The αPLL modification is shown to facilitate the redox kinetics of FIC and ferrocyanide on the SPCEs. The SPCEs coated with 0.5 mM GOx, 99.5 mM FIC, and 5 mM αPLL had better sensitivity for glucose detection due to the appreciable effect of protonated αPLL on the promotion of electron transfer between GOx and FIC. Moreover, the SPCEs coated with 0.5 mM GOx, 99.5 mM FIC, and 5 mM αPLL were packaged as blood glucose testing strips for the measurement of glucose-containing human serum samples. The glucose testing strips had good linearity from 2.8 mM to 27.5 mM and a detection limit of 2.3 mM. Moreover, the 5 mM αPLL-based glucose testing strips had good long-term stability to maintain GOx activity in aging tests at 50 °C.Second-generation glucose biosensors are presently the mainstream commercial solution for blood glucose measurement of diabetic patients. Screen-printed carbon electrodes (SPCEs) are the most-used substrate for glucose testing strips. This study adopted hydrophilic and positively charged α-poly- Diabetes mellitus is the most common endocrine disease and is a disorder of carbohydrate metabolism producing high glucose levels in the blood, leading to different dysfunctions such as nerve degeneration, kidney failure, and blindness ,2,3. Dia2) of GOx. The FIC mediator dissolves faster and more completely in water than HQ and ferrocene, resulting in increased sensitivity, a wider linear range, and shorter response time urea] and poly[oxyethylene-(dimethylimino)propyl(dimethylimino)ethyl hydrogensulfate] of quaternary ammonium; ethoxylated polyethylenimine (PEI) of secondary ammonium; and ε-poly-l-lysine (εPLL), α-poly-l-lysine (αPLL), poly  − 0.261 and a correlation coefficient (R) of 0.998 in the range of 2.8 mM to 27.5 mM calculated from three individual strips. The statistical background current, blankI, was 2.92 ± 0.12 µA/mm2, implying that the background noise was 0.12 µA/mm2. The calculated limit of detection (LOD) was 2.3 mM, based on the IUPAC recommendation (S/N > 3). This linear range can cover the requirement of self-monitoring glucometers for diabetes for use in point-of-care testing [2) measured on the testing strips exceeded that (128.7 nA/mM mm2) measured at the SPCEs modified with the GOx/FIC/αPLL layer of 0.5/99.5/5 mM in a beaker. This is attributed to the fact that the packaged strips can confine the FOC product in a limiting space to promote the glucose concentration. The optimal GOx/FIC/αPLL ratio of 0.5/99.5/5 mM was used to modify two-electrode SPCEs, and the SPCEs were packaged with a spacer and a hydrophilic cover as blood glucose testing strips by Tyson Bioresearch Inc. A 2 μL aliquot of human serum containing concentration-varied glucose was dripped onto the testing strips and then measured by chronoamperometry.  testing . Furthero day) with the number of storing days. For strips stored at 30 °C, neither the 0.5 mM αPLL- nor the 5 mM αPLL-modified strips exhibited significant I/Io day change over time. Katano also demonstrated that PLL can maintain GOx activity [o day change, but that measured for the 5 mM αPLL-modified strips was still below 10%. The result shows that 5 mM αPLL for the entrapment of GOx and FIC provides better biocompatibility for maintaining GOx activity than does 0.5 mM αPLL.The stability of testing strips is one important index in commercialized development. SPCEs coated with 0.5 mM GOx, 99.5 mM FIC, and 0.5 or 5 mM αPLL were packaged as blood glucose testing strips by Tyson Bioresearch Inc. and separately kept at 30 °C and 50 °C for different periods to assess their stability. Keeping strips at 50 °C served to accelerate the aging test. A 2 μL aliquot of 6.67 mM glucose-containing serum was used to estimate the time-lapse change in the oxidative current of FOC. In this work, the GOx/FIC/αPLL-modified SPCEs packaged as glucose testing strips were demonstrated to successfully detect the glucose concentration of serum samples and exhibit a wide linear range of 2.8–27.5 mM, which covers the ISO15197 regulation of a 2.8–22.2 mM detecting range for a commercial glucometer . Moreove−1, H2PO4−1, divalent HPO4−2, and trivalent FIC. The SPCEs coated with 0.5 mM GOx, 99.5 mM FIC, and 5 mM αPLL had greater sensitivity to glucose than did those coated with 0.5 mM GOx, 99.5 mM FIC, and 0.5 mM αPLL, suggesting that the protonated αPLL can promote electron transfer between GOx and FIC. Finally, the SPCEs coated with 0.5 mM GOx, 99.5 mM FIC, and 5 mM αPLL were packaged as blood glucose testing strips to measure glucose-containing human serum samples. The glucose testing strips had good linearity from 2.8 mM to 27.5 mM and a LOD of 2.3 mM. Moreover, the 5 mM αPLL-based glucose testing strips exhibited good long-term stability for the maintenance of GOx activity in a 50 °C aging test. The sensing properties of the glucose strips cover the requirements relating to self-monitoring glucometers for diabetes for use in point-of-care testing. However, the usage of αPLL has only been demonstrated to promote the electrochemical electron transfer efficiency between the mediator, GOx, and SPCEs. More experimental designs are required to explore the effect of the αPLL coating on the optical and electromagnetic properties of SPCEs. Furthermore, the glucose testing strips used in this study need further investigation to realize the effect of varied interferents, such as ascorbic acid, uric acid, acetaminophen, etc., and hematocrit on their sensing properties for the development of commercial products in the future.This study adopted αPLL as the entrapping matrix of GOx and FIC to construct glucose testing strips. The αPLL modification can improve the redox kinetics of FIC and FOC. In particular, tetravalent FOC has a stronger interaction with the protonated αPLL than monovalent Cl


Food safety has become a major issue, with serious environmental pollution resulting from losses of nitrogen (N) fertilizers. N is a key element for plant growth and is often one of the most important yield-limiting nutrients in paddy soil. Urea-N immobilization is an important process for restoring the levels of soil nutrient depleted by rice production and sustaining productivity. The benefits of biochar application include improved soil fertility, altered N dynamics, and reduced nutrient leaching. However, due to high variability in the quality of biochar, the responses of N loss and rice productivity to biochar amendments, especially those prepared at different pyrolysis temperatures, are still unclear. The main objectives of the present study were to examine the effects of biochar prepared at different pyrolysis temperatures on fertilizer N immobilization in paddy soil and explore the underlying mechanisms.–1). Pot experiments were performed to determine nitrous oxide (N2O) emissions and 15N recovery from paddy soil using a 15N tracer across the rice growing season.Two biochar samples were prepared by pyrolysis of maize straw at 400 °C (B400) and 700 °C (B700), respectively. The biochar was applied to paddy soil at three rates , with or without N fertilization  and 0.75 kg ha−1 (B700); low-N treatment caused a decrease in N2O emissions under B400, but this decrease was not observed under B700. An application rate of biochar of 2.1% plus 210 kg ha−1 N fertilizer substantially decreased the N fertilizer-induced N2O emission factor under B400, whereas under B700 no significant difference was observed. Biochar combined with N fertilizer treatment decreased rice biomass and grain yield by an average of 51.55 and 23.90 g pot–1, respectively, but the yield reduction under B700 was lower than under B400.Compared with the non-biochar control, biochar significantly decreased soil bulk density while increasing soil porosity, irrespective of pyrolysis temperature and N fertilizer level. Under B400 and B700, a high biochar rate decreased N loss rate to 66.42 and 68.90%, whereas a high N level increased it to 77.21 and 76.99%, respectively. Biochar also markedly decreased N15N content, while it negatively affected the 15N recovery of rice, N2O emissions from soil, rice biomass, and grain yield in the first year. Generally, a high application rate of biochar prepared at high or low pyrolysis temperature reduced the N fertilizer-induced N2O emission factor considerably. These biochar effects were dependent on N fertilizer level, biochar application rate, and their interactions.Irrespective of pyrolysis temperature, biochar had a positive effect on residual soil  Biocharentation , while aentation .2O emissions in a particular agricultural field, but the effects are still highly unpredictable. Furthermore, little is known about the effect of pyrolysis temperature and biochar application rate on urea-N fixation and N2O emissions in paddy systems.The biochar effects depend on a number of factors, such as its intrinsic characteristics and application rate . Tempera15N was used to monitor N immobilization in a rice-soil system and N uptake by rice plants. The following hypotheses were tested: (i) low pyrolysis temperature indirectly affects soil N retention for plant uptake and rice yield through increasing the surface area and lowering the pH of biochar; and (ii) higher-temperature biochar has a larger suppressive effect on soil N2O emissions because its larger pore size and higher C/N ratio resulting in lower nitrification rates compared with lower-temperature biochar.Here a pot experiment study was carried out, with two different pyrolysis temperatures, three biochar application rates, and three N fertilizer levels. Stable isotope −1. Temperature was first raised to 200 °C and then to a final temperature of 400 or 700 °C, maintained for 1 h , the straw was cut into small pieces (<2 mm) and stored in sealed plastic bags. The straw samples were then transferred to a rectangular porcelain container (150 × 100 × 50 mm) and placed in a muffle furnace for pyrolysis at a heating rate of 15 °C min for 1 h . To miniPaddy soil was collected in an experimental field  managed by the Rice Institute of Shenyang Agricultural University. The site experiences a typical semi-humid temperate continental monsoon climate, with a mean annual temperature and precipitation of 8.3 °C and 500 mm, respectively, and 183 frost-free days. The accumulated temperature (>10 °C) is 3,300–3,400 °C. Mean annual precipitation is concentrated, with slight variation in mean annual temperature . Tempera−3, total N = 1.87 g kg−1, and total C = 15.39 g kg −1. The soil were air-dried, passed through a 2-mm nylon sieve, and mixed thoroughly with biochar at different rates before use.The soil was taken from the surface layer (0–20 cm) in April 2016. The soil was classified as Histosols according to the United States Department of Agriculture (USDA) soil taxonomy. The basic properties of the soil prior to the experiment were as follows: initial pH = 6.8  , bulk density = 1.46 g cm−1; C0, C0.7, and C2.1, respectively), and three N fertilizer levels . Each treatment group included three PVC pots (30 cm diameter ×25 cm height), giving a total of 54 pots. A total of 14 kg of soil with or without biochar was packed into each pot at a depth of 20 cm.The experiment followed a 2 × 3 × 3 factorial completely randomized design. There were two pyrolysis temperatures , three biochar application rates  japonica variety ‘Shennong 265’ was cultivated in 2016. Two seedlings at the three-leaf stage were transplanted from the nursery bed to each pot on 29 May 2016. In the experiment, 36% total urea was applied before transplanting (15N-labeled urea as base fertilizer), with 24% at the active tillering stage (unlabeled urea as tillering fertilizer) and 40% at the ear primordial stage (unlabeled urea as panicle fertilizer). The 15N-labeled urea (10.18 atom% 15N abundance) was provided by Shanghai Research Institute of Chemical Industry in Shanghai, China. In addition, all treatments received the same amounts of phosphorus (615 kg P2O5 ha−1 as triple super phosphate) and potassium (200 kg K2O ha −1 as potassium chloride) as base fertilizers. Fertilizers were dissolved in water and then added into pots. All pots were watered regularly to maintain flooded conditions, with water level 5 cm above the soil surface (except for aeration at the top-tillering stage to control effective tillering). Pots were kept outdoors. To reduce the effects of precipitation, a mobile steel-framed plastic canopy (800 cm long ×300 cm wide ×200 cm high) was used.The rice , all plants were harvested and separated into grains and straw then oven-dried to a constant weight at 70 °C for 48 h and weighted to determine total yield. Grain moisture was determined using a hand-held moisture tester after drying , and grain yield was estimated with a 14.5% moisture content.15N content (% in atoms) by isotope ratio mass spectrometry. 15N analyses were performed using elementar ISO prime 100 . Stable nuclides and the natural abundance differ from the atom% excess of the element. The background value (atom %) was subtracted from the experimental value to give the atom% excess. The natural 15N abundances of the plants and soil were estimated by averaging the values of all experimental treatments, respectively. Plant N uptake, N use efficiency, and the percentage of plant N derived from urea fertilizer were calculated using the following equations: 15N-labeled urea fertilizer (mg pot−1) and Ndfs is the N in the plant derived from the soil (mg pot−1), TN is the total N content in the plant or soil (mg pot−1), and Au, Acf, and Af are the 15N abundance in the 15N-labeled urea fertilizer (10.18 atom%), natural 15N abundance in the plant or soil, and total 15N abundance in the plant or soil, respectively.Dry plant samples (grain and straw) were ground and sieved (0.15 mm) to analyze total N and 15N-labeled urea in the plant tissue or percentage retained in the soil was derived at the harvest stage using the following equation (F is the amount of 15N-labeled urea applied (mg pot−1).The recovery of equation : (4)\doc4+-N) was extracted with 2.0 M KCl solution (3) with additional samples collected at a 0–10 cm soil depth. Soil porosity was calculated as follows: ρb is the bulk density (g cm−3) and ρp is the soil specific gravity (g cm−3) in Three soil samples were obtained using a hand-operated core sampler  from each pot after harvest (October 2016). Soil samples were sealed in plastic bags and maintained on ice in an insulated box then transported to the laboratory where they were stored at −20  °C until use. Each soil sample was divided into two parts, one for analysis of water content and another for physico-chemical analyses. Soil water content was determined after oven-drying ca. A total of 5 g of samples at 105 °C for 48 h. Before analysis of other soil properties, samples were ground using a Wiley millto and passed through a 2-mm sieve to remove root detritus. Subsamples were further passed through a 0.15-mm sieve for determination of total N using an Elementar Variomax CNS Analyzer . Soil inorganic N  using static opaque chambers  and hourly emissions of N2O were determined from the slope of the mixing ratio change with four sequential samples. Quality checks were applied and N2O flux measurements were discarded if the r2 of the linear regression of the fluxes was <0.90. Cumulative emissions of N2O during the growing season were calculated using trapezoidal integration to interpolate fluxes between successive sampling days \docP <0.05. All data are expressed as the mean ± standard error (n = 3). Multiple comparisons of means were based on Fisher’s least significant difference (LSD) test at a 5% significance level unless stated otherwise.Treatment effects were assessed by three-way analysis of variance (ANOVA) using SPSS Statistics 18.0 , and significance was expressed at A significant decrease in soil bulk density was observed with increasing biochar rate, irrespective of pyrolysis temperature and N level. A high biochar rate significantly increased soil porosity to 50.43–52.54% under low-temperature biochar treatment (B400) with or without N fertilization; however, under high-temperature biochar treatment (B700), this increasing effect was only observed without N fertilization. Biochar had no significant effects on soil capillary porosity or air-filled porosity between the different treatments of pyrolysis temperature or biochar rate .4+-N decreased slightly with increasing biochar rate, irrespective of pyrolysis temperature. The lowest soil NH4+-N concentration was observed at 3.79 µg g−1 under B400 and 3.71 µg g−1 under B700 (4+-N.Under high-N treatment (N210), soil NHder B700 . Biochar15N-labeled urea in rice, irrespective of pyrolysis temperature and N level. Under B400, a low application rate of biochar resulted in significantly higher recovery of 15N in rice compared with a high biochar rate; however, under B700, no significant difference was observed in 15N recovery between the two biochar rates. Following N fertilization alone at 210 kg ha−1 (C0), N uptake percentage from 15N-labeled urea reached 15.80 and 15.30% under B400 and B700, respectively. Compared with non-biochar treatment, a high application rate of B400 decreased N uptake from 15N-labeled urea by 55.03 and 44.03% under N210 and N168, respectively; by contrast, B700 caused a smaller decrease in N210 (44.03%) compare with N168 (55.52%). Similarly, N uptake from the soil also decreased with increasing biochar rate . Overall, the highest 15N recovery in the plant-soil system was obtained with biochar application under B400 (540.56 mg pot−1) and B700 (500.77 mg pot−1). The corresponding N recovery rates in the plant-soil system under these conditions were 33.58 and 31.10%, ranking highest among all treatments, although there were no significant differences  and B700 (68.90%) following co-application of 2.1% biochar and 168 kg ha−1 N fertilizer. Under B400, a smaller 15N loss rate was observed following N fertilization at 168 kg ha−1 compared with 210 kg ha −1, irrespective of biochar rate. No significant difference between the two pyrolysis temperatures was observed in terms of 15N loss rate. Concerning biochar rate alone, a high application rate resulted in a lower N loss rate (71.82 and 72.94%) than a low application rate (78.00 and 79.69%) under both B400 and B700. In the presence of 168 kg ha−1 N fertilizer, a high biochar rate of B700 significantly decreased the 15N content in rice plants compared with non-biochar treatment (P < 0.05). Both B400 and B700 enhanced soil 15N retention at the 2.1% biochar rate, while B400 retained a larger soil 15N value compared with B700  was 0.14%, which was significantly lower than that under all other biochar treatments.The N fertilizer-induced NP < 0.05). However, biochar alone had no significant effects on rice biomass, irrespective of pyrolysis temperature. Co-application of 2.1% biochar (B400) and a high N level decreased the aboveground biomass by 21.58% compared with non-biochar treatment, whereas a low N level caused a smaller decrease of 10.15%. Under B700, the rice biomass under high-N treatment alone was significantly higher than that under co-application of 2.1% biochar and 168 kg ha−1 N fertilizer. Moreover, under 2.1% biochar (B700), high-N treatment caused an increase in biomass of 19.07 g pot−1 compared with low-N treatment. There were no significant differences in rice biomass between biochar treatments at different pyrolysis temperatures (The aboveground biomass (including straw and grain) of rice plants at harvest was significantly higher under high-N treatment alone compared with all other treatments , and therefore, the NO3−-N content in paddy soil is very low and barely detectable (4+-N concentrations occurred at relatively low levels across treatments (3.71–6.07 µg g−1), whereas 4+-N concentration (0.51 µg g−1) in the 2% biochar-treated soil in laboratory microcosms. Since we adopted single extractions with 2 M KCl solution, the results of soil NH4+-N might be underestimated. Interestingly, the soil NH4+-N concentration did not respond to increasing rate of biochar application alone (N0); nonetheless, a small decrease was observed with increasing biochar application following 210 kg ha−1 N fertilization (N210), irrespective of pyrolysis temperature  than low-temperature biochar treatment (B400) with 0.7% biochar application only (4+-N sorption ability of biochar is thought to be due to (1) difference in the ash content between biochar samples; and (2) difference in the amount of particular functional groups on the biochar surface , which is a major problem encountered in paddy fields  are as follows: (1) The N immobilization and NH4+ sorption ability of B400 was enhanced, making it a better substrate for NH3 oxidation and accumulation of NO2−, a majority substrate of N2O  B400 had a lower C/N ratio, resulting in faster N cycling  B40 cycling . Cayuela15N in rice plants between B400 and B700 treatments. 15N and subsequent plant 15N uptake in an agricultural field in Canada  was previously found to have no significant effect on rice production in the first season (−1) resulted in a 10% increase in rice yield in the first cycle and more than 9.5% increase in the second cycle. Such sustainable yield-increasing effects of biochar were also found in other experimental studies on crops. Together, these data suggest that pyrolysis temperature has only a small effect compared with biochar application rate on the short-term yield potential in paddy soil.There is a direct relationship between crop biomass and grain yield . The rest season . However15N retention in soil over the rice growing season, while negatively affecting urea-N uptake and biomass production in the first year. Soil bulk density and rice yield decreased after application of both low-temperature and high-temperature biochar, although N2O emissions from the paddy soil were markedly reduced throughout the growing season. The lowest N loss rate was obtained under low-temperature treatment with application of 168 kg ha−1 urea plus 2.1% biochar. In conclusion, application of low-temperature biochar may be an effective strategy for mitigation of N losses in paddy fields in Northeast China.This study confirms that appropriate application of biochar is an effective approach to reduce N loss in rice-soil systems. The results indicate that irrespective of pyrolysis temperature, a high biochar rate could enhance base fertilizer 10.7717/peerj.7027/supp-1Table S1Given data show mean ± SD of the B400 and B700 for each treatment. A, B400; B, B700.Click here for additional data file.10.7717/peerj.7027/supp-2Table S2Given data show mean ± SD of the B400 and B700 for each treatment. A, B400; B, B700.Click here for additional data file.10.7717/peerj.7027/supp-3Table S3Given data show mean ± SD of the B400 and B700 for each treatment. A, B400; B, B700.Click here for additional data file.10.7717/peerj.7027/supp-4Table S4Given data show mean ± SD of the B400 and B700 for each treatment. A, B400; B, B700.Click here for additional data file.10.7717/peerj.7027/supp-5Table S5Given data show mean ± SD of the B400 and B700 for each treatment. A, B400; B, B700.Click here for additional data file.10.7717/peerj.7027/supp-6Figure S1Given data show mean from May 26, 2016 to October 13, 2016 the daily maximum temperature, minimum temperature and daily rainfall.Click here for additional data file.10.7717/peerj.7027/supp-7Figure S2Given data show mean ± SD of the B400 and B700 for each treatment. A, B400; B, B700.Click here for additional data file.10.7717/peerj.7027/supp-8Figure S32O emissions mean ± SD of the B400 and B700 for each treatment.Given data show cumulative NClick here for additional data file.10.7717/peerj.7027/supp-9Figure S4Given data show mean ± SD of the B400 and B700 for each treatment. A, B400; B, B700.Click here for additional data file.


Osteoclasts are the sole bone resorbing cell in the body and their over activity is key in the development of osteoporosis. Osteoclastogenesis is mediated by receptor activator of nuclear factor κB ligand (RANKL) signalling pathways. Unsaturated fatty acids (UFA) are known to inhibit osteoclastogenesis by targeting RANKL signalling. However, the mechanisms of action remain unclear. Peroxisome proliferator activated receptors (PPARs) are a family of nuclear receptors, with three known isoforms , that are known to bind UFAs and are expressed in osteoclasts. In this study, we aimed to determine how different families of UFAs activate PPARs and how PPAR activation influences osteoclast signalling. Human CD14+ monocytes were seeded into cluster plates with RANKL and macrophage colony stimulating factor (M-CSF) in the presence of PPAR agonists or different types of UFAs. All the PPAR agonists were shown to upregulate the activity of their respective receptors. Polyunsaturated fatty acids increased PPAR-α to a greater extent than monounsaturated fatty acids (MUFAs), which favoured PPAR-β/δ activation. All PPAR agonists inhibited osteoclastogenesis. The activation of RANKL signalling pathways and expression of key osteoclast genes were downregulated by PPAR agonists. This study reveals that PPAR activation can inhibit osteoclastogenesis through modulation of RANKL signalling. Osteoclasts are large multinucleated cells responsible for the resorption of bone . TogetheOsteoclasts are formed from precursors derived from the same haematopoietic stem cells that produce monocytes and macrophages . These pPeroxisome proliferator activated receptors (PPARs) are a family of nuclear receptors found in several tissues throughout the body. Three different isoforms of PPAR have been discovered, namely: PPAR-α, PPAR-β/δ and PPAR-γ. PPARs have been studied as therapeutic targets for several diseases. PPAR-γ agonists are currently in use for the treatment of type 2 diabetes  while PPAll three PPAR isoforms are known to be expressed in osteoblasts and osteoclasts . PPAR-γ Unsaturated fatty acids (UFAs) are natural ligands of PPARs, and PPARs have been suggested as potential targets through which several UFAs exert their bone protective effects . Both moTo test whether PPARs are expressed in differentiating CD14+ monocytes, cells were exposed to RANKL and M-CSF for 1, 7 or 14 days before RNA was extracted. PPAR expression was determined by PCR. Results indicate that all three PPARs, PPAR-α, PPAR-β/δ and PPAR-γ, were expressed in differentiating CD14+ monocytes from day 1 until day 14 of culture . Gene exTo test whether the PPAR agonists or UFA had any effects on cell viability in the undifferentiated human CD14+ monocytes, a resazurin assay was conducted. CD14+ monocytes were seeded into 96 well plates in the presence of fenofibrate (10 µM), L-165041 (10 µM), troglitazone (10 µM), AA (40 µM), DHA (40 µM), EPA (40 µM), PLA (100 µM) or OA (100 µM) for 48 h. Neither PPAR agonists nor any of the UFAs showed any effect on cell viability in the human CD14+ monocytes .CD14+ monocytes were seeded into 6-well plates in complete alpha-MEM to attach overnight before cells were exposed to PPAR agonists (10 µM), AA (40 µM), DHA (40 µM), EPA (40 µM), PLA (100 µM) or OA (100 µM) in the presence of RANKL and M-CSF. After 24 hours, nuclear protein was extracted and used to determine PPAR activity. Fenofibrate (PPAR-α agonist) caused an increase in PPAR-α activity . L-16504Human CD14+ monocytes were seeded into 96-well plates in complete alpha-MEM and exposed to PPAR agonists or UFAs in the presence of RANKL and M-CSF for 14 days. Tartrate resistant acid phosphatase (TRAP) activity was determined from conditioned media using pNPP as a substrate. The cells were further fixed and stained for TRAP A. AA, DHAfter confirming the UFAs could activate PPARs in our osteoclast model, we sought to determine whether activation of PPARs could modulate RANKL signalling pathways. CD14+ monocytes were seeded into 6-well plates in complete alpha-MEM to attach overnight before cells were exposed to PPAR agonists in the presence of M-CSF. RANKL was then added for 10–20 min. Protein was isolated and used to determine activation of proteins in the RANKL signalling pathway using western blotting. The addition of RANKL led to the phosphorylation of IKK and the degradation of IκB after 10 min A. All thThe activation of MAPK proteins  was also determined B. All th−1) and M-CSF (25 ng mL−1) for 14 days. Thereafter RNA was isolated, and osteoclast specific gene expression was determined by qPCR. The absence of RANKL resulted in significantly lower levels of cFos , and all other chemicals were supplied by Sigma-Aldrich . PLA was provided by Santa Cruz Biotech . DNAse and RNAse free alpha-MEM was provided by GIBCO . Foetal bovine serum (FBS) was supplied by Amersham . Human RANKL and M-CSF were purchased from R&D Systems . LASEC  supplied all cell culture plates and other plasticware. Sigma-Aldrich  or Abcam  supplied the primary antibodies used in this study. All primers were synthesised by InqabaBiotec .DHA, AA, EPA, OA, PPAR agonists , antibiotic solution (100 μg mLStock concentrations (100 mM) of AA, DHA, EPA, PLA and OA were prepared in ethanol. PPAR agonists were prepared in DMSO at a stock concentration of 10 mM. The compounds were frozen at −70 °C in aliquots and diluted to working concentrations in culture media when required. AA, DHA and EPA were used at concentrations that have previously been shown to inhibit osteoclast formation (40 µM) . SimilarThis study received ethical clearance from the Faculty of Health Sciences Research Ethics Committee, University of Pretoria (reference number: 321/2017) .7 cells and 20 µl of MACS® MicroBeads  per 107 cells were added. The suspension was mixed carefully and incubated for 15 min on ice with mixing by gentle inversion every 5 min. Thereafter the cells were washed with PBS supplemented with 2 mM EDTA and 0.5% BSA followed by centrifugation at 300 xg for 10 min. The cells were resuspended in PBS supplemented with 2 mM EDTA and 0.5% BSA and passed through a magnetic separation column. Thereafter, 1 mL of the PBS supplemented with 2 mM EDTA and 0.5% BSA was added and the magnetically labelled cells were flushed out of the column. The CD14+ monocytes were then counted and seeded into cell cluster plates at 1.3 × 105 cells cm−2 in complete alpha-MEM .Peripheral blood was drawn from healthy male donors (age 18–30) after informed consent as previously reported . In brie5 cells cm−2 in complete alpha-MEM. After 24 hours, 7 days and 14 days, total RNA was extracted using TRI Reagent®  and reverse transcribed into cDNA using M-MuLV reverse transcriptase as previously described [PCR was conducted to determine the expression of PPAR-α, PPAR-β/δ, and PPAR-γ in the CD14+ monocytes during different stages of maturation. CD14+ monocytes were seeded into 24-well plates at 1.3 × 10escribed . PCR was5 cells cm−2 in 96-well plates in complete alpha-MEM for 24 h before exposure exposed to AA (40 µM), DHA (40 µM), EPA (40 µM), PLA (100 µM), OA (100 µM), fenofibrate (10 μM), L-165041 (10 μM) or troglitazone (10 μM) for 48 h. At the end of the culture period, media was discarded from each well and replaced with 10 µl of 0.8 μM resazurin solution in 90 μl of fresh media. The plates were then incubated at 37 °C for 4 h and absorbance was read at 570 nm using 600 nm as a reference using an Epoch micro-plate spectrophotometer .A resazurin assay was conducted to determine whether the UFAs or PPAR agonists had cytotoxic effects on human CD14+ monocytes. Resazurin is a blue dye that is converted to resorufin, a pink dye, by metabolically active cells. This colourimetric change can be used as a measure of cell viability. Cells were seeded at 1.3 × 105 cells cm−2 in 6-well plates and incubated at 37 °C overnight. Cells were then exposed to PPAR agonists or fatty acids and RANKL (30 ng mL−1) and M-CSF (25 ng mL−1) for 24 h. At the end of culture, the cells were washed in ice-cold PBS before lysis with cytoplasmic extraction buffer  NP40, 1 mM DTT and 1 mM PMSF. pH adjusted to 7.6). Cells were then incubated on ice for 3 min followed by centrifugation at 150 xg at 4 °C for 4 min. The supernatant was removed and gently resuspended in cytoplasmic extraction buffer without NP40. Following centrifugation at 150 xg at 4 °C for 4 min, the supernatant was removed, and the pellet was resuspended in nuclear extraction buffer . After 10 min incubation on ice, the extract was centrifuged at maximum speed at 4 °C for 10 min. The supernatant (containing the nuclear fraction) was then transferred to a fresh tube. CD14+ monocytes were seeded at a density of 1.3 × 10PPAR activation was determined using a PPAR  Transcription Factor Assay Kit  according to the manufacturer’s instructions. In brief, nuclear protein extracts were loaded into wells coated with PPAR-α, PPAR-β/δ or PPAR-γ consensus DNA and the plates were stored at 4 °C overnight. The plates were then washed 5 times with wash buffer to remove unbound reagents. Primary antibody was added, and the plates were incubated at room temperature for 60 min. After another wash period, goat anti-rabbit-HRP conjugate secondary antibody was added and the plates were stored at room temperature for an hour. Thereafter, transcription factor developing solution was added for 15–45 min followed by the addition of stop solution. The plates were then read at 450 nm using an Epoch micro-plate spectrophotometer .5 cells cm−2 in complete alpha-MEM in the presence of RANKL (30 ng mL−1), M-CSF (25 ng mL−1) and PPAR agonists (0.01–10 µM) or AA (40 µM), DHA (40 µM), EPA (40 µM), PLA (100 µM) or OA (100 µM). Medium and all factors were replaced every 2–3 days. Experiments were terminated on day 14.CD14+ monocytes were seeded in 96-well plates at 1.3 × 10TRAP activity in the media was measured as previously described . In brie−1 naphthol AS-BI phosphate in the acetate-tartrate solution at 37 °C for 30 min. The solution was replaced with hexazotised pararosaniline in acetate-tartrate solution for 15 min at 37 °C before counter-staining with haematoxylin for 40 sec. Osteoclasts appear as large multinucleated cells staining red. TRAP-positive stained cells with three or more nuclei per unit area were counted as mature osteoclasts [After fixation with 3.7% formaldehyde in PBS, cells were stained for TRAP as previously described . Cells weoclasts . Photomi5 cells cm−2 in 6-well plates in complete alpha-MEM supplemented with M-CSF (25 ng mL−1) and incubated at 37 °C overnight. Cells were exposed to PPAR agonists for 4 h. RANKL (30 ng mL−1) was then added and cells were incubated for 15 min.CD14+ monocytes were seeded at a density of 1.3 × 102O, pH adjusted to 8) supplemented with 0.3 M PMSF, 5% protease inhibitor cocktail and 5% phosphatase inhibitor cocktail. The lysate was centrifuged at 13 000 xg for 30 min using a Jouan Br4i centrifuge  at 4 °C for the removal of non-lysed fragments. Purified proteins were quantified using a BCA protein determination kit  according to the manufacturer’s instructions. Equal concentrations of protein were loaded in sample buffer containing 1% β-mercaptoethanol and resolved on a 12% polyacrylamide gel. Bio-Rad transfer system  was used to transfer proteins onto a nitrocellulose membrane with Tris-glycine transfer buffer . After blocking with 5% BSA powder for an hour the membranes were washed with TBS-T and then incubated with rabbit polyclonal primary antibodies against IκB, pIKK, IKK, JNK, pJNK, ERK, pERK, p38 and pp38 (1:1 000) overnight at 4 °C. After incubation with goat anti-rabbit IgG Antibody, HRP-conjugate (1:20 000)  for an hour at room temperature, the membranes were developed using a Clarity ECL Western Blotting Substrate  and visualized on a ChemiDoc MP . ImageJ software were used to quantify band densities [Cells were then washed in ice-cold PBS before being lysed in 100 µl RIPA buffer  and M-CSF (25 ng mL−1) and PPAR agonists (0.01–10 µM). Medium and all factors were replaced every 2–3 days. Experiments were terminated on day 14.To determine whether PPAR agonists influenced the expression of osteoclast specific genes, a qPCR was conducted. CD14+ monocytes were seeded at a density of 1.3 × 10−ΔΔCT method and results were normalized to the housekeeping gene (GAPDH). The primers used are shown in RNA was collected and reverse transcribed as described previously. For quantitative PCR (qPCR) the SensiFAST™ SYBR® No-ROX kit Master Mix  was used for amplification using the following cycling protocol: initial denaturation at 95 °C for 2 min, denaturation at 95 °C for 5 s, annealing at 65 °C for 10 s, and extension at 72 °C for 10 s for 40 cycles. A LightCycler® Nano System  was used for detection. Relative gene expression levels were analysed using the 2Data displayed is representative of three repeats unless otherwise stated. Results are displayed as a mean ± the standard deviation relative to the vehicle control. Data was compared to the vehicle control of the respective experiment and analysed by a one-way analysis of variance (ANOVA) followed by a Bonferroni post hoc test using GraphPad software . All p-values ≤ 0.05 were considered statistically significant.


N-acetyl-neuraminide alpha-2,8-sialyltransferase 2 (ST8SIA2) synthesizes polysialic acid (PSA), which is essential for brain development. Although previous studies reported that St8sia2-deficient mice that have a mixed 129 and C57BL/6 (B6) genetic background showed mild and variable phenotypes, the reasons for this remain unknown. We hypothesized that this phenotypic difference is caused by diversity in the expression or function of flanking genes of St8sia2. A genomic polymorphism and gene expression analysis in the flanking region revealed reduced expression of insulin-like growth factor 1 receptor (Igf1r) on the B6 background than on that of the 129 strain. This observation, along with the finding that administration of an IGF1R agonist during pregnancy increased litter size, suggests that the decreased expression of Igf1r associated with ST8SIA2 deficiency caused lethality. This study demonstrates the importance of gene expression level in the flanking regions of a targeted null allele having an effect on phenotype.ST8 alpha- N-acetyl-neuraminide alpha-2,8-sialyltransferase 2 (ST8SIA2) is one of the enzymes that synthesize polysialic acid (PSA), which is crucial for brain development4. PSA, a unique linear homopolymer of α2,8-linked sialic acid, is known mainly as a posttranslational modification of the neural cell adhesion molecule (NCAM), a member of the immunoglobulin superfamily7. Multiple processes, including synaptic plasticity, migration of neural precursor cells, and brain wiring, involve the PSA on NCAM8. The synthesis of PSA depends on the two Golgi-resident polysialyltransferases, ST8SIA2 and ST8SIA47. ST8SIA2 is predominantly involved in PSA synthesis during brain development, whereas ST8SIA4 seems to regulate PSA synthesis in the adult brain6. Complete abrogation of PSA synthesis in St8sia2/St8sia4 double homozygous null mice causes a postnatal lethal phenotype2 with ventricular hydrocephalus10; however, the knockout of St8sia2 alone has been reported to show variable developmental phenotypes. One previous report found that St8sia2−/− mouse strains are normal11, but a more recent one demonstrated that the mutant mice have mild hydrocephalus12. The origin of this phenotypic difference has not been delineated.Glycosylation is one of the important post-translational modifications of proteins. ST8 alpha-14. Because many of the resulting mutants show inconsistent behaviour16, generally, these knockout mice generated from 129 strain ES lines are backcrossed to the C57BL/6 (B6) strain two to seven times before analysis of their phenotypes. However, even after several backcrosses, several centimorgans of flanking region derived from the 129 strain remain linked to the targeted allele, potentially containing hundreds of genes14. 129 and B6 strains have many differences between their gene expression profiles, both in the bodies and brains of adults and neonates18. This phenotypic variation due to flanking gene effects has been reported most often for behavioural phenotypes21. We therefore must consider the effects of these unreplaced flanking regions, as their presence might mask the pure phenotype of the targeted genes22. However, in most cases, the mechanism by which these flanking regions alter the phenotype has not been clarified.Gene targeting in mice has helped in delineating the physiological function of many genes in mice. Most of the embryonic stem (ES) cell lines used in gene targeting have been derived from the 129 strain because of its higher efficiencySt8sia2 null mutant mice with mixed genetic background derived from backcrossing the 129 to B6 have a variable phenotype. We found that the variability in gene expression in the flanking regions of the St8sia2 locus contributed significantly to these phenotypic differences. By identifying polymorphisms allowing us to map flanking regions, it may allow the explanation of variable mutant phenotypes observed with targeting of a single gene.In this study, St8sia2 homozygous null mutant mice have almost 50% of the NCAM lacking PSA relative to the NCAM of wild-type (WT) controls1. We confirmed this observation in N7 using western blotting (WB)  activitySt8sia2−/− mice  Fig. . We furtWB) Fig.  and founWB) Fig.  and signice Fig. .  Togeth25, and lack of PSA inhibited kinesis of the sea urchin sperm and suppressed fertilization26, a subset of homozygous mutant mice was sterile  and birth rates of homozygous mutant mice . To clarify the contribution of the 129/Sv genetic background to the penetrance of the mutant phenotype, we backcrossed N17/N18 males to 129 females (129X1/SvJ)  Fig. . In thisvJ) Fig. . CrossinvJ) Fig.  and S3c,vJ) Fig.  of homozvJ) Fig. . To confvJ) Fig. . The resSt8sia2 heterozygotes using microsatellite markers on chromosome 7 . Taken together, these data suggest that one of these four genes may be responsible.To identify candidate genes related to each phenotype, we performed quantitative trait locus (QTL) mapping analysis for hydrocephalus by intercrossing e 7 Fig. . This ane 7 Fig. . The dece 7 Fig. . To furte 7 Fig. . This anIgf1r expression levels in the head yos Fig. , consistice Figs  and S5b,ice Figs  and S5b.yos Fig. . This suSt8sia2 mutant mice, it suppressed body weight reduction or delayed its timing during pregnancy, compared with that in the control group  Suppression of Igf1r expression due to the maternal B6-allele may affect embryonic lethality. To examine these possibilities, we next transferred in vitro fertilization (IVF) embryos from a heterozygous intercrossing of 129N1F1 and N18 mice to wild-type ICR female mice. As a result, the number of littermates decreased  injections daily to pregnant B6 and N18 heterozygotes Fig. . In wildoup Fig. . Moreoveoup Fig. , indicatoup Fig. . These rsed Fig. , and no sed Fig. . This suSt8sia2 homozygous mutants increased incidence of hydrocephalus and embryonic lethality after their genetic background was purified by additional backcrosses to B6 up to almost inbred mice level 6 and the resulting product, PSA-NCAM, plays an important role in the migration of neurons36. Therefore, it is possible that the two conditions worked synergistically and resulted in the abnormalities being more severe. Furthermore, the interaction between the ST8SIA2 and IGF1R might contribute to the construction of the pathological state. In this study, St8sia2 deletion did not alter IGF1R level, but decreased PSA-NCAM level 32. Therefore, it is possible that genetic variation of this region was involved in Igf1r function in B6 mice. One other possibility is the effect of intron retention. Igf1r gene has intron-retained splicing variants and isoforms (Ensemble asia), resulting in degradation of the mRNA by a monitoring system called nonsense-mediated decay (NMD)38. In mammals, this mechanism can downregulate up to 35% of alternatively spliced transcripts39. Therefore, intrinsic SNP may affect Igf1r expression through these modification on the introns.Differential analysis of SNP variations around the ion Fig. . It is pSt8sia2 homozygous mutant mice  cycle (light on 0700 h). Individual cage illumination was achieved by using LEDs  through frosted glass. All animal experiments were approved by the Committee of Animal Care and Use of Kindai University School of Medicine, and all experimental procedures were conducted in accordance with institutional guidelines for the use of experimental animals.Mice were maintained at a temperature of 23 ± 1 °C. Food  and water were provided St8sia2 subcongenic mice (B6.129-St8sia2tm1Jxm/J) backcrossed to C57BL/6J for 6 generations were obtained from Jackson Laboratory  and defined as the N7 generation. We further backcrossed these N7+/− mice with C57BL/6J  mice for 6 or 7 to 10 or 11 generations to generate N13 or N14 generations, showing hydrocephalus and embryonic lethality at N17 and N18 respectively. For rescue experiments, N17 or N18 generation mice were crossed with 129  and Balb/c  mice. We defined their pups as 129N1 and BalbN1, respectively. After intercrossing the strains mentioned above with St8sia2 heterozygotes, subsequent generations were defined as F1, F2, F3, and F4.St8sia2+/− intercrossing, pregnant mice were administered 1.2 g/L D.W. lithium chloride  in their drinking water from E10.5 (through pregnant) as previously reported24, and then, following birth, we genotyped and counted numbers of mice suffering from hydrocephalus. For rescue of embryonic lethality by N18 St8sia2+/− and B6 intercrossing, 1 mg/kg body weight of an IGF1 analogue (IGF1 LR3)  was injected i.p. from E10.5 to E18.5 into pregnant B6 and N18+/− mice42; then, after birth, we determined litter sizes. For WB analysis of PSA, brains were collected from 10-week-old male St8sia2 +/+ and −/− mice kept under LD conditions. For WB analysis of IGF1R and activated IGF1R, embryonic heads were collected from N17 St8sia2−/− mice on B6 and 129 backgrounds at Igf1r SNP (rs3698065) at E10.5, and from 129N1F1 St8sia2 +/+ and −/− mice at E11.5 respectively.Following timed matings, embryos were isolated at embryonic day 11.5 (E11.5) and fixed in 4% paraformaldehyde in PBS for 12–20 h. As treatment for hydrocephalus during N13 2+, 200 μM dNTP  and 0.4 μM of the corresponding primers . The PCR conditions were 95 °C for 10 min, followed by 35 cycles at 94 °C for 30 s, 63 °C for 1 min, and 72 °C for 1 min, with a final extension of 72 °C for 7 min. PCR products were resolved on a 2% agarose gel in Tris-Acetate-EDTA Buffer (TAE) to identify the diagnostic bands (700 bp for WT and 380 bp for KO).All mice were genotyped before and after behavioural testing by polymerase chain reaction (PCR). Tail DNA was extracted using the GenScript TissueDirect Multiplex PCR System  and subjected to PCR using a buffer containing 1.0 U/50 μL Taq DNA polymerase, 45 mM KCl, 2.5 mM MgSt8sia2−/− mice by painting hind limbs with India ink. We made the mice walk on the clear film, and then measured length and width of their steps and body width.Gait analyses were performed in six-week-old female normal and hydrocephalic St8sia2−/− animals for hydrocephalus and 43 couples of the same generation St8sia2+/− mice as controls were genotyped using 11 microsatellite markers in chromosome 7. A standard genome scan was conducted using R/qtl43 to detect loci associated with hydrocephalus and litter size. For each analysis, genome-wide thresholds for suggestive (P < 0.05) and significant (P < 0.01) QTL were determined by 1000 permutations. Transformation of the data allows for permutation-based thresholds that are the same for all traits.The entire group of 36 male N17 generation 44. In total, 14 microsatellite markers of mice chosen from the Mouse Microsatellite Data Base of Japan (MMDBJ) (http://shigen.nig.ac.jp/mouse/mmdbj/top.jsp) and ENSEMBL genome web browser (http://asia.ensembl.org/Mus_musculus/Marker) were tested by amplification of genomic DNA from C57BL/6J and 129X1/Sv mice (Supplementary Table DNA was extracted from the tail by ethanol extraction after proteinase K digestion as previously describedhttp://www.informatics.jax.org/snp) and Mouse Genomes Project - Query SNPs (Wellcome Trust Sanger Institute). In a comparative analysis of Igf1r gene between mouse strains, the 10 kbp upstream and downstream region of a gene was used. Primer sequences are described in Supplementary Table For SNP analysis, we chose markers within a restricted chromosome 7 region based on data from Mouse Genome Informatics . After genomic DNA removal by DNase I treatment (Sigma), a 0.5 μg aliquot of each total RNA preparation was reverse transcribed using ReverTra Ace (Toyobo) and 2.5 μM oligo (dT). Real-time PCR (qPCR) was performed with an ABI PRISM 7900HT, in a total volume of 10 μL using SYBR Premix Ex Taq II (Tli RNaseH Plus) , according to the supplier’s instructions. mRNA quantification was performed with two primers as described in Table 45. After 10 min of incubation on ice, lysates were centrifuged at 10,000 × g, at 4 °C for 20 min, and the supernatant was collected. Protein concentration of the supernatant was measured using the Micro BCA protein assay reagent (Thermo Fisher Scientific), and the supernatant was stored at −80 °C.Embryonic heads and adult brains  were homogenized using a pestle and sonicated with PBST  containing 1% protease inhibitor cocktail (Sigma-Aldrich) as previously described46. Immunohistochemistry to detect Igf1r was performed using a rabbit polyclonal antibody to IGF1R , a monoclonal antibody to phospho-Akt (Thr308) , and a mouse monoclonal IgM to PSA-NCAM (2-2B) , as previously reported45. For PSA-NCAM, peroxidase-conjugated goat polyclonal antibody against mouse IgM  was used as secondary antibody.Paraffin sections (5 µm) of PFA fixed embryos or hydrocephalus brains were used for haematoxylin and eosin (HE) staining and immunohistochemistry45. Membranes were incubated with the following primary antibodies: rabbit polyclonal antibodies against NCAM  , IGF1R , and phosphor- IGF1R (Tyr1161) , rabbit monoclonal antibodies against phospho-Akt (Thr308) , and Akt , and mouse monoclonal antibodies against polysialic acid (PSA)-NCAM  and GAPDH ; HRP-conjugated goat polyclonal antibody against rabbit and mouse IgG  or IgM .WB was performed as previously reported2. The fertilized eggs were then transferred to KSOM medium (Merck Millipore), cultivated overnight in a 5% CO2 incubator at 37 °C, and 50 two-cell stage embryos were then transferred into the oviducts of pseudopregnant ICR females at 0.5 d.p.c. (Nihon CLEA).The IVF was commissioned to the Animal Collaborative Center at Kindai University Faculty of Medicine. Briefly, female heterozygotes (3–4 weeks old) were super ovulated by i.p. administration of CARD HyperOva  followed 48 hr later by 7.5 IU (i.p.) human chorionic gonadotrophin (hCG). About 20 hr after hCG injection, oocytes were collected from the oviduct, and then were co-incubated with gametes for 5 hr at 38.5 °C in an atmosphere of 5% COp < 0.05 were considered statistically significant.All data are presented as mean ± SEM. Statistical comparisons were made using Excel-Toukei 2012 software . Student’s t-test was used for comparison of two groups and one-way ANOVAs with Fisher’s least significant difference (LSD) tests when more than two groups were compared. Ratios were statistically analysed by Fisher’s exact test. Differences with Supplementary information


Complex 1 exhibits phosphorescence  in contrast to its known ruthenium(II) analogue, which is non-emissive at room temperature. The complex undergoes significant oxygen-dependent quenching of emission with a 43-fold reduction in luminescence intensity between degassed and aerated acetonitrile solutions, indicating its potential to act as a singlet oxygen sensitiser. Complex 1 underwent counterion metathesis to yield [Os(btzpy)2]Cl2 (Cl1), which shows near identical optical absorption and emission spectra to those of 1. Direct measurement of the yield of singlet oxygen sensitised by Cl1 was carried out (φ (1O2) = 57%) for air equilibrated acetonitrile solutions. On the basis of these photophysical properties, preliminary cellular uptake and luminescence microscopy imaging studies were conducted. Complex Cl1 readily entered the cancer cell lines HeLa and U2OS with mitochondrial staining seen and intense emission allowing for imaging at concentrations as low as 1 μM. Long-term toxicity results indicate low toxicity in HeLa cells with LD50 >100 μM. Osmium(II) complexes based on 1 therefore present an excellent platform for the development of novel theranostic agents for anticancer activity.The complex [Os(btzpy) The complex shows significant dependence of emission intensity on the presence of oxygen. The water soluble chloride salt Cl1 has been subjected to preliminary cellular uptake and luminescence imaging studies and relevant results are reported.We report here the synthesis and characterization of the orange-emissive bis(terdentate) osmium(II) complex [Os(btzpy)1 was prepared by reaction of two equivalents of the ligand btzpy [6][NH4]2 in refluxing ethylene glycol 2][PF6]2  was also prepared.Complex nd btzpy ,38 with e glycol . After b1 were investigated by cyclic voltammetry (CV) and reveal a reversible Os(II)/Os(III) oxidation at +0.64 V (vs Fc/Fc+ = 0.0 V). This is close to that exhibited by the known model complex 2 (Eox = +0.49 V) and other related osmium(II) complexes 2+  2+ [The lowest lying triplet state of omplexes  and [Os(es [2]2+ .2]Cl2 (Cl1), was achieved by stirring a suspension of 1 in methanol with Amberlite IRA-400 ion-exchange resin (chloride form) before filtering, removal of solvent and freeze-drying from aqueous solution. Removal of the hexafluorophosphate couterion was confirmed by the lack of the corresponding resonances in the 19F- and 31P-NMR spectra. The UV-visible absorption spectrum of Cl1 m of 1Cl  in aqueoCl1 was seen to localise to the mitochondria in the cancer cell lines HeLa  and U2OS (osteosarcoma) following a short incubation time of 4 h and with clear phosphorescence seen at concentrations as low as 1 μM. Colocalisation with the mitochondrial stain MitoView 633 was seen under confocal microscopy , of complex Cl1 was measured in air-equilibrated acetonitrile against the standard perinaphthenone. A φ (1O2) of 57% was determined by direct measurement of the 1Δg state emission from 1O2 in the NIR (λem 1275 nm) under 355 nm irradiation by a pulsed Nd:YAG laser as described previously [PF6]2 in aerated MeCN, with analyte solutions being excited at a single wavelength at a point of common optical density. Thus, φem values are determined from the ratio of integrated area under the corrected peaks, with an assumed experimental uncertainty of ±20%. Cyclic voltammograms were recorded using an Autolab PGSTAT100N potentiostat with NOVA electrochemical software (version 1.10.1.9). Analyte solutions were prepared using nitrogen saturated dry acetonitrile, freshly distilled from CaH2. All measurements were conducted at room temperature under a stream of dry nitrogen at potential scan rates ranging from 20 to 500 mV·s−1. [NBu4][PF6] was used as a supporting electrolyte, being recrystallised from ethanol and oven dried prior to use, with a typical solution concentration of 0.2 mol dm−3. The working electrode was a platinum disc, with platinum wire utilised as the counter electrode. The reference electrode was Ag/AgCl, being chemically isolated from the analyte solution by an electrolyte containing bridge tube tipped with a porous frit. Ferrocene was employed as an internal reference, with all potentials quoted relative to the Fc+/Fc coupleAmmonium hexachloroosmate(IV) was purchased from Alfa Aesar  whilst all other reagents were purchased from Sigma-Aldrich , Acros Organics  or Fluorochem  and used as supplied. The ligand btzpy  and comp2  were pre4)2OsCl6], 150 mg, 0.341 mmol) and 2.5 equivalents of 2,6-bispyridine  in ethylene glycol (25 cm3) was heated at reflux overnight under nitrogen. The resulting mixture was then allowed to cool to room temperature. 2.5 equivalents of aqueous NH4PF6 was added resulting in a dark brown precipitate which was collected by filtration and washed with cold water and diethyl ether. This was then redissolved in acetonitrile, cooled in the fridge overnight and filtered to remove unreacted ligand. The solvent was removed from the filtrate and the residue recrystallized from dichloromethane/hexane to give an orange powder. Yield = 185 mg, 45%; 1H-NMR : δ 9.13 ; 8.36 ; 8.01 ; 7.49–7.60 . 13C-NMR : δ 153.26, 151.74, 138.26, 136.94, 131.12, 131.07, 124.98, 121.64, 120.18. ESI HRMS: calculated for [C42H30N14Os]2+m/z = 461.1191; found m/z = 461.1195.Ammonium hexachloroosmate(IV)  in methanol (25 cm3) was stirred with Amberlite IRA-400 ion-exchange resin  for 24 h at R.T. in the dark. The resin was removed by filtration and the solvent then removed by evaporation. The residue was then dissolved in water and the solution was freeze dried to yield Cl1 as an orange powder. Yield = 71 mg, 87 % 1H-NMR : δ 10.15 ; 8.58 ; 8.21 ; 7.66–7.72 ; 7.47–7.57 . 13C-NMR : δ 152.01, 150.32, 137.57, 135.58, 130.08, 130.00, 125.27, 120.46, 119.31. ESI HRMS: calculated for [C42H30N14Os]2+m/z = 461.1191; found m/z = 461.1192.A suspension of 1 and 2 were optimized using DFT calculations at the B3LYP [1 states were also optimized and the spin density calculated and plotted. All calculations were carried out using the NWChem 6.6 software package [The geometries of cations for complexes he B3LYP ,54 levelhe B3LYP  and 6-31he B3LYP . Optimis package  with geo2. Both cell lines were routinely checked for mycoplasma infection. Complex Cl1 was stored as a stock solution at 10 mM in DMSO.Both HeLa  and U2OS (human bone osteosarcoma) cell lines were purchased from American Type Culture Collection–LGC partnership  and used within 20 passages of purchase. Cells were cultured in Dulbecco′s modified Eagles Medium (DMEM)  with 10% fetal calf serum (FCS)  and incubated at 37 °C under 5% CO5 cells per well and allowed to adhere overnight in culture media. Complex Cl1 (1 μM) was added and incubated for 4 h, for co-localisation studies MitoView™ 633 (Biotium) was added for the final 15 min, prior to cells being washed 3 times in PBS and fixed in 4% paraformaldehyde solution in PBS at 4 °C for 20 min. Following a further wash (PBS × 3) the coverslips were mounted to microscope slides . The slides were imaged by confocal microscopy  using a 60× lens . An argon laser (405 nm and 561 nm) was used to excite complex Cl1 and a diode laser (642 nm) was used to excite MitoView™ 633. Colocalisation indices were calculated using the open source imaging software Fiji (based on ImageJ) and the coloc 2 colocalisation tool. The threshold regression chosen was Bisection.Cover glasses (22 × 22 mm) were sterilised  and placed flat in 6-well plates. Cells were seeded at a density of ~1 × 10Cl1 or DMSO control and incubated for 4 h before replacing with fresh media. After 5 days further growth, 25 μL of 3 mg cm−3 thiazoyl blue (MTT) solution was added to each well. Following incubation for 3 h the solution was removed from each well and 250 μL/well DMSO added ensuring mixing of crystals. Optical density of wells at 540 nm was recorded on a plate reader .96-well plates were seeded with HeLa cells at 1000/well and incubated overnight. Wells were treated with 0.1–100 μM complex Cl1 for 4 h before replacing with fresh media. Plates were incubated for 8–10 days to form colonies before staining with 4% methylene blue in 70% methanol and counting. Each colony was considered to represent a single surviving cell and survival fraction calculated for each condition compared to DMSO control.Six-well plates were seeded with HeLa cells at 400 cells/well and incubated overnight. Wells were treated with DMSO, 50 μM or 100 μM complex


Cx43 heterozygous (+/−Cx43) mice and 37,43Gap27, which is a pharmacological inhibitor of Cx37 and Cx43. Contraction and relaxation responses were studied in intra-lobar pulmonary arteries (IPAs) derived from normoxic mice and hypoxic mice using wire myography. IPAs from male +/−Cx43 mice displayed a small but significant increase in the contractile response to endothelin-1 (but not 5-hydroxytryptamine) under both normoxic and hypoxic conditions. There was no difference in the contractile response to endothelin-1 (ET-1) or 5-hydroxytryptamine (5-HT) in IPAs derived from female +/−Cx43mice compared to wildtype mice. Relaxation responses to methacholine (MCh) were attenuated in IPAs from male and female +/−Cx43 mice or by pre-incubation of IPAs with 37,43Gap27. Nω-Nitro-L-arginine methyl ester (l-NAME) fully inhibited MCh-induced relaxation. In conclusion, Cx43 is involved in nitric oxide (NO)-induced pulmonary vascular relaxation and plays a gender-specific and agonist-specific role in pulmonary vascular contractility. Therefore, reduced Cx43 signaling may contribute to pulmonary vascular dysfunction.Pulmonary arterial hypertension (PAH) is a chronic condition characterized by vascular remodeling and increased vaso-reactivity. PAH is more common in females than in males (~3:1). Connexin (Cx)43 has been shown to be involved in cellular communication within the pulmonary vasculature. Therefore, we investigated the role of Cx43 in pulmonary vascular reactivity using  Pulmonary arterial hypertension (PAH) is a progressive disease in which the mean resting pulmonary artery pressure rises above 25 mmHg with a mean resting capillary wedge pressure lower than 15 mmHg . Dysregu2+), and inositol 1,4,5-triphosphate (IP3), which can pass directly through the cell membrane. Hemi-channels on adjacent cells can align to form gap junctions that allow these mediators to pass directly from the cytoplasm of one cell to that of another. Connexins 37 (Cx37), 40 (Cx40), 43 (Cx43), and 45 (Cx45) are expressed in diverse networks throughout the vasculature  to which 1.5 µL proteinase K  was added. Samples were then incubated overnight at 55 °C. The next day, 84 µL of 5 M NaCl was added to each sample and samples were centrifuged for 10 min. The supernatant was collected and transferred to fresh tubes. DNA was precipitated by adding 200 µL ice cold 100% ethanol to each tube and vortexing. Samples were then centrifuged for 10 min, the supernatant was discarded, and the pellet was retained. Excess salt was removed by adding 200 µL ice cold 75% ethanol and samples were centrifuged again for 10 min. The supernatant was decanted gently and the pellet was allowed to air dry. The pellet was then re-suspended in 15 µL nuclease-free water and stored at −20 °C until use. A polymerase chain reaction (PCR) was then carried out to amplify the Cx43 (GJA1) and neomycin resistance (rneo) genes. The primers used were as follows: rneo forward 5′-GATCGGCCATTGAACAAGATG, melting temperature (Tm) = 56.4 °C, molecular weight (MW) = 6808.5, rneo reverse: 5′-CCTGATGCTCTTCGTCCAGAT Tm = 57.2 °C, MW = 6637.3, Cx43 forward: 5′-CAGTCTGCCTTTCGCTGT, Tm = 56 °C, MW = 5433; Cx43 reverse: 5′-GTAGACCGCACTCAGGCT, Tm = 58 °C, MW = 5485. All primers were purchased from Integrated DNA technologies, Belgium. PCR reactions were performed in a thermal cycler  and comprised an initial denaturation at 95 °C for 3 min followed by 40 cycles of: denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 1 min. A final extension step was carried out at 72 °C for 15 min. Samples were then run in 2% agarose gel (v/v) for 45 min at 100 V.DNA was extracted from ear notch tissues derived from WT and +/−Cx43 mice were placed in a hypobaric chamber for 14 days, which is previously described [v/v O2) slowly over two days to allow mice to acclimate. The temperature was maintained at 20–22 °C. Control littermates were kept in a normoxic environment.For induction of hypoxia, male WT and escribed . The preMice were euthanized by injection of phenobarbitone (60 mg/kg i.p.). After death was confirmed, the chest walls were opened using the mediastinal approach and the hearts and lungs were dissected freely.2 2.5, MgSO4 1.2, NaHCO3 25, KH2PO4 1.2, and d-glucose 5.5) at (37 °C) and gassed with 95%O2 / 5% CO2. All chemicals required for Krebs-Henseleit solution were purchased from Fisher Scientific except CaCl2 which was purchased from VWR International Ltd. . Following equilibration for one hour, IPAs from normoxic mice were placed under pressures of 12–15 mmHg and IPAs from hypoxic mice that were placed under pressures of 30–35 mmHg to mimic the in vivo environment described previously [37,43Gap27 (100 µM) for 30 min prior to MCh or isoprenaline-induced relaxation responses. 37,43Gap27 has previously been shown to have an IC50 of 31.5 ± 4.1 µM and to produce a maximum effect at 100 µM [37,43Gap27 at 100 µM [l-NAME (100 µM) was used to inhibit eNOS. In these experiments the l-NAME was applied for 30 min prior to pre-constriction with PE and the concentration of PE was reduced to 30 nM. It should be noted that 30 nM PE in the presence of l-NAME produced a similar contractile response to 3 µM PE alone. Changes in isometric tension were recorded on LabChart 7 software . PE, L-NAME, MCh, ET and 5-HT were purchased from Sigma-Aldrich Company Ltd. .Pharmacological experiments were carried out in third generation intra-lobar pulmonary arteries , which was previously described ,68. IPAseviously . Arteriet 100 µM . We prevt 100 µM ,71,72. ITM 2 Reverse Transcription Kit , according to the manufacturer’s protocols. qPCR was performed using Precision PLUS 2x qPCR master mix (Primerdesign Ltd.) II with taqman primers described in 2-microglobulin (B2M)  was used as the endogenous control and the sequences for the B2M were kept confidential by Primerdesign Ltd.Main and branch pulmonary arteries (1st and 2nd order) were homogenized and RNA was extracted using the Nucleospin RNA kit  as per the manufacturer instructions. RNA was then reverse transcribed to cDNA using the Precision nanoScriptTM 7 Real Time PCR System, ThermoFisher Scientific, Loughborough, UK) using the following conditions: 50 °C for 2 min and 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Gene expression was analyzed by the 2Ct∆∆ method. Samples from at least six mice were used for each group and reactions for each sample were run in triplicate. qPCR reactions were run in a real time PCR thermo-cycler machine . Sections were fixed in ice cold (−20 °C) methanol for 20 min and rehydrated in phosphate buffer saline . Sections were permeablised in PBS containing 0.1% (v/v) Triton-X100 before being blocked in 5% (w/v) skimmed milk in PBS solution for 30 min at room temperature. Sections were stained with primary antibody  and incubated at 4 °C overnight. The next day, sections were washed in PBS for 30 min at room temperature and incubated with secondary antibody  at 4 °C for 2 h. Nuclei were counter-stained with DAPI . Mounting medium was applied on tissue sections and cover slides were applied. Slides were then examined under the LSM 800 Carl ZEISS confocal microscope,  for immunoreactivity.Sagittal sections (7 µM) were cut from lung embedded at an optimal cutting temperature (OCT) compound using a cryostat , maximal contractile responses (Emax), and maximal relaxation responses (Rmax) between two different groups were analyzed by using the Student’s t-test. All data were shown as mean ± S.E.M. Data for cumulative concentration response curves (CRCs) were analyzed using GraphPad Prism 6 software . Data were fitted to a logistic equation, CRCs were generated, and EC


Alternaria tennuis). We have designed, synthesized, and characterized a derivative of dehydroaltenusin conjugated with a C12-alkyl side chain . C12 was the strongest pol α inhibitor in vitro. We introduced C12 into NIH3T3 cells with the help of a hypotonic shift, that is, a transient exposure of cultured cells in hypotonic buffer with small molecules which can not penetrate cells. The cells that took in C12 by hypotonic shift showed cell growth inhibition. At a low concentration (5 μM), DNA replication was inhibited and several large replication protein A (RPA) foci, which is different from dUTP foci. Furthermore, when C12 was incubated with aphidicolin, RPA foci were not observed in cells. Finally, these findings suggest that C12 inhibited DNA replication through pol α inhibition, and generated single-stranded DNA, resulted in uncoupling of the leading strand and lagging strand synthesis. These findings suggest that C12 could be more interesting as a molecule probe or anticancer agent than aphidicolin. C12 might provide novel markers for the development of antiproliferative drugs.Dehydroaltenusin is a selective inhibitor of mammalian DNA polymerase α (pol α) from a fungus ( The dehydroaltenusin derivative was dissolved in dimethyl sulfoxide (DMSO) at various concentrations and sonicated for 30 sec. Four microliters of each sonicated sample was mixed with 16 μl of each enzyme  in 50 mM Tris-HCl (pH 7.5) containing 1 mM dithiothreitol, 50% glycerol and 0.1 mM EDTA, and kept at 0°C for 10 min. These inhibitor-enzyme mixtures (8 μl) were added to 16 μl of each enzyme standard reaction mixture, and incubated at 37°C for 60 min. Activity without the inhibitor was considered 100%, and the remaining activity at each concentration of the inhibitor was determined relative to this value. One unit of pol activity was defined as the amount of enzyme that catalyzed the incorporation of 1 nmol of dTTP into the synthetic DNA template primer in 60 min at 37°C under the normal reaction conditions for each enzyme [Pol α was purified as described previously . For the2/95% air. For the cell viability assay, cells were plated at 1 x 103 into each well of 96-well microplates with various concentrations of dehydroaltenusin, C12 or aphidicolin. Cell viability was determined by MTT assay [NIH3T3 cells were cultured in Dulbecco's Modified Eagle’s Medium (DMEM) supplemented with 10% calf serum, penicillin (100 units/mL), streptomycin (100 mg/mL) at 37°C in a humid atmosphere of 5% COTT assay .4 cells were plated in each well of an 8-well-chamber slide (Nunc). After incubation for 16 h, the cells were washed with hypotonic buffer  [® Viability/Cytotoxicity Kit (Molecular Probes). In accordance with the manufacturer’s protocol, cells were exposed to Calcein AM (3 μM), which was hydrolyzed by intracellular esterases, and to ethidium homodimer-1 (4 μM), which binds to nucleic acids  Dead cells were treated with 70% methanol for 30 min. Culture dishes were stained and cell viability was examined under a confocal laser-scanning microscope .2.5 x 10 mM KCl)  and then4 cells were seeded per well and incubated with 3 mM thymidine for 16 h at 37°C. The cells were washed two times with medium without thymidine and hypotonic buffer, respectively, and incubated with hypotonic buffer with 10 µM FITC-dUTP (Roche), or 5 µM aphidicolin, 5 µM dehydroaltenusin, and 5 µM C12, respectively, for 10 min at 37°C, and incubated for 90 min with medium at 37°C. The cells were pre-extracted with 0.5% Triton X-100 in cytoskeleton buffer , 1 mM DTT and 0.25 mM PMSF 5 min on ice, and then fixed with 3.7% formaldehyde in PBS on ice 10 min, incubated with 50, 75 and 95% ethanol for 5 min each and image IT (Invitrogen) at RT 15 min, and 5% normal goat serum in PBS 15 min at RT. Cells were incubated with anti-p180 [Eight-well chamber slides (Nunc.) were pre-treated with acid-collagen (Koken) (1 μg/mL) for 2 h at 37˚C and 3 x 10nti-p180  and antinti-p180 .


Pisum sativum L. cv. Cysterski) seedlings and then during infestation by the pea aphid (Acyrthosiphon pisum Harris). The second objective was to verify whether the presence of lead in pea seedling organs and induction of signalling pathways dependent on the concentration of this metal trigger defense responses to A. pisum. Therefore, the profile of flavonoids and expression levels of genes encoding enzymes of the flavonoid biosynthesis pathway  were determined. A significant accumulation of total salicylic acid (TSA) and abscisic acid (ABA) was recorded in the roots and leaves of pea seedlings growing on lead-supplemented medium and next during infestation by aphids. Increased generation of these phytohormones strongly enhanced the biosynthesis of flavonoids, including a phytoalexin, pisatin. This research provides insights into the cross-talk between the abiotic (lead) and biotic factor (aphid infestation) on the level of the generation of signalling molecules and their role in the induction of flavonoid biosynthesis.The aim of this study was to investigate the effect of an abiotic factor, i.e., lead at various concentrations (low causing a hormesis effect and causing high toxicity effects), on the generation of signalling molecules in pea ( Under natural conditions we may frequently observe the effect of many stress factors acting simultaneously or sequentially. Plants demonstrate a great ability to adapt their metabolism to rapid changes in the environment  and therSince in the course of the coevolution of plants and biotic factors, including herbivores, undergo mutual adaptation, abiotic factors also significantly affect that process. Poschenrieder et al.  reportedA. pisum. This is the first report revealing the effect of lead as an abiotic factor and phytophages (A. pisum) as a biotic factor on the biosynthesis of pisatin, a phytoalexin characteristic of Pisum sativum L., which may serve a significant role in its defense strategy. Pisatin is believed to play a key role during abiotic and biotic stress responses. Jeandet et al. ABA were added. The methanol fraction was removed under reduced pressure and the aqueous phase was acidified to pH 2.0 with 12 M HCl and centrifuged at 10,000× g for 15 min to remove chlorophyll. The supernatant was partitioned three times against ethyl acetate and dried under vacuum. The dry residue was dissolved in 5 mL 1 M formic acid (FA) and loaded on a Discovery® DSC−18 SPE cartridge  pre-conditioned with 4 mL MeOH and allowed to equilibrate with 4.0 mL of 1 M FA. The column was then washed with 4 mL 1 M FA, 20% of methanol in 1M FA and finally target phytohormones were eluted with 4 mL of 80% methanol in water. The eluate was evaporated and further purified by HPLC using a SUPELCOSIL ABZ + PLUS column . The samples were reconstituted in 130 µL of 20 % methanol and chromatographed with a linear gradient of 20–80% methanol in 0.1 M formic acid for 20 min, flow rate 1.0 mL/min at 22 °C. The fractions collected at 12.5 ± 0.5 min were evaporated to dryness, methylated with diazomethane, dissolved in 30 µL of methanol and analyzed by GC/MS–SIM  using a MDN-5 column . The GC temperature program was 60 °C for 1 min, 60–250 °C at 10 °C/min, flow rate 1.5 mL/min, injection port was 280 °C and electron potential 70 eV. The retention times of ABA and [2H6] ABA were 14.07 and 14.3 min, respectively. GC/MS–SIM was performed by monitoring m/z 190 for endogenous ABA and 194 for [2H6] ABA, 130 for endogenous IAA and 132 [2H2] IAA according to the method described by Vine et al. [For ABA measurement samples consisting of approx. 1 g frozen tissue were homogenised in liquid nitrogen and extracted twice with 20 mL 80%  and sonicated for 3 min in a VirTis VirSonic 60 sonicator [Plant material, previously frozen at −80 °C was homogenised in 80% methanol  and B . Elution steps were as follows: 0–5 min 10%–30% B, 5–12 min isocratic at 30% B, 12–13 min linear gradient up to 95% of B and 13–15 min isocratic at 95% of B. Pisatin and flavonols were identified by comparing their retention times and mass spectra with the data from respective standards. The micrOToF-Q mass spectrometer consisted of an ESI source operating at a voltage of ±4.5 kV, nebulisation with nitrogen at 1.2 bar and dry gas flow of 8.0 L/min at 220 °C. The instrument was operated using the micrOTOF Control program version 2.3 and data were analysed using the Bruker Data Analysis ver. 4 package. Targeted MS/MS experiments were performed using a collision energy ranging from 10 to 25 eV, depending on the molecular masses of the compounds. The instrument operated at a resolution of minimum 15,000 full widths at half maximum.Plant extract samples were analysed using a Waters UPLC Acquity system coupled with a micrOToF-Q mass spectrometer . An Agilent Poroshell RP-C18 column  was used. During LC analyses elution was performed using two solvent mixtures: A . Total quantitative analysis was conducted based on all signals acquired in the positive MS mode using the Profile Analysis 2.1 software . Multivariate analyses were carried out by the unsupervised principal component analysis (PCA).For targeted quantitative analysis, the extracted ion chromatogram traces were used, with peaks plotted for exact monoisotopic masses of compounds. Such traces were prepared for each compound earlier and identified based on their MS2 spectra. For calibration purposes, 260/A280 ratio varied from 1.8 to 2.0. The cDNA samples for RT-PCR experiments were synthesised from 1 μg of total RNA and oligo (dT)18 primers using the High Capacity cDNA Reverse Transcription Kit . One µL of the each cDNA was used as a template for the PCR reaction with specific PCR primers. Thermal cycling conditions in the PAL, CHS and actin genes expression assay consisted of an initial denaturation at 95 °C for 5 min, followed by 25 cycles at 95 °C for 30 s, 58 °C for 30 s and 72 °C for 45 s. The PCR products were analysed by agarose gel electrophoresis (1.5%). The specific primers were used for PCR reactions: PAL F 5’-GAGCAGCACAACCAAGATG-3′ and PAL R 5’-CTCCCTCTCAATTGACTTGG-3’, CHS F 5’-AGGTCTTCGCCCATATGTGAA-3’ and CHSR 5’-GTGCTTGTCCAACAAGACTGT-3’. The fragment of Pisum sativum actin (HQ231775) coding sequence was amplified as a reference gene using actin F 5′-GCATTGTAGGTCGTCCTCG-3′ and actin R 5′-TGTGCCTCATCACCAACATAT-3′ primers.Pea seedling leaves (0.50 g) were frozen in liquid nitrogen and ground with a mortar and pestle in the presence of liquid nitrogen. For RT-PCR analyses of the target gene, total RNA was isolated from 45 mg tissue using the SV Total RNA Isolation System  according to the recommendations of the manufacturer ,113. Theg for 20 min. The mixture containing 0.2 mL phosphate buffer , 0.2 mL extract and 0.2 mL 4-nitrophenyl-β-d-glucopyranoside as substrate (2 mg·mL−1) was incubated for one hour at 35 °C. Afterwards, 0.6 mL 0.2 M Na2CO3 was added. The formation of p-nitrophenol (p-NP) was followed at 400 nm. The activity was measured in three replications and expressed as μM p-nitrophenol mg−1·protein·h−1.The activity of β 1,3-glucosidase (EC 3.2.1.21) was determined spectrophotometrically  applying the method proposed by Nichols et al.  and moditrans-cinnamic acid using a Perkin Elmer Lambda 15 UV-Vis spectrophotometer. The activity of PAL is expressed as µM trans-cinnamic acid expressed per mg protein per hour (µM trans-cinnamic acid· mg−1protein h−1).The activity of PAL (EC 4.3.1.24) was determined with a modified method of Cahill and McComb  as modif−1 Pb (certified 2.16 ± 0.23 mg·kg−1) for plant samples and 1.99 mg·kg−1 Pb for aphid samples. The Laboratory is accredited according to ISO/IEC 17025.All reagents and standards were of at least analytical grade. All solutions were prepared using ultrapure water 18.2 MΩ·cm. Root and leaf samples of pea seedlings were rinsed with ultrapure water  and air dried at room temperature. Air dried samples were then ground  and oven dried at 70 °C before weighing. Subsamples (0.5 g) were digested in 10 mL 65% nitric acid  using a closed vessel digestion microwave system . The digested solutions were diluted to 50 mL using ultrapure water. The aphid samples were washed and dried as described for the plant samples. 0.2 g subsamples of aphids were digested in 10 mL 65% nitric acid  at 80 °C until the solution became clear. The digested solutions were diluted to 25 mL using ultrapure water. The lead concentrations were determined by flame atomic absorption spectroscopy (FAAS)  and graphite furnace atomic absorption spectroscopy with the Zeeman correction (GFAAS) . Certified reference materials were analysed to verify the accuracy and precision of the measurements. Observed concentrations for the certified INCT-MPH-2 mixed Polish herbs sample  were 2.02 mg·kgt-test for equal means for all the observed traits. To account for multiple testing, we used the Bonferroni correction. Moreover, comparisons related to the following plant material variants, i.e., the control vs. the 0.075 mM Pb2+ variant; the control vs. 0.5 mM Pb2+ variant; the control vs. the +aphids variant; the control vs. 0.075 mM Pb2++aphids variant; the control vs. 0.5 mM Pb2++aphids variant;. 0.075 mM Pb2+ variant vs. 0.5 mM Pb2+ variant; 0.075 mM Pb2+ variant vs. 0.075 mM Pb2++aphids variant; 0.5 mM Pb2+ variant vs. 0.5 mM Pb2++aphids variant; 0.075 mM Pb2++aphids variant vs. 0.5 mM Pb2++aphids variant; +aphids variant vs. 0.075 mM Pb2++aphids variant; +aphids variant vs. 0.5 mM Pb2++aphids variant. In turn, comparisons related to the following aphid variants, i.e., the control vs. 0.075 mM Pb2+ variant; the control vs. 0.5 mM Pb2+ variant; 0.075 mM Pb2+ variant vs. 0.5 mM Pb2+ variant, respectively. The figures present data obtained as means of triplicates for each variant along with standard errors of mean (SE). All the analyses were conducted using the GenStat v. 17 statistical software package.All determinations were conducted within three independent experiments. Additionally, three biological replicates per experimental variant were made for a given experiment. Analysis of variance (ANOVA) was used to verify the significance of means from independent experiments within a given experimental variant. The elementary comparisons between particular levels of the analysed factor in different times (independently) were tested using the two-sample A. pisum induced generation of SA and ABA in pea seedlings. Increased generation of these phytohormones strongly enhanced the biosynthesis of flavonoids, including a phytoalexin, pisatin. Strong generation of SA and ABA found in pea seedlings as a result of the influence of lead alone, especially at a lead high concentration, and the cross-talk of lead and A. pisum may indicate the involvement of these molecules in defensive responses, with these responses being stronger at exposure to a toxic lead dose rather than at a low dose of lead.In the presented study we revealed that lead at various concentrations (low causing the hormesis effect vs. high causing the toxic effect) and the cross-talk of lead and