In both trials, plasma glucose concentrations and kinetics, insulin, C‐peptide, and glucagon were measured. Rates (mg kg−1 min−1) of glucose appearance from endogenous (RaEndo) and exogenous  sources, and glucose disappearance (Rd) were determined. We found that exercise increased RaEndo, RaOGTT, and Rd  in all groups with a tendency for a greater (~20%) peak RaOGTT value in NGT subjects when compared to IGT and T2D subjects. Accordingly, following exercise, the plasma glucose concentration during the OGTT was increased in NGT subjects (P < 0.05), while unchanged in subjects with IGT and T2D. In conclusion, while a single bout of moderate‐intensity exercise increased the postprandial glucose response in NGT subjects, glucose tolerance following exercise was preserved in the two hyperglycemic groups. Thus, postprandial plasma glucose responses immediately following exercise are dependent on the underlying degree of glycemic control.We investigated glucose tolerance and postprandial glucose fluxes immediately after a single bout of aerobic exercise in subjects representing the entire glucose tolerance continuum. Twenty‐four men with normal glucose tolerance (NGT), impaired glucose tolerance (IGT), or type 2 diabetes (T2D; age: 56 ± 1 years; body mass index: 27.8 ± 0.7 kg/m This study shows that following an exercise bout, plasma glucose concentrations during an oral glucose tolerance test are increased in subjects with normal glucose tolerance, but unchanged in subjects with impaired glucose tolerance or type 2 diabetes. While rates of glucose disappearance and rates of glucose appearance from endogenous sources and from orally ingested glucose were all increased following exercise, there was a 20% greater peak value for the rate of orally ingested glucose appearance in normal glucose tolerant subjects, when compared to IGT and T2D subjects. In summary, postprandial plasma glucose responses immediately following exercise are dependent on the underlying level of glycemic control. We hypothesized that the effect of a single bout of exercise on plasma glucose kinetics following oral glucose ingestion would be influenced by the subject's underlying level of oral glucose tolerance.n =8), IGT (n =8), or T2D (n =8), based on WHO definitions  were treated with insulin; (2) had unstable weight (>5 kg in previous 6 months); (3) had an illness that contraindicated physical activity; or (4) demonstrated any evidence of current or previous hematological, renal, hepatic, cardiovascular, or pulmonary disease. All enrolled subjects underwent a dual‐energy x‐ray absorptiometry (DXA) scan to determine whole‐body adiposity and fat‐free mass. All DXA images were analyzed by the same investigator using Encore 2004 software . Subjects also performed an exercise test on a bicycle ergometer  to determine maximal aerobic capacity (VO2max) with indirect calorimetry  reflecting fitness level, as well as maximal power output (Watt [W]max) and heart rate (HRmax) to calculate individual exercise workloads. Furthermore, subjects filled out a questionnaire reflecting habitual activity level modified from The Minnesota Leisure Time Physical Activity Questionnaire .Twenty‐four male subjects were recruited from the local area and were screened with a medical history and physical examination, blood chemistry analyses, and an oral glucose tolerance test (OGTT). Subjects were stratified as having NGT (Wmax and 60–90 rounds per minute (rpm). Power output was fixed during the entire trial. Heart rate was measured continuously and all trials were supervised by the same study investigator. For 3 days, prior to each trial, subjects were instructed by the same study investigator to complete diet records to ensure that daily energy intake  and daily macronutrient composition  were not different between trials. Diet records were analyzed by the same investigator using DanKost Sport 2000 software . During the three pretrial days, subjects were also instructed to refrain from physical activity and to pause any antidiabetic , antihypertensive (n =3), or statin drugs (n = 7).On two occasions, separated by a minimum of 14 days, each subject underwent a 180‐min OGTT immediately following an hour of exercise  or rest  in a randomized order. Exogenous and endogenous glucose kinetics were measured during the OGTT. Exercise was performed as 1 h of cycle ergometry at 50% of T =−90 min and a primed (30 μmol/kg multiplied by fasting plasma glucose/5), continuous (0.3 μmol kg−1 min−1) infusion of glucose began. During the exercise trial, exercise was commenced at T =−60 min. Immediately after rest/exercise at T =0 min, a 180‐min OGTT was started by ingestion of a 300‐mL solution containing 73 g of anhydrous glucose and 2 g of [U‐13C]glucose. Sterility and pyrogen‐tested tracers were purchased from Cambridge Isotope Laboratories  and prepared on the day of the test under aseptic conditions. Blood samples were collected at T =−90, −60, −30, 0 min and every 10 min during OGTT for determination of plasma glucose and tracer enrichment, and every 30 min for the measurement of serum insulin, C‐peptide, and plasma glucagon. Venous blood was collected into the following tubes: heparin syringes for glucose analyses; Vacuettes  containing sodium fluoride for glucose enrichment analyses, Vacuettes containing EDTA and 10,000 kiU/mL aprotinin for glucagon analyses; and serum‐separation Vacuettes for insulin and C‐peptide analyses. Blood samples for plasma collection were immediately placed on ice and subsequently centrifuged , and the plasma was separated and stored at −80°C until analyses. Samples for serum collection were allowed to clot at room temperature for 30 min before centrifugation and subsequently sent for insulin and C‐peptide analysis at the Department of Clinical Biochemistry at Rigshospitalet.Each trial was performed after an overnight fast (~10 h). When arriving in the laboratory, catheters were placed in an antecubital vein in each arm – one for blood sampling and one for tracer infusion. Baseline blood samples were drawn at 2H2]glucose and [U‐13C]glucose enrichments were quantified using liquid chromatography tandem mass spectrometry  using a hexobenzoyl derivatization method, as described previously . Serum insulin and C‐peptide were determined by electrochemiluminescence immunoassay . Plasma glucagon concentrations were determined by radioimmunoassay . Plasma adrenaline and noradrenaline concentrations were determined by RIA . Plasma glucose enrichment. Rate of exogenous oral glucose appearance (RaOGTT) was determined from plasma [U‐13C]glucose enrichment (Proietto RaEndo) was calculated as the difference between total Ra and RaOGTT. The postprandial suppression of RaEndo was determined as the incremental response during the first 20 min, calculated as delta of T =0 and T =20 (Δ0–20 min). Glucose clearance during rest/exercise (T =−90 to 0 min) and during OGTT (T =0–180 min) was determined as Rd divided by plasma glucose.Glucose, insulin, C‐peptide, and glucagon responses were calculated as area under the curve (AUC) during rest/exercise  and statistical significance was accepted when P < 0.05.One‐way analysis of variance (ANOVA) was used to compare between‐group baseline characteristics and AUC measures. Bonferroni post hoc tests were used to examine differences between group means. Two‐way repeated measures ANOVA was used to compare time and trial differences in total responses of plasma metabolites and glucose kinetics as well as pre‐/post‐exercise catecholamine levels within and between groups. Bonferroni post hoc tests were used to examine differences between trial means. Paired P = 0.05). Fasting glucose levels were highest in subjects with T2D (P < 0.05), and 2‐h OGTT glucose values were progressively higher across the groups . Furthermore, groups did not differ in habitual activity levels or VO2max corrected for fat‐free mass.Subject characteristics are presented in t‐tests between any of the groups or between trials.Daily energy intake and dietary macronutrient composition is shown in Wmax at 60–90 rpm with a mean heart rate of 116.5 ± 3.7 beats per minute (bpm) for all groups. Exercise intensity (% of Wmax) was not different between groups.All subjects finished the 1 h of cycle ergometry exercise and exercise data are presented in P > 0.05). During exercise, plasma glucose  was increased in the NGT group  while decreased in IGT subjects . Furthermore, in subjects with T2D glucose levels tended to be lower immediately after exercise compared to baseline . Two‐way repeated measures ANOVA revealed a main effect of time in all groups (P < 0.0001) and a main effect of trial in subjects with NGT . Post hoc analyses revealed that in NGT subjects, plasma glucose during OGTT was significantly higher in the exercise trial compared to the rest trial . Also, exercise increased the glucose response (AUC) to the OGTT in NGT subjects ; however, it was still lower than AUC glucose in subjects with T2D (P < 0.001). In contrast, the glucose response (AUC) to the OGTT in subjects with IGT and T2D was unaltered by exercise , and a time × trial interaction in NGT and IGT groups . Post hoc analyses showed that RaTotal was increased by exercise in all groups . Compared to the rest trial, RaTotal (AUC) was increased during exercise in subjects with NGT, IGT, and T2D  as well as immediately after exercise . Furthermore, RaTotal (AUC) during OGTT was increased by exercise in subjects with NGT, IGT, and T2D .No group differences in P < 0.000, all, anP < 0.000, all, anP < 0.0001, all), and a time × trial interaction in NGT and IGT groups . Post hoc analyses showed that Rd was increased by exercise in all groups . Compared to the rest trial, Rd (AUC) was increased during exercise in subjects with NGT, IGT, and T2D  as well as immediately after exercise . Furthermore, Rd (AUC) during OGTT was increased by exercise in subjects with NGT, IGT, and T2D .No group differences were found in the rest trial. Two‐way repeated measures ANOVA revealed an overall main effect of time and trial in subjects with NGT, IGT, and T2D P < 0.000, all, anT =40 min in NGT, T =50 min in IGT, and T =60 min in subjects with T2D. Moreover, postprandial suppression during the first 20 min (Δ0–20 min) was lower in subjects with T2D than NGT (P < 0.01). Two‐way repeated measures ANOVA revealed a main effect of time , trial , and a time × trial interaction in subjects with NGT, IGT, and T2D . Post hoc analyses revealed that RaEndo was significantly higher in the exercise trial in all groups . Compared to the rest trial, RaEndo (AUC) was increased during exercise in subjects with NGT, IGT, and T2D . Also, RaEndo (AUC) during OGTT was increased by exercise in subjects with NGT and T2D .No baseline group differences were found in the rest trial; however, postprandial suppression  occurred at T P < 0.0. Two‐wayT P < 0.0. Two‐wayP < 0.0001, all) and time × trial interaction in all groups . Post hoc analyses showed that RaOGTT was significantly greater in the exercise trial in all groups . Compared to the rest trial, RaOGTT (AUC) during the OGTT was greater following exercise in all groups , and although not statistically significant (P = 0.17), the peak RaOGTT value following exercise was ~20% higher in NGT subjects compared to subjects with IGT or T2D.No group differences were found in the rest trial. Two‐way repeated measures ANOVA revealed a main effect of time, trial P < 0.000, all andP < 0.01) and trial (P < 0.05) for glucose clearance during rest and exercise . Also, two‐way ANOVA revealed a main effect of group (P < 0.001) and trial (P < 0.0001) for glucose clearance during OGTT. Post hoc analysis showed that glucose clearance was lower in subjects with IGT and T2D in both trials when compared to NGT subjects . Also, post hoc analysis showed that glucose clearance during OGTT was increased in all groups in the exercise trial compared to the rest trial , and was still lower in subjects with IGT and T2D compared to NGT subjects .Two‐way ANOVA revealed a main effect of group . Serum insulin  was higher during exercise in subjects with T2D as compared to NGT . In IGT subjects, the insulin response (AUC) to exercise showed a trend to be decreased as compared to rest (P = 0.058) with levels being lower immediately after exercise compared to baseline . Two‐way repeated measures ANOVA revealed a main effect of time in all groups (P < 0.0001), but no main effect of trial (P > 0.05). No between‐group differences were found in the overall insulin response to the OGTT in the rest trial. Exercise did not affect the total insulin response (AUC) to the OGTT in any of the groups. However, the initial incremental insulin response (Δ0–15 min) to the OGTT was increased after exercise in all groups .Fasting levels did not differ between any of the groups or between trials (P > 0.05). Initial incremental response (Δ0–15 min) to the OGTT was lower in subjects with T2D as compared to NGT . Also, serum C‐peptide  was higher during exercise in subjects with T2D as compared to NGT . In IGT subjects, the C‐peptide response (AUC) during exercise was decreased compared to the rest trial . Two‐way repeated measures ANOVA revealed a main effect of time in all groups (P < 0.0001) and a main effect of trial in IGT subjects , indicating that C‐peptide levels were lower in the exercise trial than in the rest trial. Post hoc analyses revealed no specific between‐trial differences. However, the first‐phase incremental C‐peptide response (Δ0–15 min) to the OGTT was increased in the exercise trial as compared to the rest trial in subjects with IGT and T2D .Fasting levels did not differ between any of the groups or between trials . By systematically investigating groups representing the entire glucose tolerance continuum, for the first time these findings determine that the immediate effect of a single bout of aerobic exercise on oral glucose tolerance differs between healthy and diabetic subgroups, implying an impact of the underlying level of glycemic control.RaEndo was increased by exercise in the present study. However, in contrast to Minuk et al.  cannot explain the present group differences in exercise‐induced changes in oral glucose tolerance. Second, exercise increases muscle‐contraction‐induced glucose disposal via insulin‐independent GLUT‐4 translocation  was increased to the same extent by exercise in all three groups. This was seen when glucose clearance was normalized to either body mass  of the OGTT in NGT subjects (ΔAUC 209.8 ± 52.0 mg kg−1 min−1) than in IGT  and T2D  subjects. These group differences were found along with a nonsignificant but ~20% greater peak value of RaOGTT following exercise in NGT subjects (6.6 ± 1.7 mg kg−1 min−1) compared to IGT (5.0 ± 0.6 mg kg−1 min−1) and T2D (4.4 ± 0.3 mg kg−1 min−1) subjects. Despite being underpowered to detect these differences, in support of the findings of Rose et al.  responses of both serum C‐peptide and insulin in IGT and T2D subjects, indicating that exercise increases early‐phase glucose‐stimulated insulin secretion in hyperglycemic subjects. In contrast, in NGT subjects only initial postprandial insulin levels were increased, but not C‐peptide. In healthy subjects, it is known that exercise‐induced increases in adrenaline suppresses insulin secretion  response to the oral glucose load following exercise in subjects with IGT and T2D is an important observation. Diminution of first‐phase insulin secretion is one of the earliest detectable signs of D Gerich ; therefoThe glucose‐lowering effect of exercise found in subjects with IGT and T2D  differed between NGT and IGT, and while not significant, the absolute VO2max for T2D was substantially lower than NGT. This caused a ~30 watt difference in mean power output during exercise between NGT and IGT/T2D groups, which may have influenced our findings. However, this was not statistically different, and catecholamine levels were not different between groups also confirming that the exercise work load was similar between groups.Prior knowledge of the effects of acute exercise on glucose kinetics in subjects with different underlying levels of glycemic control is compiled from several independent studies. The present study is the first to examine the effects of a single aerobic exercise bout on immediate glucose tolerance and postprandial glucose kinetics in age‐ and BMI‐matched groups of NGT, IGT, and T2D subjects simultaneously, representing the entire glucose tolerance continuum. Thereby, a strength of our study is that we can make group comparisons while directly controlling for differences in study designs and subject characteristics. However, our NGT subjects were in fact overweight/obese and since obesity per se is associated with impaired glucose tolerance  for her technical assistance with clinical biochemistry assays. We also thank G. A. Wallis  for providing intellectual critique on our manuscript.None declared.


Toona microcarpa Harms is a tonic, antiperiodic, antirheumatic, and antithrombotic agent in China and India and an astringent and tonic for treating diarrhea, dysentery, and other intestinal infections in Indonesia. In this study, we prepared ethyl-acetate extract from the air-dried leaves of  Toona microcarpa Harms and investigated the anticoagulant activities  in vitro by performing activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) assays. Antiplatelet aggregation activity of the extract was examined using adenosine diphosphate (ADP), collagen, and thrombin as agonists, and the inhibitions of factor Xa and thrombin were also investigated. Bleeding and clotting times in mice were used to determine its anticoagulant activities  in vivo. It is found that  Toona microcarpa  Harms leaf extract (TMHE) prolonged APTT, PT, and TT clotting times in a dose-dependent manner and significantly inhibited platelet aggregation induced by thrombin, but not ADP or collagen. Clotting time and bleeding time assays showed that TMHE significantly prolonged clotting and bleeding times  in vivo. In addition, at the concentration of 1 mg/mL, TMHE inhibited human thrombin activity by 73.98 ± 2.78%. This is the first report to demonstrate that THME exhibits potent anticoagulant effects, possibly via inhibition of thrombin activity. Toona microcarpa Harms, a tree reaching 10 m, is a perennial hardwood of the family Meliaceae that is found in India, Bhutan, Laos, Malaysia, Myanmar, Papua New Guinea, Thailand, Sikkim, Indochina, and southern China. This species yields excellent timber and has long been used as a traditional Chinese medicine (TCM) for treating various conditions as its leaves, seeds, and root bark have medicinal effects. Specifically, the bark is used as a powerful astringent and purgative, and the leaf extract has antithrombotic effect and antibiotic activity against Staphylococcus, with leaf tip concoctions applied to swellings. Toona microcarpa Harms is considered a tonic, antiperiodic, antirheumatic, and antithrombotic agent in China and India and is used as an astringent and tonic for treating diarrhea, dysentery, and other intestinal infections in Indonesia [ Toona microcarpa Harms and the mechanism is unknown.Many traditional Chinese herbal medicines have been used for thousands of years in clinical practice because of their proven efficacy, wide indications, high safety profile, and low toxicity . Toona mndonesia . However Toona microcarpa Harms has some effects on activating blood circulation to dissipate blood stasis; however, the mechanism underlying its effect has been poorly studied. In this study, we prepared ethylacetate extract from the air-dried leaves of Toona microcarpa Harms and investigated its antithrombotic activity and underlying mechanism.Thrombosis is a major cause of morbidity and mortality and is closely related to activated platelet adhesion, aggregation, secretion functions, and activation of intrinsic and extrinsic coagulation systems, which cause blood coagulation and fibrin formation . In TCM, Toona microcarpa Harms were collected in February 2013 from the Jinghong region of Yunnan Province, China. The plant was identified by Dr. Rong Li, and a voucher specimen (KIB 13-02-08) was deposited in the State Key Laboratory of Phytochemistry and Plant Resources in west China, Kunming Institute of Botany, Chinese Academy of Sciences. Air-dried and powdered leaves of Toona microcarpa Harms (1 kg) were extracted with 90% ethanol (5000 mL × 2) at room temperature and concentrated in vacuo to yield crude extract. The dry extract was resuspended in distilled water (1000 mL) and extracted twice with petroleum (30–60°C) to remove pigments and lipids, followed by two more extractions with ethylacetate using liquid-liquid partitioning. After removing the solvent using a rotary vacuum evaporator, the ethylacetate fraction was used to determine its bioactivity. TMHE was dissolved in dimethylsulfoxyde to obtain stock solutions of 50 mg/mL. Working solutions were obtained by dilution with distilled water.The aerial parts of in vitro APTT assays, 50 μL normal citrated PPP was incubated with 50 μL TMHE  and 50 μL APTT reagent for 3 min at 37°C. APTT clotting time was immediately recorded after the addition of 100 μL calcium chloride (20 mM). For in vitro PT assays, 50 μL normal citrated PPP was incubated with 50 μL  TMHE for 3 min at 37°C. Clotting time was immediately recorded after the addition of 100 μL PT reagent [ in vitro TT assay, 100 μL normal citrated PPP was incubated with 100 μL  TMHE for 2 min at 37°C. Clotting time was immediately recorded after the addition of 100 μL TT reagent [Male imprinting control region (ICR) mice (28–32 g) were supplied by the animal center of Nanjing Medical University. All animal experiments were approved by the Animal Care and Use Committee of The First Affiliated Hospital of Nanjing Medical University. Blood was drawn from the eyeball of ICR mice and separately centrifuged at 3000 rpm for 15 min to obtain platelet poor plasma (PPP). ForμL PRP and 30 μL TMHE were incubated at 37°C in an aggregometer. After a 3 min preincubation, 30 μL of agonists  was added to initiate aggregation, which was monitored for 6 min. The extract solvents were used as negative controls; aspirin (1 mg/mL) and argatroban (0.005 mg/mL) were used as positive controls. The inhibition rate was calculated as follows: inhibition rate = (Av – At)/Av × 100%; Av is the platelet aggregation percent of negative control and At is the platelet aggregation percent of the TMHE group, respectively.Male New Zealand rabbits (4-5 kg) were supplied by the animal center of Nanjing Medical University. All animal experiments were approved by the Animal Care and Use Committee of The First Affiliated Hospital of Nanjing Medical University. After application of the local anesthetic lidocaine, blood was drawn from the carotid artery of male New Zealand rabbits and directly collected into vials containing sodium citrate (1 : 9 v/v) mixture. The blood samples were centrifuged at 1000 rpm for 10 min at room temperature to prepare platelet rich plasma (PRP), and the residue was centrifuged at 3000 rpm for 15 min at room temperature to obtain PPP . BrieflyμL)  and 20 μL human thrombin (5 NIH/mL)  in 20 μL Tris-HCl buffer  were incubated for 15 min in a 96-well plate. The reaction was initiated by adding 20 μL thrombin chromogenic substrate CS-01(38) (2.5 mg/mL), and the absorbance at 405 nm was recorded every 0.5 min for 5 min. The background absorbance was measured just before adding the substrate. In A-t curve, the curve slope was considered the reaction rate (v). The enzyme inhibition percentage (I) was determined as follows: I = (V0 − Vi)/V0 × 100%. V0 is the rate of extract solvents, and Vi is the rate of TMHE. Extract solvents were used as negative controls, whereas argatroban  was used as a positive control. The results were expressed as mean ± SD for three independent experiments.TMHE (20 μL human factor Xa (2.5 μg/mL) (Hyphen-BioMed) in 20 μL PBS buffer  and 20 μL CS-11(22) substrate (2.5 mg/mL). Rivaroxaban  was used as a positive control. The results were expressed as mean ± SD for three independent experiments.The same protocol as described for thrombin was followed using 20 ICR mice (18–22 g) were divided into five treatment groups  and orally administered extract solvents (control), low dose TMHE (20 mg/kg body weight), medium dose TMHE (40 mg/kg body weight), high dose TMHE (80 mg/kg body weight), and dabigatran etexilate . Blood was collected intracardially at 120 min after dosing . The APTWhole blood clotting time in mice was measured by the capillary glass tube method . ICR micICR mice 18–22 g) were divided into five groups  and orally administered extract solvents (control), low dose TMHE (20 mg/kg body weight), medium dose TMHE (40 mg/kg body weight), high dose TMHE (80 mg/kg body weight), and dabigatran etexilate (20 mg/kg body weight). Each group was administered drug for four consecutive days. Ninety minutes after the last administration, the mice tails were marked with a tag approximately 5 mm long and then cut at the mark. Then, the tip of the tail was immersed in saline at 37°C, and the time from cutting the tip of the tail to stopping the bleeding was recorded; this interval was defined as bleeding time [–22 g werThe acute toxicity of TMHE was evaluated in mice according to the description of Wang et al. . Six ICR in vitro coagulation assays, TMHE prolonged APTT, TT, and PT clotting times in a dose-dependent manner  inhibition tests were used. Thrombin and FXa are two highly validated targets that function at key steps in the coagulation cascade . Many cl via inhibition of thrombin. These results will be helpful to understand the antithrombotic mechanism of Toona microcarpa Harms.In summary, TMHE prolonged APTT, PT, and TT clotting times, inhibited the thrombin but not ADP or collagen stimulated platelet aggregation, and prolonged the whole bleeding and clotting time, possiblyRadix angelicae Sinensis), Honghua (Flos carthami), and Danshen  are examples of TCM herbs that are used to activate blood circulation to remove blood stasis [Persicae semen) and Honghua (Flos carthami) used in pair named as Taoren-Honghua herb pair have also been used for many years to promote blood circulation to dissipate blood stasis [ Geum japonicum at a concentration of 2 mg/mL has significant anticoagulant activity in the extrinsic coagulation pathway [ Rubus chingii leaves is the most antithrombotic fraction in vitro and in vivo, and flavonoids make an important contribution [ Erigeron breviscapus extract has anticoagulant activity [ Oenanthe javanica inhibit not only the activities of thrombin and FXa but also the generations of thrombin and FXa in human umbilical vein endothelial cells [ Croton zambesicus Müell. Arg exhibited both the thrombin and FXa inhibition but no antiplatelet activity [Many TCMs have been demonstrated to have anticoagulant activity. Danggui (d stasis . In addid stasis . In somed stasis . Zeng et pathway . Han et ribution . Wang etactivity . Howeveral cells . Robert activity . In this Toona microcarpa Harms may be a thrombin inhibitor that can function as an anticoagulant therapeutic. However, the anticoagulant activity of TMHE is weaker compared with positive control drugs such as heparin, argatroban, and dabigatran etexilate. It may be due to that TMHE is merely raw product which is extracted from the air-dried leaves of Toona microcarpa Harms by ethylacetate. We will perform further separation and purification for the active components.In conclusion, this is the first report to demonstrate that TMHE has anticoagulant activity, most likely via its ability to inhibit thrombin activity.


A hollow (concave) mask appears convex when viewed from beyond a certain distance even when viewed stereoscopically—this is the hollow-face illusion. At close viewing distances, the same mask is seen as hollow even when disparity information is eliminated by monocular viewing. A potential source of nonpictorial, monocular information that favors a veridical percept at close distances is accommodation in conjunction with focus blur. In this article, we used pinhole viewing to minimize this potential source of information and test whether it affects whether a hollow mask is seen as veridical (concave) or illusory (convex). Since monocular viewing also facilitates the illusory (convex) percept, it was included in the design both as a comparison and to test whether any effect of accommodation depends on vergence. Pinhole viewing was found favor the illusory percept, and its effect was at least as large as, and added to, that of monocular viewing. A control experiment using tinted glasses that attenuate illumination at least as much as the pinholes did not strengthen the illusion ruling out explanations in terms of reduced luminance. For pinhole viewing, there was no difference between monocular and binocular conditions. The results are interpreted as evidence that focus driven depth information affects perceived three-dimensional shape at close distances even when other sources of depth information are available. The lack of a difference between monocular and binocular pinhole viewing suggests that, by disrupting accommodation, pinholes may also interfere with linked vergence cues to depth. A hollow face mask is seen as convex when viewed from beyond a certain distance, even when unambiguous stereoscopic depth information from binocular disparities is available. This is the hollow face illusion. When viewed from close enough, however, the same mask is seen as hollow even if viewing is monocular and binocular disparities are no longer available . One souAccommodation potentially provides information about the absolute egocentric depth of the part of the visual scene that is being fixated, at least within the 2- to 3-m range over which it varies . AccommoIn this experiment, we used pinhole glasses to open loop , or at lAccommodation is synkinetically linked with vergence  which isWe used “flipping distance,” the distance at which perception of the hollow mask changes from convex to concave, as a measure of the strength of the illusion . ViewingIn summary, we used pinhole viewing to reduce or eliminate focus blur driven accommodation as a potential source of disambiguating depth information. If this source of depth information normally plays a role in disambiguating the hollow-face illusion at close distances, we would expect shorter flipping distances when pinhole viewing. We included monocular and binocular viewing for comparison and to test whether any effect of accommodation depends on vergence.Twenty undergraduate psychology students  between the ages of 18 and 40 (mean 21.5) took part in the experiment for course credit. The experiment was approved by the University of Wollongong Human Research Ethics Committee (ref HE12/462).http://www.grand-illusions.com. Commercially available multiple pinhole glasses were used which consisted of perforated opaque nylon “lenses” each with 114 pinholes aligned in an offset grid of nine rows . Pinholes were 1.1 mm in diameter and separated by 2.5 mm. The “lenses” were 1.1 mm thick. The pinhole arrays were fitted in black plastic spectacle frames 14.5 cm wide, 4.2 cm high. The hollow face was lit from above and behind with a goose-necked Krompton halogen lamp (KL2379) which uses two bi-pin 50 W natural color bulbs in an otherwise black and dark room. The luminance of the mask as measured using a spot photometer pointed at the bridge of the nose from a distance of 1 m was 157 cd/m2. The mask was placed 1.3 m above the ground, that is, at approximately head height facing the participant’s direction of movement. Flipping distance was measured from the base of the mask.The mask used was the unpainted 35.5 cm × 45.5 cm white plastic “Shakespeare Hollow Face” available from http://www.eyerobics.com.au/eye_chart.html) and stereo acuity using the “stereo-butterfly” stereoacuity kit . All acuity testing was undertaken both with and without pinholes. Spatial acuity was measured both binocularly and monocularly. Visual acuity was tested at 1.8 m and scored as Snellen decimals. A Snellen decimal of 1.0 corresponds to 6/6 vision with higher values indicating better acuity. The proportion correct of the next most difficult line reported was linearly interpolated to give intermediate scores.Spatial acuity was measured using a standard seven-letter Snellen chart  face.For experimental trials, participants either advanced toward the mask starting approximately 3 m away or slowly retreated from 0 m until their perception of mask depth changed from convex to concave or vice versa, respectively. More details on this method can be found in A 2 Pinholes (with/without) × 2 Eyes (monocular/binocular) × 2 Direction (approaching/retreating) fully factorial within subjects design was used. There were two repetitions for each cell. The dependent variable was flipping distance in millimetres.The distribution of data points did not differ significantly from normal in any of the conditions, and variance did not violate the assumption of homogeneity. There was no effect of repetition, and data were collapsed across this factor.F = 17.02, p < .001, partial-η2 = .47, and between pinhole viewing and direction of movement, F = 5.54, p = .03, partial-η2 = .23. These interactions are plotted in F = 18.29, p < .001, partial-η2 = .49, and pinholes, F = 37.92, p < .001, partial-η2 = .67. Both monocular and pinhole viewing significantly reduced flipping distance overall. No other main effects or interactions were significant .A 2 Pinholes (with/without) × 2 Eyes (monocular/binocular) × 2 Direction (approaching/retreating) fully factorial repeated measures analysis of variance revealed significant interaction effects between number of eyes and pinhole viewing, Follow-up analysis of the pinholes × eyes interaction showed pinhole viewing significantly reduced flipping distance for both monocular and binocular viewing whereas monocular viewing only significantly reduced flipping distance for nonpinhole viewing. For the pinholes × direction interaction, the effect of direction was only significant for pinhole viewing. The effect of pinholes was significant for both directions of movement. These significant differences are indicated in p > .1).The half luminance control trials were not significantly different from the equivalent binocular nonpinhole conditions conducted as part of the main experiment (2 through the tinted glasses and 26 cd/m2 through the pinholes (compared with 157 cd/m2 unrestricted). Run as a 3 Glasses (tinted/pinhole/none) × 2 Eyes (monocular/binocular) × 2 Direction (approaching/retreating) fully factorial repeated measures design with 12 participants , analysis of variance gave an independent main effect of type of glasses, F = 22.68, p < .001, partial-η2 = .67, and of number of eyes, F = 9.41, p = .011, partial-η2 = .46. Follow-up pairwise comparisons using Bonferroni correction on the effect of glasses showed that flipping distance (mm) was significantly less for pinholes  than for either tinted glasses  or normal viewing . Tinted and normal did not differ significantly from each other (p = .199). The effect of eyes was as expected with flipping distance lower for monocular  than for binocular  viewing. No other main effects or interactions were significant . In particular, the interactions of glasses with number of eyes F = 2.08, p = .148, partial-η2 = .16, and direction F = 2.10, p = .147, partial-2η = .16, were not significant in this control experiment . This may have been in part due to the lower number of participants, 12 as opposed to 20. Consistent with the pattern reported in the main experiment of pinholes disrupting stereo, the difference between monocular and binocular viewing was less for pinholes  than for tinted glasses  or normal . Pairwise comparisons to check consistency with the main experiment showed an effect of number of eyes for normal, t(11) = 3.93, p = .002, and tinted t(11) = 2.71, p = .020, but not for pinholes, t(11) = 0.76, p = .463. Also in line with the main experiment, the difference between approaching a retreating directions was greater for pinhole viewing  that for normal  or tinted glasses . Pairwise comparisons were again consistent with the main experiment showing a significant difference between approaching and retreating for pinholes, t(11) = 3.15, p = .009, but not normal, t(11) = 0.97, p = .355, or for tinted glasses, t(11) = 0.84, p = .417.A follow-up experiment was conducted using tinted glasses chosen to match the reduction in light reaching the eye associated with pinholes cf. . The lumNeither spatial nor stereo acuity data were normal, and both were analysed nonparametrically. One participant was off the scale for both stereo and visual acuity for binocular pinhole viewing and was excluded from these analyses. Including or excluding their flipping distance data from analysis of the main experiment reported above made no difference to the pattern of results.Z = 2.51, p = .012.A nonparametric Wilcoxon signed rank test showed that stereoacuity was slightly but significantly better without pinholes (median = 40 arcs), than with (median = 50 arcs), F = 19.28, p < .001: acuity with pinholes was M = 1.18, SE = 0.02; without pinholes was M = 1.30, SE = 0.02. There was also a main effect of number of eyes F = 5.76, p = .032: monocular acuity was M = 1.22, SE = .02; binocular acuity was M = 1.27, SE = 0.02. The main effects of both pinhole and monocular viewing correspond to a reduction in acuity. There was no interaction between pinholes and number of eyes, p = .700.For visual acuity, a 2 Pinholes × 2 Eyes resampling analysis of the nonnormal data with 5,000 repetitions showed a main effect of Pinholes, Both monocular and pinhole viewing strengthened the hollow-face illusion. The effect of pinholes is consistent with focus blur driven accommodation helping to disambiguate perceived three-dimensional (3-D) shape in a situation where other sources of depth information are available. The effect of monocular viewing was as expected and confirms that binocular disparities help disambiguate the illusion at close distances. There was an effect of pinhole viewing on monocular as well as binocular viewing—the flipping distance was reduced indicating a stronger illusion. This suggests that pinhole viewing reduces the depth information about the actual concave surface of the mask under both monocular and binocular viewing.The claim that accommodation affects depth perception contradicts the widely accepted conclusion that the human visual system makes little or no use of this potential source of depth information. The task used here was very different from the explicit judgements of absolute egocentric distance under reduced cue conditions that provide the evidence that accommodation in itself is not a strong cue to depth . In the This experiment also differs from much previous work in that we did not attempt to present accommodative depth information is isolation but rather to remove it from a situation where multiple other sources of depth information remained available. This approach, and the indirect measure of perceived depth, is more similar to Alternative explanations for the effect of pinholes include the associated reduction in retinal illuminance. A control involving halving the illumination of the mask had no effect on flipping distance but the reduction in illuminance due to pinholes is by more than a factor of two. However, a second control experiment using tinted glasses that reduced the light reaching the eye by slightly more than the pinholes had no effect on flipping distance, ruling out this explanation. The effect of monocular viewing on depth reversibility was as expected and is as has been reported before . MonoculThe effect of pinhole viewing also interacted with direction of movement. Previously experiments have shown that the flipping distance for approaching observers is lower  than that for retreating observers which is consistent with a hysteresis effect whereby the starting percept is maintained . This efThe accuracy of accommodation depends on both the spatial frequency and the contrast of the stimulus . The holIn summary, the large effect of pinholes on flipping distance suggests that they disrupt a source or sources of depth information that are usually critical for disambiguating the mask at close distances. An effect of pinholes on focus blur, accommodation and, indirectly, vergence provide a possible account for the pattern of results observed. This would suggest that focus blur and accommodation do play a role in 3-D shape perception under full cue conditions.


The object of this paper was to obtain more accurate methods for estimation of PhytOC stock in monopodial bamboo because previous studies may have underestimated it. Eight monopodial bamboo species, widely distributed across China, were selected and sampled for this study in their own typical distribution areas. There were differences (P < 0.05) both in phytolith content (Phytolith/dry biomass) across leaves, branches and culm, and in PhytOC content (PhytOC/dry biomass) across leaves and branches between species, with a trend of leaf > branch > culm. The average PhytOC stored in aboveground biomass and PhytOC production flux contributed by aboveground biomass varied substantially, and they were 3.28 and 1.57 times corresponding dates in leaves, with the highest in Phyllostachys glauca McClure and lowest in Indocalamus tessellatus (Munro) Keng f. It can be concluded that it could be more accurate to estimate PhytOC stock or PhytOC production flux by basing on whole aboveground biomass rather than on leaf or leaf litter only. The whole biomass should be collected for more estimation of bamboo PhytOC sequestration capacity in the future.Bamboo plants have been proven to be rich in phytolith-occluded carbon (PhytOC) and play an important role in reducing atmospheric concentrations of CO With further global economic development, the annual CO2 emissions will increase further. It is also worth noting that the continuous increase in global average temperature has directly increased soil respiration, accelerating the release of large amounts of carbon from soil562 content: to reduce emissions and to increase carbon sinks. Studies have shown that soil is the largest carbon pool in terrestrial ecosystems7911Increasing greenhouse gas emissions and continuous rising of atmospheric COPoaceae, Bambusoideae and Cyperaceae plants. Amorphous silicon in soil is absorbed by these plants and gradually accumulates in plant cells and cell gaps1315161820Phytolith is a complex silicon-coated carbon compound formed mainly during the growth of Bambusoideae plants have been shown to have exceptional capacity for phytolith accumulation227 ha. In China, bamboo forests occupied an area of 6.73 × 106 ha in 2010 with the area occupied by Phyllostachys heterocycla (Carr.) Mitford cv. Pubescens (Ph. Pubescens) forests was 4.48 × 106 ha. The total area of other monopodial bamboos was 7.46 × 105 ha. Monopodial bamboo forests accounted for 77.71% of the total area of bamboo forests. In addition, with the development of the bamboo economy, the area of monopodial bamboo forests is projected to increase at a rate of 2.0 × 105 ha per year6 ha, accounting for over 80% of the total area of bamboo forests. The forest carbon sink is one of the most important ways to reduce atmospheric CO2242526et al. and Li et al. estimated the amount of PhytOC sequestered in bamboo forests worldwide in 2010 and in bamboo forests in China in 2013, respectively17et al.et al.2230As part of the forest carbon sink, bamboo forest PhytOC has been studied by a number of scholars in recent years17222829p < 0.05) except for the PhytOC content in the culm. Both the phytolith and PhytOC contents showed the following trend: leaf > branch > culm. The leaf phytolith content was highest in the triennial Phyllostachys heteroclada Oliver (122 ± 4 g kg−1), and lowest in the triennial Pleioblastus amarus (Keng) Keng f. (37 ± 1 g kg−1)  Keng f. However, the amount of PhytOC stock per ha within the same organ varied among species and significant differences were observed between certain species (Both phytolith contents (Phytolith/dry biomass) and PhytOC contents (PhytOC/dry biomass) varied substantially in the leaf, branch and culm samples among the eight monopodial bamboo species . Differe g kg−1) . The Phy3 g kg−1 . The Phy3 g kg−1 , except  species .−1, with the highest PhytOC stockpile being 24.7 kg ha−1 for Phyllostachys glauca McClure, and the lowest being 5.1 kg ha−1 for I. tessellatus , phytolith content, and C content in phytolith (PhytOC/phytolith) and PhytOC content in monopodial bamboo leaves, we performed a statistic analysis based on results for all leaf samples. It was found that there was significant linear correlation between silicon and phytolith content . The PhytOC production flux for Ph. pubescens was 26.4 kg ha−1 yr−1 for leaves and 44.7 kg ha−1 yr−1 for aboveground biomass, respectively −1)−1)−1)−1)−1)Gramineae) as bambooPlants with different morphological types have different abilities to enrich nutrients in the soil2 sequestered as PhytOC in bamboo leaves, branches and culm is stable over time, either in bamboo ecosystems or in harvested bamboo timber, because PhytOC is thought to be a stable organic carbon. The PhytOC stock of leaves accounted for 19.7% to 60.4% of the aboveground PhytOC stock, with an average of 30.1% and 27.1% for the widely distributed Ph. Pubescens bamboo (−1 yr−1) was much more than that of leaves (21.1 kg ha−1 yr−1). The value of leaf PhytOC flux in this study was generally similar to previous reports  for Ph. Pubescens this study was somewhat higher than that (21.8 kg ha−1 yr−1) by Parr et al.The amount of PhytOC sequestered in the bamboo branches and culms has been largely overlooked in earlier studies. Previous researchers estimated the PhytOC production flux in bamboo forest ecosystems based on the PhytOC content in the leaves of a one bamboo species1720s bamboo . When Phs bamboo , with le reports , e.g. thPh. glauca, Ph. prominens, Pl. amarus, Bambusa piscatorum McClure, Ph. heteroclada Oliver, Pseudosasa amabilis (McClure) Keng f., I. tessellatus and Ph. Pubescens, were selected for sampling in Zhejiang and Anhui Provinces . Origin 8.5 was used to plot the figures. The related formulas are as follows:The leave PhytOC production flux was calculated by reference the ratio of living leave biomass to annual litter amount3031X: The ages of different bamboo species−1 yr−1) = (5) + (6) + (7)Biomass PhytOC production flux .


World Health Organisation (WHO) recommends weight‐for‐length/height (WFL/H), represented as a Z score for diagnosing acute malnutrition among children aged 0 to 60 months. Under controlled conditions, weight, height and length measurements have high degree of reliability. However, the reliability when combined into a WFL/H Z score, in all settings is unclear. We conducted a systematic review of published studies assessing the reliability of WFL/Hz on PubMed and Google scholar. Studies were included if they presented reliability scores for the derived index of WFL/Hz, for children under 5 years. Meta‐analysis was conducted for a pooled estimate of reliability overall, and for children above and below 24 months old. Twenty six studies on reliability of anthropometry were identified but only three, all community‐based studies, reported reliability scores for WFL/Hz. The overall pooled intra‐class correlation coefficient (ICC) estimate for WFL/Hz among children aged 0 to 60 months was 0.81 (95% CI 0.64 to 0.99). Among children aged less than 24 months the pooled ICC estimate from two studies was 0.72 (95% CI 0.67 to 0.77) while the estimate reported for children above 24 months from one study was 0.97 (95% CI 0.97 to 0.99). Although WFL/Hz is recommended for diagnosis of acute under nutrition among children below 5 years, information on its reliability in all settings is sparse. In community settings, reliability of WFL/Hz is considerably lower than for absolute measures of weight and length/height, especially in younger children. The reliability of WFL/Hz needs further evaluation.The  Childhood undernutrition is responsible for approximately 3.1 million child deaths each year  represented as a Z score or a percentile is recommended.There are more published articles reporting the inter‐observer reliability estimates for absolute weight and height/length measures than reliability estimates for WFL/Hz.All studies reported high reliability scores for absolute measures of weight and length/height but this did not translate to high scores for the combined index of WFL/Hz.Future studies on reliability of anthropometric measures to present data on derived indicators of undernutrition to validate their usability across different settings.To evaluate the inter‐observer variation of the WHO recommended anthropometric criteria for assessing acute undernutrition (weight‐for‐length/height) in children aged 0 to 60 months.The search was conducted on PubMed using the following search terms: ‘use OR reliability OR repeatability OR “inter‐observer” OR “inter‐observer” OR “inter observer”) and (anthropom* OR “weight for height” OR “weight for length”) and (infants OR children)’. From the search, relevant titles and abstracts were identified while those obviously irrelevant were excluded. The remaining articles were reviewed to identify those reporting reliability scores for derived indicators of wasting in children. Additional studies not picked up by the search strategy were identified from reviewing the list of references from the full texts.Reliability studies were considered in this review if they reported a reliability estimate for WFL/H using either the technical error of measurement TEM  and/or et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. Of the 390 articles identified in the initial search, 23 reported the reliability of weight and or height/length among children. Three additional studies were identified from reviewing the reference list of the identified articles  appear to be lower than that of WFHz among older children (24 to 59 months); the confidence interval for pooled ICC on younger children; <24 months, and the ICC for older children; >24 months, did not overlap. This observation indicates increased levels of measurement error among the younger children likely because they cannot co‐operate with measurements as well as the older children  through a strategic award (084538) and personal fellowship (083576) from the Wellcome Trust and is published with the permission of the Director of KEMRI.The authors declare that they have no conflicts of interest.The study was conceived, designed and executed by MM under the supervision of JB. Both authors were involved in data acquisition, analysis, interpretation and manuscript writing. They have read and approved the final manuscript.


Metabolic syndrome (MS) increases a risk of developing cardiovascular disease (CVD) and type 2 diabetes mellitus (T2DM). The Vitamin D Receptor gene (VDR) may be important for developing MS. The aim of this study is to investigate the correlation between the VDR gene polymorphisms and MS in North China.A case–control study included 391 participants with MS according to the MS diagnostic criteria of International Diabetes Federation 2005 (IDF2005) and 400 controls was conducted on the basis of a cross sectional study which was performed from 2008 to 2012 in Ningxia Hui Autonomous Region, China. Anthropometric data, blood pressure and blood samples were collected in the field investigation. Blood biochemistry analyses were carried out in the laboratory. Two single-nucleotide polymorphisms (SNPs) in the VDR gene, BsmI (rs1544410 A > G) and FokI (rs 2228570 C > T), were genotyped.The difference in the occurrence of genotypes in BsmI between individuals with MS and the control group was significant. Compared with genotype Bb/bb and allele b, genotype BB and allele B showed higher frequencies in MS cases than controls, which suggested they were risk factors. In addition, the genotype BB carriers with MS presented a higher waist circumference, while genotype FF for the FokI polymorphism was correlated with lower BMI in subjects with MS.Our study suggests that the VDR gene polymorphisms appear to be associated with MS in the Northern Chinese population. Allele B and BB genotype for BsmI are risk factors for MS. The BsmI polymorphism seems to influence waist circumference, while the FokI polymorphism influence BMI in subjects with MS. Metabolic syndrome (MS) includes a variety of metabolic components in abnormal pathological state, which contains abnormal plasma glucose, dyslipidemia, high blood pressure, and high cholesterol . PatientThe Vitamin D receptor (VDR) is a member of the steroid hormone receptor family that acts as a transcriptional activator of many genes. The DNA polymorphisms that have been often reported for the VDR gene are (described in both restriction sites and dbSNP info): BsmI (rs1544410 A > G), FokI (rs 2228570 C > T), TaqI rs731236 T > C), and ApaI (rs 7975232 C > T). In past years, many studies have found that these polymorphisms are related to bone mineral density(BMD) 36 T > C,, tubercu3 and the disorders of MS has been previously demonstrated [Although the molecular mechanisms of VDR in components of MS remain unclear, a relationship between 25(OH)Dnstrated –23. Studnstrated , 25. HowTherefore, we suppose the two single-nucleotide polymorphisms (SNPs), BsmI (rs1544410 A > G) and FokI (rs 2228570 C > T), of the VDR gene intricately affect the development of MS. This study was designed to determine the contribution of VDR polymorphisms to MS in a population from North China.A cross-sectional study was conducted from 2008 to 2012 in Guyuan County, Qingtongxia County and Pingluo County, of Ningxia Hui Autonomous Region, China. Stratified cluster sampling was applied to select two villages (one Hui ethnic village and one Han ethnic village) in each county, totaling six villages. During the study period, 4614 subjects aged 24–75 were interviewed with a questionnaire, followed by anthropometric measurements , and blood pressure determination. Pregnant or breastfeeding women, patients with severe mental disease and other serious illness were excluded from the study. The subjects enrolled did not report to have chronic viral infection, cold or flu, acute respiratory infection, or any type of surgery in the week preceding the study. Meanwhile, 2615 participants had blood drawn via venipuncture for laboratory measurements, and finally 1169 blood samples were selected for genotyping by mechanical sampling (k = 1). Based on diagnostic criteria , 391 cases with MS and 400 controls were selected from the same population to conduct the case–control study. The study protocol had been approved by the Medical Ethics Review Committee of Ningxia Medical University. All participants signed the consents on enrollment after they received written and verbal information about the study.All participants were interviewed by trained persons with a standard closed-ended questionnaire. The contents of the questionnaire included general demographic characteristics, blood pressure, blood lipid, blood sugar, smoking, physical activity status, drinking and related diseases .2.Obesity was defined as BMI ≥ 30. Arterial blood pressure was measured three times in sitting position using an electronic sphygmomanometer . All participants were at rest for at least 30 minutes before the measurement was collected. Patients with average blood pressures ≥140/90 mmHg or taking antihypertensive medication in 2 weeks were classified as being hypertensive.Standing height was measured using a portable ruler. Body weight was also measured using a weight scale . All measurements were performed by well-trained investigators. The reading of measurements was accurate within 0.1 cm or 0.1 kg, and the average of the two collected measures was recorded for further analysis. Body mass index (BMI) was calculated as kg/m2) along with any two of the following four factors: triglycerides ≥ 150 mg/dL (1.7 mmol/L); HDL-cholesterol < 40 mg/dl (1.03 mmol/L) in males, < 50 mg/dL(1.29 mmol/L) in females; blood pressure, systolic BP ≥ 130 or diastolic BP ≥ 85 mmHg; fasting glucose ≥5.6 mmol/L (100 mg/dl) [Blood samples were drawn from the antecubital vein at the hours of 6 to 8 AM, after at least 10 hours of fasting and avoidance of alcohol. Two sets of fasting blood samples were collected separately from each subject in sodium fluoride potassium oxalate tubes (for glucose) and lithium heparin vacuum tubes (for lipids).The latter collections were then centrifuged and kept at −80°C until analysis. Fasting glucose was immediately determined by One Touch Ultra 2 . Serum levels of HDL- cholesterol, LDL- cholesterol, and triglycerides were measured by enzymatic assay . All analyses were carried out through an automatic biochemical analyzer . Subjects were further divided into two groups, cases with MS and controls without MS, according to the IDF definition. For a subject to be defined as having MS, they must have: Central obesity . After extraction, 30 samples of the extracted material were randomly selected for validation by agarose gel electrophoresis. SNP genotyping of BsmI and FokI in the VDR gene was performed using a technology known as SNPscan™ –28 and descriptive data were presented by mean and standard deviation. The Kolmogorov-Smirnov test was used to analyze normality of the distribution of each variable. Comparisons between any two groups were performed using independent t-tests. Chi-square analysis was applied to examine the variation of sex, genotype and gene frequency in different MS groups. Chi-square analysis was also used to test Hardy–Weinberg equilibrium (HWE) for the genotypes in all groups of subjects. ANOVA analysis was used to analyze clinical variables in different genotypes. Odds ratios (ORs) and their 95% confidence interval (CI) were computed for the risk alleles from logistic regression analysis. Statistical significance was set at P < 0.05.P, 0.762 and 0.376, respectively) . However, there was no difference in FokI polymorphism.As shown in Table Ptrend = 0.02). The frequencies of genotype BB and combined Bb/bb in individuals with MS were 89% and 11% respectively, in the control group were 82% and 18% respectively, which suggested that the genotype BB was a risk factor . Compared with genotype BB, genotype Bb was likely to be a protective factor . The incidences of B and b alleles for the BsmI polymorphism in the two group were statistically significant , which indicated allele b could be a protective factor. However, it seemed no significant evidence to be risk factors or protective factors to FokI polymorphism in individuals with MS .Moreover, the genotypes of BsmI and FokI were combined and generated as 9 new genotypes like BBFF to analyze the cumulative effect of the polymorphisms, as shown in Table Table The results showed that VDR polymorphisms may influence the emergence of MS. The VDR BsmI (rs1544410 A > G) polymorphism appeared to associate with MS. Specifically, allele B and genotype of BB in BsmI were associated with MS, while allele b and genotype Bb seemed to play a protective role. The role of genotype bb was unclear, because of its limited frequency. When discussed VDR gene polymorphisms with the components of MS, we found the genotype BB carriers with MS presented a higher waist circumference. In addition, genotype FF for the VDR FokI (rs2228570 C > T) polymorphism was correlated with lower BMI in MS. Our findings were different with previous researches which demonstrated the VDR FokI polymorphism was related with triglycerides and HDL-cholesterol levels .A few studies have demonstrated that VDR polymorphisms were related to obesity, diabetes, insulin sensitivity and insulin secretion , 19, 29.2D3[The VDR gene is not a main influencing factor to the variability of circulating levels of vitamin D, according to genome-wide association (GWA) studies , 35, 36.2D3 and in i2D3, 38. Ser2D3 and inci2D3. Further2D3. UnfortuThis study revealed a significant association of VDR BsmI polymorphism with MS in the Northern Chinese population. Allele B and genotype BB for BsmI are risk factors for MS. The BsmI polymorphism seemed to influence waist circumference, while the FokI polymorphism influence BMI in subjects with MS. Thus, we propose that the BsmI and FokI polymorphisms of VDR are potential prognostic variables which may predict the risk of developing MS. Even so, further examination should be carried out on large population. Also, genome wide association studies are still needed to evaluate the direct effect of these polymorphisms on MS.Yi Zhao and Sha Liao Co-first authors.


This study evaluated the possibility of pretreating selected solid fraction of an anaerobic digester treating food waste to lower the hydraulic retention time and increase the methane production. The study investigated the effect of different pretreatments  for enhanced methane production from cottage cheese. The most effective pretreatments were thermal and enzymatic. Highest solubilisation of COD was observed in thermal pretreatment, followed by thermochemical. In single enzyme systems, lipase at low concentration gave significantly higher methane yield than for the experiments without enzyme additions. The highest lipase dosages decreased methane yield from cottage cheese. However, in case of protease enzyme an increase in concentration of the enzyme showed higher methane yield. In the case of mixed enzyme systems, pretreatment at 1 : 2 ratio of lipase : protease showed higher methane production in comparison with 1 : 1 and 2 : 1 ratios. Methane production potentials for different pretreatments were as follows: thermal 357 mL/g VS, chemical 293 mL/g VS, and thermochemical 441 mL/g VS. The average methane yield from single enzyme systems was 335 mL/g VS for lipase and 328 mL/g VS for protease. Methane potentials for mixed enzyme ratios were 330, 360, and 339 mL/g VS for 1 : 1, 1 : 2, and 2 : 1 lipase : protease, respectively. Food waste is the single largest component of waste stream by weight. About 135.5 million tons per year of municipal solid waste is generated in India and food waste alone constitutes about 30–40% . AnaerobFood waste is characterized by its high organic content, most of it being composed of easily biodegradable compounds carbohydrates, proteins, and, in some cases, small amount of lipids. The anaerobic biodegradability of organic matter depends on its composition and the amount of methane produced depends on the biochemical nature of the waste . For ins3 of biogas/day. The food waste for this plant is from the institute's cafeteria catering to 2500 students. The commonly seen undigested solid fractions in the outlet of the digesters are cottage cheese, whole potatoes, and whole eggs. Studies have shown that digestate can still contain a high biogas potential, mainly as a consequence of residual and undigested volatile solids [We had set up a horizontal plug flow type of anaerobic digester handling one ton of food waste/day and generating 60 me solids . Digestee solids . Utilizie solids . An atteThese pretreatments could improve the waste stabilization and methane production but their application should be proved to be commercially viable in relation to the additional processing costs . Due to 2·6H2O 13 g/L, KCl 4 g/L, CaCl2·H2O 1 g/L, NaHCO3 0.2 g/L, NH4Cl2 g/L, FeCl3·6H2O, KH2PO4 0.5 g/L, pH 7.0 inoculum density 2% (v/v). For production of protease skim milk was used as substrate and for lipase olive oil was used as substrate.The first stage in this study was screening of extreme halophiles isolated from solar saltern for production of protease and lipase. The cultures were grown on NTYE  medium. The culture medium for enzyme production was composed of (w/v) NaCl 200 g/L, MgClThe organisms were allowed to grow on agar plates containing substrate for particular enzymes, that is, skim milk for protease and olive oil for lipase, and incubated at room temperature. The enzymes used in this study and their reported activities were protease, 125 units/mL, and lipase, 150 units/mL. The activity of protease was checked with skim milk as substrate and activity of lipase was checked with olive oil as substrate. For measuring protease and lipase activity the raw enzyme used for the assay was isolated from the culture broth following separation of cells. The culture medium was centrifuged at 8,000 rpm for 20 minutes at 4°C and the cell-free supernatant was used as the source of enzyme.The cell-free supernatant (CFS) of the isolates obtained as described above was subjected to ultrafiltration. Briefly, 50 mL of the CFS obtained was concentrated 10 times  of its original volume using nominal molecular weight cut-off (NMWCO) 3.0 kDa cellulose membrane in a stirred ultrafiltration cell .Cottage cheese separated from the undigested solid fraction food waste was used as the substrate. The cottage cheese was collected from the outlet of the plug flow digester treating food waste. Thermal: the substrate was autoclaved at 15 psi and 120°C for 20 minutes. Chemical: the substrate was treated with sodium hydroxide 0.5 M. Thermochemical: the substrate was first autoclaved at 15 psi and 120°C for 20 minutes and then 0.5 M sodium hydroxide was added. Enzymatic: first, the single enzyme hydrolysis of cottage cheese using protease and lipase was carried out at different concentrations from 0.02 to 0.5% (v/v) with each enzyme. Secondly, three levels of mixed enzyme ratio were tested with 0.04% of mixed enzyme ratio: 1 : 1, 1 : 2, and 2 : 1 of protease and lipase, respectively, to determine the optimal enzyme mixture ratio.Methane potential was determined in batch assays as described in –19. The Total solids and volatile solids were measured in accordance with standard methods . The CheFour strains were selected and screened for production of lipase and protease enzymes. It was found that BK-11 and BK-20 were shown to produce larger zone of clearance . These tThe main characteristics of cottage cheese used in the experiment are reported in Four different pretreatments  were used in order to hydrolyse cottage cheese. The soluble COD of cottage cheese increased with each pretreatment compared to the untreated sample. The most effective pretreatments were thermal and enzymatic. Chemical and thermochemical pretreatments were less effective in terms of solubilisation. Cottage cheese consists of 25–27% of fats. Sodium hydroxide has been reported more efficient in hydrolysing proteins and carbohydrates than lipids  which coFor the different pretreatments studied, biogas production started immediately with thermal pretreatment, but thermochemical and chemical pretreatments showed a lag phase of around 15 days. The highest cumulative biogas production was obtained in thermochemical pretreatment, 615 mL/g VS, followed by thermal treatment 602 mL/g VS . For theIn single enzyme treatment with lipase, biogas production followed same pattern across all samples except the samples with 0.02%, 0.04% and 0.06% lipase wherein the maximum biogas production was faster . With thIn single enzyme treatment with protease, biogas production followed same pattern across all samples except the samples with 0.08% and 0.5% protease wherein the maximum biogas production was faster . In caseFor mixed enzyme pretreatment two enzyme combinations with three different ratios 1 : 1, 1 : 2, and 2 : 1 (lipase : protease) were investigated using an equivalent dosage of 0.04% (v/v) to evaluate the effect of mixed enzyme ratio on methane production. 526, 571, and 539 mL/g VS CH4 were observed at 1 : 1, 1 : 2, and 2 : 1 lipase : protease, respectively. For all three mixed enzyme ratios, methane production was higher than those of single enzyme treatments. Although different ratios of enzyme additions did not show much significance, the biogas production rate and biogas yield were higher compared with the control . As expeIn single enzyme pretreatments, we observed that the average methane production was similar, that is, 335 mL and 328 mL for lipase and protease, respectively . HoweverThe pretreatments studied  effectively hydrolysed cottage cheese into soluble organic compounds. Enzymatic and thermochemical pretreatments were the most effective pretreatments for cottage cheese. Chemical pretreatment showed the poorest performance in terms of both solubilisation and biogas production. High temperature required for thermochemical pretreatment would likely raise the economic and energy dynamics of the process. Moreover, the enzymes were especially suitable for protein and lipid rich cottage cheese with low dose requirement. Cell-free enzymes offer several advantages in the treatment of waste especially to reuse the separated solid fraction as a feedstock for methane production.


The purpose of the investigation was to compare and evaluate pharmacokinetics and biodistribution of ZnY and ZnSO4 in rats. ZnY or ZnSO4 were orally administered to rats at a single dose of 4 mg Zn/kg and Zn levels in plasma and various tissues were determined using inductively coupled plasma-optical emission spectrometry. Maximum plasma concentration values were 3.87 and 2.81 μg/mL for ZnY and ZnSO4, respectively. Both ZnY and ZnSO4 were slowly eliminated with a half-life of over 7 h and bone had the highest Zn level in all tissues. Compared to ZnSO4, the relative bioavailability of ZnY was 138.4%, indicating that ZnY had a significantly higher bioavailability than ZnSO4.Zinc-enriched yeast (ZnY) and zinc sulfate (ZnSO Zinc . Nitric acid and perchloric acid were the guaranteed reagents. All other reagents were of analytical grade and obtained through commercial sources.ZnY was provided by Angel Yeast Co. Ltd. . ZnSO4 at a single dose of 4 mg Zn/kg. At designated time-points, blood was collected in a heparin-coated tube and plasma was separated by centrifugation at 5,000 g for 10 min. Supernatant was then transferred to a clean tube and immediately stored at −80°C until analysis. Immediately after the last time-point blood collection, rats were sacrificed using carbon dioxide and tissues  were immediately excised. All tissue samples washed with tridistilled water were frozen at −80°C until analysis.All procedures involving animals were approved by our Institutional Animal Care and Use Committee. Adult Sprague-Dawley rats, weighing 250 ± 11 g, were obtained from Jackson Laboratory  and housed in standard cages and allowed free movement and access to food and water during the whole experiment. The rats were randomly assigned into two groups and orally given either ZnY or ZnSOμL of plasma or weighted tissue specimen was pretreated with the mixture of nitric acid and perchloric acid  overnight and heated at the temperature of 180–200°C until samples were completely digested, that is, until the solutions were colorless and clear. The remaining acid solution was cooled and diluted with tridistilled water to an appropriate concentration for the assay. Quality control samples were employed for the validation of analytical method and the analytical error was less than 11%.Zn levels in plasma, liver, heart, pancreas, spleen, kidney, and bone were determined using inductively coupled plasma-optical emission spectrometry . Samples were prepared as follows: 100 4 were calculated using noncompartmental analysis of WinNonlin software . Results are presented as mean ± standard deviation (SD). The comparison for difference between parameters or between groups was analyzed using one-way analysis of variance (ANOVA). Differences were considered statistically significant at P < 0.05.Pharmacokinetic parameters of ZnY and ZnSO4, and Zn oxide are often employed for food fortification; however there are serious shortcomings for those compounds. Zn chloride causes some adverse effects, ZnSO4 renders unpalatable flavor, and water-insoluble Zn oxide precipitates in liquid foods; therefore, those compounds are used only in low quantities and in solid foods. ZnY has high solubility and a soft taste, and it does not modify the sensorial characteristics of food.Although food is the major source of Zn, low Zn content in diet and many adverse nutritional factors are barriers to prevention and treatment of Zn deficiency , 9. Zn c4 in plasma is shown in Cmax) and the time to reach Cmax (Tmax) were 3.87 μg/mL and 2 h for ZnY and 2.81 μg/mL and 4 h for ZnSO4, respectively. Cmax, AUC0–24 h, and AUC∞0– ratio values of ZnY to ZnSO4 were 137.7%, 138.4%, and 139.2%, respectively, indicating that ZnY had a significantly higher bioavailability than ZnSO4. Both ZnY and ZnSO4 were slowly eliminated in vivo with respective tλz1/2, values of 7.68 and 7.93 h. During ZnY fermentation in the presence of Zn chloride or ZnSO4, a specific strain of yeast produced specific zinc compounds conjugated with proteins, peptides, and amino acids, which helped improve bioavailability of ZnY compared to inorganic Zn via enhanced intestinal absorption. Our findings with ZnY are consistent with previous studies in which animal proteins could improve Zn absorption [The concentration-time profile of ZnY and ZnSOsorption , which c4 in which bone had the highest Zn level, and ZnY showed higher Zn level than ZnSO4 in each tissue. The results are in agreement with previous report [Zn levels in liver, heart, pancreas, spleen, kidney, and bone are presented in s report . Zn is es report . Zn can s report –15; thers report .4; disposition and biodistribution of Zn in ZnY were similar to those of other sources of Zn.In conclusion, Zn from ZnY was more bioavailable than zinc from inorganic zinc salts ZnSO


The spinal cord is extremely complex. Therefore, trans‐spinal direct current stimulation (tsDCS) is expected to produce a multitude of neurophysiological changes. Here, we asked how tsDCS differentially affects synaptic and nonsynaptic transmission. We investigated the effects of tsDCS on synaptically mediated responses by stimulating the medullary longitudinal fascicle and recording responses in the sciatic nerve and triceps and tibialis anterior muscles. Response amplitude was increased during cathodal‐tsDCS (c‐tsDCS), but reduced during anodal‐tsDCS (a‐tsDCS). After‐effects were dependent on the frequency of the test stimulation. c‐tsDCS‐reduced responses evoked by low‐frequency (0.5 Hz) test stimulation and increased responses evoked by high‐frequency (400 Hz) test stimulation. a‐tsDCS had opposite effects. During and after c‐tsDCS, excitability of the lateral funiculus tract (LFT) and dorsal root fibers was increased. However, a‐tsDCS caused a complex response, reducing the excitability of LFT and increasing dorsal root fiber responses. Local DC application on the sciatic nerve showed that the effects of DC on axonal excitability were dependent on polarity, duration of stimulation, temporal profile (during vs. after stimulation), orientation of the current direction relative to the axon and relative to the direction of action potential propagation, distance from the DC electrode, and the local environment of the nervous tissue. Collectively, these results indicate that synaptic as well as axonal mechanisms might play a role in tsDCS‐induced effects. Therefore, this study identified many factors that should be considered in interpreting results of DCS and in designing tsDCS‐based interventions. There are two plastic mechanisms operating in different regions in the nervous system: synaptic‐mediated mechanisms and intrinsic excitability mechanisms. This study indicates that direct current stimulation affects both synaptic and intrinsic mechanisms of plasticity. Trans‐spinal direct current stimulation (tsDCS) modulates the activity of spinal pathways and circuits in both humans and animals. The main goal of tsDCS is to ameliorate disease sequelae locally at the spinal cord  or brain . Studies have shown that tsDCS can exert polarity‐dependent quantitative control over somatosensory inputs in rats ? How does it modulate synaptic‐mediated responses? Are its effects polarity‐dependent? The answers to these questions will form the experimental foundation to guide and broaden tsDCS research and clinical applications. In addition, determining rules of stimulation by quantifying the effect of DCS on neural systems will allow scientists and clinicians to predict behavioral effects based on DC location, magnitude, orientation, and polarity. An important step toward establishing rules of tsDCS is to test its specific effects on synaptic transmission compared to effects on axonal excitability. Thus, in this study, we hypothesized that tsDCS would differentially affect synaptically and nonsynaptically mediated spinal responses.To assess synaptically mediated activity, we recorded from the sciatic nerve and associated muscles in response to electrical stimulation of the medullary longitudinal fascicle (MLF). We also studied the effects of tsDCS on activation of the spinal lateral funiculus tract (LFT). Finally, a simpler sciatic nerve preparation was used to further characterize the effect of subthreshold DCS on axonal excitability. Together, these investigations revealed complex responses of spinal and peripheral neural tissue to DCS.n = 103; 35–40 g), which were housed under a 12:12‐h light–dark cycle with free access to food and water. Experiments were carried out in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Protocols were approved by the Institutional Animal Care and Use Committee of the College of Staten Island.This study used adult male CD‐1 mice . TS were carefully separated from the surrounding tissue. The TS tendon was threaded with a hook‐shaped 0–3 surgical silk, which was then connected to force transducers. Muscle length was adjusted to yield maximal response. In studies testing the LFT and MLF, muscle force was recorded from two muscles: tibialis anterior (TA) and triceps surae (TS). Tissue surrounding the distal part of the sciatic nerve was removed.Animals were placed in the stereotaxic frame, and a large craniotomy was made to expose the cerebellum. The cerebellum was removed to allow access to the brain stem Fig. A. Relatiμm (31 ga); tip, rounded (standard); inner pole, 125 μm), which was selected instead of a monopolar electrode to reduce interference with the tsDCS electrode. The stimulator consisted of a PowerLab system and stimulus isolator unit (FE180), which produces constant‐current pulses. Recording electrodes were placed on the LFT at L6 and on the sciatic nerve. Muscle twitch force was simultaneously recorded from TS and TA muscles. Test stimulation was performed before, during, immediately after, and 10, 20, 30, and 40 min after tsDCS offset.Two distinct laminectomies were performed to expose the spinal cord at T9 and L6. A spinal DC electrode  was placed over the intact spinal column region between those two laminectomies Fig. B. tsDCS Differential recordings for MLF and LFT were collected using a NeuroAmp Ex Headstage. The headstage has three input connections: single input (+), single input (−) or reference, and ground. The reference electrode was inserted in the adjacent abdominal skin on the left side of the body. The ground electrode was attached to the contralateral abdominal skin (right side of the body). The signal was filtered , digitized at 4 kHz, and stored in the computer for further processing. A power lab data‐acquisition system and LabChart 7 software (AD Instruments) were used to acquire and analyze the data.μm; width, 7 mm; length, 15 mm). In exploratory experiments, we determined that the width of the DC electrode, not its length, was crucial to produce consistent results. Electrodes narrower than 7 mm were not effective to produce the effect observed in this study. The electrode was glued on top of a piece of silicone rubber  was used to stimulate the sciatic nerve. This electrode was located 4‐mm rostral to the DC electrode. The recording electrode was a hook electrode that was custom‐made of tungsten  and attached to the nerve about 3‐mm distal to the DC electrode. The reference electrode was a needle electrode inserted into a flap of the skin in the left hindlimb paw. The ground electrode was attached to the abdominal skin on the right side of the body. The signal was passed to a differential amplifier with an active headstage , filtered , digitized at 4 kHz, and stored in the computer for further processing. A power lab data‐acquisition system and LabChart 7 software (AD Instruments) were used to acquire and analyze the data. Sciatic nerve stimulation was performed using a Digitimer DS7AH constant current stimulator . A Grass stimulator and a constant current isolation unit  were used to deliver subDCS. In all experiments, subDCS was ramped for 10 sec.To test the effect of subthreshold direct current stimulation (subDCS) on local excitability of the sciatic nerve, we first used a single DC electrode configuration. Specifically, one electrode was placed underneath the sciatic nerve, and the reference electrode was connected to a flap of abdominal skin, as shown in ber Fig. A, which μA current was 0.0014 A/M2 or 1.4 mA/cm2. Threshold was determined by gradually increasing DC strength until nerve spike activity began to appear, as shown in n = 7) or cathodal electrode (n = 6) and the nerve segment facing the DC anodal electrode (n = 6) or cathodal electrode (n = 5). Baseline CAP amplitudes were identical in groups for which the same polarity was tested.Given the width of the plate electrode and the 1‐mm diameter of the proximal sciatic nerve, current density produced by 10‐n = 5/group) were used. In one group, cathodal sub‐DC (−10 μA) was applied for 3 min to induce long‐lasting inhibition of nerve excitability, followed 10 min later by anodal sub‐DC (+10 μA) applied for 3 min. In a second group, a‐subDCS was applied first, followed 10 min later by cathodal sub‐DC. In these experiments, only proximal test stimulation was used.To test reversibility of subDCS effects, 10 animals (μm) were used  and when the nerve was brought closer to one of the electrodes . Similarly, between‐electrode test stimulation was used when the nerve was centered (n = 5) and when the nerve was brought closer to one of the electrodes (n = 5). Long‐term effects of this arrangement were tested in three groups of animals: (1) proximal test stimulation was used when current was passed lateral to medial with the nerve centered between the two electrodes (n = 6), and (2–3) between‐electrode test stimulation was used when the nerve was brought closer to the cathode (n = 5) or anode (n = 5).Four groups of animals were used to test the short‐term effects of subDCS in a parallel electrode arrangement. Currents were passed in either the lateral to medial or medial to lateral direction. Intensities, tested in a pseudorandom order for each direction, were 10, 15, and 20 μm) were aligned normally relative to the sciatic nerve and glued to a piece of silicone rubber 5 mm apart  and proximal to distal subDCS effects (n = 5). Similarly, between‐electrode test stimulation was used to test distal to proximal subDCS effects (n = 5) and proximal to distal subDCS effects (n = 5).Four groups of animals were used to test the long‐term effects of perpendicular electrode subDCS arrangement. Proximal test stimulation was used to test distal to proximal subDCS effects  ANOVA with a Holm–Sidak post hoc correction to test differences across time points. Independent variables were time course and stimulation condition; dependent variables were muscle force, nerve CAP amplitude, and nerve response latency. Pearson correlations were used to test correlations between sciatic nerve or spinal potentials (waves) and muscle twitch force. Statistical analyses were performed using SigmaPlot (SPSS). Muscle force, sciatic nerve potentials, and spinal potentials were measured using LabChart software (ADInstruments). Data in all graphs represent means ± SEM, except latency data, which represent medians. The critical level of significance was set at F = 23.6, P < 0.001; n = 6). Latency of the short‐latency response was reduced during c‐tsDCS , but increased during a‐tsDCS  compared to baseline (11.8 ± 0.6 ms) . Latency was reduced during c‐tsDCS , but increased during a‐tsDCS  compared to baseline (53.5 ± 2.6)  Fig. , the cur.6) Fig. .F = 14.7; P < 0.001; n = 5) , but was not changed during a‐tsDCS  compared to baseline (14.6 ± 0.3) (Holm–Sidak method).Next, in a different group of animals, a high‐frequency testing procedure was used . As shown in  5) Fig. D. ResponLonger tsDCS (3 min) interacted with the test stimulus to change the after‐effect outcomes. Sciatic nerve responses and muscle twitch forces were increased after c‐tsDCS and decreased after a‐tsDCS (data not shown). In general, this is consistent with previous findings using a high‐frequency test procedure , second , and third spinal waves , as shown in n = 8). Compared to baseline, the first wave was increased during and across the 40 min after a‐tsDCS. The second wave was reduced during and 20 to 40 min after a‐tsDCS. The third wave was reduced during and across the 40 min after a‐tsDCS.RM ANOVA revealed significant effects of a‐tsDCS on the first , second , and third sciatic waves (F = 5.6. P < 0.001), as shown in There were significant effects of a‐tsDCS on the first  and TA muscle twitch force , as shown in There were significant effects of a‐tsDCS on TS , second,  and third spinal waves , as shown in n = 7). All three spinal waves were increased during c‐tsDCS and after offset. Similarly, there were significant main effect of c‐tsDCS on the first , second , and third  sciatic waves, as shown in F = 25.8, P < 0.001) and TA muscle  across 40 min, as shown in There were significant main effects of c‐tsDCS on the first . The amplitude of the CAP was decreased during a‐subDCS (P < 0.001), but increased for at least 25 min after offset , as shown in P < 0.001; n = 5). The amplitude of the CAP was increased during cathodal subDCS , then decreased for at least 25 min after offset   on CAP  and c‐subDCS , as shown in P < 0.01). During c‐subDCS, the amplitude of the CAP was decreased, and this effect persisted for at least 25 min after offset .RM ANOVA showed significant main effects of a‐subDCS (P = 0.007) and a significant decrease in latency during c‐subDCS (1.7 ± 0.1) compared to baseline (2.1 ± 0.1) .Latency of sciatic nerve potentials was also measured in these experiments. There was a significant increase in latency during a‐subDCS (2.25 ± 0.07 ms) compared to baseline (1.6 ± 0.1 ms) , c‐subDCS was applied for 3 min, then 15 min after its offset, a‐subDCS was applied for 3 min, as shown in F = 25.3, P < 0.001) because c‐subDCS decreased CAP , and subsequent application of a‐subDCS increased CAP relative to baseline (P < 0.003) and to c‐subDCS (P < 0.01) (Holm–Sidak method). In the second group of experiments (n = 5), a‐subDCS was applied first. RM ANOVA showed a significant effect  because a‐subDCS increased CAP , and a subsequent application of c‐subDCS (782.4 ± 46.3 μV) decreased CAP compared to baseline (P < 0.05) and a‐subDCS (P < 0.001) (Holm–Sidak method). Note that the opposite polarity of subDCS did not simply reverse the effect of the previous polarity, but it induced a change that was significantly different from baseline.Next, we tested whether the long‐lasting effects of subDCS could be reversed by applying the opposite subDCS polarity. Proximal test stimulation was used in these experiments. In the first group of experiments (n = 6), the nerve was centered between the two DC electrodes, and sub‐DC current was passed in the lateral to medial direction  , then reverted to baseline values after subDCS offset . Passing the current in the opposite direction  had similar effects. RM ANOVA showed significant effects on CAP  , then reverted to baseline values after subDCS offset .In this group of experiments  , then reverted to baseline value after subDCS offset (P > 0.05). In the same experiments, the current direction was switched so that the nerve was closer to the cathode. RM ANOVA showed significant effects when the nerve was closer to c‐subDCS  , then reverted to baseline values after subDCS offset .The nerve was brought closer to either the anode or the cathode (1 mm relative to 4 mm), and a series of subDCS strengths were passed between the electrodes. RM ANOVA showed significant effects when the nerve was closer to a‐subDCS ( 5) Fig. C. Compar01) Fig. D. ComparF = 72.4, P < 0.001; n = 5). At all strengths, CAP was increased compared to baseline , then reverted to baseline values following subDCS offset . Passing the current in the opposite direction  produced a similar effect on CAP . At all strengths, CAP increased compared to baseline , then reverted to baseline values after subDCS offset .F = 33.6, P < 0.001; n = 5). Compared to baseline, CAP increased (P < 0.001), then reverted to baseline values after a‐subDCS offset . F = 10.4, P < 0.001). Compared to baseline, CAP decreased (P < 0.001), then reverted to baseline value after c‐subDCS offset .μA) was passed between the two polarizing electrodes for 3 min. The centered nerve preparation always produced a persistent increase in nerve excitability regardless of the location of test stimulation; therefore, results from only one of these experiments will be shown here . RM ANOVA showed significant effects of subDCS on excitability of the centered nerve preparation . As expected, CAP decreased during subDCS (P < 0. 05), but increased at all time points following subDCS offset  . Compared to baseline, CAP was decreased during subDCS (P < 0.001) and for 0–10 min after subDCS offset , then increased from 15 to 25 min . Finally, when the nerve was placed closer to anode, RM ANOVA showed a significant effect of subDCS on CAP . Compared to baseline, CAP increased during subDCS and at all time points after sDC offset  . Compared to baseline, CAP was decreased (61.9 ± 1.8%). Following subDCS offset, CAP was decreased at three time points  and showed nonsignificant but numerical decreases at the other three time points  (Holm–Sidak method)  was tested. RM ANOVA showed a significant effect . Compared to baseline, CAP was increased during subDCS and at all time points afterward  , then rebounded and increased at all time points after offset . RM ANOVA showed significant effects of distal to proximal subDCS . Compared to baseline, CAP amplitude increased during subDCS (P < 0.001) and remained increased at all time points following offset . Overall, these findings emphasize that the effects of subDCS are dependent on the polarity of the subDCS electrode closest to the test point , or voluntary movements. Supraspinal systems and sensory inputs play an important role in modulating spinal circuits to adapt to a particular type of movement. For example, increasing the intensity of inputs from the mesencephalic locomotor region to the central pattern generator patterning circuit shifts the gait pattern from slow walking  to galloping (synchronous pattern). This shifts the activity of hindlimb muscles from out‐of‐phase  to in‐phase (co‐contraction). The pattern of locomotor activity largely depends on the excitability of spinal inhibitory interneurons. Therefore, to explain the present findings, we propose that c‐tsDCS inhibits spinal inhibitory interneurons. This allows production of synchronous activity of antagonistic motor neurons, as shown in this study Fig. . Thus, wLow‐frequency test stimulation showed that longer duration a‐tsDCS caused amplification of MLF‐evoked sciatic nerve responses after current offset, however, LFT‐evoked sciatic nerve responses were decreased after the same protocol. This difference indicates that a‐tsDCS affects local tract excitability. This is supported by the observation that the second wave Fig. A was decThis study demonstrated that subDCS can induce short‐ and long‐lasting changes in the excitability of nerve fibers. During stimulation, single electrode anodal subDCS increased nerve excitability in segments further away from the electrode and decreased excitability of segments overlaying the electrode, and cathodal subDCS had opposite effects. It should be emphasized that in single‐electrode application of subDCS, the return electrode was attached to abdominal skin. However, in exploratory experiments (data not shown), the return electrode was attached to the paw, tail, or abdominal skin on the same or opposite side of the body. This did not alter the effects of the sciatic DC electrode. This indicates that the return electrode position is only meaningful if it is placed near or in direct contact with neural tissue, as shown in the parallel and perpendicular electrode experiments. Regardless of the site of test stimulation, after current offset, anodal subDCS caused long‐lasting increases in nerve excitability, and cathodal subDCS caused long‐lasting decreases in nerve excitability. The results shown in One of the most important findings in this study was that the long‐lasting effects of subDCS were reversible. Not only did subDCS of one polarity reverse the effects of the opposite polarity, but it significantly changed CAP amplitude relative to baseline. This has three implications: 1) subDCS did not damage the nerve fibers, 2) the mechanism(s) underlying the effects of subDCS can be changed in either direction (increase or decrease), similar to synaptic mechanisms of plasticity . Effects on axonal excitability were determined by polarity, duration of stimulation, temporal profile (during vs. after stimulation), orientation of axon relative to current direction, current direction relative to action potential propagation direction Fig. B, and diIn addition, an important finding in this study was that the neuronal structures located directly under the electrode respond differently during DCS than the structures surrounding the electrode Fig. . AlthougNone declared.