In this study, we try to evaluate the effects of acupuncture stimulation with different amounts at Sanyinjiao (SP6) on uterine contraction and uterus microcirculation in rats with dysmenorrhea of cold coagulation syndrome and to explore whether there is direct relativity between “De qi” and needle stimulus intensity. Diestrus female rats were randomly divided into 4 groups, including saline control group, model control group, “A” stimulus group (with strong stimulus), and “B” stimulus group (with weak stimulus). We found that dysmenorrhea rats of the cold coagulation syndrome present a high intensity in uterine tension and high contraction of microvascular diameter. Acupuncture applied with two different stimuli could relieve the symptoms, but, compared with “B” stimulus, “A” stimulus leads to better outcomes on reducing uterine contraction and increasing diameter of uterine microvascular; moreover, hand manipulation during needling mediates the curative effect on the microvascular diameter. Our finding indicates that using thick needles and deep insertion with hand manipulation are more effective and achieve desired level of “De qi” in dysmenorrhea rats. Miraculous Pivot-Nine Types of Needle and Twelve Yuan-Primary Points, the relationship between “De qi” and curative effect is mentioned that the key point of acupuncture therapy is to wait for the “qi” arrival, and to use manipulations to induce the needle sensation if the “qi” has not arrived. If the “qi” has arrived, stop stimuli and withdraw the needle [“De qi” is a main concept in traditional Chinese acupuncture theory , 2. It he needle . Previoue needle . Concerne needle . Miraculous Pivot-Ni Shun Fei Shou has mentioned, “Because young children have weak physique and insufficient “qi” and blood, it is suggested to use thin needles, shallow needling without needle retention”, “Skinny patients are easy to suffer from deficiency of “qi” and blood. They are suggested to use shallow needling with no retention of needles [On the other hand, some studies have found that the intensity of “De qi” is not only affected by the body state, the specificity of acupoints, and the intervention methods, but also closely related to the intensity of stimuli and the needle thickness, the depth of needling, and different manipulating methods –8. Recen needles .” Theref needles . Clinica needles . TherefoIn this experiment, we will use the dysmenorrhea of the cold coagulation syndrome rats and two different intensities of stimuli (“A” stimulus group with strong stimulus and “B” stimulus group with weak stimulus) to evaluate the relationship between the stimulation intensity (an indirect indicator of “De qi” sensation) and the treatment effect. We believe that the result obtained will enhance our understanding of the association among “De qi,” uterine contraction, and microcirculation changes.This experimental scheme was approved by the Beijing University of Chinese Medicine Ethics Committee on April 13, 2012 . The animal experiment of laboratory animal care was complied with the United States National Institutes of Health Advocacy, using the guidelines of reduction, replacement, and refinement of the animal experiment “3R” principle: adoption of clean grade Sprague-Dawley rats (SD), female, rats aged 3 months, weighing (240 ± 20) g, and sexual maturity but without mating; rearing conditions: indoor temperature (23 ± 1)°C, humidity (45 ± 5)%, general feed, and free drinking water.The equipment and reagents used were estradiol benzoate injection, produced by Ningbo Sansheng Pharmaceutical Co. Ltd., batch number: 120207. oxytocin injection, produced by Shanghai Hefeng Factory, batch number: 120302, self-control of methylene blue staining solution, 20% Urad injection: batch number T20080530, BC/BD-379HB horizontal freezer (Qingdao Haier Group), BL-420F Biological Function Experimental System, Chengdu Taimeng Technology Co. Ltd, FT-100 Type Tension Transducer, Chengdu Taimeng Technology Co. Ltd, XW-B-3 Cold Light Microcirculation Tester, Nanjing Sheng Photoelectric Medical Instrument Co., Ltd, KEL-2000 Type Microscope Temperature Controller, Nanjing Sheng Photoelectric Medical Instrument Co., Ltd, and SG-H1 Type Constant Temperature and Humidity Test System, Nanjing Sheng Photoelectric Medical Instrument Co., ltd.After screening the estrous cycle through vaginal smear test , 64 femaThe cold coagulation syndrome model is improved from Xu Shuyun's pharmacological experiment , 16. FirOn the basis of the morphological, anatomical, and physiological characteristics of the rat model and referring to Lin Wen-zhu's experimental acupuncture , standarFor “A” stimulus group with a thick needle (Φ 0.25 × 40 mm), acupuncture was applied to the rat's Sanyinjiao (SP6) in the depth of 4-5 mm with twirling for 30 seconds at the beginning and 10 minutes after insertion of the needle. For “B” stimulus group with a thin needle (Φ 0.18 × 13 mm), acupuncture was applied to the rat's SP6 in the depth of 1-2 mm but did not use any hand manipulation. After observing the effectiveness index of the rat's uterine contraction and microcirculation for 20 min, the needle was withdrawn.On the 10th day after the injection of estradiol benzoate, the rats were given 20% urethane (0.7 mL/100 g) intramuscular injection for anesthesia; then an incision of 2-3 cm length was made, 0.5 cm apart from the abdominal midline. After pulling out the uterine, a thread line was penetrated 1 cm above the uterine horn bifurcation. The free-end of the line was attached to the tension sensor and BL-420F Biological Function Experimental System Recorder was used to record the uterine contraction wave. The model control group and the treatment group were directly infused with 2U oxytocin, and the saline group was infused with 2U normal saline. Results were recorded 20 min after acupuncture intervention for the treatment group.The rats were given intramuscular injection of 20% urethane (0.7 mL/100 g) for anesthesia. After they were completely anesthetized, incision of 2-3 cm length was made, 0.5 cm apart from the abdominal midline, and one side of the uterine and its ligament were pulled out. The model group and the treatment group had uterine infusion, 2U oxytocin, and the saline group had uterine infusion, 2U normal saline. Acupuncture was applied to the treatment group. After fixing the uterine and ligament to a constant temperature and humidity box, XW-B-3 Cold Light Microcirculation tester was used to observe the parameters every 5, 10, and 20 min values in fixed visual field.The experiment of uterine contraction observation index includes uterine contraction wave number, uterine contraction wave peak, and uterine activity (uterine contraction wave number × peak); uterus microcirculation, cap diameter, and blood flow status were observed under 40x magnification vision of the microvascular diameter. Flow status score was taken according to Tian's  standardR ≤ 1, using P < 0.05 as the significant difference between the standard.SAS 9.3 software was used for statistical analysis. Excel was used for graph generation. Data was represented into P < 0.01)  and uterine activity (P < 0.05) significantly decreased in “A” stimulus group. This result showed significant difference compared with “B” stimulus group (P < 0.05) in the decrease of uterine contraction wave number. Compared with the model control group, the uterine contraction wave number and uterine activity decreased in “B” stimulus group, but the difference had no statistical significance . Compared with the model group, “A” stimulus group had significant difference in uterine microvascular diameter expansion (P < 0.01). After acupuncture intervention, “B” stimulus group also had microvascular diameter expansion, but the difference was not statistically significant (P > 0.05). “A” stimulus group had significant difference compared with “B” stimulus group in every period, 5 min and 10 min (P < 0.01) and 20 min (P < 0.05)  in every period. Rat models in “A” stimulus group, compared with the model group, had significant expansion in uterus diameter of capillaries immediately after acupuncture treatment, 5 min (P < 0.05) and 10 min and 20 min (P < 0.01). Although “B” stimulus group had expansion too, the difference was not statistically significant (P > 0.05). After acupuncture stimulus, the expansion in uterine diameter of capillaries of “A” stimulus group, compared with “B” stimulus group, had significant difference: 5 min and 20 min (P < 0.05) and 10 min (P < 0.01). This indicated the effect of hand manipulation of needle in “A” stimulus group ; compared with the model control group, “A” stimulus group improved significantly in uterine microvascular blood flow after acupuncture stimuli (P < 0.01). In “B” stimulus group, although the blood flow velocity increased, the difference was not statistically significant (P > 0.05). After acupuncture treatment, the microvascular blood flow of “A” stimulus group was significantly faster than “B” stimulus group, (P < 0.01)  .P < 0.01). In “B” stimulus group, although the blood flow velocity increased, the difference was not statistically significant (P > 0.05).r < −1). This proposes that, under dysmenorrhea condition, contraction of uterus and microcirculation are directly connected (The relationship of uterine microvessel capillary diameter and the contraction of uterus showed negative correlation (onnected .r < −1).Dysmenorrhea refers to painful menstruation, that is, recurrent abdominal pain or pain in the lumbosacral region in periods, before or after periods. According to the theory of Chinese medicine, it is induced by either “qi” stagnation and blood stasis, or cold coagulation and blood stasis, belonging to the heat accumulation type, liver and kidney injury type, and “qi” and blood deficiency type. The syndrome of cold coagulation and blood stasis is mostly seen . Intense2 and PGFα2; PGFα2 releases more than PGE2 which combined with PGFα2 receptor in the uterine spiral artery wall and executes the contraction of uterine smooth muscles. According to researches [α2 : PGE2 ratio is significantly greater as well. This results in uterine local vasoconstriction, blood flow decrease, blood supply deficiency, muscle ischemia, metabolite accumulation, pain, and uterine pressure exceeding the average arterial blood pressure. Consequently, the uterine cavity pressure increases and the uterus wall blood flow reduces, so the microcirculation gets disordered and finally causes dysmenorrhea [During the menstrual period, because of the lysosomal instability, it is easy to cause endometrial cell lysis, resulting in endometrial tissue necrosis and release of large amounts of PGEsearches , the conenorrhea .r < −1). The uterine microvascular and capillary contraction decreases the uterine microvascular blood flow and increases uterine contraction wave number. This furthermore explains that dysmenorrhea could cause uterine muscle spasm, increase contraction, and produce microcirculation disorder. Accordingly, it proves that uterine contraction and uterus microcirculation are closely related to dysmenorrhea environment.According to the data, this study has found that dysmenorrhea of the cold coagulation syndrome presents a high intensity of uterine tension and high contraction of the microvascular diameter, the uterine microcirculation, and tension have negative correlation , which means that using thick needles, deep insertion, and hand manipulation can alleviate spasmodic contraction of uterine muscles and can easily achieve “De qi.” In addition, we have found that, although the therapeutic effect in “B” stimulus group compared to the “A” stimulus group is not significantly different, there still some therapeutic effects, which explains the reason why some superficial needling, such as abdominal needle therapy, is also effective [Based on this experimental study, the “A” stimulus group has better effectiveness than the “B” stimulus group in relieving dysmenorrhea of the cold coagulation syndrome in rats, such as uterine microcirculation diameter, blood flow, and the contraction of uterus and uterine activity. The “A” stimulus group receiving acupuncture for 5 minutes immediately presents some effects on tiny blood vessels, capillaries diastolic, and blood flow, which is greater than “B” stimulus group. After 10 minutes, the expansion of diameter of “A” stimulus group is better than “B” stimulus group, and has significant statistic difference. This further explains that hand manipulation can help the microvessel to relax. Besides, the “A” stimulus group can noticeably reduce uterine contraction and activity (ffective .Dysmenorrhea of the cold coagulation syndrome presents a high intensity of uterine tension and high contraction of the microvascular diameter. Acupuncture applied with two different stimuli could relieve the symptoms, but, compared with “B” stimulus, “A” stimulus leads to better outcomes on reducing uterine contraction and increasing diameter of uterine microvascular. Moreover, we have found that hand manipulation can help to expand the microvascular diameter, which implies that hand manipulation is one of the important factors affecting the curative effect. Our finding highlights using thick needles and deep insertion with hand manipulation to cause appropriate intensity of stimuli to achieve “De qi” state in acupuncture treatment of dysmenorrhea. Results of this experiment remind us that in the treatment of dysmenorrhea with acupuncture appropriate but comparatively strong stimuli with particular hand manipulation can have better therapeutic effect.


Introduction: Lipoprotein (a) [Lp(a)] is considered as a risk factor for coronary atherosclerotic disorder and an increase in plasma Lp(a) concentration is usually seen in patients with kidney failure.Objectives: We aimed in this study to evaluate the impact of plasma Lp(a) level on early changes of atherosclerotic vessels in stable hemodialysis (HD) patients.Patients and Methods: In this clinical study 61 patients (50 non-diabetic (F=20 M=30) and 11 diabetic HD patients), receiving maintenance hemodialysis were included and serum Lp(a) was measured. B-mode ultrasonography of carotid intima-media thickness (cIMT) was also determined.Results: The mean ±SD of patients Lp(a) was 58.5±19 mg/dL. The mean ±SD of diabetic patients’ Lp(a) was 62±12mg/dL and for nondiabetic ones was 57.7± 20mg/dL. In this study the thickening of Intima-media complex in diabetic group was more than non-diabetics, and no significant difference was found of Lp(a) between diabetic and non diabetic HD patients. There was a significant positive association between serum Lp(a) and cIMT.Conclusion: Accelerated atherosclerosis seen in diabetic hemodialysis patients. Lp(a) might have an important role in progression of atherosclerosis to accelerate progressive atherosclerosis in these patients. Accelerated atherosclerosis seen in diabetic hemodialysis patients. Lipoprotein (a) might have an important role in progression of atherosclerosis to accelerate progressive atherosclerosis in these patients.Lipoprotein (a) is a macromolecule consisting a glycoprotein apolipoprotein (a) that is linked to apolipoprotein B-100 on LDL core . EpidemiAtherosclerosis has been shown to be accompanied by some changes in arterial structure. Subtle structural changes such as arterial intima-media thickening (IMT) may occur early in the atherosclerotic disorder process ,4. UsingThere is a significant association between conventional risk factors and increased arterial wall thickness ,4, howevIn this study we, therefore, aimed to evaluate the effects of plasma Lp(a) level on early structural atherosclerotic vascular changes in a group of HD patients under maintenance hemodialysis.This cross-sectional study was conducted on sixty-one patients on maintenance hemodialysis. Cigarette smoking, taking lowering drugs, recent MI, vascular diseases, body mass index (BMI) more than 25, as well as active or chronic infection were considered as exclusion criteria. Lp(a) was measured in patients by enzyme immunoassay (ELISA) method using immuno-biological laboratories (IBL) kits (Germany).Carotid sonography (Honda-Hs-2000 Sonograph with 7.5 MHZ linear probe) was done at the end of diastolic phase by a sonologist who was blind to history or lab data of patients. For examination, patients were placed in supine position with neck hyperextension and rotation of head to facilitate the performance of the procedure. The sites of measurements were at the distal common carotid artery, first proximal internal carotid artery and area of bifurcation. IMT was measured at the plaque free areas ,4. IMT w(1) The research followed the tenets of the Declaration of Helsinki; (2) informed consent was obtained; (3) the research was approved by the institutional review board.For analysis we  measured the mean of right and left  cIMTs. Data were expressed as Mean±SD. Comparison between groups was done using, Mann-Whitney U test, Kruskal Wallis test and Fisher's exact test. To evaluate the correlations we used spearman`s rho test, partial correlation test with adjustment for age, also Phi  & Cramer's V test and Eta test were used. All statistical analysis was performed using SPSS 11 . P of less than 0.05 was considered as significant.The total patients were 61 , consisting of 11 diabetic hemodialysis patients (F=3 and M=8) and 50 non diabetic hemodialysis patients (M=30 and F=20). et al. observed a lot of vascular plaques in patients suffering from Uremia. He concluded that the process of advance atherosclerosis should be started with initiating of renal failure. They suggested that HD treatment might not be a potential factor for accelerating arthrosclerosis. In addition, they concluded that, progression of atherosclerosis should be related to atherogenic factors which exist before regular dialysis  between diabetic and non-diabetic HD patients. Pascasio dialysis . To invedialysis . The cordialysis -9. They dialysis -9. In agdialysis . Likewispatients . Similapatients . Finallypatients .In the present study there was a significant positive association between serum Lp(a) and cIMT. Although, the extraordinary high mortality of HD patients are due to cardiovascular disease, there are some interests toward nontraditional atherosclerotic cardiovascular diseases risk factors such as Lp(a) which are high in HD patients . This needs more attention because of its effects on acceleration of rapid progressive atherosclerosis seen in HD patients.Diabetic HD patients have more accelerated atherosclerosis. Lp(a) as a nontraditional factor in progression of atherosclerosis can have a more important role in acceleration of rapid progressive atherosclerosis observed in these patients.We would like to thank research deputy of Shahrekord University of Medical Sciences for financial support and Dr. M. Mowlaei (sonologist) for carotid ultrasonographies.HN designed and performed the research. MRK wrote some parts of paper. MRK edited the draft. HN prepared the final draft.The authors declared no competing interests.Ethical issues  have been completely observed by the authors.None.


Sitobion avenae, is a major pest species of wheat crops; however, certain varieties may have stronger resistance to infestation than others. Here, we investigated 3 classical resistance mechanisms  by 14 wheat varieties/lines to S. avenae under laboratory and field conditions. Under laboratory conditions, alatae given the choice between 2 wheat varieties, strongly discriminated against certain varieties. Specifically, the ‘Amigo’ variety had the lowest palatability to S. avenae alatae of all varieties. ‘Tm’ (Triticum monococcum), ‘Astron,’ ‘Xanthus,’ ‘Ww2730,’ and ‘Batis’ varieties also had lower palatability than other varieties. Thus, these accessions may use antibiosis as the resistant mechanism. In contrast, under field conditions, there were no significant differences in the number of alatae detected on the 14 wheat varieties. One synthetic line  and Triticum turgidum (var. durum) hybridization) had low aphid numbers but high yield loss, indicating that it has high antibiosis, but poor tolerance. In comparison, ‘Amigo,’ ‘Xiaoyan22,’ and some ‘186Tm’ samples had high aphid numbers but low yield loss rates, indicating they have low antibiosis, but good tolerance. Aphid population size and wheat yield loss rates greatly varied in different fields and years for ‘98-10-35,’ ‘Xiaoyan22,’ ‘Tp,’ ‘Tam200,’ ‘PI high,’ and other ‘186Tm’ samples, which were hybrid offspring of T. aestivum and wheat related species. Thus, these germplasm should be considered for use in future studies. Overall, S. avenae is best adapted to ‘Xinong1376,’ because it was the most palatable variety, with the greatest yield loss rates of all 14 wheat varieties. However, individual varieties/lines influenced aphid populations differently in different years. Therefore, we strongly recommend a combination of laboratory and long-term field experiments in targeted planting regions to identify varieties/lines that consistently show high resistance to S. avenae infestation.The English grain aphid,  Triticum aestivum L.) is a major grain crop worldwide. Cereal aphids cause serious wheat crop yield losses by reducing the number of grains and grain weight of wheat spikes. In addition, they are vectors of the barley yellow dwarf virus (BYDV), with the joint occurrence of the virus and aphids further increasing yield losses [http://www.natesc.moa.gov.cn/). The English grain aphid, Sitobion avenae (Fab.), is the dominant cereal aphid species, causing most damage at the wheat filling stage. This species is documented annually in the wheat fields of northern China, including the central part of Shaanxi Province [Wheat (d losses –2. In ChProvince .S. avenae infestation on resistant wheat varieties. This phenomenon has been detected in England [Schizaphis graminum (Rodani), and the Russian wheat aphid, Diuraphis noxia (Kurdjumov) [Insecticide application is currently the main method used to control cereal aphids in China. However, this technique generates “3R” problems: residues, resistance, and resurgence , in addi England , the Cze England , Pakista England , France  England , 10, and England . The plardjumov) . Even whrdjumov) , which prdjumov) –16. A firdjumov) .Secale cereale L.). Amigo has a single dominant resistant gene located on chromosome 7D, which originates from chromosome 1A of rye [In the USA during the 1950s, hybridization techniques were used to breed a wheat variety that was resistant to the green bug; specifically ‘Amigo,’ which is a hybrid of wheat variety ‘Largo’ and rye , antibiosis, and tolerance , 51. DifHere, we conduct a comprehensive evaluation on the resistance mechanisms of 14 wheat varieties using the 3 classical resistance mechanisms. This research is expected to provide baseline information about the mechanisms that cause resistance in these varieties, and a theoretical basis on breeding resistant wheat varieties to reduce the impacts of aphids for integrated management of aphid.The experiments were performed in a climate chamber laboratory (25 ± 0.5°C (day) and 22 ± 0.5°C (night), with a 16 h light:8 h dark 114 photoperiod, and approximately 70 ± 10% relative humidity (R.H.)) and the experimental fields of Northwest A&F University .T. aestivum and T. monococcum or T. turgidum (var. durum). An einkorn wheat line was used as a resistant control.The 14 wheat varieties (lines) used in this study are listed in S. avenae, and less than 5% are bird cherry-oat aphid Rhopalosiphum padi L. and greenbug aphid Schizaphis graminum (Rondani) [S. avenae aphids in our experiment.In the wheat field population of Shaanxi Province, China, more than 95% aphids are Rondani) . TherefoS. avenae aphids used in the antixenosis experiment was originally obtained from a single wingless individual that was found on a winter wheat plant in the experimental fields. The stock population was maintained in a separate cage on wheat seedlings (variety ‘Xiaoyan22’) in a climate chamber.The stock population of S. avenae alatae .Antixenosis represents a clear non-preference of a plant to serve as a host to an arthropod. Specifically, 3 experiments were conducted to determine the antixenosis of wheat varieties to 2 area. The blocks were 4 m long, and consisted of 20 rows with 25 cm spacing . Before planting, 10 kg/666.7 m2 of nitrogen (ammonium nitrate) and 25 kg phosphate fertilizer (potassium dihydrogen phosphate) were applied to the experimental plots. An additional top-dressing of 10 kg/666.7 m2 nitrogen was added at the stem elongation stage (March) of winter wheat. To avoid assessing the mutual effect between different wheat varieties/lines planted in neighboring plots, data were only collected from a 12 m2 (3 m × 4 m) area in the center of each repeated block. Natural infestation by adult S. avenae alatae was counted 3 times in the 12 m2 center area of each repeated block, with 1 week separation between the end of March to mid-April, This is because, during this period, alatae migrated from wheat fields in southern China [The first antixenosis field experiment was conducted from 2003 to 2006. The wheat varieties/lines were sown on October of the previous year (2003 and 2005), and were harvested on June of the next year (2004 and 2006). Two fields were planted in 2003 and harvested in 2004, marked as 2004-A and 2004-B. A single, large field was planted in 2004 and harvested in 2005. This large field was divided into 3 sections, marked as 2005–1, 2005–2, and 2005–3. One field was planted in 2005 and harvested in 2006, and was marked as 2006. Over the 3-year period, each variety/line had a total of 6 replications in 6 fields/sections. Each replicated block of each variety/line covered a 20-mrn China , 54.The second antixenosis experiment was performed under laboratory conditions from September 2011 to November 2011. In this experiment, aphid alatae were allowed to choose among the 14 wheat varieties. Five seeds from each variety/line were planted in separate plastic pots . The 14 pots containing wheat seedlings at the 2-leaf stage were randomly arranged in a circle, and were covered with a 2 m × 2 m × 1.5 m rectangular gauze cage to stop the alatae from escaping. One-hundred adult aphid alatae were deprived of food for 24 h, and were then released into the center of the experimental plants in the cage. The number of adult aphid alatae that established on each variety/line was counted and recorded after 1, 2, 4, 8, and 24 h. This experiment was repeated 3 times. The randomly layout of pots within the three repeats was different.The third antixenosis experiment was a palatability experiment, which was performed under laboratory conditions from February 2012 to May 2012. In this experiment, aphid alatae were allowed to choose between 2 wheat varieties. The 14 wheat varieties/lines were combined as 91 pairs (14 × 13 ÷ 2). The leaves of each paired combination of wheat leaves were cut to a length of 3 cm, and were placed on opposite sides of a 9-cm-diameter Petri dish. The bottom of the Petri dish was covered with filter paper, on which 2 ml distilled water was added to keep the leaves moist. Subsequently, 20 alatae aphids that had been deprived of food for 24 h were released in 2 batches onto the center of the Petri dish. After 1, 2, 4, 8, and 24 h, the number of aphids that had established on the leaves of each cultivar/line was counted and recorded. This experiment was repeated 3 times.Antibiosis is the antagonistic association between 2 species, in which at least 1 of the 2 species is adversely affected. It is a heritable quality possessed by a plant that adversely affects the life history or biology of the insect , 51. In 2 central area of each repeated block. Five 1 m long rows were sampled per block. In this antibiosis experiment, all aphids  from 5 points in each block were counted once a week from mid-March to the end of May . Direct counts were used before the end of April, and scale counts, which were the average aphid number of 10 heads multiplied by count wheat head number in 1 m long rows, were used following the end of April. The maximum aphid number  and the aphid cumulative count (ACC) were integrated to elucidate aphid population development. The sum of the MAN and ACC at the 5 points in each repeat block was used as a single sample. The index of aphid susceptibility (IAS) was counted as:IAS = the number of aphids at each point/ the average number of aphids at all points combined Data collection was performed in the 12 mIAS ≤ 0.3 indicated high resistance, 0.3 < IAS ≤ 0.6 indicated intermediate resistance, 0.6 < IAS ≤ 0.9 indicated low resistance, 0.9 < IAS ≤ 1.2 indicated low susceptibility, 1.2 < IAS ≤ 1.5 indicated intermediate susceptibility, and IAS ≥ 1.5 indicated high susceptibility.loss ratio = / control value Tolerance is a genetic trait of a plant that enables it to tolerate higher pest populations than a susceptible cultivar before damage occurs , 51. At 2 core area, but within the 20-m2 area of each block. The control points were sprayed 3–5 times with imidacloprid  at the start of April and end of May to eliminate all aphid infestation. When spraying, both sides of the control line were protected with cardboard to prevent the pesticide from reaching the other wheat plants [The control points were 1 m long rows. These points were located outside the 12-mt plants , 29.There were 2 fields in 2004, 3 sections in 2005, and 1 field in 2006. The results for each field or section were assumed to be independent in different years; thus, the 6 fields/sections were treated as a single factor in the field experiments when using ANOVA. The number of adult aphid alatae was very small in early spring; thus, the total number of alatae obtained from each repeated block was combined as a single sample for each wheat variety/line. Then, two-way (14 wheat varieties and 6 fields/sections) ANOVA without duplication was used in the first antixenosis field experiment. The number of aphid alatae was analyzed using a repeatable one-way ANOVA in the second and third antixenosis laboratory experiments. MAN and ACC were transformed using natural logarithm transformation to ensure homogeneity of variance. The loss ratios of TY, AY, and KW were transformed using arcsine transformation to reduce variance. IAS were not transformed. Then, these parameters were analyzed using a two-way (14 wheat varieties and 6 fields/sections) ANOVA with the duplication of random factors model. All of the parameters were analyzed using ANOVA with General Linear Model (GLM). The means were separated by Tukey’s test under α = 0.05 . All figures were drawn with SigmaPlot 12.1 .F = 298; D.F = 13, 28; p < 0.001). After 8 h, aphid choosing behavior plateaued. After 24 h, the highest number of alatae was attracted to ‘Xinong1376,’ ‘Xiaoyan22,’ ‘PI high,’ ‘Tp,’ ‘Tam200,’ and ‘186Tm’ (from high to low), higher number of alatae was observed on ‘Ww2730,’ ‘98-10-35,’ and 98-10-30.’ The lowest number of alatae was observed on ‘Amigo,’ lower number of alatae attracted to each plant was observed on ‘Astron,’ ‘Tm,’ ‘Batis,’ and ‘Xanthus’ (from low to high) in 14 wheat varieties/lines  . However, there were significant different between 6 fields/portions  , significant differences in the average number of alatae on each variety/line were detected after 24 h Figs  and 2 (Fes/lines . ANOVA i = 0.18) . The sec= 0.014) . The totS. avenae population were similar among all 14 wheat varieties/lines in all 6 fields/sections over the 3-year study period . In contrast, ANOVA indicated no variation among the 6 fields/sections . Higher mean numbers of MAN and ACC were observed for ‘Tp,’ ‘Tam200,’ ‘186Tm,’ ‘98-10-35,’ ‘PI high,’ and ‘Xiaoyan22.’ These varieties/lines were considered to be susceptible to aphid infestation. Lower mean numbers of MAN and ACC were observed on ‘98-10-30,’ ‘Astron,’ ‘Xanthus,’ ‘Ww2730,’ and ‘Tm.’ These varieties/lines were considered to be relatively resistant to aphid infestation. There were significant interactions between the wheat varieties and replications . When checking the 14 wheat varieties, we found the MAN and ACC of 3 resistant  and 1 low susceptibility (‘Xinong1376’) lines/varieties were not significantly different among the 6 fields/sections (In the ANOVA-based random model with 2 factors (14 wheat varieties and 6 fields/sections) variation existed among the 14 wheat varieties/lines with respect to MAN and ACC  and interactions between the wheat varieties and replications . There was no significant difference among the 6 fields/sections. The IAS identified ‘Tm’ and ‘Ww2730’ as stable resistant wheat germplasms in 6 fields/sections over the 3 years. ‘Xinong 1376’ was also identified as a stable wheat germplasm, but with low susceptibility over the same period. The IAS of ‘98-10-30,’ ‘Astron,’ ‘Xanthus,’ and ‘Batis’ was relatively stable, ranging between 0.5 and 1.2. The IAS of ‘Xiaoyan22,’ ‘Amigo,’ ‘Tam200,’ ‘Tp,’ ‘186Tm,’ ‘PI high,’ and ‘98-10-35’ fluctuated broadly across the 6 fields/sections over the 3 years.The ANOVA based on the random model with 2 factors (14 wheat varieties and 6 fields/sections) showed a significant difference for IAS based on MAN and ACC among the 14 wheat varieties , TY , and AY  among the 14 wheat varieties . The interactions between the wheat varieties and replications for KW  was significant. However, a significant relationship was not detected for the interactions of the wheat varieties with TY and AY . The highest average KW, TY, and AY yield loss rates were obtained for ‘98-10-30,’ ‘Xinong1376,’ ‘PI high,’ and ‘98-10-35.’ The lowest average KW, TY, and AY yield loss rates were obtained for ‘Xanthus,’ ‘Amigo,’ ‘Astron,’ ‘Ww2730,’ and ‘Tm.’ Intermediate average KW, TY, and AY yield loss rates were obtained for ‘186Tm,’ ‘Tp,’ ‘Batis,’ and ‘Xiaoyan22.’The ANOVA based on the random model with 2 factors (14 wheat varieties and 6 fields/sections) showed significantly different average loss rates for KW . Liu et al. reported that the IAS of S. avenae is about 1.0 on ‘Batis’ and ‘Ww2730’ at the adult plant stage in other fields in Yangling, Shaanxi Province [S. avenae populations, whereas ‘Batis’ was susceptible [S. avenae salivated more in the sieve elements of ‘Batis’ than ‘Ww2730,’ and spent more time passive sucking in the sieve elements of ‘Batis’ than ‘Ww2730,’ and ‘Ww2730’ also has other factors in the epidermis, mesophyll, and phloem that restrict feeding by aphids [S. avenae.We found that the average IAS of Province . In Germceptible , 78. ‘Asy aphids . Thus, tT. aestivum (var. Chris) and T. turgidum (var. durum) hybridization. Both lines have high hydroxamic acid, DIMBOA  content [S. avenae under laboratory conditions [T. aestivum and wheat related species, ‘Tam200,’ ‘Tp,’ ‘Xiaoyan22,’ ‘PI high,’ and ‘98-10-35, even among 3 repeats in 2005.’ Thus, we conjecture that the quantitative trait locus (QTL) influences resistance in heterozygote and control varieties/lines, in addition to variation in meteorological conditions.The synthetic wheat lines ‘98-10-30’ and ‘98-10-35’ are the offspring of  content , and appnditions . In our nditions . The resnditions . Highly Triticum monococcum) as a resistant germplasm in all repeats over the 3 years. The resistance mechanism of this variety is antibiosis and non-preference. Seventy percent of S. avenae alatae preferentially fed on ‘Xinong1376’ after 24 h when given the choice between 2 wheat varieties. ‘Xinong1376’ contained large numbers of aphids, and had high yield loss rates in most repeats. This result was similar to that obtained by Li et al. and Liu et al. [S. avenae in China [S. avenae on‘Xinong1376’ was shorter compared to other varieties, with greater fecundity and intrinsic rate of increase than other varieties [Our results identified ‘Tm’ , which, in turn, is controlled the by micro effect of multiple genes, with resistance levels being influenced by environmental factors and aphid adaptability.We detected high variation in the IAS and yield loss rate variations of ‘186Tm,’ ‘Tp,’ ‘Tam200,’ and ‘PI high’ in different fields and years, similar to that detected for ‘90-10-35.’ The resistance of some wheat varieties to nt genes  or dominnt genes , 83–85. Our results showed that the yield loss rate of some wheat varieties/lines broadly varied across years. However, such variation was less for ‘Amigo,’ ‘Xiaoyan22,’ and some sample points of ‘186Tm’ compared to other varieties subject to similar or higher aphid numbers . In gene2, with the sample area for the 5-point sampling being 0.25 m2, in which 80–130 wheat plants were planted.Screening for resistant germplasm in the laboratory may produce different results to field screening. Field screening is used to determine the resistance level of wheat germplasm to aphids based on natural aphid numbers that occur on wheat varieties . This teS. avenae aphids may differ between laboratory and field experiments, due to the different settings. In addition, under field conditions, the resistance of different varieties differs across fields and years due to the way in which aphids aggregate, as well as differences in the genetic mixing of aphid stocks across years and climatic conditions. Therefore, we strongly recommend a combination of laboratory and long-term field experiments in the target planting regions to identify varieties/lines that consistently show high resistance to S. avenae infestation. Our study suggests that ‘Amigo’, ‘Astron,’ ‘Xanthus,’ and ‘Batis’ use antibiosis as the resistant mechanism. ‘98-10-30,’ ‘Astron,’ ‘Xanthus,’ ‘Ww2730,’ and ‘Tm’ also use antibiosis. ‘Amigo,’ ‘Xiaoyan22,’ and some ‘186Tm’ samples had good tolerance. Aphid population size and wheat yield loss rates greatly varied in different fields and years for ‘98-10-35,’ ‘Tp,’ ‘Tam200,’ ‘PI high,’ and other ‘186Tm’ samples, all these germplasm should be considered for use in future studies.In conclusion, our study demonstrates that the detected resistance of wheat varieties to S1 Table(DOCX)Click here for additional data file.


Understanding which factors affect the distribution pattern of extremely small populations is essential to the protection and propagation of rare and endangered plant species. In this study, we established 108 plots covering the entire Hainan Island, and measured the appearance frequency and species richness of plant species with extremely small populations, as well as the ecological environments and human disturbances during 2012–2013. We explored how the ecological environments and human activities affected the distribution pattern of these extremely small populations. Results showed that the extremely small populations underwent human disturbances and threats, and they were often found in fragmental habitats. The leading factors changing the appearance frequency of extremely small populations differed among plant species, and the direct factors making them susceptible to extinction were human disturbances. The peak richness of extremely small populations always occurred at the medium level across environmental gradients, and their species richness always decreased with increasing human disturbances. However, the appearance frequencies of three orchid species increased with the increasing human disturbances. Our study thus indicate that knowledge on how the external factors, such as the ecological environment, land use type, roads, human activity, etc., affect the distribution of the extremely small populations should be taken for the better protecting them in the future. Plant population is the most basic level of plant ecology, connecting plant individuals with communities and ecosystems. The ecological patterns of a plant population are characterized by its population dynamics, as well as the relationships between the population and the external environments Both internal and external factors cause plants to become vulnerable to extinction. Internal factors refer to the plant biological characteristics, including failures in heritability, reproduction, viability, and adaptability Hainan is the largest island of the Indo Burma Biodiversity Hotspot, and has the best preserved and the most extensive tropical forests in China. It contains rich rare and endangered plant species, and therefore, becomes a priority for species conservation in China The State Forestry Administration of China has been carrying out the “Conservation Program for Wild Plants with Extremely Small Populations in China (from 2011 to 2015)”. In this plan, 24 wild plant species with extremely small population in Hainan are recorded. The term “extremely small population” refers to a population with a narrow geographical distribution that has been disturbed and constrained by external factors over a long time, and contains less individuals than the minimal number requirements inhibiting from extinction. Some of these plants are qualified as the biological resources important to the country's ecology, science, culture, and economics 2. The island is high at its center and relatively low around its perimeter. The highest peak is Wuzhishan Mountain, with an altitude of 1,867 m  and covers a total area of 33,900 km 1,867 m . CurrentA total of 108 plots were established covering areas of tropical rainforest, tropical monsoon forest, mangrove forest, tropical coniferous forest, tropical evergreen and semi-evergreen shrubs, semi-natural vegetation, and artificial vegetation. Plots were mainly distributed in the central and southern mountainous areas because of their rich rare and endangered wild plants . Each plA comprehensive survey was performed to identify plant species names, and measure the all individuals across the entirety of Hainan Island during 2012–2013. The survey of wild plants with extremely small populations in Hainan is permitted and approved by the Ministry of Agriculture of the People's Republic of China, and the Wildlife Protection Bureau of Hainan Province. Our work was conducted with a focus on the protection of wildlife and causing no threats or damage to wildlife, especially to endangered or protected species.Cycas changjiangensis, Michelia odora, Chieniodendron hainanense, Ilex kaushue, Horsfieldia kingii, Paranephelium hainanense, Sonneratia hainanensis, Lumnitzera littorea, Hopea hainanensis, Bulbophyllum hainanense, Ceratostylis hainanensis, Cymbidium eburneum, Cymbidium insigne, Oxystophyllum changjiangense, Dendrobium hainanense, Dendrobium sinense, Dendrobium strongylanthum, Doritis pulcherrima, Pinalia quinquelamellosa, Dendrolirium tomentosum, Gastrochilus acinacifolius, Phaius hainanensis, Sunipia hainanensis, and Thrixspermum odoratum. We found that four species  that had previously been recorded in Hainan Island were not found in our field survey. Thus, further investigation is required to determine whether these species have disappeared from the wild. Among the remaining 20 species with extremely small populations, we recorded the distributed area, individual number and habitat characteristics  with a spatial resolution of 0.0083° (approximately 1 km2 at the equator) LAI 4 Ring (LAI), is the effective leaf area index integrated over the zenith angles 0–60°. Canopy density is the percentage of shelter sky seen from beneath a forest canopy. A hemispherical photograph was taken in each 20×20 m-quadrat using a Nikon 4500 Coolpix Digital camera  fitted with a Nikon FC-E8 fisheye converter  We measured 13 variables which were divided into five types. (1) Topographical: altitude, slope aspect, slope gradient, and exposure of rock. Altitude, slope aspect, and slope gradient were measured using the global positioning system, a compass, and a slope measuring instrument, respectively. And the exposure of rock was estimated by the ratio of the exposed surface stone area to the total area of the quadrates. (2) Climate: annual average temperature and average annual precipitation. These variables were extracted for each site from the WorldClim database  was first used to analyze the 13 variables to reduce the dimension of factors. The top eight variables with absolute eigenvalues more than 0.620 were selected. Then the Canonical correspondence analysis (CCA) and the bivariate correlation analysis were performed to examine the relationship of these selected variables with the appearance frequency of each extremely small population.We compared the species richness of the extremely small populations that were found at the different levels of the each selected variables using one-way ANOVA.j = C/(A+B−C) We divided the altitude values into six levels from 0 to 1800 m: 0–300 m (8 samples), 300–600 m (22 samples), 600–900 m (16 samples), 900–1200 m (44 samples), 1200–1500 m (10 samples), and 1500–1800 m (8 samples), and then analyzed the species similarity between neighboring altitudes using the following Jaccard similarity coefficient index: CWe divided the slope aspect into eight ranges: north slope , northeast slope , east slope , southeast slope , south slope , southwest slope , west slope , and northwest slope , and then calculated the frequency for each plant species with extremely small populations occurred at the eight slope aspects. Before the CCA analysis, the slope aspect was cosine transformed, and resulted in values ranged from 0 (south) to 2 (north) We divided the slope gradient into six levels ranged from 0° to 90°: 0°–15° (15 samples), 15°–30° (34 samples), 30°–45° (40 samples), 45°–60° (9 samples), 60°–75° (4 samples), and 75°–90° (6 samples). Canopy density was divided into six levels from 0.4 to 1.0: 0.4–0.5 (8 samples), 0.5–0.6 (7 samples), 0.6–0.7 (12 samples), 0.7–0.8 (28 samples), 0.8–0.9 (24 samples), and 0.9–1.0 (29 samples). Distance correlation analysis was then performed to calculate the Pearson similarity coefficient, using the presence/absence data of each extremely small population for the six levels of slope gradient or canopy density. Distance between population and road was divided into five levels: 0–200 m (25 samples), 200–400 m (26 samples), 400–600 m (21 samples), 600–800 m (16 samples), and 800–1000 m (20 samples). Road quality, surrounding population density, and land use type were each divided into four levels according to their 1–4 scales.PCA, bivariate correlation analysis and distance correlation analysis were performed using SPSS 19.0. CCA was performed using CANOCO version 4.5 for Windows, and a Monte Carlo permutation test was conducted with 1000 randomizations for the significance of both the variables and the ordination axes PCA showed that the accumulative contribution rate of the three principal components were 58.380% . Thus thP = 0.002) between the eight variables and the ordination axes, using the Monte Carlo permutation test. The appearance frequencies of all species were affected by the eight factors, with a decreasing order of importance: altitude, land use type, distance between population and road, road quality, slope aspect, surrounding population density, canopy density, and slope gradient , but were significantly negatively correlated with surrounding population density and agricultural activities (P<0.01). Three species with less than 5 distribution sites and more than 100 individuals (see P<0.05). Three species with more than 5 distribution sites and 100 individuals (see P<0.05), but were significantly and positively correlated with agricultural activities (P<0.05)  .P<0.01), but were significantly positively correlated with distance between population and road (P<0.05). One species with less than 5 distribution sites and more than 100 individuals (see P<0.05). Two species with more than 5 distribution sites had significant positive correlations with altitude, and distance between population and road (P<0.05), but had extremely significant negative correlations with agricultural activities (P<0.01)  .F = 43.43, P<0.001; Road Quality: F = 5.86, P = 0.001; F = 32.55, P<0.001; Agricultural Activities: F = 12.22, P<0.001; The species richness of plant species with extremely small populations decreased with the increasing levels of road quality, agricultural activities, and surrounding population density, but increased with increasing distance of population and road . The speF = 3.98, P = 0.002; The species richness of extremely small populations showed a unimodal curve with the increasing altitude. The species richness among six levels of altitudes were significantly different  = 2.16, P = 0.012; Chieniodendron hainanense, Dendrobium hainanense) were found on all slope aspects. Three species  were mainly observed on the north, northeast, and northwest aspects. Seven species  mainly occurred on the south, southeast, and southwest aspects  = 3.99, P = 0.002; The species richness among the six levels of slope gradient were significantly different  = 13.34, P<0.001; The species richness among the six levels of canopy density were significantly different  with extremely small populations were not found in our field survey, and the remaining 20 species underwent human disturbances and threats. Thus the population size of these species are smaller than the minimum number requirements inhibiting from extinction, suggesting that these extremely small populations face the threat of extinction if no protective actions are taken. Our findings have been demonstrated by the view that the main factors causing rare and endangered plants susceptible to extinction are habitat destruction, plant overexploitation and the ecological environment deterioration As expected, we found that plant species with extremely small populations often lived in a fragmental habitat . For exaAlthough there is one or several factors causing plant species endangerment, these factors may be completely different for different species For most of the endangered plants, the direct factors leading them susceptible to extinction are human activities Dendrobium hainanense, Oxystophyllum changjiangense, Dendrolirium tomentosum) were significantly and positively correlated with roads and agricultural activities Click here for additional data file.


Acid-sensing ion channels (ASICs) are proton-gated cation channels that mediate acid-induced responses in neurons. ASICs are important for mechanosensation, learning and memory, fear, pain, and neuronal injury. ASIC2a is widely expressed in the nervous system and modulates ASIC channel trafficking and activity in both central and peripheral systems. Here, to better understand mechanisms regulating ASIC2a, we searched for potential protein motifs that regulate ASIC2a trafficking.We identified a LLDLL sequence in the C-terminal juxtamembrane region of ASIC2a. Deleting or mutating the LLDLL sequence increased total expression and surface levels of ASIC2a in CHO cells. Mutating either of the two LL motifs had a similar effect. We further assessed ASIC2a localization in organotypic hippocampal slice neurons. The LL motif mutants exhibited increased dendritic trafficking and elevated targeting to dendritic spines. Consistent with an efficient trafficking, the LL motif mutants increased acid-activated current density. In addition, mutating the second LL motif increased pH sensitivity of the channel. These data identify the LL motifs as a negative regulator of ASIC2a trafficking and function, and suggest novel regulatory mechanisms in acid signaling. Acid-sensing ion channels (ASICs) are the main class of proton receptors in brain neurons. Previous studies have shown that ASICs are important for neuron physiology and synaptic plasticity, and play critical roles in fear- and anxiety-related behavior in mice , 2. In a50 and faster rate of desensitization [ASIC2 gene altered acid-activated current properties in CNS neurons, and reduced acid-activated calcium rise in hippocampal slices [The major ASIC subunits expressed in the brain are ASIC1a, 2a and 2b . ASIC1a tization . In the tization . Deletinl slices –15. Consl slices . In addil slices .The current data showed that ASIC2 is important in regulating the outcome of acid signaling in the brain. Moreover, ASIC2a contributed to a large proportion of functional ASICs (primarily in the form of ASIC1a/2a hetermoers) in the brain . These d462LLDLL” in the C-terminal juxtamembrane region. Previous studies have shown that the di-leucine (LL) motif is important for ER retention, intracellular sorting, and/or surface expression of multiple proteins [p = 0.0504). In contrast, the DAA mutant exhibited a bigger effect, and led to a 27 % increase (p = 0.003) in surface:total ratio of ASIC2a. These data suggest that the second LL motif is more important for ASIC2a surface trafficking.To identify potential motifs in ASIC2a, we examined the intracellular tail of ASIC2a. We noticed the presence of “proteins –24. Therproteins , but preNext, we performed immunofluorescence in transfected CHO cells to visualize the localization of WT and mutant ASIC2a. Compared to the soluble GFP, WT ASIC2a showed a membranous localization as expected Fig. , althougASIC2−/− mice [p < 0.01, ANOVA followed by Turkey’s HSD test). These results indicate that mutating the LL motifs led to increased dendritic trafficking and spine targeting of ASIC2a.In a previous study, we have shown that ASIC2a exhibited a somatodendritic distribution and was enriched in dendritic spines . The inc−/− mice . We tran−/− mice , ASIC2a N-glycosylation of ASIC1a and ASIC2a was important for their trafficking [N-glycosylation. To test this hypothesis, we treated CHO cell lysates with two endoglycosidases: Endo H and PNGase F. PNGase F removes all N-linked glycans. In contrast, Endo H only removes the core-glycans added in endoplasmic reticulum (ER) but cannot cleave more complex (“mature”) glycans that have been modified in mid- to late-Golgi. The removal of N-linked glycans resulted in a faster migrating population on the gel . One general technical consideration for most patch clamp studies is that it is harder to obtain good space clamp with large currents . Although this may affect the exact values of current amplitude and pH50, it does not alter our main conclusion that the mutants had increased acid-activated current and the DAA mutant showed an increased pH sensitivity.The increase in surface ASIC2a level suggests an increase in acid-activated current. In addition, besides regulating channel biogenesis and trafficking, protein motifs may also alter channel function. To address these issues, we studied acid-activated current of CHO cells transfected with WT or mutant ASIC2a. Cells expressing either WT or mutant ASIC2a exhibited typical ASIC-type current in response to pH 4.5 stimulation Fig. . Furtherp = 0.049). These data, together with our current recordings, indicate that most of the effect on ASIC2a surface trafficking and channel function was mainly mediated by the second LL motif. We speculate that the exact location of the LL motif may contribute to the differences observed between mutating the two LL motifs. It remains unclear as to the exact mechanism of how the LL motifs regulate ASIC2a. Our data here showed that the AADAA and DAA mutants increased the maturation of N-linked glycans. N-glycosylation is an important process in protein sorting and trafficking [N-linked glycans regulated ASIC1a trafficking [Previous studies have identified a number of mutants and/or motifs that affect ASIC channel trafficking and/or function –34. Mostfficking . We and fficking , 36. Thefficking –39. Besifficking , 40. It Do LL motifs affect the response of ASIC2a to various pharmacological inhibitors or modulators of ASICs? The LLDLL sequence locates inside the cell, right after the second transmembrane domain of ASIC2a. Therefore, mutating the LL motifs is unlikely to have a direct effect on most pharmacological reagents that bind to the extracellular side of ASICs, e.g., amiloride, diminzenes, or mitTx –43. Inte, we identified a LLDLL sequence, which contains two LL motifs, in the C-terminus of mouse ASIC2a. Both LL motifs were involved in ASIC2a expression and trafficking. Mutating either one resulted in increased ASIC2a surface trafficking and dendritic targeting, and elevated acid-activated current. Mutating the second LL motif also increased pH sensitivity of the channel. These data indicate that the LL motifs play a critical role in modulating ASIC1a/2a heteromer trafficking and function. These data suggest potential novel mechanisms on the regulation of acid-activated responses through regulating the LL motifs in ASIC2a.In summaryASIC2−/− mice on a congenic C57 background was kindly provided by Dr. Michael Welsh. Wild-type (WT) and knockout mice were maintained as described earlier . PostnatWild-type mouse ASIC2a constructs  have been described previously , 48. TruCHO-K1 cells were purchase from ATCC. CHO cell culture and lipofectamine 2000 mediated transfection were performed as described earlier . For immSurface biotinylation, NeutrAvidin pulldown, and de-glycosylation were performed similar to what was described earlier . The samOrganotypic hippocampal slice was isolated from postnatal day 6–7 ASIC2a−/− mice and cultured as described earlier , 27. MedConfocal images were captured using a Nikon A1 laser scanning microscope. Illumination was provided by an argon  and a 561 diode laser. Green and red channels were imaged sequentially, using 488 nm excitation and a 525/50 emission filter and 561 nm excitation and a 595/50 emission filter, respectively. Images were captured with a 20x or a 63x PL APO water lens. Dendritic and spine images were exported and analyzed with NIH ImageJ as described previously .2, 10 HEPES, 11 EGTA, 2 TEA, 4 MgCl2, pH 7.3, adjusted with CsOH, 290–300 mOsm. Extracellular solution contained: 140 NaCl, 5.4 KCl, 20 HEPES, 10 Glucose, 2 CaCl2, 1 MgCl2, pH 7.4 or 4.5, adjusted with NaOH and HCl, 320–330 mOsm. Whole-cell currents were recorded using Axopatch 200B amplifier  and pCLAMP software. Signals were filtered at 2 kHz, and digitized at 5 Hz using Digidata 1322A (Axon Instruments). The recordings with an access resistance of less than 10 MΩ and a leak current less than 100 pA at −60 mV were included for data analysis. Extracellular acidic solution was applied for 4 s with an interval of 1 min.Whole-cell patch-clamp recordings were performed similar to what was described previously . CHO celt-test. For multiple comparisons, we used one way ANOVA followed by Turkey’s HSD test. For electrophysiological data, we used Kruskal-Wallis One Way Analysis of Variance on Ranks followed by Dunnett’s test. Data were reported as mean ± s.e.m. for the number of samples indicated. Differences were considered significant if p < 0.05.For comparing two groups, we used paired


Interestingly, α-synuclein and tau share striking common characteristics suggesting that they may work in concord. Tau and α-synuclein are both partially unfolded proteins that can form toxic oligomers and abnormal intracellular aggregates under pathological conditions. Furthermore, mutations in either are responsible for severe dominant familial neurodegeneration. Moreover, tau and α-synuclein appear to promote the fibrillization and solubility of each other in vitro and in vivo. This suggests that interactions between tau and α-synuclein form a deleterious feed-forward loop essential for the development and spreading of neurodegeneration. Here, we review the recent literature with respect to elucidating the possible links between α-synuclein and tau.The accumulation of α-synuclein aggregates is the hallmark of Parkinson’s disease, and more generally of synucleinopathies. The accumulation of tau aggregates however is classically found in the brains of patients with dementia, and this type of neuropathological feature specifically defines the tauopathies. Nevertheless, in numerous cases α-synuclein positive inclusions are also described in tauopathies and  To date, PD and AD remain incurable and only very limited palliative treatment options exist [Age-related neurodegenerative disorders like Alzheimer’s disease (AD) and Parkinson’s disease (PD) take an overwhelming toll on individuals and society . AD and ns exist . The etins exist .Interestingly, at the molecular level, protein misfolding, accumulation, aggregation and subsequently the formation of amyloid deposits are common features in many neurological disorders including AD and PD. Thus neurodegenerative diseases are sometimes referred to as proteinopathies . The exiPD is pathologically characterized by the presence of Lewy bodies in the subcortical regions of the brain, which are composed of aggregated and phosphorylated alpha-synuclein protein (αsyn) Figures  and 3 55. Hence The concept of the existence of a continuum between pure synucleinopathies and tauopathies has emerged and is supported by clinical observations of a high comorbidity and overlap between neurodegenerative disorders Figure , in partSNCA) mutations [PARKIN or LRRK2 gene mutations, the inconsistent accumulation of tau, αsyn, neither, or both proteins has been observed [There are many exceptions to the classical view that αsyn and tau pathology are the hallmarks of PD and AD , the obvutations –34. In aobserved , 35.In PD and PDD cases with tauopathy, phospho-tau is restricted to striatal tissues and dopaminergic neurons , 37 and MAPT) or SNCA genes can phenotypically present with a combination of both parkinsonism and dementia. For instance, familial forms of parkinsonism due to αsyn pathogenic substitutions  or due to the duplication or triplication of the wild-type (wt) SNCA gene commonly present with additional atypical clinical signs such as hallucinations, cognitive impairment, and dementia [It is fascinating to observe that familial cases carrying mutations in the microtubule-associated protein tau  and  and GAK,ies . HoweveMAPT locus with disease susceptibility was found in a recent genome-wide association study [SNCA single nucleotide polymorphism (SNP) rs2572324 has been correlated with the extent of neocortical Lewy body and neurofibrillary pathology [MAPT/tau with PD. For instance, a SNP located within the RIT2 gene, was recently nominated through a meta-analysis of genome-wide association studies. GTP-binding protein Rit2 binds to calmodulin 1 , which also binds to both tau and αsyn [GSK3β gene, an established tau kinase [SNCA and MAPT loci. An additive or even multiplicative effect between polymorphisms in SNCA and MAPT would be expected if both genes interact within the same pathogenic pathway. One study did suggest a synergistic increase in the susceptibility of developing dementia in patients with PD when a SNCA risk allele was analyzed with MAPT H1/H2 inversion polymorphism [SNCA and MAPT (or LRRK2) polymorphisms were found to increase PD susceptibility in two other epidemiological studies [In DLB, although no significant association of the on study , correlaon study . The SNCathology . These oand αsyn . Increasu kinase , althougu kinase . Interesmorphism . Convers studies , 85 and  studies .MAPT and SNCA, toxin-based rodent models characterized in vivo parkinsonism research. The discovery that dopaminergic mid-brain neurons are especially sensitive to oxidative stress inducers such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 6-hydroxydopamine (6-OHDA), rotenone, and paraquat resulted in the creation of toxin-based rodent models to study parkinsonian phenotype in vivo . Interer review –90. In rr review . Insolubr review  and Dukar review . Later, r review . Howeverr review . These asubstantia nigra of rats, overexpression of human wt and P301L tau, but not αsyn, provoked dopaminergic neurodegeneration, reduced striatal dopamine content, and motor deficit as measured by amphetamine-stimulated rotational behavior [The effect of targeted human αsyn and tau protein expression has been investigated using viral vectors-based models. In these models, co-occurrence of tau and αsyn pathologies has also been observed. For instance, in rats, αsyn overexpression induced by stereotaxic injection of lentivirus increases phospho-tau levels . On the behavior . In thisbehavior .Numerous genetically modified mice that overexpress the human tau and/or αsyn proteins have been generated and are used to model specific aspects of the human diseases. Interestingly, tau transgenic models not only develop cognitive changes but also motor dysfunction. Mice overexpressing the mutant K369I tau develop L-DOPA sensitive parkinsonism , and oveLikewise, cognitive deficits and tauopathy have been observed in αsyn-overexpressing PD models –103. IntSome groups also crossed different transgenic mouse models to trigger the co-occurrence of tauopathy and synucleinopathy with the final aim to better model complex human disorders like DLB. For instance, a quadruple transgenic mouse line has been generated by crossing a triple transgenic mouse that overexpresses human AD-causing M146V presenilin-1, APP Swedish mutation, and the FTDP-17 T-causing P301L tau with a transgenic mouse that overexpresses human PD-causing A53T αsyn . Co-oversubstantia nigra[Several tau- and αsyn- deficient mouse lines have been generated to determine if any particular phenotype or resistance to neurodegeneration might be present. Generally, tau- or αsyn-deficient mice are viable with only minor phenotypic differences –112. Remia nigra, but couin vivo models have been developed. These models work surprisingly well and present many practical aspects [Caenorhabditis elegans, expression of the human αsyn or tau protein in neurons recapitulates key features of the human diseases such as motor deficits and neuronal and dendritic loss [Drosophila melanogaster also induces neurotoxicity as well as L-DOPA-sensitive motor deficits and formation of αsyn-positive fibrils [D. melanogaster but not in C. elegans, tau expression conversely leads to the formation of insoluble inclusions in C. elegans but not in D. melanogaster[D. melanogaster were rescued without suppressing the presence of αsyn inclusions by co-expression of the Hsp70 chaperone [D. melanogaster has been shown to induce motor dysfunction, dopaminergic denervation, cytotoxicity, formation of abnormal ubiquitin positive inclusions, axonal transport disruption, and cytoskeletal and synaptic disorganization [D. melanogaster was linked to severe cytoskeletal and axonal disorganization and subsequent synaptic alterations rather than αsyn–promoted tau hyperphosphorylation [In addition to rodents, non-vertebrate αsyn and/or tau transgenic  aspects , 115. Fotic loss . Human α fibrils . Remarkanogaster. Nonethehaperone . More renization . Tau affrylation .Saccharomyces cerevisiae, overexpressing human tau does not induce significant toxicity [Interesting findings have also been made in yeast models. In toxicity , 121. Hotoxicity , 122. Fitoxicity , 122.SNCA and MAPT in genetic studies sections) and reinforces the idea that the interplay between αsyn and tau are pivotal in the neurodegenerative process. Nonetheless, results from these in vivo models have to be interpreted with caution. For instance, there is no ortholog gene of the human SNCA in the fly, worm or yeast, whereas in rodents the endogenous wt αsyn carries the A53T mutation without deleterious effect [In various transgenic or toxin-induced PD models ranging from mice to yeast, the existence of a deleterious and emulative action between tau and αsyn has been repetitively shown. This corroborates what has been observed in humans  and the microtubule binding region of tau 192 to 383) . Accordi to 383 . binding . No effe binding , but lat binding . Convers binding . On the  binding . Consequin vitro study showed that αsyn promotes tau phosphorylation at S262 and S356 residues via PKA [in vitro[The promotion of tau hyperphosphorylation by αsyn has been demonstrated in several studies and could be a mechanism that explains how αsyn triggers tauopathy Figure . An in v via PKA . Later,  via PKA , 132. Th via PKA , 105 and via PKA . However via PKA , 107. Inin vitro. Followiin vitro. Interesin vitro, 134 andin vitro, linkingαSyn may trigger tauopathy through the destabilization of the tau-tubulin interaction, which results in both tau aggregation and cytoskeleton disorganization Figure . Interespost mortem observation that αsyn aggregates spread to healthy transplanted neurons in PD patients [in vitro and in vivo using polymerized αsyn and tau [In the last few years, effort has been directed at unravelling the mechanisms by which neurodegeneration progresses in the brain. In PD and AD, neurodegeneration and protein aggregation overlap with each other and seem to follow a preset path giving the impression of spreading . Moreovepatients , 144. Co and tau –154. Mor and tau , 156.in vitro. Whereas the six alternative spliced variants of tau were able to aggregate in the presence of full length wt αsyn, tau aggregation was neither promoted by the delta71-82 truncated form of αsyn, nor by β-synuclein, nor by amyloidogenic Aβ peptide [in vitro when compared to wt αsyn [In this perspective, a major difference between tau and αsyn is that αsyn is prone to self-aggregate, whereas tau cannot aggregate by itself and requires an inducing agent . This ha peptide . These o wt αsyn . Convers wt αsyn .3+ and Fe3+ or DMSO [in vitro findings in vivo by demonstrating that exogenous αsyn can be taken up by neurons and induce the formation of intracellular Lewy body-like structures [Using the fluorescent intensity distribution analysis technique (FIDA), Nübling and colleagues have shown that tau and αsyn can form co-oligomers and that co-aggregation happens even at nanomolar concentrations but only in the presence of a cationic aggregation inducer such as Al or DMSO . Moreove or DMSO . However or DMSO . This is or DMSO  and suggructures , 160 andructures , 161. Foructures , 161. Coructures .in vitro and in vivo observations that tau and αsyn interact highly suggest that αsyn and tau play as teammates, however how this interaction occurs and affects neurodegenerative processes is still not fully elucidated and several scenarios are possible (Figure The overlap and numerous similarities between synucleinopathies and tauopathies suggest that therapeutic strategies that target common processes of tau and αsyn aggregation could benefit patients across a spectrum of neurodegenerative disorders, and may be particularly relevant for the treatment of secondary symptoms such as cognitive impairment in PD or secondary parkinsonism in dementia. In the present review we have compiled data from the literature linking tau and αsyn. The repeated e Figure .vice versa. Reciprocal promotion of phosphorylation is probably a key player, suggesting that kinases could be used as targets. GSK3β inhibition for instance was concomitantly able to decrease tau phosphorylation, αsyn accumulation and cell death in a cellular MPTP model [αSyn was initially shown to bind to tau and interfere with the normal interaction between tau and tubulin . The disTP model . In a P3TP model .A mechanistic cross-seeding effect based on templating of a pathological β-sheet conformation is also highly suspected , 159–161in vitro[Observations suggest that αsyn being a trigger of tauopathy is more plausible than the opposite scenario. For instance, whereas the presence of tau only accelerated αsyn polymerization, co-incubation with αsyn was necessary to trigger tau aggregation in vitro. Consistin vitro. Furtherin vitro, 105 andin vitro, 33, 34,in vitro. Howeverin vitro–29, 37.Nonetheless, even if some gray areas persist regarding the mechanisms and roles of the interaction between tau and αsyn, applications and future directions are already emerging. One future development is the identification of reliable biomarkers for efficient diagnosis of neurodegenerative disorders at the prodromal stage. In addition to other proteins, αsyn and tau are presently being developed as cerebrospinal fluid biomarkers for improved clinical diagnoses. Cerebrospinal fluid levels of Aβ, total and phospho-αsyn, and total and phospho-tau change differentially depending on the nature of the disease. Consequently, looking at the ratios between these proteins could enable clinicians to determine the risk of developing PD, AD, or a mixed disorder such as DLB and provide them the needed therapeutic window to start preventive and tailor-made treatments .


The creation of licensed medical countermeasures against Select Agents such as Ebola virus (EBOV) is critically dependent on the use of standardized reagents, assays, and animal models. We performed full genome reconstruction, population genomics, contaminant analysis, and characterization of the glycoprotein gene editing site of historical United States Army Medical Research Institute of Infectious Diseases (USAMRIID) nonhuman-primate challenge stock Ebola virus Kikwit “R4368” and its 2014 replacement “R4415.” We also provide characterization of the master stock used to create “R4415.” The obtained data are essential to understanding the quality of the seed stock reagents used in pivotal animal studies that have been used to inform medical countermeasure development. Furthermore, these data might add to the understanding of the influence of EBOV variant populations on pathogenesis and disease outcome and inform attempts to avoid the evolution of EBOV escape mutants in response to current therapeutics. Finally, as the primary challenge stocks have changed over time, these data will provide a baseline for understanding and correlating past and future animal study results. Ebola virus disease (EVD) is a frequently lethal human viral hemorrhagic fever caused by four distinct ebolaviruses . EVD occurs sporadically and usually affects no more than several hundred people during an outbreak (reviewed in ). In 201Zaire ebolavirus in the genus Ebolavirus  [Taxonomically, EBOV is the only member of the species viridae) . The EBOviridae) , 4. For viridae) ) differsviridae) , 7) and viridae) ) by lessviridae) .in vitro experiments [The development, evaluation, and final licensure of medical countermeasures (MCMs) against EVD in the US is critically dependent on standardized animal models of filovirus infection and standardized assays and reagents –12, incleriments , 13. Eboeriments . Unfortueriments .GP genes to access three partially overlapping open reading frames (ORF) [GP gene that typically consists of seven consecutive uridylyls (7U). Read-through results in the transcription of a cognate 7 adenylyl mRNA (7A mRNA) and thereby in the expression of pre-sGP, a protein precursor that is proteolytically processed to a secreted homodimeric glycoprotein (sGP) and a probably monomeric secreted peptide (Δ-peptide). Stuttering of the ebolavirus RNA-dependent RNA polymerase (L) at the editing site results in transcription of mRNAs that contain various numbers of adenylyls. The majority of non-7A mRNAs contain eight adenylyls, leading to the expression of the homotrimeric ebolavirus spike glycoprotein GP1,2, or six or nine adenylyls, leading to expression of the small soluble glycoprotein (ssGP) [1,2:ssGP = 75%:25%:5% [All ebolaviruses make use of co-transcriptional editing of their glycoprotein-encoding es (ORF) , 16. Edin (ssGP) –20. The n (ssGP) . Editing%:25%:5% , 22.in vitro and in vivo environments [GP gene editing sites. For instance, during serial passage of a 7U EBOV/Yam-May isolate in Vero E6 cells, a viral population evolved that predominantly contained an 8U editing site. The opposite occurs in vivo: 8U EBOV/Yam-May populations converted to 7U populations in guinea pigs [1,2 and/or sGP. Preliminary evaluations indicate that a 7U→8U mutation in EBOV does not alter pathogenesis in guinea pigs or nonhuman primates [1,2 in different environments . In addition, a recent study reported subtle, but significant, differences in disease course and severity between nonhuman primates exposed to 7U or 8U virus stock variants [EBOV, Sudan virus (SUDV), and possibly other ebolaviruses adapt to different ronments , 23–25. nea pigs . These cvariants . All of Here, we report the coding-complete genome sequence  was obtained from an EBOV-infected 65-year old female patient during an EVD outbreak that occurred in 1995 around Kikwit, Zaire . The patient’s disease onset was recorded as April 29, 1995. She was hospitalized on May 1, 1995, and died on May 5, 1995. The clinical specimen, most likely plasma or serum, was obtained from the patient on May 4, 1995. How the patient became infected and what medical care she may have received is unclear. Unfortunately, chain-of-custody records that further detail the origin of 9510621 or its shipment to the CDC are not available anymore, and EBOV titration was not attempted from this specimen prior to cell-culture passage.Chlorocebus aethiops) Vero E6 cells (ATCC #CRL-1586). The multiplicity of infection (MOI) used for this passage is unknown. Virus was harvested after a 6-day incubation period on May 19, 1995, but the method of harvest  is not indicated in the available records. A second passage of virus, was conducted at the CDC, again using Vero E6 cells, and resulted in VSP 807224 (MOI unknown). Virus was harvested after an 8-day incubation, using a “freeze/thaw” method, on May 29, 1995. A titer of 3.2E+06 was determined for “VSP 807224” in January 2008 using the TCID50/Reed-Munch viral titration method. “VSP 807224” was transferred from CDC between late May and late June 1995 to USAMRIID.The first passage of EBOV/Kik-9510621, designated “virus seed pool (VSP) 807223” , was conet al. using classical dideoxynucleotide sequencing. “134” (passage 3) is cell culture-adapted, consisting predominantly of viruses with an 8 uridylyl (8U) glycoprotein (GP) gene editing site. A coding-complete genome for “134” was deposited on July 24, 2003, in GenBank under accession #AY354458. “135” (passage 2) was amplified at USAMRIID at least three more times (and in the process was depleted), leading to three separate NHP challenge stocks: “R4367” , “R4369” , and “16502” . The genomic characterization of stock “16502” (passage 3) and its evolution in a nonhuman primate study over several days was previously described [At USAMRIID, “VSP 807224” was stored frozen at -70°C and designated there as “135” (passage 2). After an additional passage in Vero E6 cells, the virus, now designated “134” (passage 3), was sequenced by Chain escribed  Fig 1)..et al. uescribed . Each ofIndependently at UTMB, “VSP 807223” was passaged one time on Vero E6 cells, harvested after a 10-day incubation period on February 21, 2012, and stored in 1.0-ml aliquots as “WRC000121” . A titer of 1.8E+07 pfu/ml was determined (titration method unknown). On May 1, 2012, UTMB transferred “WRC000121” to USAMRIID, where the virus was stored at -70°C and designated as “R4414” (passage 2). This material was passaged at an MOI of 0.001 on Vero E6 cells to prepare NHP challenge stock “R4415” (passage 3), which was harvested on June 11, 2013, after a 12-day incubation period (time to develop 2–3+ CPE) with a titer of 1.31E+06 pfu/ml determined by agarose-based plaque assay [Vials (1 ml) of EBOV/Kik-9510621 challenge stocks were stored and maintained at USAMRIID at -70°C. “R4368” (passage 4) was stored since July 24, 2011, and thawed on March 2, 2012; “R4414” (passage 2) and “R4415” (passage 3) were stored since June 11, 2013, and were thawed in January 2014. An aliquot of 100 μl of each thawed virus stock was placed in 3:1 TRIzol . Nucleic acids were isolated from TRIzol-treated material, and genome sequence was determined on Illumina technology (MiSeq or HiSeq) with EBOV-specific oligonucleotides following sample preparation performed as described in [ATTACCAGAGTTGATTAGTGTG; 5’ RACE (inner primer): TTAAATAACGAAAGGAGTC; 3’ RACE (outer primer): TGAATCTCCAATCCTCTAAGTA; 3’ RACE (inner primer): AAGGGATTTTCAACTGAGCACACT. Amplification primer removal, duplicate removal, low average quality exclusion (≤ Q30), and quality trimming was performed. Viral assemblies were completed in DNAStar Lasergene nGen  with ≈4×105 reads. Only single nucleotide polymorphism (SNPs) present in the population above the 2% threshold are presented in this report . Considering this threshold, a target depth of 200 requires an SNP to have ≥ 4 supporting reads prior to being called a SNP. The depths are reported in the tables presented in this text for all samples . A consensus change is defined here as a change relative to the published sequence for EBOV/Kik-9510621 “134” (GenBank accession # AY354458) present in ≥50% of the population. Below that threshold, SNPs are considered subclonal substitutions and part of a minority subpopulation of the virus.For rapid amplification of cDNA ends (RACE), “R4415” (passage 3) RNAs were extracted with Zymo Direct-Zol  from cell-culture supernatant in TRIzol according to the manufacturers' instructions. SMARter RACE 5’/3’ kit  was used to amplify both 5' and 3' untranslated regions (UTRs) of the virus genome from the extracted RNAs. The kit's two-stage nested PCR protocol was found to be optimal. The gene-specific primers for each RACE experiment are as follows 5’ RACE (outer primer): Research has been reviewed for compliance with dual-use guidelines and approved for publication by the USAMRIID Institute Biosafety Committee (IBC) and the Operational Security office.Compared to EBOV/Kik-9510621 “134” (passage 3), “R4368” (passage 4) acquired only one consensus-level substitution . Six intrahost single nucleotide variants (iSNVs) (≥2% of the population) were detected and are reported in in vitro and in vivo Ebola virus experimentation, including pathogenesis studies and candidate medical countermeasure evaluation. “R4415” (passage 3) replaced “R4368” (passage 4) in December 2014 and has been used for most pathogenesis and medical countermeasure evaluation research since then. This work provides a framework for genomic comparison between past experiments as challenge stocks are replaced to address propagation issues and depletion. Characterization of these NHP challenge stocks was completed to the level of “Coding Complete” plus population-level characterization in the case of “R4368” (passage 4) and “Finished” in the case of “R4415” (passage 3) [We have provided here a concise report on the history and genomic characterization of the USAMRIID Ebola virus/H.sapiens-tc/COD/1995/Kikwit-9510621 NHP challenge stocks “R4368” (passage 4) and “R4415” (passage 3), as well as the “R4415” (passage 3) predecessor, “R4414 (passage 2).” “R4368” (passage 4) was used between July 2011 and December 2014 in both ssage 3) . This chssage 3) .


Apolipoprotein E (ApoE) gene has been reported to be associated with migraine and tension-type headache (TTH), but the results are conflicting. This study aimed to evaluate the association of ApoE with migraine by a meta-analysis.MEDLINE, ISI Web of Knowledge, The Cochrane Central Register of Controlled Trials, and EMBASE databases were searched to identify eligible studies published in English from 2000 to 2014. Data were extracted using standardized forms. The association was assessed by relative risk (RR) with 95 % confidence intervals (CIs) using a fixed or random effects model.Four studies, comprising 649 migraineurs, 229 TTH subjects and 975 controls, met all the criteria and were included in the meta-analysis. No significant difference was found comparing genotypic and allelic frequencies in the case of migraineurs versus controls and TTH subjects versus controls. Only when migraineurs and TTH subjects were considered as a whole group, ApoE4 was found to increase the relative risk of headache by 1.48 , compared to controls.ApoE ε4 allele is not associated with migraine susceptibility, but is positively related to headache (including migraine and TTH). Migraine is a common neurovascular disorder, characterized by recurrent episodes of headache and the dysfunction of the autonomic nervous system, affecting 10–20 % of the population . In partApolipoprotein E (ApoE) is a 299 amino acid protein, encoded by a gene located on chromosome 19cen-q13.2 , 7. ApoETherefore, to define further the disease risk associated with APOE polymorphism, in this study we performed a meta-analysis of all related studies that evaluated allelic and genotypic frequencies of ApoE polymorphism in migraine.Eligible studies were identified by searching on web-based databases  with no language restrictions. Search terms were “apolipoprotein E”, “APOE”, “ɛ2”, “ɛ3”, “ɛ4”, “APOE2”, “APOE3”, “APOE4”, “migraine”, “headache” and “Polymorphism”. Potentially relevant studies were retrieved and reviewed by 2 reviewers. All case–control studies with extractable data were included. Included studies were published as full-length articles in peer-reviewed journals.Studies were included for meta-analysis if they met the following criteria: (1) the association between APOE and migraine was examined by using a population-based, case–control design; (2) migraine was diagnosed using an international standard; (3) genotype or allele frequencies were reported in both cases and controls; (4) the genotype frequencies in control groups were consistent with Hardy-Weinberg equilibrium.2 test (I2 = (Q− df) / Q × 100 %. I2 values of above 25 %, 50 %, and 75 % were taken as indicators of mild, modest, and high heterogeneities, respectively. When the effects were assumed to be homogeneous , the fixed-effects model was used; otherwise, the random-effects model was more appropriate. The meta-analyses were performed using Stata Version 12.0 software .Heterogeneity across the eligible studies was tested using Q-test, and it was considered statistically significant when P < 0.1. Heterogeneity was also quantified with IAs a result of our literature search, a total of four published articles reported on the relationship between APOE SNP and migraine and met the inclusion criteria. Rainero et al. divided the subjects into three subgroups: migraine with aura; migraine without aura; and mixed headaches (migraine associated with tension-type headache) . Stuart Total 649 migraineurs, 229 TTH subjects and 975 controls were genotyped for APOE polymorphism. In the four studies, the distribution of the genotypes in control groups was in Hardy–Weinberg equilibrium (P < 0.05).2 = 74.1 %). This significance disappeared with random effect . While E4 gene increased the relative risk of headache by1.48 , they did not show any effect when migraineurs and TTH subjects were taken as a reference category  plays an essential role in the pathogenesis of both migraine and TTH , 15. MorThere are some limitations in our study. First, since migraine is a complex and heterogeneous disorder with a wide clinical spectrum, patients with different clinical phenotypes may not sufficiently capture this variability and may result in misclassification. Second, we only chose three genotypes in our study, despite various genotypes of APOE. Third, the lack of individual data has restricted further adjustments for the subgroup analysis for TTH. Fourth, the analysis was performed on a relatively small number of retrospective case–control studies, and the subjects included in these studies are also limited. Further population-based studies with larger sample size are necessary to confirm our conclusions.In summary, the evidence from the present meta-analysis showed that APOE4 is not associated with migraine or TTH, but is related to headache (including migraine and TTH), suggesting a nonspecific role for the risk of headache.