterone). However, because the post-exercise response in T and C is not expected to differ between women who use hormone-based contraceptives and those who do not [n = 1) and personal reasons (n = 1). Another male participant could not supply a sufficient saliva sample on WK1.Five, male  and five, female  recreationally trained HIFT practitioners who were enrolled to compete in the CFO were recruited for this study. Following an explanation of all procedures, risks and benefits, each participant provided his/her written informed consent to participate. This investigation was approved by the University’s Institutional Review Board. All participants were free of any physical limitations . Participants were identified as being “recreationally trained” because they had been regularly participating (at the time of recruitment) in CrossFito do not ,25, thisSaliva samples were collected prior to and following exercise on each week of the competition. During the first four weeks (WK1–WK4), data for all competition workouts were collected at mid-day (12:00–3:00 p.m. EST (Eastern Standard Time)). The same four participants began these workouts on each Friday at ~2:00 p.m. EST, while the remaining participants always began these workouts on each Saturday at ~12:00 p.m. EST. The final competition workout (WK5) was initiated by all participants at night, within 30 min of its release (~8:00 p.m. EST). Saliva samples were analyzed for concentrations of T and C.® affiliate in front of an official judge.Competition officials released CFO workouts individually on each Thursday (5:00 p.m. pacific time) of the 5-week competition and competitors could complete multiple attempts before submitting their best attempt by the following Monday (5:00 p.m. pacific time). Each competition workout consisted of two or more exercises performed for a prescribed (Rx) number of repetitions using standardized technique and/or resistance. A “Scaled” option, which consisted of modified technical requirements and/or reduced resistance, was also available. Regardless, competition workouts were scored by the number of repetitions completed within a set time frame (WK1–WK4) or by time to completion . All competition workout descriptions, standards, and scoring criteria are available on the competition website  and are ® affiliate approximately 30–40 min before attempting the competition workout and having refrained from consuming any food, drinking hot fluids, or brushing their teeth for two hours prior to their arrival. Upon their arrival, participants were asked to remain seated for 15 min before providing their resting sample . Subsequently, all competitors completed a self-selected warm-up for approximately 10–15 min before attempting the competition workout. IP saliva samples were taken within one minute of workout completion. Participants were then asked to remain in a relaxed position  while at the affiliate leading up to the 30P and 60P collection time points. The participants could drink water ad libitum while waiting but were asked to refrain from drinking water within 10 min of any post-exercise collection time point.Saliva samples were obtained prior to exercise (PRE), immediately post-exercise (IP), 30 min post-exercise (30P), and 60 min post-exercise (60P). Competitors reported to the CrossFitApproximately 2 mL of saliva were collected into a cryovial  in duplicate at each time point using the passive drool method and stored at −80 °C until assay. Concentrations of T and C were assessed via commercially available, enzyme-linked immunosorbent assays (ELISA) and a spectrophotometer . To eliminate inter-assay variance, all samples were thawed once and analyzed in duplicate in the same assay run by a single technician, with an average coefficient of variation (CV) of 9.92% for T and 9.76% for C.p < 0.05.Changes in concentrations of T, C, and the TC ratio were separately examined across time using a generalized linear mixed model with maximum likelihood estimation and an autoregressive-heterogenous repeated covariance to account for the dependent relationships existing between time points. Due to known effects of sex on steroid hormone concentrations ,28, sex n = 1), WK2 (n = 3), WK3 (n = 2), WK4 (n = 1), and WK5 (n = 4). In contrast, all male participants completed all workouts using Rx standards. The selected workout format and performance scores for each competitor across each week of the CFO are presented in Throughout the CFO, participants did not report any changes to their normal dietary or training habits. The number of participants completing each workout version  varied each week. Specifically, the number of female competitors who used Rx standards varied between WK1 (p = 0.318) or main effect for sex  were observed for T concentrations. However, a significant main effect for time was observed  where T concentrations were elevated from PRE-values at IP on WK2–WK5 , at 30P on WK3  and WK5 , and at 60P on WK3  and WK5 . On specific time points between weeks, T concentrations were significantly higher (p = 0.036) at IP on WK2 (487 ± 113 pg·mL−1) compared to WK5 (229 ± 49 pg·mL−1), and significantly higher (p = 0.023) at 30P on WK4 (318 ± 79 pg·mL−1) compared to WK5 (150 ± 29 pg·mL−1). Additionally, compared to WK5, trends were noted for higher T concentrations at PRE on WK3 , at IP on WK4 , and at 30P on WK2 . No other differences were found in T concentrations within or between each week. Individual values for T concentrations across each time point are presented in Neither a significant sex x time  or main effect for sex  were observed for C concentrations. However, a significant main effect for time was observed  where C concentrations were elevated from PRE-values at IP on WK1–WK5 , at 30P on WK1–WK5 , and at 60P on WK1  and WK5 . Compared to WK5, PRE-concentrations of C were significantly higher (p ≤ 0.017) on WK1–WK4 (mean difference: 4.7–7.6 ng·mL−1). At 30P, C concentrations were significantly higher (p = 0.004) on WK1 (24.1 ± 2.9 ng·mL−1) compared to WK3 (14.0 ± 1.7 ng·mL−1). Likewise, higher C concentrations were observed at 60P on WK1 (19 ± 3.1 ng·mL−1) compared to WK2 (11.8 ± 2.0 ng·mL−1) and WK3 (10.2 ± 1.2 ng·mL−1). Additionally, a trend was noted for higher C concentrations at IP on WK1 (25.8 ± 3.9 ng·mL−1) compared to WK3 . and WK5 , lower C concentrations at 30P on WK3 (14.0 ± 1.7 ng·mL−1) compared to WK4  and WK5 , and lower (p = 0.084) C concentrations at 60P on WK3 (10.2 ± 1.2 ng·mL−1) compared to WK5 (16.2 ± 3.1 ng·mL−1). No other differences were found in C concentrations within or between each week. Individual values for C concentrations across each time point are presented in Neither a significant sex x time  or main effect for sex  were observed for the TC ratio. However, a significant main effect for time was observed  where the TC ratio was significantly reduced from PRE-values at 30P on WK5  and at 60P on WK1 . Trends were also noted for reduced TC ratio on WK1 at 30P  and on WK5 at IP  and 60P . Compared to WK5, a significantly higher TC ratio was observed at 30P on WK2  and trends for a higher TC ratio were observed on WK3  and WK4 . Compared to WK1, significantly higher TC ratios were observed at 60P on WK2  and WK3 . No other differences were found in the TC ratio within or between each week. Differences between post-exercise TC and PRE-values during each week of the CFO, as well as differences between weeks at specific time points, are illustrated in Neither a significant sex x time . Given the consistent variation among each workout, it is not surprising that the available data on this training style also suggests variability in the acute physiological responses of different workouts . Fernand athlete . In thatAs an anabolic hormone, the physiological role of testosterone is to increase protein synthesis through a variety of mechanisms , but itsIn the present investigation, significant T elevations were observed on each week except WK1. Though it might be assumed that maximal effort was given on each week, it cannot be confirmed. Therefore, the presence of (or lack of) overload may have been responsible for this inconsistency. Each competitor was given the option of completing the Rx or the scaled version of each workout based on their ability to perform the required exercises and do their best. However, differences between loads prescribed and exercise modifications within each workout version were not based on a specific metric . Rather, the prescribed modifications were standard, which could reduce the available options to being impossible or not sufficiently challenging for individual competitors. An example of this occurred on WK3, where a participant attempted the Rx challenge but could not complete a “muscle-up” which prohibited her from continuing the workout. The lack of a significant T response on WK1 may have been the consequence of athlete workout selection and the differences between Rx and scaled intensities. On WK1, four female competitors chose to complete the scaled version of the workout, which included modified intensity and complexity for each exercise. While four females also chose to complete the scaled version of WK4 and a significant T response was observed, modifications were only made for two of the four prescribed exercises. Comparatively, the scaled version of WK4 also appeared to require more work than the scaled version of WK1. For instance, the first exercise of WK4 required participants to complete 55 deadlift repetitions at 135 lbs.  and all participants finished this portion. In contrast, the only exercise on WK1 that required participants to lift external weight was the walking lunge while holding a 35-pound barbell in the front rack position. The female participants, who completed the scaled version of WK1, averaged ~216 repetitions  which would roughly equate to a total volume load of 2975 lbs. Had they chosen to complete the Rx version of WK 1  and completed the same number of repetitions, they would have nearly doubled their total volume load. Though speculative, the increased volume load combined with more difficult body weight exercises (chest-to-bar pull-ups vs. jumping chin-ups and bar-over burpees vs. burpees) may have elicited a more robust T response.Differences in the T response were also noted between weeks, where elevations in T on WK2 (at IP) and WK4 (at 30P) were ~2.1 times greater than those seen on WK5 at each respective time point. While differences between workouts may have played a role, the reduced sample on WK5 and its occurrence at night prohibit any definitive conclusions. On WK5, two of the male competitors were not available to complete the workouts due to personal reasons. Across all time points, the average T concentrations in these two individuals were approximately 2.4 times greater than those observed in their remaining male counterparts and 1.7 times greater than the combination of all other participants. Previously, greater T concentrations following resistance exercise have been observed in men compared to women ,28, and Elevations in C were seen following exercise on each week of the CFO, while specific differences were noted between weeks prior to and following exercise. Prior to exercise, lower C concentrations were observed on WK5 compared to all other weeks. Although it is possible that psychological factors related to stress  may have played a role , the cleAt rest, the TC ratio is believed to be indicative of an individual’s current anabolic status because of T and C’s interaction along the hypothalamic-pituitary-adrenal axis  and theiAlthough HIFT constantly varies the design, intensity, and complexity of workouts, practitioners can modify workouts to best suit their skillset. Within the context of the CFO, these options are limited and may influence the endocrine response to exercise. This presents a challenge when attempting to study responses in recreational competitors . Given their status, these individuals cannot be expected to be proficient in all possible exercises and/or always able to lift the specific loads prescribed during the CFO. Unless competition workouts are known in advance or a much larger sample is accessible, allowing participants to select between Rx and scaled versions of each challenge may be unavoidable in future endeavors. Despite that limitation, our findings suggest that CFO workouts that provide an overload will stimulate a T response, whereas a lesser (or no) response may occur if modification is inappropriate. Regarding C, each workout elicited an elevated response, but longer duration workouts may elicit a greater response. When examined together, the TC ratio was mainly consistent throughout the CFO. Inconsistent responses in T, C, or TC may be indicative of inappropriate selection of workout difficulty, overstress, or both. Considering the potential influence of post-exercise concentrations in these hormones on performance, HIFT practitioners who continue their normal training should consider monitoring their changes during the CFO. Athletes and their coaches may find this useful for selecting between Rx and scaled workouts, and for modifying programming between weekly CFO workouts. However, these findings are inconclusive, possibly due to sample size limitations, the competitive environment, and time of day. Future research on these hypotheses is warranted.


Aedes aegypti, to honeydew odorants from the green peach aphid, Myzus persicae, and the pea aphid, Acyrthosiphon pisum, feeding on fava bean, Vicia faba. We used solid phase micro-extraction and gas chromatography-mass spectrometry to collect and analyze headspace odorants from the honeydew of A. pisum feeding on V. faba. An eight-component synthetic blend of these odorants and synthetic odorant blends of crude and sterile honeydew that we prepared according to literature data all attracted female A. aegypti. The synthetic blend containing microbial odor constituents proved more effective than the blend without these constituents. Our study provides the first evidence for anemotactic attraction of mosquitoes to honeydew and demonstrates a role for microbe-derived odorants in the attraction of mosquitoes to essential plant sugar resources.Plant sugar is an essential dietary constituent for mosquitoes, and hemipteran honeydew is one of the many forms of plant sugar that is important to mosquitoes. Many insects rely on volatile honeydew semiochemicals to locate aphids or honeydew itself. Mosquitoes exploit volatile semiochemicals to locate sources of plant sugar but their attraction to honeydew has not previously been investigated. Here, we report the attraction of female yellow fever mosquitoes,  Honeydew is a sugar-rich liquid  secretedPlant sugar is an essential basic food for adult male and female mosquitoes . MosquitInflorescence odorants are the most important cues that guide mosquitoes to floral nectar ,24,25. NAphid honeydew and floral nectar contain sugars and amino acids ,29,30 thAedes aegypti, is a widely distributed mosquito that can vector many arboviruses including dengue, yellow fever, chikungunya, and Zika [Ae. aegypti have been observed to imbibe honeydew from pea aphids, Acyrthosiphon pisum, and green peach aphids, Myzus persicae, colonizing broad beans, Vicia faba . Working with broad bean-colonizing pea and green peach aphids and Ae. aegypti as model organisms, we tested the hypothesis that Ae. aegypti females are attracted to (i) natural aphid honeydew odorants, (ii) a synthetic blend of these odorants, and (iii) the microbe-produced constituents of this blend.The yellow fever mosquito, and Zika ,42,43,44ad libitum with a 10% sucrose solution. Once a week, DP fed female mosquitoes on his arm , 3 days later giving them access to a water-containing 354 mL cup  with a paper towel  lining its sides. We transferred strips of paper towel carrying Ae. aegypti eggs into a small circular glass dish (10 cm diameter × 5 cm high), filled with water and inoculated with brewer’s yeast . Upon larval hatching (2–4 days later), we transferred the larvae with the water to water-filled trays (45 × 25 × 7 cm high) and provisioned them with NutriFin Basix tropical fish food . Daily, we transferred pupae via a 7-mL plastic pipette  to water-containing 354-mL Solo cups (Solo Cup Comp.) covered with a mesh lid. We aspirated eclosed adults into separate Solo cups, fitted with a cotton ball soaked in a 10% sucrose solution. Simon Fraser University’s Office of Research Ethics advised that ethics approval is not required for this study.We reared mosquitoes in the insectary of Simon Fraser University (SFU) at temperatures of 23–26 °C, a photoperiod of 14L:10D, and a 40–60% RH. We maintained adult mosquitoes in mesh cages (30 × 30 × 46 cm high) and provisioned them We grew fava beans from seed  in a greenhouse at SFU  under a 16L:10D light regime, watering plants every other day. We kept colonies of green peach aphids and pea aphids on fava bean plants in separate bug dorms (61 × 61 × 61 cm)  under these same conditions.−1 via a mechanical pump, thus carrying volatiles towards the mosquito that could now enter the Y-tube. For each replicate, we employed a clean Y-tube, a new female mosquito, and new test stimuli. We recorded the lateral arm of the Y-tube that a mosquito entered first, and considered all mosquitoes making no decisions within 5 min as non-responders, which we excluded from the statistical analyses.To determine whether mosquitoes are attracted to aphid-infested or mechanically injured plants, we ran bioassays in Y-tube olfactometers  inclined at 45° ,18,19) tWe assigned potted bean plants with 6–10 “true” leaves to a treatment or a control group and placed them in separate plastic cages (21 × 26 × 32 cm). We released 20 green peach aphids or 20 pea aphids onto treatment plants but not control plants, allowing honeydew to accumulate on treatment plants over seven days. Over this time, colonies of green peach aphids and pea aphids grew to a mean size of 31 and 103 individuals, respectively. To account for the possibility that mechanical injury-related plant odorants in addition to honeydew odorants affect the mosquitoes’ responses, we mechanically injured each plant  by cuttiTo determine whether plant odorants derived from mechanical injury suffice to attract mosquitoes, we mechanically injured plants (see above), and in Y-tube olfactometers offered mosquitoes a choice between two non-infested bean plants (each inside an oven bag) that we had, or had not (control) mechanically injured (see above) , Exp. 3.To separate the effects of aphid feeding and aphid presence on the attraction of mosquitoes, we offered mosquitoes a choice between two intact bean plants (each inside an oven bag) that we paired with a mesh-covered Petri dish containing, or not (control), 100 non-feeding pea aphids , Exp. 4.−1) as the carrier gas with the following temperature program: 40 °C (5 min), 10 °C min−1 to 280 °C (held for 10 min). We identified volatiles by comparing their retention indices (RI) relative to n-alkane standards [We collected (commonly discoloured) droplets of honeydew from plants heavily infested with pea aphids, using a 10-µL glass capillary fitted with a rubber bulb. We collected a total of 50 µL of honeydew and expelled it into a 4-mL glass vial with a rubber septum lid. Through this lid, we inserted a carboxen-polydimethylsiloxene-coated solid-phase micro extraction (SPME) fibre , allowing absorption of honeydew odorants on this fibre for 24 h at room temperature. Prior to each odorant collection, we conditioned the fibre at 280 °C for 5 min in a gas chromatograph (GC) injection port. We desorbed odorants from the fibre in the hot (250 °C) injection port of the GC, and analyzed odorants by GC-mass spectrometry (MS) using a Saturn 2000 Ion Trap GC-MS fitted with a DB-5 GC-MS column  in full-scan electron impact mode. We used a flow of helium (35 cm standards  and theitandards  and with1), and in a previous study (CHD2) [1 at doses equivalent to 2.5 × 101 µL and 2.5 × 100 µL of crude honeydew , the CHD2 at honeydew equivalent doses of 2.5 × 106 μL, 2.5 × 105 μL, 2.5 × 104 μL, 2.5 × 103 μL, 2.5 × 101 µL, and 2.5 × 100 µL (Exps. 8–15), and the SHD at honeydew equivalent doses of 2.5 × 106 μL and 2.5 × 105 μL . The dose equivalents tested in our bioassays are biologically relevant, considering that 2.5 × 101 µL of honeydew is approximately the amount of honeydew produced by 25 pea aphids per day [2 [We prepared three blends of synthetic honeydew odorants. Two blends reflected the composition of crude honeydew collected and analyzed in this study (CHDy (CHD2)  Table 21, and iny (CHD2)  . Each cage (77 × 78 × 104 cm) was wrapped with black cloth except for the top, allowing light entry from above. We provided illumination with a shop light housing  fitted with two conventional 1.22-m fluorescent tubes . The cage housed two burette stands separated by 25 cm, each stand carrying a Delta trap 50 cm above the cage floor . We prep1 at a dose of 2.5 × 101 µL honeydew equivalents (Exp. 5), and the CHD2 at doses of 2.5 × 106 μL, 2.5 × 105 μL, 2.5 × 104 μL, 2.5 × 103 μL, and 2.5 × 101 µL honeydew equivalents (Exps. 6–10). To compare the relative attractiveness of crude and sterilized honeydew, we tested the CDV2 vs. the SHD at doses of 2.5 × 106 μL and 2.5 × 105 μL honeydew equivalents .We dissolved all synthetic honeydew blends in a 1-mL mixture of pentane (50%) and ether (50%), pipetted treatment and solvent control stimuli into separate 4-mL glass vials with a 2-mm hole in the lid, and randomly assigned the treatment and the control vials to one trap in each pair. We tested the CHDWe analyzed behavioral data using SAS statistical software version 9.4 , excluding experimental replicates with no mosquitoes responding. We analyzed data from Y-tube olfactometer experiments (Exps. 1–4) using a two-tailed exact-goodness-of-fit test. For cage experiments 5–15, we compared the mean proportions of responders to paired test stimuli using a binary logistic regression model and worked with back-transformed data to obtain means and confidence intervals.p = 0.007; Exp. 2: z = −2.56, p = 0.017; p = 0.82; p = 0.52)  or pea aphids (Exp. 2) attracted 81% and 77.3% of responding mosquitoes, respectively, significantly more than aphid-free control plants  , Exp. 4.Desorption and GC-MS analyses of SPME collected honeydew headspace odorants consistently revealed eight compounds , includi1 , but not at a dose of 2.5 × 100 µL honeydew equivalents (Exp. 6), attracted significantly more mosquitoes than the corresponding solvent control stimuli  at a do2 , 2.5 × 105 μL , 2.5 × 104 μL , 2.5 × 101 μL , and 2.5 × 100 μL .The CHDure data ) attract2 and the SHD were tested head-to-head at honeydew dose equivalents of 2.5 × 106 μL (Exp. 14) and 2.5×105 μL (Exp. 15), CHD2 at the lower dose, but not the higher dose, attracted more mosquitoes than the SHD  honeydew [Lasius niger [Some of the odorants found in natural crude honeydew may originate from the bacterium odorants . This inhoneydew . Other ohoneydew . Odorantus niger , and appus niger . Well knus niger ,36,37,38us niger . Here, wA. fabae, feeding on fava bean plants [Megoura viciae, feeding on fava bean plants [Crude aphid honeydew seems to have common odor constituents. In crude honeydew of pea aphids feeding on fava bean plants, the same five odorants  were found by us and a previous study , one odon plants , and thrn plants . At leasn plants ,61.Consumption of honeydew by mosquitoes in the field ,11 contrWe show that sugar-foraging females of the yellow fever mosquito are attracted to bean plants infested with green peach aphids or pea aphids. Mosquito females respond to the honeydew expelled by aphids but not to the physical presence of aphids or the mechanical damage inflicted on plants. The attractiveness of honeydew is due to its odorants. A synthetic blend of honeydew odorants tested at doses equivalent to those of honeydew-soiled plants did attract mosquitoes. At the lowest dose tested, the synthetic blend with microbial odor constituents was more attractive than the blend without these constituents. By responding to honeydew odorants, mosquitoes can locate and exploit honeydew and substitute it for floral nectar when nectar is scarce or honeydew is particularly abundant. Our study may lead to the development of a trap lure that combines mammalian-, inflorescence- and aphid-derived odorants for trapping both sugar- and blood-seeking mosquitoes.


Taenia solium (T. solium). Several drugs for the treatment of cysticercosis, such as praziquantel, albendazole, and mebendazole, have certain toxicity and side effects. Considering that there is no vaccine available, we studied a new vaccine for cysticercosis in this study. The complete TSOL18 gene and the optimized SP-TSOL18 gene fragments were obtained using PCR-based accurate synthesis method. The secretory and intracellular recombinant pMG36e-SP-TSOL18/Lactococcus lactis (L. lactis) and pMG36e-TSOL18/L. lactis vaccines of T. solium were prepared. Immune responses in mice orally immunized with these two recombinant L. lactis vaccines were analyzed by the determination of specific antibodies  in serum, spleen lymphocyte proliferation, and cytokines  in spleen lymphocyte culture supernatant. Our results showed that, after the first immunization, in these two recombinant L. lactis vaccine groups, the levels of serum specific IgG, IgG2a, and IgG1 increased on 14–56 d and reached the highest level on days 42, 42, and 28, respectively. The level of specific sIgA of intestinal mucosa also increased on 14–56 d and reached the highest level on day 42. The level of spleen lymphocyte proliferation increased on 14–56 d and reached the highest level on day 42. The levels of IL-2, IFN-γ, IL-4, and IL-10 in spleen lymphocyte culture supernatant increased on 14–56 d and reached the highest level on days 42, 42, 28, and 28, respectively. These results indicated that the recombinant pMG36e-SP-TSOL18/L. lactis and pMG36e-TSOL18/L. lactis vaccines can induce specific cellular, humoral, and mucosal immune responses in mice with oral vaccination. More importantly, the recombinant pMG36e-SP-TSOL18/L. lactis vaccine has a better immune effect. In summary, these results demonstrated the possibility of using L. lactis strain as a vector to deliver protective antigens of T. solium.Cysticercosis is a cosmopolitan zoonotic parasitic disease infected by larval of  Cysticercosis is a zoonotic parasitic disease that seriously harms human health and is distributed in many developing countries or areas in Latin America, Africa, and Asia –3. A larTaenia solium (T. solium) oncosphere, which has good immunogenicity and immunoprotection. The TSOL18 gene is considered to be the most promising candidate vaccine gene and has been studied extensively [Lactococcus lactis (L. lactis) is an important probiotic in intestine of human and animal. It is generally recognized as safe (GRAS) food grade microorganism and naturally present in milk foods, which has functions of regulating microecological balances, inhibiting tumor growth, reducing cholesterol, delaying aging, and improving immunity [L. lactis and pMG36e-TSOL18/L. lactis vaccines and investigate their induced immune responses in mice. Kunming mice were immunized orally with these two recombinant L. lactis vaccines, and then antibodies of serum and intestinal mucosa, proliferation and cytokines of spleen lymphocytes were determined at different time points of postvaccination.TSOL18 is a specific antigen of ensively , 14. Lacimmunity . With thimmunity –21. The TSOL18 gene sequence (Accession No. AF017788), using the L. lactics as a host system for gene optimization, the TSOL18 gene was synthesized using a PAS (PCR-based accurate synthesis) method. The signal secretion protein SPUSP45 was added at its N-terminus to synthesize the SP-TSOL18 target gene. Restriction enzyme digestion was performed using SacI and HindIII for the TSOL18 gene fragment and plasmid pMG36e to construct recombinant plasmids pMG36e-TSOL18 and pMG36e-SP-TSOL18. And then transferred them into Top10 competent cells, respectively. Positive clones were selected to perform restriction enzyme digestion and sequencing identification.According to the 600) value of the bacteria solution reached 0.2 to 0.3.MG1363 bacteria solution was inoculated in 1 mLG/L-SGM17 (M17 medium + 0.5 M sucrose + 2.5% glycine + 0.5% glucose) liquid culture medium and cultured at 30°C for 72 hours. After obvious turbidity appeared, this culture was inoculated into 5 mLG/L-SGM17 liquid culture medium, incubated at 30°C for 24 hours, and 5 mL of this culture was diluted into 50 mLG/L-SGM17 culture medium and cultivated for 24 hours. Then, 50 mL of the culture was diluted in 400 mL of G/L-SGM17 medium and continually cultured for 3 to 5 hours until the optical density (ODμL) and placed in an −80°C freezer.The culture was transferred into a 50 mL centrifuge tube and centrifuged at 4000 rpm at 4°C for 20 minutes, and the supernatant was discarded. The pellet was resuspended in 400 mL of 4°C precooled 0.5 M sucrose containing 10% glycerol, thoroughly shaked, centrifuged, and then discarded the supernatant. Then, the pellet was resuspended in 200 mL of 4°C precooled 0.5 M sucrose containing 10% glycerol and 0.05 M ethylenediaminetetraacetic acid (EDTA), placed in ice water for 15 minutes. The cooled culture was centrifuged again at 4000 rpm at 4°C for 20 minutes, and the supernatant was discarded. The pellet was resuspended by adding 100 mL of 4°C precooled 0.5 M sucrose containing 10% glycerol and shaken well. The culture was centrifuged again, and the supernatant was discarded. The pellet was resuspended in 4 mL of 0.5 M sucrose containing 10% glycerol. After shaking, the final culture was separated into 100 tubes  was immediately added. The cultures were placed on ice untouched for 10 minutes, then allowed to resuscitate at 30°C for 2–3 hours. The bacteria solution were centrifuged at 4000 rpm and the supernatant was discarded, and the pellet was concentrated in 100 μL GMMC recovery medium. The solution was then spreaded on 10 μg/mL Erythromycin GM17 agar plates, cultured at 30°C for 2–3 days. Plates were kept in a relatively closed environment, observed for colony growth, and small circular white opaque colonies formed in about one week.The previously obtained plasmids pMG36e-TSOL18 and pMG36e-SP-TSOL18 were separately mixed with competent μg/mL Erythromycin liquid culture medium, which was then incubated at 30°C for 72 hours until the solution appeared cloudy.Positive single colony was picked and placed into 1 mL of G/L-SGM17+ 5 μL of double-distilled water, placed in a boiling water bath for 10 minutes, then placed in an ice bath for 2 minutes, centrifuging again, and the supernatant was retained for extracting genomic DNA. A 579 bp region of the T. solium activated oncosphere TSOL18 gene, based on the sequence reported by Gauci et al. (1998), was amplified using the forward primer 5′-ATGGTTTGTCGTTTTGCTT-3′ and the reverse primer 5′-TTATGAACGACGAACCTTTTTA-3′. After a positive clone was confirmed, it was prepared for use as an expression strain.The abovementioned cultured bacteria solution was centrifuged at 10000 rpm for 10 minutes, and the supernatant fluid was discarded. The bacteria solution was centrifuged and washed three times with double-distilled water, and the supernatant fluid was discarded each time. The pellets were resuspended in 30 L. lactis MG1363 bacteria were cultured in GM17 liquid medium. Colonies that were identified as positive were separately selected and inoculated in GM17 liquid medium containing Erythromycin. After stationary culturing at 30°C for 72 hours, the culture was centrifuged at 6000 rpm and 4°C for 15 minutes together with the transformed bacterial solution. The precipitate and supernatant were collected separately for later use. Precooled phosphate-buffered saline solution (PBS) was used to resuspend the precipitate. This culture was then placed in an ice bath and ultrasonicated (300 watts) for 20 minutes, alternating 4 s of ultrasonication with 8 s wait intervals. An equal volume of 2x SDS loading buffer  was added, and the culture was then placed in a boiling water bath for 4–8 minutes. The total 20 μL samples were prepared after cooling and then were loaded in SDS-PAGE and Western blot gel electrophoresis plates to separately detect the expression of supernatant and intracellular components.Untransformed Eighty specific-pathogen-free (SPF) Kunming mice  were purchased from Experimental Animal Center, Daping Hospital, Third Military Medical University, China 2012-0005). All mice were 6–8 weeks old, each weighed about 20 g. All experimental procedures involving the mice were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals approved by the State Council of People's Republic of China.L. lactis vaccine. In group 2, the mice were immunized with recombinant pMG36e-TSOL18/L. lactis vaccine. In group 3 and 4, the mice were immunized only with L. lactis bacteria and PBS as control groups, respectively. The immunization doses were 3 × 109 CFU [The Kunming mice were randomly divided into four groups with each group containing twenty mice. The mice in group 1 were immunized with recombinant pMG36e-SP-TSOL18/ 109 CFU  and giveμg/mL recombinant TSOL18 antigen. The horseradish peroxidase (HRP) conjugated goat anti-mouse IgG (1 : 100000 dilution), IgG1 (1 : 10000 dilution), IgG2a (1 : 10000 dilution), IgA (1 : 10000 dilution). A diaminobenzidine (DAB) chromogenic substrate was used for staining, and absorbance (OD450) values were measured by a microplate reader. Each assay was performed in duplicate.Four mice were taken from each group on days 0, 14, 28, 42, and 56 after the first immunization. Blood was collected from the orbital vein and let stand for 12 hours at 4°C, then centrifuged at 2000 rpm for 10 minutes to separate the serum. At the same time, mice colons were aseptically removed, cut into pieces, and placed in ice saline solution then ground to homogenate and centrifuged at 3500 rpm and 4°C for 10 minutes. The supernatant was collected and frozen at −20°C for future investigation. The serum specific IgG, IgG1, IgG2a, and intestinal mucosa sIgA  were eva6 cells/mL in RPMI 1640 containing 10% fetal bovine serum. After the number of viable cells was above 90%, penicillin (100 U/mL) and streptomycin (100 U/mL) were added.Spleens were also aseptically removed from the four mice per group on days 0, 14, 28, 42, and 56 after the first immunization. The spleen lymphocytes were isolated according to the instructions of the mouse spleen lymphocyte separation kit. Spleen lymphocyte suspension was prepared and adjusted to 5 × 106 cells/mL) were dispensed in 24-well culture plates. Three wells were set for each specimen and contained 1 mL stock solution, 1 mL stock solution combining recombinant TSOL18 antigen (10 μg/mL), and 1 mL stock solution combining ConA (10 μg/mL). The cells were incubated in a 5% CO2 incubator at 37°C for 48 hours. Two hours before the end of the incubation period, 100 μL of CCK-8 solution was added to each well. Then, absorbance values (OD450) were measured for each well with a microplate reader. Each assay was performed in duplicate.The cell counting kit CCK-8 detection method was used. Spleen lymphocytes (2 × 106 cells/mL) were dispensed in 24-well culture plates using the method as described in γ, IL-4, and IL-10 cytokines using a commercial ELISA kit according to the manufacturer's manual. Each assay was performed in duplicate.Spleen lymphocytes . ANOVA models were used for multigroup comparisons, and comparison between groups was performed using the least significant difference method (LSD). Values of Recombinant plasmids pMG36e-TSOL18 and pMG36e-SP-TSOL18 were constructed following Figures TSOL18 gene fragment and pMG36e vector fragment were digested by restriction enzymes SacI and HindIII. The results of 1% agarose gel electrophoresis were shown in Figures TSOL18 gene and pMG36e vector.The amplified L. lactis MG1363 bacteria containing pMG36e-TSOL18 and pMG36e-SP-TSOL18 was used to perform PCR identification. The results showed that lanes 1–6 were the PCR products of L. lactis MG1363-positive bacteria containing pMG36e-TSOL18 and pMG36e-SP-TSOL18. Both are consistent with the expected results . The level of each antibody in recombinant pMG36e-SP-TSOL18/L. lactis group was significantly higher than that of recombinant pMG36e-TSOL18/L. lactis group (p < 0.05)  levels in mice were increased from 14 to 56 days after the first immunization. sIgA reached the highest level on day 42 which was significantly higher than L. lactis and PBS control group (p < 0.05). The level of sIgA in recombinant pMG36e-SP-TSOL18/L. lactis group was significantly higher than that of recombinant pMG36e-TSOL18/L. lactis group (p < 0.05)  see .L. lactis and pMG36e-SP-TSOL18/L. lactis groups, the spleen lymphocyte proliferation levels in mice were increased from 14 to 56 days after the first immunization. Spleen lymphocyte proliferation reached the highest level on day 42, which was significantly higher than L. lactis and PBS control group (p < 0.05). The level of spleen lymphocyte proliferation in recombinant pMG36e-SP-TSOL18/L. lactis group was significantly higher than that of recombinant pMG36e-TSOL18/L. lactis group (p < 0.05)  see .L. lactis and pMG36e-SP-TSOL18/L. lactis groups, the levels of IFN-γ, IL-2, IL-4, and IL-10 in spleen lymphocyte culture supernatant were increased from 14 to 56 days after the first immunization. Each cytokine reached the highest level on days 42, 42, 28, and 28, respectively, which was significantly higher than L. lactis and PBS control group (p < 0.05). The level of each cytokine in recombinant pMG36e-SP-TSOL18/L. lactis group was significantly higher than that of recombinant pMG36e-TSOL18/L. lactis group (p < 0.05) (see Figures As compared to the level on day 0, in both recombinant pMG36e-TSOL18/T. solium in the humans and pigs and led to serious health and economic consequences [T. solium infection [T. solium primarily infect hosts through ingestion, L. lactis as an oral vaccine for T. solium infection may be a more effective as well as practical new vaccine for the prevention and control of cysticercosis [T. solium eggs or gravid proglottid contamination, and oncospheres are hatched and developed into cysticerci, which would bring great harm to the host. Oncosphere was the key stage in the invasion of host, thus, developing an effective candidate vaccine from oncosphere antigens may be an economic and effective means.Cysticercosis is a zoonotic parasitic disease caused by the larvae of equences , 25. Theequences . Therefonfection , 27. Beccercosis . CysticeTSOL18 gene was successfully cloned from the T. solium oncosphere for the first time. Its coding sequence was highly homologous to other corresponding protective antigens in the tapeworm family, and it was highly conserved among different strains and between different clones [TSO18 gene of T. solium, and the TSO18-GST protein was successfully expressed in E. coli [L. lactis is a good candidate for the delivery of heterologous proteins in foods, which have many advantages such as safety, simplicity, affordability, easiness to prepare, and practicality [L. lactis vaccine of T. solium.Several recombinant antigens have been expressed and evaluated as potential vaccine candidates such as 45 W, 18 ku, and 16 ku –31. Amont clones , 34. Sub E. coli . There h E. coli –40, but ticality . As far L. lactis was a major secretory protein and was currently a signal peptide which improved the efficiency of exogenous protein secretion [L. lactis and pMG36e-TSOL18/L. lactis were successfully prepared and the TSOL18 protein expressed in extracellular supernatant and intracellular precipitation has specific antigenicity. It is proved that the signal peptide SP and propeptide fusion sequence LEISSTCDA can effectively realize the extracellular expression of TSOL18 and increase the success rate of protein secretion [The data showed that the signal peptide SPUSP45 derived from ecretion , 43. Theecretion , 45. ThiL. lactis and pMG36e-SP-TSOL18/L. lactis vaccines. Our results demonstrate that these two recombinant vaccines can induce significant immune responses compared to the levels at the time of nonvaccination at day 0, including antibody isotypes, cytokines associated with activation of both CD4+ Th 1 and Th 2 cells, and CD4+ T-cell proliferation.It is important to investigate the immune responses generated in mice by recombinant pMG36e-TSOL18/L. lactis and pMG36e-TSOL18/L. lactis might induce a MHC classII restricted CD4+ T cell response. The CD4+ T cell may play a role in B-cell differentiation, proliferation, and isotype regulation [+ T cells proliferate and differentiate into effector Th cells. This is consistent with the generation of specific cellular immune responses we observed by recombinant L. lactis vaccination [The lymphocyte proliferation test is an important indicator of cellular immunity. As our results showed, spleen lymphocytes showed a strong proliferative response upon stimulation with antigen or mitogen, and the responsiveness in orally immunized mice peaked at day 42, suggesting that recombinant pMG36e-SP-TSOL18/gulation . Activatcination –49. In aγ, and TNF-α are indicators of Th1 response, which promote the production of IgG2a and IgG2b [γ, IL-2, IL-4, and IL-10 in spleen lymphocytes from all immunized groups. As shown in L. lactis vaccines resulted in stimulation of both Th1 and Th2 immune responses. Data showed that the intestinal mucosa is a site that induces an effective mucosal immune response, and the immunization method is simple and easy to operate [L. lactis vaccines could induce a mucosal immune response in all immunized mice.Cytokines and expression of specific isotypes have important role besides regulating the balance between Th1 and Th2 responses . It is knd IgG2b , 52, whend IgG2b –55. As s operate . sIgA is operate . Our resL. lactis is a potential vaccine vehicle to deliver T. solium antigens.In conclusion, we have demonstrated that an oral live vaccine prepared in this study is capable of inducing specific humoral immune responses, cellular immune responses, and mucosal immune responses in mice and that


Rosa26rtTA; tet(o)sFgfr2b), we conditionally inhibited FGF10 signaling in vivo in E12.5 embryonic lungs via doxycycline IP injection to pregnant females, and in vitro by culturing control and experimental lungs with doxycycline. The impact on branching morphogenesis 9 h after doxycycline administration was analyzed by morphometry, fluorescence and electron microscopy. Gene arrays at 6 and 9 h following doxycycline administration were carried out. The relationship between FGF10 and ß-catenin signaling was also analyzed through in vitro experiments using IQ1, a pharmacological inhibitor of ß-catenin/EP300 transcriptional activity. Loss of FGF10 signaling did not impact proliferation or survival, but affected both adherens junctions (up-regulation of E-cadherin), and basement membrane organization (increased laminin). Gene arrays identified multiple direct targets of FGF10, including main transcription factors. Immunofluorescence showed a down-regulation of the distal epithelial marker SOX9 and mis-expression distally of the proximal marker SOX2. Staining for the transcriptionally-active form of ß-catenin showed a reduction in experimental vs. control lungs. In vitro experiments using IQ1 phenocopied the impacts of blocking FGF10. This study demonstrates that FGF10/FGFR2b signaling on distal epithelial progenitor cells via ß-catenin/EP300 controls, through a comprehensive set of developmental genes, cell adhesion, and differentiation.This study demonstrates that FGF10/FGFR2b signaling on distal epithelial progenitor cells, via ß-catenin/EP300, controls, through a comprehensive set of developmental genes, morphogenesis, and differentiation. Fibroblast growth factor (FGF) 10 signaling through FGF receptor 2b (FGFR2b) is mandatory during early lung development as the deletion of either the ligand or the receptor leads to lung agenesis. However, this drastic phenotype previously hampered characterization of the primary biological activities, immediate downstream targets and mechanisms of action. Through the use of a dominant negative transgenic mouse model ( In mice, the first morphological evidence of lung development is seen at embryonic day (E) 9.5 with the budding of the ventral foregut endoderm, forming the tracheal primordium ventrally and the esophagus dorsally. Concomitantly, distal to the tracheal primordium, two primary lung buds form, initiating the early stages of pseudoglandular development (E9.5-E12.5) . Lungs were incubated at 5% COin vivo and in vitro experiments were captured either on a Leica MZ 125 stereoscopic dissecting microscope using a Spot Insight 2.0 Mp Color Mosaic camera and Spot 4.5.9 imaging software, or were obtained from live imaging experiments using a Leica DM6000B inverted microscope, DFC 305FX camera, and Leica Application Suite Advanced Fluorescence imaging software.Brightfield images of lungs from To assess the expression of genes in the epithelial and mesenchymal compartments of distal E12.5 lung tips, C57BL/6 wild type embryos were used. Embryonic lungs were isolated in culture medium, and the distal epithelial buds, along with the surrounding mesenchyme and mesothelium, were carefully dissected with fine-tipped pincers. The dissected tips were then immediately transferred to 500 μl undiluted dispase  where they were incubated for 20–30 min on ice. The dispase-digested samples were then transferred to pure FBS, and incubated for 15 min on ice, thus blocking the enzymatic activity of dispase. Using tungsten microdissection needles, the epithelium was gently separated from the surrounding mesenchyme. Separated tissues were then prepared for total RNA extraction and microarray analysis.Freshly dissected E12.5 lungs were washed in sterile PBS (2 × 5 min), fixed in 4% PFA for 20 min on ice, and then washed again (3 × 5 min). Lungs were dehydrated by successive washes in a graded ethanol series  for 5 min each, and stored in 100% ethanol at −20°C.To embed the lungs in paraffin, they were first washed in Xylol , incubated for 1 h at 60°C in a 1:1 Xylol/paraffin mixture, washed in pure paraffin (3 × 20 min) at 60°C, and then stored in pure paraffin overnight at 60°C. Lungs were then embedded in paraffin blocks and sectioned to a thickness of 4 or 5 μm. Sections were placed in a 40°C water bath for ~30 min, and then placed on glass slides and incubated at 37°C overnight.Before antibody staining, sections were first washed with gentle shaking in Xylol (3 × 10 min), and then in serial dilutions of ethanol  for 3 min each, and finally in distilled water for 5 min. For each stain an antigen retrieval step was performed, which involved incubating the slides in 75–90°C citrate-based antigen unmasking solution  for 15 min and then cooling on ice for ~30 min. Sections were then washed with PBST . Blocking solution (1x PBS + 3% BSA + 0.4% TritonX) was then added atop each section for 1 h at room temperature. Primary antibodies were added to incubation buffer (1x PBS + 1.5% BSA + 0.2% TritonX) and samples were incubated overnight at 4°C , and anti-LEF1 were added at 1:100 dilution; anti-CDH1, anti-LAMA1, and anti-SOX9 were added at 1:200 dilution; see Table Sections were imaged on a Leica DM 5500B upright fluorescent microscope system, with a DFC 360FX camera, and Leica Application Suite Advanced Fluorescence imaging software. Signal intensity was optimized to either a control or experimental sample for an experiment, and the acquisition and intensity values were similarly applied to each sample in that experiment, thus ensuring valid comparisons.To assess the morphology of intact distal lung buds in control and experimental embryos, whole mount immunofluorescence followed by confocal imaging was performed.E12.5 lungs were dissected and fixed in 4% PFA for 20 min on ice. Samples were washed in PBS + 1% TritonX (3 × 10 min), and incubated in blocking buffer (1x PBS + 1% TritonX + 10% FBS) for 1.5 h at room temperature, followed by two washes in blocking buffer. Samples were then incubated for 2 h with FITC-conjugated anti-CDH1 (Dilution: 1:200) diluted in 1/4 blocking buffer and PBS, at 4°C in the dark. Lungs were then washed in PBS (3 × 10 min), and transferred to custom made imaging dishes  and a 10,0/1 mm rubber washer fixed to the middle of the dish with a suitable adhesive, thus creating an ideal well to mount and image the sample). ProLong Gold antifade reagent with DAPI was added to each well and covered with a glass coverslip.Z-stacks of distal lung buds were obtained on a Leica TCS SP5 confocal microscopy system using Leica Application Suite X software. For each bud, the first optical section of the z-stack was acquired at the basal edge of the epithelium. Z-stack images were taken at 0.5 μm increments through the bud, until imaging was no longer possible due to complete loss of signal intensity. Compensation of intensity loss through the bud was obtained using the linear compensation by AOTF option. 3-D reconstructions and movies were created using Leica Application Suite X software.To identify the effects of FGFR2b ligand inhibition on the ultrastructure of distal epithelial and mesenchymal cells, control and experimental E12.5 lungs were prepared for transmission electron microscopy.The chest cavities of freshly harvested E12.5 embryos were gently opened by incising from the lower abdomen, through the sternum, to just under the chin using a pair of fine dissection scissors. An incision was made along the diaphragm from the midline to the spine. The embryos were then immediately placed, ventral side up, in an immersion fixative solution containing 4% PFA + 2% sucrose + 0.05% calcium chloride + 1x PIPES buffer  in a 50 ml Falcon tube, such that each sample was immersed in 5X the volume of fixative in its own tube. Tubes were placed on ice and gently shaken for 2 h, after which the fixative was removed and replaced by 4% PFA + 0.05% glutaraldehyde , 2% sucrose, 0.05% calcium chloride + 1X PIPES buffer . Samples remained in this fixative overnight at 4°C.The next morning, the samples were processed for routine transmission electron microscopy. Briefly, the fixed lungs were dissected and placed into molten agar, which was allowed to harden before the samples were cut longitudinally in half. Each half was fixed for 30 min in 1.5% GA fixative containing 2% sucrose + 0.05% calcium chloride + 1X PIPES buffer . The fixative solution was then removed and samples were washed with 1X PIPES (3 × 5 min). Samples were then incubated for 1 h at room temperature in reduced osmium fixation solution containing 0.15% sodium hexacyanoferrate(II) and 2% reduced osmium, then washed very briefly with distilled water, and dehydrated via washes in a graded ethanol series , 3 times 10 min each step. Samples were then embedded by immersion in propylene oxide (3 × 5 min), in 1:1 propylene oxide:Agar 100 epoxy resin (1 × 30 min) following the manufacturer's instructions to produce blocks of medium hardness , and finally in pure Agar 100 epoxy resin in a desiccation chamber at room temperature overnight.The Agar 100 resin-penetrated lungs were then flat embedded into fresh Agar 100 resin and polymerized at 60°C for at least 2 days, or until complete polymerization was achieved. Ultrathin sections were then prepared and micrographs were obtained using a Zeiss LEO 906 transmission electron microscope equipped with a TRS slow-scan 2K CCD camera and ImageSP software.Rosa26rtTA  and tet(o)sFgfr2b . The PCR reaction mix was calculated for 20 μl per reaction, and included 10 μl 2xTaq PCR Master Mix , primers at a final concentration of 0.2 μM, RNase-free water, and up to 1 μg of genomic DNA. PCRs were performed in a C1000 Thermocycler (Bio-Rad). The cycling protocol to amplify Rosa26rtTA was as follows: initial denaturation at 94°C for 3 min; 35 cycles of denaturation at 94°C for 1 min, annealing at 63°C for 1 min, and extension at 72°C for 1.5 min; final extension at 72°C for 5 min; and hold at 4°C. The protocol to amplify tet(o)sFgfr2b was as follows: initial denaturation at 95°C for 2 min; 35 cycles of denaturation at 95°C for 5 seconds, annealing at 58°C for 5 s, and extension at 72°C for 20 s; final extension at 72°C for 2 min; and hold at 4°C. Capillary gel electrophoresis was performed using a QIAxcel Advanced capillary electrophoresis system (Qiagen).DNA was isolated from the tails and hind limbs of E12.5 embryos. Gene-specific primers were used to detect the presence of Whole embryonic lungs or separated epithelium and mesenchyme used for total RNA isolation were first put in 700 μl QIAzol Lysis Reagent . For tissue disruption and homogenization, the samples were transferred to gentleMACS M Tubes and homogenized in a gentleMACS Dissociator (Miltenyi Biotec) for 1 min. Total RNA was then isolated using the miRNeasy Mini Kit , and eluted in 30 μl RNase-free water. RNA amount and purity was assessed with a NanoDrop 2000c (Thermo Scientific). Up to 1 μg of total RNA for each sample was then reverse transcribed using the QuantiTect Reverse Transcription Kit .https://www.ncbi.nlm.nih.gov/tools/primer-blast/; last accessed, 01–08–2018). Primers were further validated by PCR-based gel electrophoresis  following the kit instructions. Per reaction, 200 ng of total RNA was used. The Cy3-labeled aRNA was hybridized overnight to 8 × 60 K 60mer oligonucleotide spotted microarray slides .For experiments where samples yielded <50 ng/μl of RNA, the SPIA-protocol was utilized. In this protocol, purified total RNA was amplified using the Ovation PicoSL WTA System V2 kit . Per sample, 2 μg amplified cDNA was Cy-labeled using the SureTag DNA labeling kit (Agilent Technologies). The Cy3-labeled aRNA was hybridized overnight to 8 × 60 K 60mer oligonucleotide spotted microarray slides .For each protocol, hybridization, and subsequent washing and drying of the slides were performed following the Agilent hybridization protocol. The dried slides were scanned at 2 μm/pixel resolution using the InnoScan is900 (Innopsys). Image analysis was performed with Mapix 6.5.0 software, and calculated values for all spots were saved as GenePix results files. Stored data were evaluated using the R software (version 3.3.2) and the limma package (version 3.30.13) from BioConductor. Gene annotation was supplemented by NCBI gene IDs via biomaRt (last accessed 08–03–2018).Proliferation in E12.5 lungs was assessed using the Click-iT EdU Imaging Kit . 5-ethynyl-2′-deoxyuridine (EdU), a nucleoside analog of thymidine incorporated into DNA during DNA synthesis, was injected (i.p.) 2 h before pregnant females were sacrificed (Dosage: 0.005 mg EdU / g mouse weight). Embryonic lungs were harvested, paraffin embedded, sectioned and placed on glass slides. Sections were then deparaffinised and stained for EdU according to the manufacturer's protocol.Apoptosis was assessed using the TdT-mediated dUTP Nick-End Labeling (TUNEL) assay. The assay was performed using the DeadEnd Fluorometric TUNEL System . E12.5 lungs were harvested, paraffin embedded, sectioned and placed on glass slides. The assay was performed according to the manufacturer's protocol.in vivo studies was entered in genepaint. The whole embryo section displaying the clearest gene expression in the lung, along with a magnification of the lung itself, was chosen for the figures.To assess the expression patterns of genes in early stage embryonic lungs (E14.5), the online database genepaint.org was used (last accessed 01–08–2018). Each of the genes significantly regulated in our Using still images of lungs, mesothelium and airways were traced in Adobe Illustrator CS6 (version 16.0.4) to create skeletal outlines. These outlines were exported and lengths and areas were quantified either using MetaMorph (version 1.5.0) or FIJI (version 2.0.0-rc-68/1.52g) software.t-tests. All data are presented as mean ± SEM. Values of p < 0.05 were considered significant. The number of independent samples (n) can be found in the figures.Significance was determined by unpaired two-tailed Student's ΔCt and ΔΔCt values were calculated according to the following formulas:Note, this equation accounts for the fact that Ct is proportional to the –log of gene expression. ΔCt is therefore positively related to the expression of the gene of interest.t-tests were performed on the ΔCt values, which can be assumed to be normally distributed. Number of “n” and significance level is indicated either in the figures or in the figure legends.Unpaired two-tailed Student's t-test on a moderated t-statistic, and heatmaps were generated displaying genes according to descending p-values. Gene set tests were done on the ranks of the t-values, using the function “geneSetTest” in the limma package from BioConductor. The number of independent samples (n) can be found in the figures. Gene sets were either user defined or, for pathway analyses, according to the KEGG database (last accessed 08–03–2018). The data from the microarray experiments have been deposited in the NCBI's gene expression omnibus (GEO accession GSE124157).Mean spot signals were background corrected with an offset of 1 using the NormExp procedure on the negative control spots. The logarithms of the background-corrected values were quantile-normalized. The normalized values were then averaged for replicate spots per array. From different probes addressing the same NCBI gene ID, the probe showing the maximum average signal intensity over the samples was used in subsequent analyses. Genes were ranked for differential expression using an unpaired two-tailed Student's SB, MJ, JZ, PM, and CC: concept and design; MJ, SaD, AL, SoD, JW, GC, and RM: acquisition of data; SB, MJ, JW, EB-V, and DA: analysis and interpretation; MJ, SaD, SB, and JZ: drafting and editing of the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


NUDT15 R139C and thiopurine S-methyltransferase (TPMT) on azathioprine (AZA) induced leukopenia in patients with autoimmune hepatitis (AIH) and related cirrhosis. A total of 149 Chinese AIH patients with a history of AZA treatment were retrospectively evaluated. The clinical and epidemiological characteristics of the patients were obtained from an electronic database and reviewed. NUDT15 (rs116855232) and ∗3CTPMT (rs1142345) SNPs were genotyped using a PCR method. Twelve patients developed leukopenia, and this adverse drug reaction was significantly associated with the T risk allele in NUDT15 , with the sensitivity and specificity of 91.67 and 89.05%, respectively. The median maintenance dosages for patients with the rs116855232 CC and CT genotypes were 1.23  mg ⋅ kg−1 ⋅ d−1 and 0.96  mg ⋅ kg−1 ⋅ d−1, respectively (P = 0.028). In contrast, no significant association was observed for ∗3CTPMT genotypes. Notably, subgroup analysis of the 13 patients with leukopenia before therapy, these white blood cell (WBC) counts did not show further reduction after AZA treatment and maintenance dosage was 1.13  mg ⋅ kg−1 ⋅ d−1. Therefore, NUDT15 polymorphism is significantly associated with thiopurine-induced leukopenia in Chinese patients with AIH and related cirrhosis. Adjusting the AZA dosage should be considered in patients according to the NUDT15 R139C genotypes.The aim of this study was to investigate the influence of  However, most recent studies implied that neither Thiopurine S-methyltransferase (TPMT) heterozygosity nor 6-mercaptopurine (6-MP) metabolite levels were reliable predictors of AZA efficacy or toxicity in patients with AIH and related cirrhosis, and cytopenia is frequently encountered due to advanced fibrosis (Autoimmune hepatitis (AIH) is a chronic self-perpetuating inflammatory liver disease characterized by increased serum aminotransferase levels and autoantibodies, elevated serum immunoglobulin G (IgG) concentration, lymphoplasmacytic infiltration of the portal tract, and interface hepatitis. Misdiagnosis or treatment failure can lead to liver cirrhosis, liver cancer, liver transplantation and rapid death . The firfibrosis .9/L, or platelet counts below 50 × 109/L are severe hematological side effects that justify the discontinuation of the AZA treatment  counts below 3 × 10reatment . Leukopereatment . The genactivity . This suactivity  and thatactivity .NUDT15) gene results in a p.Arg139Cys substitution and is strongly associated with thiopurine-induced myelosuppression and thiopurine tolerance dose with underlying race-specific genetic polymorphisms; this result has been replicated by multiple independent studies : 6.13–10.08) increased risk of developing leukopenia with a high specificity (91.74%) and sensitivity (43.19%) and a lower thiopurine intolerance dose (P < 0.00001) (NUDT15risk/riskTPMTwt/risk genotype (NUDT15 (rs116855232) and TPMT∗3C (rs1142345) SNPs on AZA-induced leukopenia in Chinese patients. Additionally, we sought to confirm the impact of NUDT15 heterozygosity on thiopurine-induced leukopenia and the tolerance dose to guide the clinical usage of thiopurines in this patient cohort.A genome-wide association study found that a c.415 C-to-T transition (rs116855232) variant of the nucleoside diphosphate-linked moiety X motif 15 ( studies . We prev0.00001) . However0.00001) , inflamm0.00001) , and oth0.00001) . No prevgenotype . As the West China Hospital is a 4950-bed tertiary teaching hospital affiliated with Sichuan University; it has a liver transplant unit  and is the leading medical center in West areas of China. In total, 149 consecutive patients with AIH or AIH- primary biliary cholangitis (PBC) variant syndrome who prescribed AZA for more than 3 months were recruited at the West China Hospital between October 2013 and October 2017. Individuals were included if they met the criteria for definite or probable AIH based on the revised criteria published by the International AIH Group in 1999; AIH – PBC variant syndrome was diagnosed according to the Paris criteria . AccordiComplete blood counts were performed weekly during the 1st month of AZA treatment, biweekly in the following 2 months, and monthly thereafter. The researchers performed follow-up via telephone and internet communication.9/L. For patients whose WBC counts were less than 3 × 109/L before therapy, leukopenia was deemed progressive deterioration after treatment. The secondary endpoint was drug withdrawal for persistent or intolerant leukopenia. The dose was reduced in patients who developed leukopenia. If any laboratory abnormality did not subside, AZA was discontinued. Decisions regarding discontinuation or dose adjustment were made by the physician responsible for the personalized therapy.The primary endpoint was leukopenia, which is defined as a WBC count less than 3 × 10This study was approved by the Ethics Committee of West China Hospital. Written informed consent was obtained from all enrolled patients.∗108 RBCs, as previously described  according to the manufacturer’s instructions. Genotypes of rs116855232 and rs1142345 were determined by polymerase chain reaction (PCR) and Sanger sequencing. The sequences of the forward and reverse primers for rs116855232 were AAGCAAATGCAAAGCATCAC and GGCTGAAAGAGTGGGGGATA, and the sequences of the forward and reverse primers for rs1142345 were ATAGGCATAATCTTTCAC and CAGCCAATTTTGAGTATTT. PCR amplification for rs116855232 starts with an initial denaturation at 94°C for 5 min, followed by 35 cycles of 95°C for 30 s, 65°C for 30 s and 72°C for 60 s. Final extension was performed at 72°C for 10 min, and samples were stored at 12°C. The PCR reaction for rs1142345 starts with an initial denaturation at 94°C for 5 min, followed by 35 cycles of 95°C for 30 s, 54°C for 30 s and 72°C for 60 s. Final extension was performed at 72°C for 10 min, and samples were stored at 12°C. Measurement of AZA metabolites uses high performance liquid chromatography and results are reported in pmol/8escribed . All proThe treatment responses were classified according to biochemical remission. AIH biochemical remission was defined as the normalization of transaminases and IgG, as determined using existing guidelines.U-test was performed to compare continuous variables. Chi-square tests or Fisher’s exact tests were performed to analyze differences in categorical variables between two independent groups as appropriate. The odds ratios (OR) and 95% CIs associated with rs116855232 were determined by performing logistic regression analyses assuming log-additive, dominant and allele genetic models of inheritance using Review Manager 5.3 software. A two-side P-value less than 0.05 were considered statistically significant. Statistical analysis was performed using the SPSS software package .Continuous data are presented as medians and quartiles, and categorical data are expressed as percentages. The Mann–Whitney n = 1), bone aches (n = 1), and herpes zoster (n = 1). For detailed patient characteristics, see P > 0.05) , 3 of whom also had transient gastro-intestinal reactions, and 2 of whom developed mild hair loss. Other side effects included pancreatitis ( > 0.05) .NUDT15 . Twenty-four subjects were NUDT15 heterozygotes , and two subjects were homozygotes , respectively. The variant allelic frequency of rs116855232 was 9.4%, while ∗3CTPMT alleles were only observed in four subjects including 3 patients who were heterozygotes (TC) and 1 patient who was homozygous (CC). Significant differences were observed in the rs116855232 genotypes between the individuals with or without leukopenia (P < 0.05).Of the 149 analyzed subjects, 123 subjects displayed wild-type NUDT15 genotypes, see For detailed patient characteristics about association between the rs116855232 genotypes and leukopenia according to −1 ⋅ d−1 prescription and reached the diagnostic criteria for grade 4 myelosuppression; this patient also carried a TPMTwt/risk genotype. Although the AZA treatment was immediately halted and human granulocyte colony-stimulating factor were prescribed to increase blood cell counts, the patient suffered a life-threatening progressive decrease in blood cell number. Subsequently, a second-line immunosuppressant (mycophenolate mofetil) was recommended after recovery from the myelosuppression. The other patient with the TT genotype was reduced to a 0.2 mg ⋅ kg−1 ⋅ d−1 prescription after the WBC count from 5.29 × 109/L dropped to 1.94 × 109/L at the 0.9 mg ⋅ kg−1 ⋅ d−1 prescription; also, WBC counts gradually increased to a satisfactory value (3–4.5 × 109/L) after the dosage adjustment.Therapy-induced leukopenia was observed in 9/24 (37.5%) patients with the CT allele and 2/2 (100%) patients with the TT allele. However, only one patient with wild-type homozygote allele experienced leukopenia. All cases of leukopenia developed within the first 60 days following therapy initiation or dose increase. Three of the nine (33.3%) patients developed leukopenia during remission induction, while the other 6 (66.7%) developed leukopenia during the maintenance therapy. One patient with the TT genotype experienced lethal levels of myelosuppression in the first 4 weeks of remission induction with a 1.09 mg ⋅ kgP < 0.00001, odds ratio = 20.41; 95% CI ], with the sensitivity and specificity of 91.67 and 89.05%, respectively  vs. 145.3 pmol/8∗108 RBC] (6-Thioguanine nucleotide concentrations were significantly higher in the patients of 108 RBC] .P = 0.22) when evaluated with a clinical therapeutic dose of 1.0 mg ⋅ kg−1 ⋅ d−1. However, the median maintenance dosages for patients with the NUDT15 CC and CT genotypes were 1.23  mg ⋅ kg−1 ⋅ d−1 and 0.96  mg ⋅ kg−1 ⋅ d−1, respectively (P = 0.028)  .In total, 93 patients have achieved biochemical remission and began maintenance therapy as of February 2018. The AZA dose did not significantly differ between CC and CT genotypes (= 0.028) . The rat= 0.013) . The mai∗108 RBC, and median 6-TGN concentration was 149.1 pmol/8∗108 RBC. The associations of concentrations of 6-TGN with different groups were analyzed in P = 0.149), and no significant difference was observed between two groups, regarding to gender, AZA maintenance dosage and severity of liver disease .The 6-TGN concentration of 61 patients ranged from 49.74 to 1583.38 pmol/89/L, but these WBC counts did not show further reduction after AZA. Only one patient was NUDT15 heterozygous (CT) but did not show progressive leukopenia after AZA therapy. Only one patient discontinued AZA because of gastrointestinal reactions; mycophenolate mofetil was subsequently administered. The maintenance dosage for the 13 patients with pre-therapy leukopenia was 1.13  mg ⋅ kg−1 ⋅ d−1)  × 101 ⋅ d−1) .P = 0.361) or the median time to biochemical remission  between the genotypes.Among the 93 patients who reached biochemical remission, 75 (80.6%) were wild-type homozygotes, and 17 (18.3%) subjects were heterozygotes. No differences were observed in the percentage of patients who achieved biochemical remission   . This pr (∼0.9%) . Hence, NUDT15 SNPs, including rs147390019 (induces p.Arg139His)  . Pharmacd NUDT15 .NUDT15 polymorphism that was significantly associated with thiopurine-induced leukopenia in East Asian ALL and IBD patients, suggesting its potential as a promising biomarker for leukopenia following the initiation of thiopurine therapy in Asian populations. In, NUDT15 allele frequencies were 10.4% in Korean IBD patients. Additionally, an immunochip-based 2-stage association study revealed that rs116855232 was strongly associated with thiopurine-induced early leukopenia . This variant showed a sensitivity and specificity of 89.4 and 93.2% in Korean IBD patients, respectively (−3) was replicated in our Japanese IBD cohort , although the association with late leukopenia (>8 weeks) was not confirmed. Another study involving 253 Chinese IBD patients found that the NUDT15 variant was correlated with the early, middle (8–24 weeks) and late (>24 weeks) phases of leukopenia   . In our ctively) . The rs1ctively) . Hence, t manner . We alsoNUDT15 homozygotes. However, further prospective validation studies with larger sample sizes are warranted to provide more robust evidence for this subgroup.In addition, no study has addressed the issues of cirrhosis, previous leukopenia, and genotype heterozygosity together in AIH and related cirrhosis. It is an independent and first verification in Chinese AIH patients. It seems that in patients with liver disease fibrosis stage may influence AZA metabolism, yet our data implied that leukopenia was not associated with advanced fibrosis or splenomegaly, although TPMT remains the most widely accepted predictor of thiopurine-induced leukopenia to date; current guidelines, including the clinical practice guidelines for AIH by EASL and AASLD, recommend TPMT genotyping prior to the initiation of AZA treatment  was significantly associated with AZA-induced leukopenia in Chinese patients with AIH and related cirrhosis. For patients with the heterozygous variants, low-dose AZA (0.96 mg ⋅ kg−1 ⋅ d−1) is safe for maintaining remission and achieving efficacy comparable to that observed in wild-type patients. Even for patients with previous leukopenia or cirrhosis, AZA treatment may not be contraindicated if NUDT15 genotypes permitted.We found that a This study was carried out in accordance with the recommendations of the ethical guidelines and approved by the Ethics Committee of the West China Hospital, Sichuan University (no. 2013221) with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki.XF and DY wrote the manuscript. LY and HX designed the research. XF, DY, and RM performed the research. XF, DY, and RM analyzed the data.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Topoisomerases have been shown to have roles in cancer progression. Here, we have examined the effect of cryptolepine, a plant alkaloid, on the growth of human non-melanoma skin cancer cells (NMSCC) and underlying mechanism of action. For this purpose SCC-13 and A431 cell lines were used as an in vitro model. Our study reveals that SCC-13 and A431 cells express higher levels as well as activity of topoisomerase (Topo I and Topo II) compared with normal human epidermal keratinocytes. Treatment of NMSCC with cryptolepine  for 24 h resulted in marked decrease in topoisomerase activity, which was associated with substantial DNA damage as detected by the comet assay. Cryptolepine induced DNA damage resulted in: (i) an increase in the phosphorylation of ATM/ATR, BRCA1, Chk1/Chk2 and γH2AX; (ii) activation of p53 signaling cascade, including enhanced protein expressions of p16 and p21; (iii) downregulation of cyclin-dependent kinases, cyclin D1, cyclin A, cyclin E and proteins involved in cell division  leading to cell cycle arrest at S-phase; and (iv) mitochondrial membrane potential was disrupted and cytochrome c released. These changes in NMSCC by cryptolepine resulted in significant reduction in cell viability, colony formation and increase in apoptotic cell death. Cryptolepis sanguinolenta (Lindl.). The aqueous extract from the roots of this plants have been traditionally used for the treatment of malaria, rheumatism, urinary tract infections, upper respiratory tract infections and intestinal disorders in Central and West African countries like Ghana and Nigeria [2 signaling and inhibition of other promotors of inflammation including TNFα and iNOS [Cryptolepine A is an a Nigeria ,2. Crypt Nigeria , anti-ba Nigeria , anti-fu Nigeria , and ant Nigeria ,7 activi Nigeria ,9. The aand iNOS ,9,10,11.and iNOS ,13,14. Hand iNOS ,16,17. Topoisomerases are highly specialized nuclear enzymes involved in the removal of superhelical tension on chromosomal DNA, correction of topological DNA errors during replication, transcription, recombination and chromosomal condensation ,19. TopoFirst we determined and compared the basal levels and activities of topoisomerases (I and II) in NMSCs cells (SCC-13 and A431) and data were compared with the NHEK and immortalized HaCaT cells. Western blot analysis revealed that basal levels of topoisomerases (Topo I and Topo IIα) were higher in SCC-13 and A431 cells compared to NHEK B. IntereIt has been suggested that higher expression and activity of topoisomerases in cancer cells may facilitate enhanced and uncontrolled proliferative potential and survival of these cells ,20,23, tp < 0.05 to p < 0.001) which is reflected from the comet tail length in cryptolepine- treated cells compared to non-treated control cells and this effect was dose-dependent , a nuclear serine/threonine protein kinase and crucial component of the DNA double-strand break repair machinery, is known to be activated upon association with DNA in response to DNA damage ,27. TherDNA damage response is initiated by variety of protein kinases and accessory factors such as ATR, ATM, and DNA-PK. These kinases/factors have ability to sense the DNA damage. In the event of DNA damage, these molecules respond by phosphorylating and activating downstream factors such as Chk1, Chk2, p53 and associated protein factors involved in the process of DNA repair, cell cycle progression and apoptosis. Therefore, we determined the effect of cryptolepine on phosphorylation of these DNA damage response factors. We found that cryptolepine treatment induced phosphorylation of ATM, ATR, BRCA1, γH2AX, Chk1 and Chk2 in SCC-13 and A431 cells in a dose-dependent manner A. RepresThe tumor suppressive protein p53 plays a crucial role in DNA damage response, cell cycle progression and apoptosis ,29. ExprAs we found a significant DNA damage in NMSC cells after a treatment with cryptolepine, we determined the possible inhibitory effect of cryptolepine on cell cycle progression in SCC-13 and A431 cells.Representative data are produced from two separate experiments. As summarized in In the event of DNA damage, activated p53 activates transcription of pro-apoptotic protein Bax and thus disrupt the balance of Bax/Bcl-2 protein ratio in cells and that results in release of cytochrome c from mitochondria leading to apoptosis ,33,34. Ip < 0.05 to p < 0.001) in cell viability, and it ranged from 17% to 45% after 24 h, 47% to 85% after 48 h of treatment. More or less similar effects of cryptolepine were obtained on treatment of A431 cells (p < 0.05 to p < 0.01) on the viability of the NHEK cells after 48 h of treatment. Moreover, the cryptolepine-induced inhibition of cell viability in NHEK cells at this dose and time point was significantly less (p < 0.01 to p < 0.005) than the effects of the same dose of cryptolepine on NMSC cells at the same time point  for 24 and 48 h. When compared with control treated cells, treatment of SCC-13 cells with cryptolepine resulted in a significant reduction . These data suggest that at least under the experimental conditions used in this study, cryptolepine is significantly less toxic to normal skin cells. Further, the cytotoxic effect of cryptolepine was also assessed in skin cancer cells using colony formation assay. As shown in p < 0.001) in both SCC-13 and A431 cell lines after the knock-down of Topo IIα, as analyzed by MTT assay.Further to verify the role of Topo IIα in NMSC cell growth, the level of Topo IIα was knocked down in the NMSC cells using siRNA kit, and cells were subjected to the analysis of cell growth/viability using MTT assay. We also checked the level of Topo IIα after its knock-down. Western blot analysis revealed that the levels of Topo IIα in both cell lines were decreased substantially after its knock down D. As shoTopoisomerases are known to play a crucial role during DNA replication and cell proliferation, and their functions become irregular in cancer cells. Because of this reason, inhibition of topoisomerases activity is a central mechanism of action of various anticancer drugs, such as camptothecin, etoposide and doxorubicin, used in cancer chemotherapy ,23. ThesThe tumor suppressor protein p53 is a crucial component of cellular machinery that regulates various signaling pathways including oncogenic processes, cell cycle, apoptosis and DNA damage responses under different conditions. Under normal conditions, in unstressed cells, the expression and function of p53 are tightly regulated and maintained in low levels with short half-life ,39. HoweIn summary, cryptolepine, a pharmacologically active plant alkaloid, possesses strong anticancer potential against non-melanoma skin cancer cells in an in vitro model. The anticancer activity of cryptolepine is mediated through topoisomerase inhibition and subsequently induction of DNA damage. Cryptolepine induced DNA damage response signaling through activation of p53 pathway leading to cell cycle arrest and induction of apoptosis in skin cancer cells, as depicted and summarized in ® 488 (Alexafluor488) kit from Molecular Probes .Cryptolepine with ≥98% purity was purchased from Sigma-Aldrich . Topoisomerase I and II assay kits were obtained from TopoGEN, Inc. . Primary antibodies specific for topoisomerase I and IIα were purchased from Abcam . Phospho forms of DNA damage specific primary antibodies of ATM, ATR, BRCA1, Chk1, Chk2, γH2AX, p53 and, acetylated-p53 were purchased from Cell Signaling Technology . Antibodies for total p53, mdm2, DNA-PK, cytochrome c, Cdc25a, Cdc25b, p16, p21, CDK2, cyclin A, cyclin D1, cyclin E, vinculin, β-actin, HRP-conjugated anti-mouse, anti-rabbit, and anti-goat were purchased from Santa Cruz Biotechnology, Inc. . Apoptotic cells were analyzed using Annexin-V Alexa Fluor2 at 37 °C. NHEK cells were grown in dermal cell basal media (ATCC® PCS-200-030) supplemented with keratinocyte growth factors (ATCC® PCS-200-040) and maintained under standard cell culture conditions. Cell lines were authenticated by the vendor and each cell line was passaged up to 3–4 times for experimental purposes. The pathological information of cancer cell lines are as follows: A431 cells are epidermoid carcinoma cells with epithelial morphology. These cells were isolated from the skin tumor tissue of female patient. SCC-13 cells were isolated from the skin squamous cell carcinoma from a female patient. Cryptolepine was dissolved in DMSO with final concentration of DMSO in media was not more than 0.1% (v/v). Equal volume of DMSO was added in control group of cells.The human squamous cell carcinoma cell lines (SCC-13 and A431), immortalized human skin keratinocyte cell line (HaCaT) and primary normal human epidermal keratinocytes (NHEK) were purchased from the American Type Culture Collection . SCC-13, A431 and HaCaT cells were cultured as monolayers in Dulbecco’s modified Eagle’s medium supplemented with 10% heat inactivated fetal bovine serum, 100 μg/mL penicillin–streptomycin, and maintained in a humidified atmosphere of 95% air and 5% COg for 3 min at 4 °C. Cell pellets were resuspended in 3 mL of ice cold TEMP buffer . Suspensions were centrifuged at 800× g for 3 min at 4 °C and cell pellets were resuspended in 3 mL of ice cold TEMP buffer and left on ice for 10 min. After incubation, suspensions were homogenized by 8–10 strokes in Dounce tight fitting homogenizer. Homogenates were centrifuged at 1500× g for 10 min at 4 °C to pellet the nuclei. Nuclear pellets were resuspended in 1 mL of cold TEMP buffer in a microcentrifuge tube. Tubes were centrifuged at 1500× g for 2 min at 4 °C, pellets collected and resuspended in a small volume (~3 pellet volume) of TEP buffer  extracts. Topoisomerase activity assays were performed on same day or extracts were aliquoted and stored at −80 °C. Protein concentration was determined using a Bio-Rad protein assay kit .Cells from treated and untreated groups were harvested and collected into 5 mL medium in 15 mL tubes. Suspensions were centrifuged at 800× To determine the topoisomerase I enzyme activity, cell extracts containing 10 µg protein from treated or untreated groups were mixed with 2 µL of 10x topoisomerase I assay buffer  and 1 µL (0.25 µg/ µL), and supercoiled DNA  as a substrate. Reaction volumes were made up to 20 µL using distilled water. Reaction mixtures were incubated for 30 min at 37 °C. Reaction was stopped by adding 5 µL of 2% SDS. Proteinase K (0.5 mg/mL) 5 µL was added and incubated for 15 min at 37 °C. Reaction mixture with 10x loading dye  was loaded to 1% agarose gel in TAE buffer. Gel was run at 1–2.5 V/cm for 2 h. Gel was stained with 0.5 µg/mL ethidium bromide and destained in distilled water for 15 min at room temperature and photographed using UV transilluminator from Bio-Rad. Comparative reactivity of the enzyme among different groups is represented by the band intensity. 2, 5 mM dithiothreitol, 300 µg bovine serum albumin/mL) and Buffer B (200 mM ATP in sterile distilled water). One µL (0.25 µg/ µL) supercoiled pHOT1 DNA  was added as a substrate. Reaction volumes were made up to 20 µL using distilled water. Reaction mixtures were incubated for 30 min at 37 °C. Reaction was stopped by adding 5 µL of 2% SDS. Proteinase K (0.5 mg/mL) 5 µL was added and incubated for 15 min at 37 °C. Reaction mixture with 10x loading dye  was loaded to 1% agarose gel in TAE buffer. Gel was run at 1–2.5 V/cm for 2 h. Gel was stained with 0.5 µg/mL ethidium bromide and destained in distilled water and photographed using UV transilluminator from Bio-Rad. Comparative reactivity of the enzyme among different treatment groups is represented by the band intensity.To determine the topoisomerase II enzyme activity, cell extracts equivalent to 10 µg protein each from treated or untreated groups were mixed with 4 µL of 5x complete topoisomerase II assay buffer prepared freshly by mixing equal volume of Buffer A . Transfection was performed according to the manufacturer’s instructions. Briefly, 2 × 10Cryptolepine-induced DNA damage in SCC-13 and A431 cells was determined using comet assay, as described in detail previously ,50. DNA After 24 h treatment with or without cryptolepine, cells were harvested and cell lysates were prepared as described previously ,52. Brie4 SCC-13 or A431 cells/well were seeded in four well chambered slides. Next day, cells were treated with cryptolepine  for 24 h. After incubation, cells were washed twice with chilled PBS, fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton-X 100 in PBS for 3 min. Nonspecific binding was blocked by incubating cells with 3.0% BSA in PBS for 30 min. Cells were incubated with specific primary antibodies over night at 4 °C. Cells were washed with PBS and incubated further for 1 h with fluorochrome conjugated secondary antibodies. After washing with PBS, slides were mounted with Vectashield® mounting medium  containing DAPI, analyzed and imaged using an Olympus microscope.Approximately 5 × 10NMSC cells (SCC-13 or A431) were treated with different concentrations of cryptolepine  for 24 h. The cells were then harvested, and processed for cell cycle analysis, as described previously . Briefly5 SCC-13 or A431 cells were treated with different doses of cryptolepine  for 24 h. Cells were incubated with rhodamine 123 for 30 min and then harvested, washed with PBS and resuspended in PBS for analysis of mitochondrial membrane potential using an Accuri Q6 flow cytometer.Retention of rhodamine 123 dye by mitochondria was performed for determining the change in mitochondrial membrane potential, as described previously . Approxi4 cells/well were plated in 96-well culture plates. The cells in each treatment group were plated at least in 8 replicates. Next day, cells were treated with different concentrations of cryptolepine  for 24 and 48 h. After incubation with indicated time periods, media was replaced with 50 µL fresh medium containing 5 mg/mL MTT and incubated for 2 h in incubator. The resulting formazan crystals were dissolved in 200 µL DMSO. Absorbance was recorded at 540 nm with a reference at 650 nm serving as the blank. The effect of cryptolepine on cell viability was presented in terms of percent of vehicle-treated control cells. The viability of control cells were arbitrarily considered as 100%.The MTT assay was employed to determine the effect of cryptolepine on cell viability, as described previously . Briefly6 cells were treated with cryptolepine  for 24 h. After incubation, cells were harvested, washed with PBS and incubated with Alexa488 and propidium iodide. The apoptotic cells were analyzed by an Accuri C6 flow cytometer.Quantitative analysis of cryptolepine-induced apoptosis in SCC-13 and A431 cells was determined by flow cytometer using Annexin V-conjugated Alexa fluor488 (Alexa488) Apoptosis Detection Kit following the manufacturer’s protocol, and as described previously by us ,55. BrieThe effect of cryptolepine on long-term cell proliferation/viability  was determined by colony formation assay, as described previously . Brieflywww.graphpad.com). In each case, p < 0.05 was considered as statistically significant.The statistical significance of the difference between the values of control and treatment groups was determined using student-t test and one-way analysis of variance (ANOVA) using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, CA, USA;