Eucalyptus clones, E. grandis × E. urophylla ‘G9’ and E. grandis × E. urophylla ‘DH32-29’were subjected to six treatments (two levels of Al stress and three levels of P). Under excessive Al stress, root Al content was higher, whereas shoot and leaf Al contents were lower with P application than those without P application. Further, Al accumulation was higher in the roots, but lower in the shoots and leaves of G9 than in those of DH32-29. The secretion of organic acids was higher under Al stress than under no Al stress. Further, under Al stress, the roots of G9 secreted more organic acids than those of DH32-29. With an increase in P supply, Al-induced secretion of organic acids from roots decreased. Under Al stress, some enzymes, including PEPC, CS, and IDH, played important roles in organic acid biosynthesis and degradation. Thus, our results indicate that P can reduce Al toxicity via the fixation of elemental Al in roots and restriction of its transport to stems and leaves, although P application cannot promote the secretion of organic acid anions. Further, the higher Al-resistance of G9 might be attributed to the higher Al accumulation in and organic acid anion secretion from roots and the lower levels of Al in leaves.Under acidic conditions, aluminum (Al) toxicity is an important factor limiting plant productivity; however, the application of phosphorus (P) might alleviate the toxic effects of Al. In this study, seedlings of two vegetatively propagated  Melaleuca cajuputi and Acacia mangium. However, under acidic conditions, Al toxicity is an important factor limiting plant productivity [3+. Secretion of organic acids from plant roots has previously been confirmed to be an important mechanism whereby plants relieve Al toxicity [Triticum aestivum) roots [Phaseolus vulgaris) [Zea mays) [Cassia tora) [Nicotiana tabacum) [Glycine max) [Aluminum (Al) is the most widespread metal and the third most common element in the earth’s crust . In milductivity . Plant suctivity . Inhibituctivity –8. Althotoxicity ,10. Sincm) roots , the resulgaris) , maize (ea mays) ,14, sickia tora) ,16, tobatabacum) , and soyine max) , secreteine max) ,20. Furtine max) . The P uine max) . Even inine max) . Previouine max) , snapbeaine max) , buckwheine max) , and sorine max) .Eucalyptus are among the most widely planted trees and have many advantages as a plantation crop. They are used as raw materials in the paper industry, because of the following advantages: strong adaptability, rapid growth, a short rotation period, and the ability to resist drought, and thus provide high economic benefits [Eucalyptus, acid-induced Al toxicity being one of the most important. Al stress might have detrimental effects on root growth of Eucalyptus seedlings, although Al application could promote the secretion of organic acids [Eucalyptus clones showed different responses to acid-Al stress, which is consistent with the findings of many previous studies [Eucalyptus gummifera can use insoluble phosphate  phosphate) under acidic conditions [3+ [E. globulus and E. urophylla, but only at the highest Al3+ level (greater than 500 μM), whereas E. grandis and E. cloeziana showed more than 20% reduction in root growth at Al3+ levels higher than 256 μM [E. camaldulensis than in other herbaceous plants [Eucalyptus species might be associated with the exudation of organic acids [Eucalyptus [3+ tolerance mechanisms and clones (genotypes) has been reported in the literature [Eucalyptus seedlings. Previous studies have indicated that Al plays an important role in inducing the secretion of organic acids, which might chelate Al outside the plasma membrane, thereby preventing its uptake [Arabidopsis thaliana improves growth in P limited soils because of the enhanced citrate excretion from the roots[Cassia tora[Species of benefits . Howeverbenefits . Many faic acids ,30. Yang studies . For exanditions , and thaions [3+ . Silva en 256 μM . The ress plants . Silva eic acids . Conversterature . Howevers uptake ,35. Orgas uptake . These es uptake . Koyama the roots. Yang etssia tora.Eucalyptus seedlings. We compared the effects of Al–P interactions on seedling growth; Al and P contents in the roots, stems, and leaves; enzyme activities in the roots; and Al-induced secretion of organic acid anions from the roots of two fast-growing Eucalyptus clones under acid-Al treatments.This study aimed to investigate the physiological mechanisms of Al tolerance and Al–P interactions in The study was conducted in Nanning, the capital of Guangxi Province, China . The area has a subtropical humid climate with an annual average temperature of 21.6°C, ranging from 12.8°C (January) to 28.2°C (July to August). On average, each year, the area has 1827 h of sunshine, a frost-free period of 340 d, relative humidity of 77%, and rainfall of 1304.2 mm. Experiments were conducted in the nursery garden at Guangxi University.Eucalyptus clones, E. grandis × E. urophylla ‘G9’ and E. grandis × E. urophylla ‘DH32-29’, were used as the experimental materials . These two clones have been widely cultivated in Guangxi Province [st, we selected healthy and uniformly sized 2-month-old tissue-cultured seedlings. Saplings were grown in porous plastic pots of 250 mm (radius) × 400 mm (height), each containing 50 kg sand. Each pot was watered daily and supplied with 1 L of nutrient solution every week after transplanting. The nutrient solution contained the following macronutrients (in mg/L): CaH8N2O10, 945; KNO3, 506; NH4NO3, 80; KH2PO4, 136; MgSO4, 493; iron salt solution , 2.5 mL/L; and microelement solution (Formula (in mg/L): KI, 0.83; H3BO3, 6.2; MnSO4, 22.3; ZnSO4, 8.6; Na2MoO4, 0.25; CuSO4, 0.025; CoCl2, 0.025), 5 mL/L.This study was conducted in a greenhouse under the natural photoperiod occurring at Guangxi University. Two Province . The exp3·6H2O (0 and 5 mM) × three levels of P as KH2PO4 . The concentrations of Al (0 and 5 mM) used in the present study were determined based on the following information: (1) In a previous field investigation, the soil soluble Al concentration in 5-year-old Eucalyptus plantations was approximately 4.4 mM[Eucalyptus camaldulensis[Helianthus annum L.), subterranean clover (Trifolium subterraneum L.), and alfalfa (Medicago sativa L.) used P concentrations of 0, 0.8, 8, 80, and 160 μM (as NaH2PO4-2H2O) [2PO4) [One month after transplanting, treatments were initiated and applied for 20 weeks. The study consisted of six treatments: two levels of Al stress as AlClly 4.4 mM. (2) Ikkldulensis. The conO4-2H2O) . (2) Fur) [2PO4) . (3) Ano) [2PO4) . (4) Mor) [2PO4) .2SO4. The pH of the treatment solutions was adjusted to 4.1 or 4.2 by using 2 mol/L HCl or NaOH. Before the treatment was initiated, each pot was supplied with 1 L of nutrient solution every week, and then the pots were supplied with 1 L of nutrient solution every 2 weeks and 1 L of treatment solution every week during the treatment period.Ninety seedlings of each clone  were arranged in a randomized block design and subjected to the six treatments in three blocks, with five pots (one plant per pot) in each treatment in each block . PotassiThe biomass of seedlings was measured after harvesting at the end of the study (on September 20). Nine plants per treatment from different pots (one plant per pot) were harvested. The plants were divided into different parts , and then oven-dried for 48 h at 80°C for dry weight (DW) determinations.3:HClO4 . Mineral (P and Al) contents were then determined using inductively coupled plasma-atomic emission spectroscopy (Agilent Technologies) and the Mo-Sb colorimetric method [After the samples were dried to a constant weight (on September 23), all samples were ground in a mortar to pass through a sieve having pore size of 0.02 mm. Samples were digested with a mixture of HNOc method –48.2 (pH 4.1–4.2). After three rinses (each for 20 min), the root tips were transferred to 2-mL centrifuge tubes containing 1 mL control solution in the presence of 0.5 mM AlCl3·6H2O (pH 4.1–4.2). The tubes were positioned vertically on a shaking platform in the dark for 12 h. After the exudates were filtered through 0.45-μm microporous membranes, the organic acids  secreted from roots were analyzed using an ICS-5000 chromatography system .On September 20, root exudates were collected according to the method of Ryan et al. . ShearedPreparation of enzyme solution: On September 20, fresh root  was placed in a precooled mortar, to which 2 mL grinding buffer  was added, followed by grinding on an ice bath. The preparation was the centrifuged at 4000 × g and 4°C for 20 min, and the supernatant was mixed with extraction buffer  to a constant volume of 5 mL. The resultant supernatant was then treated as follows: (1) 2 mL of supernatant was centrifuged at 15,000 × g and 4°C for 15 min, and then the resulting supernatant was mixed with extraction buffer  to a constant volume of 4 mL to yield the cyt-aconitase (ACO) enzyme solution, next, it was precipitated with the extraction buffer to a constant volume of 2 mL to yield the NAD–isocitrate dehydrogenase (NAD–IDH) enzyme solution. (2) The remaining 3 mL of supernatant was added to 3 mL of extraction buffer for the determination of NAD-malic acid dehydrogenase (NAD-MDH) and NADP-malic enzyme (NADP-ME); 4 mL of this solution was dialyzed against a large volume of extraction buffer overnight at 4°C, and the resulting dialysate was used for the determination of phosphoenolpyruvate carboxylase (PEPC) and citrate synthase (CS).Determination of enzyme activities in roots: PEPC, NAD–MDH, NADP–ME, CS, ACO, and NAD–IDH were extracted and assayed according to Chen and Hirai with some modifications [ications ,51.Reactions were performed in a 3-mL volume, and absorbance was determined immediately after the addition of the reaction substrates by using a Lambda 35 UV-Vis ultraviolet spectrophotometer, with continuous scanning for 3 min and measuring absorbance changes of 0.001 per minute for 1 U of enzyme. Enzyme activity is represented in units per gram fresh weight per minute (U/g FW min). The compositions of the reaction systems used for the analysis of the different enzyme activities are as follows:3: 150 μL; 20 × 2 mM MgCl2: 150 μL; 20 × 0.5 mM GSH: 150 μL; 20 × 0.15 mM NADH: 150 μL; Enzyme liquid: 600 μL; 2 × 2 mM oxaloacetate (initiated reaction): 1500 μL; colorimetric wavelength: 340 nm.NAD-MDH: 20 × 40 mM Tris-HCl (pH 8.2): 300 μL; 20 × 10 mM KHCO4: 150 μL; 20 × 0.17 mM NADP: 150 μL; ultrapure water: 300 μL; enzyme liquid: 600 μL; 2 × 2 mM malate (initiated reaction): 1500 μL; colorimetric wavelength: 340 nm.NADP-ME: 20 × 40 mM Tris-HCl (pH 7.4): 300 μL; 20 × 0.2 mM MnSO4: 150 μL; ultrapure water: 1350 μL; enzyme liquid: 1200 μL; 20 × 2 mM sodium isocitrate (initiated reaction): 300 μL; colorimetric wavelength: 340 nm.NAD-IDH: 20 × 40 mM Hepes (pH 8.2): 150 μL; 20 × 0.8 mM NAD: 150 μL; 20 × 0.2 mM MnSO3: 150 μL; 20 × 2 mM MgCl2: 150 μL; 20 × 0.5 mM GSH: 150 μL; 20 × 0.15 mM NADH: 150 μL; ultrapure water: 150 μL; enzyme liquid: 600 μL; 2 × 1 mM PEP (initiated reaction): 1500 μL; colorimetric wavelength: 340 nm.PEPC: 20 × 40 mM Tris-HCl (pH 8.5): 150 μL; 20 × 10 mM KHCOCS: 20 × 40 mM Tris-HCl (pH 9.0): 150 μL; 20 × 0.04 mM DTNB: 150 μL; 20 × 0.04 mM AcCoA: 150 μL; ultrapure water: 1800 μL; enzyme liquid: 600 μL; 20 × 2 mM oxaloacetate (initiated reaction): 150 μL; colorimetric wavelength: 412 nm.ACO (enzyme liquid and the same volume of 2 mM GSH in a 30°C water bath culture for 1 h before the determination of enzyme activity): 20 × 40 mM Tris-HCl (pH 7.5): 150 μL; 20 × 0.1 mM NaCl: 150 μL; ultrapure water: 1080 μL; enzyme liquid: 1200 μL; 20 × 0.2 mM cis-aconitate (initiated reaction): 420 μL; colorimetric wavelength: 340 nm.Y is the individual growth/physiological observation; μ is the overall mean; Bi is the effect of the ith block (fixed); Alj is the effect of the jth aluminum treatment; Pk is the effect of the kth phosphorus treatment; Cl is the effect of the lth clone (genotypes); Al*Pjk is the effect of the statistical interaction between the jth aluminum treatment and the kth phosphorus treatment; Al*Cjl is the effect of the statistical interaction between the jth aluminum treatment and the lth clone; P*Ckl is the effect of the statistical interaction between the kth phosphorus treatment and the lth clone; Al*P*Cjkl is the effect of the statistical interaction among the jth aluminum treatment; the kth phosphorus treatment and the lth clone; and ε is the residual error.The linear model we used for data analysis is as follows:P ≤ 0.05.All results are expressed as means ± standard deviations. The data were analyzed using analysis of variance (ANOVA) with three factors. When the ANOVA identified differences among groups, multiple comparisons among means were performed using Duncan’s new multiple-range test. The significance level was set at Eucalyptus clones. Furthermore, a significant effect of clone was noted on the root, stem, and leaf DWs. Further, significant interactions were noted between Al and P in terms of the biomasses of root, stem, and leaf.The results of variance analysis  indicateThe results of this study indicated that the application of P increased, whereas the application of Al decreased, the root, stem, and leaf DW of DH32-29 and G9 . RegardlEucalyptus clones. Moreover, a significant interaction between Al and P was noted on the total Al and P contents in the roots, stems, and leaves of the two Eucalyptus clones, except P content in the roots, which did not change significantly.The results of variance analysis  indicateAl stress caused a significant increase in the Al content in seedlings . In the The results of variance analysis  indicateIn the absence of Al stress, the Al-induced secretion of malate from DH32-29 and G9 roots was significantly higher after the application of 50 and 200 μM P than after the application of 0 μM P . In contThe results of variance analysis  indicateIn neither clone, the PEPC activity was significantly affected by P supply without Al stress, whereas it was significantly lower after the application of 50 and 200 μM P than after the application of 0 μM P with Al stress . In bothThe change in the trend of NADP–ME activity was similar to that of PEPC activity. NADP–ME activity did not change significantly regardless of P supply in the absence of Al stress, but decreased significantly with increasing P supply under Al stress .In both the clones, NAD–MDH activity showed no significant change irrespective of P supply in the absence of Al stress . FurtherIn both the clones, CS activity did not change significantly with or without P supply in the absence of Al stress . Under AThe change in IDH activity showed a different trend to that of the aforementioned enzymes ; Al streThe change trend in ACO activity was similar to that of IDH activity. In both the clones, ACO activity was not significantly affected by P supply without Al stress. Furthermore, ACO activity increased significantly with increasing P supply under Al stress . Like foEucalyptus species have different degrees of resistance to Al toxicity. Moreover, differences in tolerance to Al toxicity between the Eucalyptus clones were detected by Yang et al. [Eucalyptus seedlings ; different nutrient solutions (Hoagland nutrient solution with some modifications vs. Yoshida nutrient solution); different cultivation modes (sand culture vs. hydroponic assay); different Al treatment levels (5 mM vs. 200 μM); and different age of plants before exposure to Al (3 months vs. 24 days). Previous studies have shown that plant resistance to Al toxicity might be mediated by not only a decrease in Al uptake but also a relatively lower proportion of Al being distributed from roots to shoots and leaves [Eucalyptus seedlings with increasing P supply under Al stress, resulting in decreased transport of Al to stems and leaves. This could be a strategy that enables plants to tolerate Al toxicity. Several studies in other species have revealed similar results [Eucalyptus following the application of P: (1) accumulation of Al by P present in the roots and restriction of Al transport to the stems and leaves; and (2) an increase in P concentration in the roots, stems, and leaves that alleviates elemental deficiency.In our study, the P concentration in both l stress . Conversfication ,54. The d leaves ,56. In od leaves . In this results ,45. InteEucalyptus clones. Studies such as those conducted by Gaume et al. [NAC transcription factor genes might be regulated by Al [NAC domain proteins in the E. grandis genome, one of the largest NAC domain families known [NAC gene family members in the two clones of E. grandi.The Al-induced exudation of organic acids from plant roots has been proposed to be an important mechanism for Al tolerance . The rese et al. , Liang [e et al.  and Sun e et al.  have obte et al. . We alsoed by Al . Hussey es known ,60. FurtEucalyptus seedlings .(DOCX)Click here for additional data file.S2 TableNote: The abbreviations RAL, SAL, and LAL and RP, SP, and LP represent aluminum content in root, stem, and leaf and phosphorus content in root, stem, and leaf, respectively. Differences between the two Al levels were analyzed by ANOVA. Different letters in each row indicate significant differences .(DOCX)Click here for additional data file.S3 TableNote: Differences between the two clones were analyzed by ANOVA. Different letters in each row indicate significant differences .(DOCX)Click here for additional data file.S4 TableNote: The abbreviations MA, OX, and CI represent Al-induced secretion of malate, oxalate, and citrate from roots, respectively. Differences between the two Al levels were analyzed by ANOVA. Different letters in each row indicate significant differences .(DOCX)Click here for additional data file.S5 TableNote: Differences between the two clones were analyzed by ANOVA. Different letters in each row indicate significant differences .(DOCX)Click here for additional data file.S6 TableNote: The abbreviations PE, ME, MD, CS, ID, and AC represent the activities of PEPC, NADP-ME, NAD-MDH, CS, NAD-IDH, and Cyt-ACO in roots, respectively. Differences between the two Al levels were analyzed by ANOVA. Different letters in each row indicate significant differences .(DOCX)Click here for additional data file.S7 TableNote: Differences between the two clones were analyzed by ANOVA. Different letters in each row indicate significant differences .(DOCX)Click here for additional data file.


Tobacco mosaic virus (TMV) causes severe plant diseases worldwide; however, effective antiviral agents for controlling TMV infections are not available. This lack of effective antiviral agents is mainly due to the poor understanding of potential targets associated with TMV infections. During infection, the coat protein (CP), which is delivered by viral particles into susceptible host cells, provides protection for viral RNA. Here, we found that Ningnanmycin (NNM), a commercially used plant antibacterial agent, inhibits the assembly of the CP by directly binding several residues. These interactions cause the disassembly of the CP from discs into monomers, leading to an almost complete loss of pathogenicity. Substitutions in the involved binding residues resulted in mutants that were significantly less sensitive to NNM. Thus, targeting the binding of viral CPs through small molecular agents offers an effective strategy to study the mechanism of NNM. Plant viral diseases cause significant losses in agriculture and low-cost agents capable of effectively controlling such diseases are in great need. Antiviral compounds that target RNA and chemicals that aim at stimulating plant disease resistance have received intensive attention in recent years , 2. Howein vitro through physical regulation  and 10 mmol/L β-mercaptoethanol). The cells were thawed, lysed using a supersonic device, and centrifuged at 13,400 ×g for 30 min at 4°C. After passing through a 0.22-mm syringe filter , the supernatant was loaded onto a Ni2+ Sepharose High-Performance Column , washed with five column volumes of 40 mmol/L imidazole and eluted with 400 mmol/L imidazole. The elute was concentrated using an Amicon Ultra centrifugal filter device (Millipore) with a 10 kDa MW filter and then loaded onto a HiLoad 16/60 Superdex 200 pg column equilibrated with a dialysis solution (10 mM sodium phosphate buffer and 100 mM sodium chloride solution [pH 7.2]). After thrombin digestion of 6×His tags at 4°C overnight, TMV CP was further purified by SEC using a Superdex 200 10/300 GL column  and a buffer containing 10 mM sodium phosphate and 100 mM sodium chloride solution (pH 7.2). Proteins were then concentrated to 8.7 mg/mL for biochemistry experiments using amicon ultra centrifugal filter units (Millipore) with a 10 kDa MW cutoff. The target proteins were briefly stored at 4°C. TMV (common strain) was isolated from infected tobacco leaves (grown in the greenhouse at Guizhou University) and purified [To prepare TMV CP discs, TMV was purified using the method described by Gooding and modified by Shire , 26. TMVpurified , 26. The2+ beads and the tags were cleaved using thrombin. TMV CP was examined for the formation of oligomers with SEC methods. Discs were formed by incubating the oligomers in 10 mM sodium phosphate buffer containing 100 mM sodium chloride (pH 7.2) at 22°C for more than 12 h [The recombinant proteins with 6×His tags were purified using Niβ-lactoglobulin (35 kDa), ribonuclease A (13.7 kDa), cytochrome (13.6 kDa), aprotinin (6.51 kDa) and vitamin B12 (1.36 kDa). The protein was monitored by measuring the absorbance at a wavelength of 280 nm. For TEM [2O. The grid was stained with 20 μL of 2% aqueous solution of tungstophosphoric acid for 90 s as a negative stain. Images were obtained at the Electron Microscope Lab of Zunyi Medical University using a Hitachi H-7650 transmission electron microscope  with 80 kV accelerating voltage. The 17% native PAGE [−1 (ΔH), binding constant in mol−1 (K) and the number of molecules per TMV CP proteins (n) were floating during the fit. The binding free energy, ΔG, and reaction entropy, ΔS, were calculated using the equations, ΔG = −RTlnK  and ΔG = ΔH−TΔS. The Kd was calculated as 1/K. Then, the binding was calculated for MST [N. tabacum cv. K326 were mechanically inoculated with equal volumes of TMV (0.5 mg/mL). After 72 h, 1-cm diameter leaf discs were removed. The leaf discs were floated on solutions of NNM and on buffer  as a negative control. Discs of healthy leaves were floated on buffer as a mock. All of the leaf discs were kept in a culture chamber at 28°C for 48 h, and then, the TMV concentration in the leaf disc was determined. Leaf discs were ground in 5 × protein loading buffer , and then, 5 μL of sample were loaded on a polyacrylamide gel (5% stacking gel and 12% separating gel). After SDS-PAGE, TMV protein bands were transferred at 90 mA for 1 h onto a polyvinylidene fluoride membrane  using an electrotransfer system (Bio-Rad). The membrane was washed in TBST  and blocked with 5% nonfat milk powder in TBST for 1 h at 37°C. The membrane was washed three times, each time for 3 min with TBST, and reacted with a mixture of 1:30,000 alkaline phosphadase-conjugated anti-rabbit IgG  and 1:200 polyclonal antibody of TMV for 2 h at 37°C. After it was washed three times, each time for 3 min with TBST, the membrane was incubated in substrate buffer  with 330 μL/mL nitrotetrazolium blue chloride and 165 μL/mL 5-bromo-4-chloro-3-indolyl phosphate for 3–5 min in the dark until the bands of the CP were clear. For the MD simulation, the X-ray crystal structure of TMV CP was downloaded from (Protein Data Bank) PDB (PDB ID: 4GQH). The initial structure was revised by adding lost residues and hydrogen atoms and checking bonds and bumps. Subsequently, the energy was minimized for 2,000 steps of the steepest descent calculations and 2,000 steps of conjugated gradient calculations by using Sybyl 7.0  and Gaussian03 program at the HF/6-31+G* level [NNM and TMV CP binding was initiated by adding 5 mM anti-TMV compounds to 0.5 mM (8.7 mg/mL) TMV CP discs and incubating for 1 h. Then, 0–10 mM NNM was added to the reactions for 1 h. The oligomer status of TMV was then analyzed by SEC . SEC washrome 13. kDa, apr For TEM , 23, selive PAGE -31 was pive PAGE  were recive PAGE  were per for MST , 35 Monoin B12 1. kDa. TheG* level . The atoG* level . During −1Å−2. Water and counterions were minimized. The backbone atoms of the protein were then fixed with the ligand, side chains, and other atoms that are free to move. Finally, the entire system was fully minimized without any constraint. In each step, energy minimization was first performed using the steepest descent algorithm for 2,000 steps, and subsequently, the conjugated gradient algorithm was used for another 3,000 steps. The MD simulation was performed under periodic boundary conditions using the sander module of the Amber9 program. First, the system was fixed to heat only the water and counterions for 10 ps to make sure the solute was fully solvated. Then, the entire system was gradually heated from 10 K to 300 K using the weak-coupling method and equilibrated for 100 ps with the protein backbone fixed [Based on the docking results, two binding models were selected for MD simulation. Prior to the simulation, the electrostatic potential and partial atomic charges were determined by performing an electrostatic potential fitting according to the Merz–Singh–Kollman scheme with the Gaussian-optimized geometries , 41. Thene fixed . Lastly,ne fixed , 46, whine fixed . The timTo verify the results of the MD simulation, four mutations , S15G, SNicotiana glutinosa (N. glutinosa) were mechanically inoculated with the reconstructed full-length virus or the reconstructed mutated virus (see the TEM methods). The local lesion numbers were recorded 3–4 days after inoculation.Leaves of


PDGFRα on IMF deposition are still unclear. Here, PDGFRα is moderately expressed in porcine longissimus dorsi muscle (LD), whereas it highly expressed in white adipose tissue (WAT). Moreover, PDGFRα-positive cells were located in the gaps of LD fibers which there were IMF adipocytes. Compared with 180-day-old and lean-type pigs, the levels of PDGFRα were much higher in one-day-old and fat-type pigs. Meanwhile the levels of PDGFRα gradually decreased during IMF preadipocyte differentiation. Furthermore, PDGFRα promoted adipogenic differentiation through activating Erk signaling pathway. Based on PDGFRα upstream regulation analysis, we found that the knockdown of FoxO1 repressed lipogenesis by downregulating PDGFRα, and miR-34a inhibited adipogenesis through targeting PDGFRα. Collectively, PDGFRα is a positive regulator of IMF deposition. Therefore, we suggest that PDGFRα is a possible target to improve meat quality.Suitable intramuscular fat (IMF) content improves porcine meat quality. The vital genes regulating IMF deposition are necessary for the selection and breeding of an IMF trait. However, the effect and mechanism of  The content of intramuscular fat (IMF) is considered as a crucial indicator of porcine meat quality . IncreasPDGFRα) is important for embryonic organogenesis and development by effecting the differentiation and function of specialized mesenchymal cells [PDGFRα positive cells are cardiac progenitors, which contribute predominantly to the mesenchymal compartments and vascular [PDGFRα expressed in adipogenic stromal cells of adipose tissues, it has been implicated in the adipocyte lineage [PDGFRα and may contribute to fat deposition in skeletal muscle [PDGFRα may be a potential cell surface biomarker of IMF preadipocyte progenitors. However, the effect and regulatory mechanism of PDGFRα on IMF deposition is still unclear.Platelet-derived growth factor receptor α  are non-coding RNAs, approximately 18–22 nucleotides in length, which regulate gene expression at the post-transcriptional level [PDGFRα/β ; miR-34a [PDGFRα . PDGFRα or cells . TherefoFoxO transcription factors have a wide range of biological functions, including the regulation of cell proliferation, apoptosis, and differentiation [FoxO family members are widely expressed in the mammalian organizations. FoxO proteins are responsible for maintaining the basal expression levels of PDGFRα, as well as the accumulation of PDGFRα under serum starvation condition [FoxO proteins in neuroblastoma cells results in a disregulated differentiation phenotype, which can be rescued by PDGFRα expression. PDGFRα is a critical downstream target gene of the FoxO1 proteins [PDGFRα and FoxO1 needs further clarification in porcine IMF deposition.The ntiation . FoxO faondition . Depletiproteins . HoweverPDGFRα on pig IMF deposition. Therefore, we suggested that PDGFRα could serve as a positive regulator associated with meat quality for pig selection and breeding of IMF trait.In this study, we elucidated the effect and regulatory mechanism of PDGFRα in pigs, the levels of its mRNA and protein were detected in various tissues including heart, liver, spleen, kidney, LD and WAT. The results showed that the levels of PDGFRα, both mRNA and protein, were abundant in WAT, moderate in LD and the least in the liver .Fat-type breed Bamei pigs (180 days old) were obtained from Huzhu prefecture of Qinghai province. Lean-type breed Yorkshire adult pigs (180 days old) and piglets (one day old) were provided by the experimental farm of Northwest A&F University. All pigs were killed at a slaughterhouse under the guidelines of Northwest A&F University Animal Care Committee. The heart, liver, spleen, kidney, LD and SAT were dissected, and rinsed with PBS. Samples for western blot and real-time qPCR were frozen in liquid nitrogen until analysis. For the frozen section, the samples were fixed with 4% paraformaldehyde and kept at room temperature until use.5 cells per 35-mm culture dish in DMEM/F12 medium with 10% fetal bovine serum  supplement with penicillin (100 U/mL) and streptomycin (100 U/mL). After 1 h, cells were rinsed with DMEM/F12 medium to wipe off unadhered cells. At day 2, and after cells reach to 100% confluence, cells were cultured with DMEM/F12, supplemented with 5 μg/mL (872 nM) insulin, 1 μM dexamethasone and 0.5 mM isobutyl methylxanthine , for 2 days to induce differentiate. Then the medium was changed by DMEM/F12 with 10% FBS and 5 μg/mL insulin for another 4 days to maintain differentiation. At day 0, 3 and 6, cells were harvested for further analysis.The LD of piglets were separated sterilely, visible connective tissue was removed, then finely minced. Preadipocytes were isolated based on previously studies. In brief, muscle tissues were digested in a digestion buffer consisting of 0.2% collagenase I  and medium/F12 (DMEM/F12)  in a shaking water bath for 1.5 h at 37 °C. The digest sample was filtered aseptically through 70 and 200 µm nylon mesh filters to isolate cells. Then the filtered cells were washed three times with DMEM/F12 by centrifugation at 1500 rpm for 5 min. Cells were seeded at a density of 2.5 × 10To detect the expression of miRNA and genes associated with adipogenic differentiation, the total RNA was extracted with Trizol reagent . The concentration of total RNA was measured by the NanoDrop 2000 . Then we used reverse transcription kits  to synthesize cDNA. For miRNA analysis, specific reverse transcription primers and procedures were used, whereas the normal process was performed for mRNA analysis. In real-time quantitative PCR, every reaction performed in triplicate using SYBR green kits on a Bio-Rad iQTM5 system. The expressions of all genes were normalized to GAPDH, but U6 small RNA was internal reference when examined the level of miR-34a. The primer sequences used for qPCR were shown in PDGFRα , FABP4 , β-actin , β-tubulin . The secondary antibodies were anti-rabbit, anti-goat and anti-mouse antibodies . The targeted proteins were detected using the Gel Doc XR System  as the instructions of the manufacturer.The various tissue samples of the pigs and Cells were split by radio immunoprecipitation assay (RIPA) buffer  add with protease inhibitor . The total protein sample was pointed into and separated in the SDS-polyacrylamide gel. Then transferred it into a PVDF membrane . Nest, the membrane was blocked in 5% defatted milk for 2 h. After that, the membrane was incubated with primary antibodies at 4 °C overnight followed by a secondary antibody at room temperature for 1.5 h. Protein bands were exposure by chemiluminescence reagents  and quantified using the Image Lab Image Document. Following primary antibodies were used: Fixed tissues were dehydrated in 30% sucrose and sectioned (10 µm) through sliding microtome . Hematoxylin-eosin staining was realized as previously reported . Then, the sections were observed and took pictures with microscope  in 100× magnification.2O2 for 10 min to eliminate endogenous oxidase activities and washed three times with PBS. Then, sections were treated with 0.5% TritonX-100/TBS at room temperature for 10 min, and washed three times using distilled water. Antigens were unmasked by high-temperature antigen retrieval (10 min boiling in 0.05 mM Tris/EDTA buffer (pH = 9) and cooling slowly at room temperature. They were then washed three times with TBS. The 5% BSA/TBS as a blocking solution was incubated for 2 h at room temperature. Sections were incubated with primary antibodies of PDGFRα (1:100), and diluted in blocking solution at 4 °C overnight. They were then washed three times with TBST and incubated with anti-rabbit red fluorescent secondary antibody (1:1000)  for 1 h. Then sections were washed with TBST three times for 5 min each. For nuclear visualization, DAPI  was incubated 10 min, then the section was rinsed with TBS. After treatment, the sections were observed under a fluorescence microscope .Frozen sections were treated with 3% HAfter being fixed in 4% paraformaldehyde solution, incubated with 0.5% Oil Red O for 30 min, and washed three times with PBS, the myoblast cells were visualized by phase-contrast microscopy . Oil Red O dissolved in lipid droplets was extracted with 100% isopropanol and its relative concentrations were determined by measuring the absorbance at 510 nm.Frozen sections were treated with 0.5% TritonX-100/TBS at room temperature for 10 min and washed three times with TBS. They were then stained with BODITY  for 10 min; the sections were washed with TBST three times for 5 min each. For nuclear visualization, DAPI  was incubated for 10 min, then the section was rinsed with TBS. After treatment, the sections were observed under fluorescence microscope .https://rnaidesigner.invitrogen.com/rnaiexpress/). PDGFRα and FoxO1 shRNA vectors were constructed. They were annealed and inserted into pLentiHI plasmids at BamHI and XhoI sites, and confirmed by sequencing.Vectors were constructed with lentiviral plasmid pLentiHI  and the inserted shRNAs were designed by online Invitrogen RNAi Designer , combined with 6 μg Δ8.9 packaging plasmid and 9 μg VSVG envelope protein plasmid, were cotransfected into HEK293T packaging cells (2 × 105 cells per well) with the calcium phosphate method. Forty-eight hours after transfection, the supernatant containing virus particles was collected and passed through a 0.45 μm filter to remove cellular debris. When the cells reached 70–80% confluence, the viral suspension of pLentiHI-PDGFRα shRNAs, pLentiHI-FoxO1 shRNAs or scrambled shRNA was added respectively.The pLentiHI-Preadipocytes were seeded in 12-well or 6-well plates, and miR-34a mimics or negative control (NC)  were transfected into cells at 80% density in 50 nM using X-tremeGENE siRNA Transfection Reagent (Roche) and Opti-MEM (Gibco) culture medium according to the manufacturer’s protocol and the culture medium was changed to fresh medium after 24 h. When the cells grew to confluence after transfection, adipogenic differentiation was initiated by switching to differentiation medium.p < 0.05 was considered to be significant.Statistical analyses were performed using SPSS18.0 software . Data represented mean ± SEM. Multiple comparisons were performed by one-way ANOVA followed by Dunnett’s tests. PDGFRα-positive cells were porcine IMF adipocyte and PDGFRα contributed to IMF deposition. Mechanically, PDGFRα regulated by miR-34a and FoxO1 promotes adipogenensis in porcine intramuscular preadipocytes through activating Erk signaling pathway.Taken together, our findings indicated that


Aedes aegypti is the primary urban mosquito vector of viruses causing dengue, Zika and chikungunya fevers –for which vaccines and effective pharmaceuticals are still lacking. Current strategies to suppress arbovirus outbreaks include removal of larval-breeding sites and insecticide treatment of larval and adult populations. Insecticidal control of Ae. aegypti is challenging, due to a recent rapid global increase in knockdown-resistance (kdr) to pyrethroid insecticides. Widespread, heavy use of pyrethroid space-sprays has created an immense selection pressure for kdr, which is primarily under the control of the voltage-gated sodium channel gene (vgsc). To date, eleven replacements in vgsc have been discovered, published and shown to be associated with pyrethroid resistance to varying degrees. In Mexico, F1,534C and V1,016I have co-evolved in the last 16 years across Ae. aegypti populations. Recently, a novel replacement V410L was identified in Brazil and its effect on vgsc was confirmed by electrophysiology. Herein, we screened V410L in 25 Ae. aegypti historical collections from Mexico, the first heterozygote appeared in 2002 and frequencies have increased in the last 16 years alongside V1,016I and F1,534C. Knowledge of the specific vgsc replacements and their interaction to confer resistance is essential to predict and to develop strategies for resistance management. Aedes aegypti, the principal vector of dengue, chikungunya, Zika and yellow fever viruses. The lack of vaccines for most of these arboviruses results in a tremendous reliance on chemical control. Unfortunately, intense application of pyrethroids has resulted in pyrethroid resistance in Ae. aegypti populations worldwide5. A major mechanism underlying pyrethroid resistance is known as knockdown resistance (kdr), which is caused by mutations in the voltage-gated sodium channel (vgsc)6. Knowledge of the specific vgsc mutations that confer resistance is essential to predict the rise of pyrethroid resistance and to develop strategies for resistance management.Pyrethroids are the most common class of insecticides used to suppress adult populations of vgsc mutations have been identified in Ae. aegypti and in most cases, have been linked to conferring some degree of pyrethroid resistance. These include G923V, L982W, I1,011 M and V1,016 G first identified in 20037, I1,011 V and V1,016I in 20078, D1,763Y in 20099, S989P and F1,534C in 201011, T1,520I in 201512 and V410L in 201713. These kdr mutations are usually confined to specific geographic areas and co-occurrence of certain mutations is a common phenomenon sometimes associated with higher levels of phenotypic resistance14.Globally, eleven vgsc sensitivity to pyrethroids, including S989P (IIL5–6), I1,011 M, V1,016 G (IIS6), F1,534C (IIIS6)14 and most recently V410L (IS6)13. Computer modeling predicts that pyrethroids bind to two homologous lipid exposed interfaces between domains: one binding site (PYR-1) is formed by the linker helix connecting S4 and S5 in domain II (IIL45) and domains IIS5, IIS6, and IIIS6, and the second binding site (PYR-2) is formed by the linker helix connecting S4 and S5 in domain I (IL45) and domains IS5, IS6, and IIS617.To date, only five mutations have been functionally confirmed to reduce Ae. aegypti populations carry at least two vgsc mutations, V1,016I which is linked to permethrin survival8 and F1,534C. Interestingly, F1,534C reduces permethrin binding to vgsc channels whereas V1,016I has no effect in pyrethroid binding14. However, both replacements have co-evolved in Mexican populations; V1,016I and F1,534C allele frequencies have increased in the last 16 years18 and F1,534C has reached fixation in several locations in Southern Mexico19. The co-occurrence of V1,016I and F1,534C has been reported in several countries in Latin America, including Brazil21, Venezuela22, Colombia23, Martinique Island24, Puerto Rico25, Grand Cayman Islands4, Cuba26, Haiti27 and Jamaica28.In Mexico, pyrethroid-resistant Ae. aegypti from Brazil13. Alone or in combination with F1,534C, V410L reduced the sensitivity of mosquito sodium channels expressed in Xenopus oocytes to both type I (eg. permethrin) and II pyrethroids (eg. deltamethrin). Interestingly, authors did not detect this mutation in field populations from Pernambuco, Brazil, concluding the novel mutation may not yet be widespread in nature. In contrast, we identified high frequencies of V410L alongside V1,016I and F1,534C in a genome-wide association study of Ae. aegypti from Mexico. To extend this observation, we screened the frequencies of V410L in different temporal and geographical Ae. aegypti collections made over the last 16 years in Mexico. We compared the evolution and linkage disequilibrium of V410L alongside the V1,016I and F1,534C replacements, which occur in different domains IS6, IIS6 and IIIS6, respectively . Figure 19, L410 also increased in frequency from 2000 to 2016, noting that at each of these time points L410 and I1,016 alleles changed frequencies in parallel from 0.00 to 0.71 , χ2 and associated probabilities obtained between pairwise loci. Fourteen out of 25 collections had alleles segregating at loci 410–1,016 with Rij values ranging between 0.53–0.99 among collections; overall Rij was 0.96 (p = 0.0001). For loci 410–1,534, only five collections had mutant alleles segregating, and four were in significant linkage disequilibrium with Rij ranging from 0.33 to 0.99; the overall Rij coefficient was 0.76 (p = 0.0001). At loci 1,016–1,534, segregating alleles from six collections were in significant linkage disequilibrium, with Rij coefficients ranging from 0.31 to 0.99; overall Rij coefficient was 0.76 (p = 0.0001).Pairwise linkage disequilibrium analyses were performed between locus 410-1,016, 410–1,534 and 1,016–1,534. Table 410/VV1,016/FF1,534) occurred at a high frequency (0.99) whereas a genotype including a heterozygote at loci 1,534  had a frequency lower than 0.01  and the 1,534 heterozygotes decreased to frequencies lower than 0.06. Twelve additional combinations occurred, the four most frequent genotypes were VV410/VV1,016/CC1,534 (frequency = 0.2), VL410/VI1,016/FC1,534 (0.18), VL410/VI1,016/CC1,534 (frequency = 0.26) and the triple resistant homozygote LL410/II1,016/CC1,534 (frequency = 0.18)  . Also, genotypes including heterozygotes at locus 410 and homozygotes at locus1,016 and vice versa  were observed at 10-fold lower frequencies  than expected (Supplementary Table Out of 27 genotype combinations (3 genotypes at 3 loci), we found 20 tri-locus genotype combinations in 1,176 individual mosquitoes collected from 2000 to 2016. Figure .01 Fig. . By 2002.01 Fig. . By 200618) Fig. . Observe18) Fig. . By 201234) Fig. . Observe410, heterozygote was VL410 and resistant was LL410. Table 410) were knockdown resistant, 40% recovered and only 7% died. In the heterozygote group (VL410) 4% was knockdown resistant, 28% recovered and 64% died following permethrin exposure. For deltamethrin, 72% of the resistant homozygotes (LL410) were knockdown resistant whereas 23% recovered and only 5% died. Note that the phenotype outcome is very similar between genotypes at loci 410 and 1,016. The most striking difference at locus 1,534, is that more than 92% of the heterozygotes died after exposure to permethrin or deltamethrin. Across all analyses, strong correlations were detected between phenotype and genotype.L410 was present at a frequency of 0.69 in Viva Caucel mosquitoes used for our phenotype/genotype association study. We used a dose of permethrin or deltamethrin to discriminate three phenotypes: knockdown resistant, recovered and dead. The susceptible genotype at locus 410 was VV1,534 or CC1,534 were associated with the dead phenotype. The double heterozygotes at locus 410 and 1,016 in presence of FF1,534 or FC1,534 were also associated with the dead phenotype for both pyrethroids. The double heterozygote at locus 410 and 1,016 with CC1,534  was associated with the dead phenotype for permethrin exposure but was associated with knockdown resistance and recovery in the deltamethrin exposure group.Because our results indicated that L410, I1,016 and C1,534 do not occur independently, we analyzed the phenotype outcome by tri-locus genotype combinations. In the Viva Caucel population, 13 tri-locus genotype combinations were identified. Figure vgsc of several pyrethroid resistant insect species. Specifically, V410L was associated with deltamethrin resistance in the common bed bug Cimex lectularis29. However, replacements V410M in the tobacco budworm30 and V410A, V410G and V410M in the earworm31 have also been reported. In Ae. aegypti, V410L was recently detected in a pyrethroid resistant insectary strain from Brazil13, which demonstrated that V410L alone or in combination with the F1,534C reduced the sensitivity of mosquito sodium channels expressed in Xenopus oocytes to both type I and II pyrethroids. In the same study, V410L was not detected in a small field survey in the State of Pernambuco, Brazil, and authors suggested that V410L was not yet widespread in the field. Importantly, our results show that V410L has existed for at least 16 years in Mexico, the first heterozygote was detected in 2002 in Coatzacoalcos, Veracruz, and has increased gradually to high frequencies in 2016 (up to 0.92). Interestingly, we found L410 to be in greater linkage disequilibrium with I1,016 than with C1,534. Our previous study measured linkage disequilibrium between I1,016 and C1,534 and we proposed a sequential model, wherein C1,534 first occurred (providing low levels of resistance) and then the replacement I1,016 occurred with this haplotype, providing even higher levels of resistance. V410L challenges this sequential model, in which both V410L and V1,016I might have occurred independently on a C1,534 haplotype and then became cis to C1,534 by recombination. An alternative model assumes the three mutations arose independently at very low frequencies, and then by two recombination events, came to occur in a cis arrangement. Our results indicate that C1,534 was at a frequency of 0.004 in collections from 2000 whereas L410 and I1,016 were below limits of detection. By 2002–2005, C1,534 was at higher frequency (0.055), while L410 and I1,016 appeared at lower frequencies . During this period, we identified heterozygotes at 410 and 1,016 occurring independently in a FF1,534 background. Also, the triple heterozygote was identified in low frequencies (5/346). By 2006–2008 heterozygotes VL410 and VI1,016 on a FC1,534 heterozygote or CC1,534 homozygote were favored by selection, whilst mutant genotypes at 410 and 1,016 never occurred independently or were at very low frequencies.Different replacements at residue V410 have been reported in the 13. In contrast, we show large linkage disequilibrium between L410 and I1,016, except among very few individuals collected early in 2002–2005; however, this genotype combination was no longer detected in following years. Whether L410 remains independent of I1,016 in Brazil will provide evidence of the mutations arising independently at a local level in the sequential model. However, I1,016 and C1,534 are already widespread in several regions of Brazil, and due to high migration rates among Ae. aegypti populations, we would expect I1,016 and C1,534 to recombine with L410-C1,534 in future years. However, an alternative scenario is that as in Mexico, L410 is already present at high frequencies in Brazilian collections previously genotyped with I1,016-C1,534 but simply has not yet been detected.A recent study found that V410L and F1,534C occurred without V1,016I in a deltamethrin resistant laboratory strain originated from Rio de Janeiroet al. 201613. L410 alone or in combination with C1,534 confers high levels of resistance, however, it remains to be seen if it is fit in the field. We found 4 out of 1,176 individuals with L410 and C1,534 occurring independently , and this genotype became extinct in Mexican populations. In our phenotype and genotype studies, the triple homozygote resistant individuals had better survival (knockdown resistance and recovery) following either permethrin or deltamethrin exposure. One particular genotype, VL410/VI1016/CC1,534 had different outcome depending of the specific pyrethroid, with this genotype associated with dead in mosquitoes following permethrin exposure. In contrast, this genotype was mostly associated with survival (knockdown and recovery) in mosquitoes exposed to deltamethrin. Apparently, the presence of heterozygotes at loci 410 and 1,016 was sufficient for deltamethrin survival.The selection of the triple homozygote resistant genotypes detected in our data suggests higher fitness of this genotype in the presence of pyrethroids. The role of L410 and C1,534 in conferring pyrethroid resistance was determined in Haddi Ae. aegypti from Asia reduces vgsc whilst the V1,016I replacement found in the Americas does not16. In contrast, V410L is located in DIS6 but does not form part of the PYR-2 receptor site. It has been suggested that the reduction of sodium channel sensitivity to permethrin and deltamethrin by V410L might result from changes in the gating properties of vgsc without inhibiting molecule docking13. Because pyrethroids prefer to bind to sodium channels in the open state, kdr mutations that deter the open state would counteract the pyrethroid effects13. In recent structural models, pyrethroids make multiple contacts with helices IIL45, IIS5, IIS6, and IIIS6, as well as IL45, IS5, IS6, and IIS6 that would maintain vgsc in an open state32. Simultaneous binding of pyrethroids to both PYR-1 and PYR-2 is thought to effectively prolong the opening of vgsc14. It is possible that co-occurrence of V410L and V1,016I, although in different receptor sites, provide fitness advantages in the presence of pyrethroids, thus favoring co-selection. The interaction of both mutations in electrophysiology experiments will address if the co-occurrence of these mutations is compensatory or synergistic in the presence of pyrethroids.F1,534C is located in the PYR-1 receptor site and is responsible for reducing vgsc sensitivity to permethrin. Although residue 1,016 is located in the PYR-1 receptor site, only the V1,016G replacement occurring in vgsc in Ae. aegypti are shown in Supplementary Figure 20, 1.1 µl of each primer (at 50 pmol/µl) and 1 µl genomic DNA (~25 ng). PCR conditions were 3 min at 94 °C, followed by 35 cycles of 30 sec at 94 °C, 30 sec at 60 °C and 1 min at 72 °C and a final extension of 5 min at 72 °C. Products were purified using the MinElute PCR purification columns  and sequenced using the primers 410_ex10fw 5′-TACGATCAGCTGGACCGTGG and 410rev targeting a fragment of 174 bp in exon 10. Sequences were analyzed using the Geneious software .Primer positions at exon 9 and 10 of GTA = Val) and T (TTA = Leu) alleles at locus 410, we designed an allele-specific PCR system to detect individual genotypes by melting curve analysis8. Each reaction contained 50 µM of each forward primer V410fw (5′-GCGGGCAGGGCGGCGGGGGCGGGGCCATCTTCTTGGGTTCGTTCTACCGTG-3′), and L410fw (5′-GCGGGCATCTTCTTGGGTTCGTTCTACCATT-3′) and 100 µM of reverse primer 410rev (5′-TTCTTCCTCGGCGGCCTCTT-3′), 10 µl Sybr Green Master , 9.7 µl ddH20 and 1 µl of genomic DNA (~25 ng). PCR and melting curve analysis was run in a CFX-96 (BioRad) following 3 min at 95 °C, 39 cycles of 10 sec at 95 °C, 10 sec at 60 °C, 30 sec at 72 °C followed by a melting curve from 65 °C to 95 °C with increments of 0.2 °C during 10 sec. The products consisted of  a single 133 bp amplicon for a VV410 homozygote, a single 113 bp amplicon for a LL410 homozygote (resistant) and the presence of both amplicons in heterozygote individuals (VL410)  around these frequencies were calculated in WINBugs2.0 following 1,000,000 iterations. Departure from Hardy-Weinberg expectations were expressed as Wright’s inbreeding coefficient (FIS) and a χ2 test was used to test the hypothesis FIS = 0 (d.f. = 1). Pairwise linkage disequilibrium between alleles at loci 410 and 1,016 or between loci 410 and 1,534 was calculated using LINKDIS following Vera-Maloof et al.19. Composite disequilibrium between resistant alleles was tested and a χ2 test determined if significant disequilibrium existed among alleles at both loci.V410L allele frequencies were calculated from genotypic frequencies following Garcia 50) in 3–4 days old adults of the F3 generation. The insecticide treatment consisted of a 1 h exposure in an insecticide coated bottle, transfer of mosquitoes to recovery chambers and mortality scored at 24 h after treatment. We assessed the levels of permethrin and deltamethrin resistance in Viva Caucel relative to the New Orleans (NO) reference strain. The permethrin LC50 was 47.9-fold higher than NO  and the deltamethrin LC50 was 47.6-fold higher than NO (10.49 µg vs 0.22 µg).To determine phenotype/genotype associations, we used the Viva Caucel  collection from Yucatan, Mexico made in 2011. First, we calculated the permethrin and deltamethrin (Chem Service) lethal concentration that killed 50% of the mosquitoes  and inactive mosquitoes were transferred to separate containers. To ensure correct categorization, we phenotyped mosquitoes 4 h after treatment. We observed activity and if the mosquitoes were capable of flight, they were scored as ‘knockdown resistant’. In the inactive group we separated the newly recovered mosquitoes from the inactive mosquitoes and scored them as ‘recovered’ and ‘dead’, respectively. Supplementary Table Once the LC33 and resuspended in 150 µl of TE buffer . For the Viva Caucel mosquitoes exposed to permethrin we randomly selected 95 knockdown-resistant, 95 recovered and 95 dead mosquitoes. For deltamethrin we randomly selected 111 knockdown-resistant, 67 recovered and 92 dead mosquitoes. We conducted genotyping at locus 410 using the V410L melting curve system described above. For V1,016I and F1,534C genotypes, we used previously described methodologies11. A contingency table was used to test for association between the phenotypes  and genotypes  at each locus separately  and for the 27 (3 × 3 × 3) tri-locus genotype combinations.A subsample of mosquitoes from each group was individually frozen; DNA was isolated by the salt extraction methodThe findings and conclusions in this paper are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.Supplementary files


ABCB1 levels did not differ significantly throughout individual livers and showed 4.4-fold protein variation between subjects. Neither mRNA nor protein levels varied with sex, ethnicity, obesity or triglycerides in lysates or S9 (that showed the same relationships), but protein levels were lower in pediatric S9 (p < 0.0001), with 76% of adult ABCB1 present at birth and predicted to mature in 5 years. Pediatric total liver lysates were not available. In summary, opportunistic collection for studying human hepatic ABCB1 is acceptable. Additionally, ABCB1 may be lower in children, indicating differential potential for toxicity and response to therapy in this special population.The ATP Binding Cassette B1 (ABCB1) transporter has critical roles in endo- and xenobiotic efficacy and toxicity. To understand population variability in hepatic transport we determined ABCB1 mRNA and protein levels in total liver lysates sampled from 8 pre-defined sites ( Interest in active transporters, such as the ATP Binding Cassette (ABC) proteins, has peaked in recent years because they play crucial roles in drug, chemical, hormone, and nutrient disposition . ClinicaOne of the important ABC transporters is ABCB1 . The ABCB1 protein is present on the plasma membrane of almost all tissues of the human body  unless otherwise stated.n = 24). n = 87). Lysates were prepared from approximately 5 g of frozen tissue. Tissues were thawed and weighed and homogenized 1:3 weight:volume in 0.1 M Tris-HCl containing 5 mM MgCl2 and 2 mM freshly prepared Phenylmethylsulfonyl fluoride. Tissues were homogenized to total lysate in 50 mL falcon tubes using a Tissue Tearor electrical homogenizer . Total lysates were aliquoted and frozen at −80 until use.Liver S9 and lysate samples were released from the Hawaii Biorepository, with approval from the Institutional Review Board for Human Ethics at the University of Hawaii CHS15844 and the Review Ethics Board at the University of British Columbia H14-00092. The cohort was supplemented with samples purchased commercially from Cellzdirect , Puracyp , and Xenotech . Total RNA (up to 5 µg) was extracted from human liver S9 (100 µL at 10–30 mg protein/mL). RNA (up to 6 µg) was also extracted from human liver lysates (50 µL at 4–50 mg protein/mL) using the RNeasy Mini kit according to the manufacturer’s instruction . The RNA purity and concentration were determined by Nanodrop , then RNA was aliquoted stored at −80 °C until use. Only samples with 90% pure RNA or higher (OD260/280 > 0.8) were used for reverse-transcription with an ABI High Capacity Reverse Transcription Kit .Primer sequences for ABCB1 were (F): 5′-CAC CCG ACT TAC AGA TGA TG-3′ and (R) 5′-GTT GCC ATT GAC TGA AAG AA-3′ with predicted amplicon length of 81 and can be retrieved from NM_000927 . The priCT) was set in the exponential phase of amplification and quantified by normalization to 18S rRNA. Analysis was performed on StepOne™ software version 2.3 , with CT values converted to fold change differences using the 2CT−ΔΔ method for relative quantitation [Real-time PCR was performed on an ABI Step-one-plus real time PCR system (Life Technologies) using cDNA template . First, the forward and reverse primers concentrations were optimized to give best signal-to-noise amplification and were 300 nM for both forward and reverse primers for 18S and 300 nM forward and 500 nM reverse for ABCB1. Detection was with PerfeCTa SYBR Green SuperMix for IQ . Cycling conditions were: 1 cycle 30 s at 95 °C, 40 cycles of 5 s at 95 °C, 15 s at 60 °C, and 10 s at 70 °C then melt curve of 15 s at 95 °C, 60 s at 60 °C, and 15 s at 95 °C. The threshold value detection (titation .n = 9. Samples were semi-quantified with Image J 1.48v (http://imagej.nih.gov/ij) with background subtraction.For western blotting of ABCB1, SDS-page gels (7%) were used to resolve 20 µg of liver lysate or S9 and each sample was analyzed on at least 3 separate gels as previously described . PrimaryThe triglyceride colorimetric assay kit  was used to determine the levels of triglycerides (mg/g of liver) as per manufacturer’s instructions. t-tests (two-tailed) were performed between groups. For binned continuous data , one way ANOVA with Tukey’s post hoc analysis was performed. Pediatric samples (<18 years) were not included in the BMI analysis, as BMI is not an appropriate measure for obesity in children. Where body weight was known, appropriate categories were assigned using the National Center for Health Statistics weight-for-age growth charts for children: underweight, <5th percentile; ideal weight, 5–85th; overweight, 86–94th; obese, >95th percentile .All data sets were analyzed for normality with D’Agostino-Pearson tests. For sex or ethnicity (binary tests), student’s thcharts , Table 2CT values (±SD) for 18S and ABCB1 were 13.3 ± 2.2 and 32.6 ± 2.8, respectively. The ABCB1 mRNA levels had an average 30-fold variability in mRNA levels. Similarly, there were no significant differences in protein expression of ABCB1 throughout the liver  or protein .Similarly, for triglycerides mean levels were 15.4 ± 1.2 mg/g of liver (mean ± SD). There were no regional differences in triglyceride levels a, but trn = 79) was detectable from birth, and a significant negative correlation with age was observed , although the correlation coefficient suggested that this was only a moderate association, with 8% of the change in mRNA expression being attributed to age as a covariate (CT ± SEM of 22.7 ± 0.4) had lower ABCB1 mRNA levels than adults . A representative Western blot image for ABCB1 detection is presented in F-test; and fit parameters of AICc, 15.56, Sum of Squares 0.77, p < 0.001, Subsequently, we performed the same analysis for ABCB1 protein levels . The BMI parameter is frequently used as a measure for obesity; however, this can be flawed ,28, and Finally, because we did not have pediatric or significant numbers of elderly liver lysates, we used S9 to determine ontogenetic differences in ABCB1. Children (≤18) have significantly lower and more variable expression of hepatic ABCB1 protein than adults, with ~76% of adult ABCB1 protein levels present at birth, reaching full maturity by five years of age. The strength of the data presented is that in every other category tested, S9 results were reflective of liver lysates, albeit with lower absolute levels of mRNA and protein present. In addition, all of the S9 samples were treated the same way i.e., similarly depleted of membranes, so the relative variability of the ABCB1 proteins should be preserved, even though absolute levels are not. Relatively few studies of pediatric ABCB1 and ontogeny exist. Miki et al., 2005, reported no differences in hepatic ABCB1 mRNA but significant decreases in mRNA expression in the lungs of the elderly . MoreoveIn summary, investigators who collect human liver samples opportunistically, as well as those who collect uniformly from a single site, likely have a cohort representative of population variability in ABCB1 that can be accurate for extrapolation. Additionally, children under five express less hepatic ABCB1 than the general population, which could cause xeno- and endobiotic toxicity including altered responses to therapy, but this requires confirmation in a larger study, preferably using total liver lysates. Better understanding of the natural expression patterns of ABCB1 in the human liver can assist in translating work from human tissues into understanding of the mechanisms of drug and chemical efficacy, toxicity, and resistance.


Linum usitatissimum L.) plants under Al stress. Flax seedlings of resistant (TMP1919 and G1071/4_k) and sensitive (Lira and G1071/4_o) to Al cultivars and lines were exposed to AlCl3 solution for 4 and 24 hours. Twelve small RNA libraries were constructed and sequenced using Illumina platform. In total, 97 microRNAs from 18 conserved families were identified. miR319, miR390, and miR393 revealed expression alterations associated with Al treatment of flax plants. Moreover, for miR390 and miR393, the alterations were distinct in sensitive and resistant to Al genotypes. Expression level changes of miR319 and miR390 were confirmed using qPCR analysis. In flax, potential targets of miR319 are TCPs, miR390–TAS3 and GRF5, and miR393–AFB2-coding transcripts. TCPs, TAS3, GRF5, and AFB2 participate in regulation of plant growth and development. The involvement of miR319, miR390, and miR393 in response to Al stress in flax was shown here for the first time. We speculate that these microRNAs play an important role in Al response via regulation of growth processes in flax plants.Acid soils limit agricultural production worldwide. Major reason of crop losses in acid soils is the toxicity of aluminum (Al). In the present work, we investigated expression alterations of microRNAs in flax ( Toxicitso forth –5. via miRNA was revealed as one of the mechanisms of response to Al in different plant species [Linum usitatissimum L.). Flax fiber is utilized in textile industry; flax seeds are used for production of oil, linoleum, food, and pharmaceutical products [MicroRNA (miRNA) negatively regulates gene expression and in this way controls numerous biological processes in plants , includi species –12. Howeproducts –15. Flaxproducts –20. In tproducts  and exceproducts –24 was sIn the present work, we performed high-throughput sequencing of flax small RNAs under normal conditions and Al exposure to identify miRNAs, whose expression was altered in response to aluminum stress, and suggested potential targets of these miRNAs in flax to speculate on affected signaling pathways.L. usitatissimum plants of resistant (TMP1919 and G1071/4_k) and sensitive (Lira and G1071/4_o) to aluminum cultivars and lines were used in the present study. Seeds germinated on filter paper soaked with distilled water for 5 days. Then seedlings were transferred to falcon tubes with filter paper soaked with a 0.5 mM CaCl2 solution at pH 4.5 for 24 h before being exposed to a 0.5 mM CaCl2 solution (pH 4.5) containing 0 (N) or 500 μM AlCl3 for 4 (Al-4) and 24 (Al-24) hours. Roots were cut off and immediately frozen in liquid nitrogen. Plant samples were stored at −70°C.Total RNA was extracted from roots of flax plants using RNA MicroPrep kit . RNA quality and concentration were determined by Qubit 2.0 fluorometer  and Agilent 2100 Bioanalyzer . For further analysis, only RNA samples with RNA Integrity Number (RIN) value not less than 8.0 were used.Library preparation was performed using Illumina TruSeq small RNA preparation kit  in compliance with manufacturer's protocol. Twelve libraries from pooled plant samples were obtained: N, Al-4, and Al-24 for each of four cultivars/lines . Library quality was evaluated using Agilent 2100 Bioanalyzer (Agilent Technologies). The sequencing was performed on Genome Analyzer IIx (Illumina). L. usitatissimum transcripts [Low-quality reads and adapter reads were removed from raw sequencing data using Trimmomatic . For furnscripts , 28. fold change parameter: FC = RPM in Al-4 or Al-24/RPM in N. P values were calculated using χ2 test with Benjamini-Hochberg multiple testing correction. Changes were considered significant if FC or 1/FC were 1.5 or higher, that is, absolute value of log2FC ≥ 0.58.miRNA levels were normalized to obtain reads per million (RPM) values. The comparison of miRNA expression levels in Al-4 and Al-24 libraries with N library was performed usingμL reaction containing 1x RT primer (Thermo Fisher Scientific), 200 U of RevertAid Reverse Transcriptase (Thermo Fisher Scientific), 1x Reverse Transcription Buffer, 250 nM of dNTPs, and 10 ng of total RNA using the following program: 16°C for 30 min, 42°C for 30 min, and 85°C for 5 min. QPCR was performed using the 7500 Real-Time PCR System in a 20 μL reaction mix containing 1x PCR mix , 250 nM of dNTPs, 2 U of polymerase (GenLab), Rox dye, and RT product using the following program: 95°C for 15 min, 40 cycles of 95°C for 15 s, and 60°C for 60 s. Three technical replicates were performed. For the evaluation of expression level alterations, ΔΔCteff values, which are directly proportional to the expression level changes, were calculated [ ETIF3H and ETIF3E were chosen as the reference genes for the qPCR data analysis [2FC) and qPCR (ΔΔCteff) expression data was evaluated using Spearman's correlation coefficient.We performed qPCR analysis to evaluate expression of miR319 and miR390. TaqMan MicroRNA Assays aau-miR319 and ath-miR390a  were used. Reverse transcription was performed in 15 lculated , 30. ETIanalysis , 29, 31.analysis , 32. CorSeedlings of resistant (TMP1919 and G1071/4_k) and sensitive (Lira and G1071/4_o) to Al flax cultivars and lines were exposed to Al for 4 and 24 hours. Twelve small RNA libraries were constructed and sequenced on Illumina GAIIx. In total, about 40 million raw reads were obtained. All the sequences were deposited in the European Nucleotide Archive, accession number PRJEB15342.https://doi.org/10.1155/2017/4975146). Among these miRNAs, the search for Al responsive miRNAs was performed. To reveal common trends specific to flax plants, we evaluated expression alterations after 4 and 24 hours of Al treatment using pooled data for all examined cultivars and lines  upregulation of miR164, miR319, miR393, and miR394 and downregulation of miR159, miR167, and miR408. After 24 hours of Al exposure, significant expression decrease was revealed for all 18 miRNA families except miR393. Thus, after 4 hours of Al treatment, miRNA levels of different families were increased, decreased, or stable. However, after 24 hours of Al exposure, expression was decreased for almost all miRNA families.After 4 hours of Al exposure, we observed significant . log2FC varied from 0.22 to 1.16) and decreased after 24 hours , but increased in resistant genotypes , but strong downregulation in sensitive cultivar and line (log2FC was −3.04 for G1071/4_o and −1.77 for Lira). miR393 was upregulated in resistant to Al genotypes (log2FC was 1.66 for TMP1919 and 1.10 for G1071/4_k), but slightly decreased in sensitive to Al genotypes (log2FC was −0.46 for G1071/4_o and −0.10 for Lira) after 4 hours of Al exposure (2FC was 0.20 for TMP1919 and 0.07 for G1071/4_k) and deregulated in sensitive to Al genotypes (log2FC was −1.43 for G1071/4_o and 0.51 for Lira).Definite regularities were revealed for expression alterations of miR319, miR390, and miR393 families. The level of miR319 was changed in a similar way in resistant and sensitive to Al cultivars and lines: expression was increased after 4 hours of Al exposure ; Teosinte Branched/Cycloidea/PCF transcription factor 3 (TCP3) superfamily protein; TCP family transcription factor 4; ABC transporter. Within predicted targets of miR319, Lus10002195 and Lus10000463 transcripts encoding TCP3 and TCP4 are the most interesting. It was previously shown that miR319 targets mRNA of TCP transcription factors, which control plant growth and development –42. We s L. usitatissimum unigene library, was also predicted as potential target of miR390. In other plant species, miR390 initiates tasiRNA  biogenesis via cleavage or interaction with TAS3 transcript. TAS3 tasiRNAs negatively regulate ARFs (auxin response factors) that is necessary for proper plant development [For miR390, predicted targets in flax were transcripts encoding leucine-rich receptor-like protein kinase family protein; protein kinase superfamily protein; growth-regulating factor 5 (GRF5); root hair specific 10 (RHS10); poor homologous synapsis 1 (PHS1); leucine-rich repeat (LRR) family protein; TAS3. Transcript Lus10009533 encoding GRF5 was one of the potential targets of miR390. GRFs play important role in plant developmental processes and growth under adverse environments and could be regulated by TCP4 . TAS3-coelopment –48. We sAuxin signaling F-box 2 (AFB2), zinc finger (C3HC4-type RING finger) family protein, and pol-like 5 (PLL5) transcripts were predicted as targets of miR393 in flax. Lus10031991 and Lus10035160 encoding AFB2 are the most interesting targets of miR393. It was previously reported that targets of miR393 are AFB1 (auxin f-box protein1), AFB2, and AFB3, which are involved in auxin signaling , 49–51. Thus, expression alterations of miR319, miR390, and miR393, which were revealed as Al-responsive in flax, could affect expression level of a number of key transcripts involved in plant growth and development. L. usitatissimum plants. Moreover, we revealed diverse alterations of miR390 and miR393 levels in resistant and sensitive to Al genotypes. We concluded that, in flax, potential targets of miR319 are TCPs, miR390–TAS3 and GRF5, and miR393–AFB2. Thus, we speculate that miR319, miR390, and miR393 play an important role in Al stress response via regulation of growth and development processes in flax plants.High-throughput sequencing and qPCR analyses of flax small RNAs allowed us to reveal miRNAs with expression alterations under Al exposure. Our results suggest the involvement of miR319, miR390, and miR393 in Al response inMicroRNAs in flax plants under aluminum stress .


Male breast cancer (MBC) is rare. We assembled 446 MBCs on tissue microarrays and assessed clinicopathological information, together with data from 15 published studies, totalling 1984 cases. By immunohistochemistry we investigated 14 biomarkers  for survival impact. The main histological subtype in our cohort and combined analyses was ductal , grade 2; , respectively. Cases were predominantly ERα  and PR positive , respectively, with HER2 expression being infrequent , respectively. In our cohort, advanced age (>67) was the strongest predictor of overall (OS) and disease free survival (DFS) . Node positivity negatively impacted DFS (p = 0.04). FOXA1 p = 0.005) and AR p = 0.009) were both positively prognostic for DFS, remaining upon multivariate analysis. Network analysis showed ERα, AR and FOXA1 significantly correlated. In summary, the principle phenotype of MBC was luminal A, ductal, grade 2. In ERα+ MBC, only AR had prognostic significance, suggesting AR blockade could be employed therapeutically. With over 1.6 M cases diagnosed in females in 2012 alone, breast cancer is the most common cancer worldwide for women3The genesis of MBC is yet to be elucidated fully, with many studies suffering from small number of cases available, with published reports on as few 16 cases910111291011129101213101012101113Through international collaboration, we accumulated a series of 446 MBC and evaluated and compared their clinicopathological characteristics with 15 published studies reporting ≥30 MBC cases. Using immunohistochemistry, we evaluated biomarkers with well-established roles in female breast cancer, represented on tissue microarrays (TMAs). These included ERβ1, ERβ2, ERβ5, PR, AR, Bcl-2, HER2, p53, E-cadherin, Ki67, survivin, prolactin and Forkhead box protein A1 (FOXA1). Our aim was to identify their expression and associate this with known clinical or pathological prognostic variables to determine potential prognostic roles in MBC.Leeds (West) Research Ethics Committee granted ethical approval (06/Q1205/156). TMAs comprising 446 cases were constructed as previously describedTM Polymer Detection System kit was used for visualization of primary antibodies following the manufacturer’s instructions . Slides were washed in TBST and stained in haematoxylin before dehydration in graded ethanol. Slides were mounted in DPX . Scoring criteria were selected according to previously reported studies  were analysed . Hazard ratios were determined by Cox regression. Follow up information was at least 10 years and was updated in June 2013 and survival periods calculated. Patients were censored at the last day they were known to be alive. Variables were entered in univariate and multivariate analysis ; these included the biomarker of interest, grade, nodal status and tumour size as is routinely used in analysis of breast cancer datasets. P values reported for univariate and multivariate analysis were calculated using Cox proportional hazards regression model in PASW (v21).These are illustrated in Comparatively our cohort characteristics are virtually identical to those from the combined data. The main histological subtype in our cohort and in the combined analyses was ductal , grade 2 , respectively. Lobular carcinoma was the second most common histology in both cohorts. The “other” category included cribriform, papillary, intraductal papillary, micropapillary, mucinous and DCIS. Cases were predominantly ER  and PR positive . HER2 expression was infrequent in our cohort (2%), and seen in 10% of cases from the combined cohort. Grade break down was very similar: grade 1 , grade 2 , grade 3 . Despite missing/unavailable for grade (19%), node status (42%) and HER2 (33%) in our cohort, with the possible exception of node status, this was fairly typical of the other studies, illustrating the representativeness of our cohort. While this work was under review, data from the International Male Breast Cancer Pooling Project was reportedWe performed univariate analysis on the whole cohort (n = 446) and in ERα + cases only (n = 375), considering the effects of grade, age, lymph node status and tumour size. This is shown in ERα, ERβ1, ERβ2, ERβ5, PR, AR, Bcl-2, HER2, p53, E-cadherin, Ki67, survivin, prolactin and FOXA1 expression were all evaluated . In termIn the ERα+ cohort, Kaplan Meier survival analysis showed that FOXA1 was significantly associated with better OS and DFS . AR was As FOXA1 is emerging as a critical player in breast cancer biology we examined the impact of its co-expression with AR and ERβ isoforms on survival in an ERα+ background. Here, co-expression of AR and FOXA1 , in concordance with the average age of diagnosis of MBC, which is typically 10 years older than that seen for female breast cancer7, in conAs MBC is characteristically predominantly ERα-positive we evaluated the effect on outcome in ERα-positive cases only (84% of our cohort), investigating several biomarkers for their impact on survival. We found FOXA1 expression was positively prognostic for both OS and DFS. Often described as a pioneer factor, FOXA1 is emerging as a critical player in hormone dependent cancer, including breastFOXA1 has the ability to bind to compacted chromatin, making these regions more accessible to other transcription factors, notably ERα and ARFOXA1 typically works in cooperation with another transcription factor, GATA3. While we did not evaluate GATA3 in our work, a previous study has shown GATA3 positivity in only 6 out of 19 MBC (32%), compared to 82% of female breast cancerIn the current study and previous work , AR expression was associated with better outcome in MBCWe also identified a significant correlation of ERα, AR and FOXA1 expression using hierarchical clustering and correlation network analysis, with weaker, although still statistically significant, association with PR. We previously identified that ERα clustered with AR and ERβ in male but not female breast cancer which clustered with ERα and PRIn line with our findings, a previous study examining survivin in MBC showed no effect on survival, irrespective of its cell locationIn conclusion, the majority of MBC are luminal A, ductal grade 2 with nodal positivity in approximately half of all cases with HER2 expression being rare. While MBC expresses many of the same biomarkers as female breast cancer, of those examined, we found only AR remained significant upon multivariate analysis, providing potential for AR blockade to be employed therapeutically.How to cite this article: Humphries, M. P. et al. Characterisation of male breast cancer: a descriptive biomarker study from a large patient series. Sci. Rep.7, 45293; doi: 10.1038/srep45293 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Background: Approximately 60% of patients experience moderate-to-severe pain after neurosurgery, which primarily occurs in the first 24–72 h. Despite this, improved postoperative analgesia solutions after neurosurgery have not yet been devised. This retrospective study was conducted to evaluate the effect of intra- and post-operative infusions of dexmedetomidine (DEX) plus sufentanil on the quality of postoperative analgesia in patients undergoing neurosurgery.Methods: One hundred and sixty-three post-neurosurgery patients were divided into two groups: Group D  and Group ND (no DEX infusion during surgery). Patient-controlled analgesia was administered for 72 h after surgery  in this retrospective study. The primary outcome measure was postoperative sufentanil consumption. Hemodynamics, requirement of narcotic, and vasoactive drugs, recovery time and the incidence of concerning adverse effects were recorded. Pain intensity [Visual Analogue Scale (VAS)], Ramsay sedation scale (RSS) and Bruggemann comfort scale (BCS) were also evaluated at 1, 4, 8, 12, 24, 48, and 72 h after surgery.Results: Postoperative sufentanil consumption was significantly lower in Group D during the first 72 h after surgery (P < 0.05). Compared with Group ND, heart rate (HR) in Group D was significantly decreased from intubation to 20 min after arriving at post anesthesia care unit (PACU), while mean arterial pressure (MAP) in Group D was significantly decreased from intubation to 5 min after arriving at PACU (P < 0.05). The intraoperative requirements for sevoflurane, remifentanil, and fentanyl were approximately 35% less in Group D compared with Group ND. VAS at rest at 1, 4, and 8 h and with cough at 12, 24, 48, and 72 h after surgery were significantly lower in Group D (P < 0.05). Compared with Group ND, patients in Group D showed lower levels of overall incidence of tachycardia, hypertension, nausea, and vomiting (P < 0.05). There were no significant differences between the two groups in terms of baseline clinical characteristics, recovery time, RSS, and BCS (P > 0.05).Conclusions: DEX (0.02 μg·kg−1·h−1) plus sufentanil (0.02 μg·kg−1·h−1) could reduce postoperative opioid consumption and concerning adverse adverse effects, while improving pain scores. However, it did not influence RSS and BCS during the first 72 h after neurosurgery. A previous study reported that 60% of patients experience moderate-to-severe pain during the first 72 h after neurosurgery . Patients provided informed consent. Patients who underwent neurosurgery from January 2015 to December 2015 were enrolled in this study if they met the following inclusion criteria: age between 35 and 65 years, American Society of Anesthesiologists (ASA) grade I–II. Exclusion criteria included a history of endocrinological disease, hypertension (diastolic blood pressure >160 mmHg), ischemic heart disease, second or third degree heart block, pregnancy, long-term abuse of alcohol (>6 months), opioids, or sedative—hypnotic drugs, DEX allergies, nausea, and vomiting after previous surgery, neuropsychiatric diseases, operation time shorter than 1 h or longer than 6 h, blood loss greater than 1200 mL, reoperation during the first 72 h after neurosurgery, or emergency operation.Patients were divided into two groups: Group D and Group ND. A total of 153 medical records were reviewed retrospectively, 76 from Group D and 77 from Group ND. Electronic chart and DoCare clinic electronic anesthesia recording system data were utilized.2, and temperature were continuously monitored using an automated system (Philips IntelliVue MP50) after patients arrived at the operating room, then two peripheral intravenous catheters were placed in all patients before induction. A forced-air warming device  was used in both groups to maintain normothermia.Electrocardiography, arterial blood pressure, pulse-oximetry, end-tidal CO−1) over a period of 10 min before endotracheal intubation, which was then adjusted to 0.3 μg·kg−1·h−1 until incision suturing. Both groups were induced using the same agents: fentanyl (2 μg·kg−1), lidocaine (1.5 mg·kg−1), propofol (2 mg·kg−1), and cisatracurium (0.2 mg·kg−1). General anesthesia was maintained with a 1–1.2 minimum alveolar concentration of sevoflurane in an air/oxygen mixture, and a remifentanil (0.05–0.1 μg·kg−1·min−1) infusion and intermittent boluses of cisatracurium (0.05 mg·kg−1) for muscle relaxation. Positive pressure ventilation and oxygenation were maintained with endotracheal intubation to achieve an arterial partial pressure of carbon-dioxide of 35–40 mmHg. The concentration of sevoflurane and rate of remifentanil were adjusted according to the hemodynamic limits and bispectral Idex. (40–60).Patients in Group D received intravenous DEX  was used if hypertension was persistent for 3 min, and if persistent after fentanyl treatment, urapidil (10–15 mg) was administered. Hypotension was defined as a >30% decrease from baseline and was treated by decreasing the inspired sevoflurane concentration by 0.2% or remifentanil by 0.02 μg·kg−1·min−1 in a stepwise titration, and if persistent, by administration of ephedrine ephedrine (6–12 mg) or phenylephrine (20–80 μg). DEX and sevoflurane were discontinued approximately 15 and 5 min before completion of the surgery, respectively. All patients received 5 mg of tropisetron and underwent routine reversal of neuromuscular blockade. After extubation in the operating room, all patients were transferred to the post anesthesia care unit (PACU).During the operation, bradycardia and tachycardia were defined as HR < 45 bpm or a >30% increase from baseline and were treated using atropine (0.2–0.5 mg) or esmolol (20–30 mg), respectively. Hypertension was defined as a >30% increase from baseline and was treated by increasing the inspired sevoflurane concentration by 0.2% or remifentanil by 0.02 μg·kg−1·h−1, a bolus dose of 0.02 μg·kg−1 with a 5-min lockout interval and a 1 h limit of 16 mL, while patients in Group D received both sufentanil and DEX under the same regimen as those in Group ND after arriving at the PACU.Patients in Group ND received sufentanil at a continuous dose of 0.02 μg·kgPatients were encouraged to self-control the pump when they experienced pain during coughing at a severity of Visual Analogue Scale (VASc) score >4. If patients demonstrated a poor response to the patient controlled analgesia (PCA) (VASc scores of >6), or an obvious PCA-associated adverse effect took place, 30 mg of ketorolac was administered. If the rescue analgesia was ineffective 30 min after administration, 100 mg of tramadol was administered.The intraoperative hemodynamic data  were obtained from the Phillips IntelVue monitor at the following points: arrival at the operating room (T0), just before intubation (T1), after intubation (T2), during head pinning (T3), during surgical incision (T4), extubation (T5), arrival at the PACU (T6), and 5 min (T7), 10 min (T8), 15 min (T9), and 20 min (T10) after arriving at the PACU. The requirement of narcotic and vasoactive drugs, fluid requirements, and estimated blood loss, incidence of complications during the operation and in the PACU were recorded. The length of the PACU stay was also recorded based on the Aldretes criteria Aldrete, .The cumulative amount of self-administered sufentanil was recorded until 72 h after the surgery. Postoperative pain intensity , the Ramsay Sedation Scale , and the Bruggemann comfort scale  were evaluated at 1, 4, 8, 12, 24, 48, and 72 h after surgery. The number of rescue analgesia and adverse effects  was also recorded at the end of the study.The data from a preliminary study showed that the requirement of sufentanil in patients not treated with DEX was approximately 165.34 ± 23.13 μg during the first 72 h after neurosurgery. A 25% reduction in sufentanil use was considered clinically feasible. For a study power of 80% , assuming a dropout rate of 15%, the required sample size per group was calculated to be 75, with a total of 150 patients allowed for adequate data collection .post-hoc multiple comparisons. The non-parametric Wilcoxon—Mann—Whitney test was used for variables that were not normally distributed. Categorical data were expressed as frequency and percentage and analyzed using chi-squared tests or Fisher's exact tests when appropriate. Probability (P) values of <0.05 were considered statistically significant. Statistical analysis was performed using SPSS for Windows Version 18.0 .The Kolmogorov–Smirnov test was used to assess the distribution of variables. Homogeneity of variance was determined using Levene's tests. Quantitative data were expressed as mean and standard deviation or median and inter-quartile range. Inter-group comparisons were performed using repeated-measures analysis of variance. Bonferroni's correction was used for The patient demographics are illustrated in a patient enrollment flow diagram Figure . One thoThe two groups were comparable regarding age, sex, BMI, ASA grade, history of comorbidity and tumor location Table .P > 0.05; Figure P < 0.05; Figure Baseline HR and MAP were not statistically different between the two groups , fentanyl (3.24 ± 1.13 vs. 4.72 ± 1.41 μg·kg−1P < 0.01) and estimated blood loss  were significantly decreased in Group D , phenylephrine  and esmolol , while the requirements for ephedrine  and atropine  were similar , P < 0.05, Figure P < 0.05, Figure P = 0.02, Table P > 0.05, Figure The total dosage and dosage per body weight of sufentanil were significantly lower in Group D than in Group ND at 4, 8, 12, 24, 48, and 72 h after surgery (P = 0.04), hypertension , delirium , nausea  and vomiting  than patients in Group ND. However, there are no statistically significant differences between the two groups in terms of the incidence of bradycardia  and hypotension . Although more patients in Group ND suffered seizures during the period of this study than those in Group D, the difference was not statistically significant  as PCA in patients who underwent neurosurgery could decrease both the total dosage and dosage per body weight of sufentanil from 4 to 72 h after surgery and improve postoperative analgesia during the 72 h after surgery. It was also found that HR and MAP; the requirement of sevoflurane, remifentanil, and fentanyl; and the estimated blood loss were significantly decreased in Group D during the operation (P < 0.05). VAS at rest at 1, 4, and 8 h and with cough at 12, 24, 48, and 72 h after surgery were significantly lower in Group D than in Group ND (P < 0.05). More patients in Group ND required urapidil, phenylephrine, and esmolol during surgery and postoperative rescue analgesia (P < 0.05). However, there were no significant differences between the two groups in terms of baseline clinical characteristics, RSS, BCS, and main adverse events with the exception of tachycardia, hypertension, delirium, nausea, and vomiting.In this retrospective trial DEX plus sufentanil . At the same time, the number of patients in Group D who needed urapidil, phenylephrine, and esmolol was significantly decreased (P < 0.05). A potential explanation is the better effect of the DEX-opioid combination used in Group D during neurosurgery in terms of sympathetic response and antinociceptive properties, as previous studies have shown that DEX demonstrates nociceptive-modulating effects through both the central and spinal cord α2 receptor without significant respiratory depression . Compared with Group ND, the analgesic effect was better in Group D. However, significant differences between the two groups in terms of RSS and BCS during the first 72 h after surgery were not observed (P > 0.05).For a long time, craniotomy was thought to be less painful than other surgical procedures. The perioperative pain management of patients undergoing neurosurgery has drawn more attention recently, and an increasing number of studies have reported that most patients experience moderate-to-severe pain after neurosurgery , hypertension (P = 0.02), and delirium (P = 0.01).Previous studies have found that the most common side effects associated with DEX are bradycardia and hypotension  after neurosurgery, and different dosages of DEX-sufentanil should be be further investigated.We acknowledge that the present study has some limitations. First, this study is a retrospective trial, and a multi-center large sample prospective study is necessary to more rigorously verify the feasibility of DEX-sufentanil treatment for postoperative analgesia. Second, operation time varies according to the type of surgical procedure; however, the duration of surgery and anesthesia was not significantly different between the two groups. Third, DEX was administered at a rate of 0.5 μg·kg−1·h−1) plus sufentanil (0.02 μg·kg−1·h−1), which could reduce postoperative sufentanil consumption and improve pain scores, while not improving RSS and BCS scores during the first 72 h after neurosurgery. However, more multi-center prospective studies are still required to determine the optimal dosage of DEX-sufentanil to be applied during the perioperative period of neurosurgery.In summary, to our best knowledge, this is the first report regarding the application of DEX (0.02 μg·kgSS and CR conceived and designed the trial; HZ analyzed the data; SS and CR collected the data, ZL and ZZ wrote the paper. ZL and ZZ contributed equally to this trial and should be considered as co-corresponding authors.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.