In alpine regions cows are often equipped with bells. The present study investigated the impact of wearing a bell on behaviour and heart rate variability in dairy cows. Nineteen non-lactating Brown-Swiss cows with bell experience were assigned to three different treatments. For 3 days each, cows were equipped with no bell (control), with a bell with inactivated clapper (silent bell) or with a functional bell . The bells weighed 5.5 kg and had frequencies between 532 Hz and 2.8 kHz and amplitudes between 90 and 113 dB at a distance of 20 cm. Data were collected on either the first and third or on all 3 days of each treatment. Whereas duration of rumination was reduced with a functional bell and a silent bell compared with no bell, feeding duration was reduced with a silent bell and was intermediate with a functional bell. Head movements were reduced when wearing a silent bell compared with no bell and tended to be reduced when wearing a functional compared to no bell. With a functional bell, lying duration was reduced by almost 4 hours on the third day of treatment compared with the first day with a functional bell and compared with no bell or a silent bell. All additional behavioural measures are consistent with the hypothesis of a restriction in the behaviour of the cows wearing bells, although this pattern did not reach significance. There was no treatment effect on heart rate variability, suggesting that the bells did not affect vago-sympathetic balance. An effect of experimental day was found for only 1 out of 10 behavioural parameters, as shown by a decrease in lying with a functional bell on day 3. The results indicate behavioural changes in the cows wearing a bell over 3 days, without indication of habituation to the bell. Altogether, the behavioural changes suggest that the behaviour of the cows was disturbed by wearing a bell. If long-lasting, these effects may have implications for animal welfare. Noise is described as an acute, chronic or intermittent sound , which cIn alpine regions cows are often equipped with a bell throughout the summer season, i.e. for several months, to ensure that farmers can locate their animals on the wide alpine pastures, many areas of which are obstructed from the view. Various types of bells are available, ranging from small ones for goats and calves to large and heavy ones that are used for traditional purposes like seasonal cattle drives (‘Alpabzug’) or exhibitions. Whereas cows are equipped with the larger and heavier bells during these traditional purposes, the smaller bells are used on pasture. However, although the bells used on pasture are smaller than the ones used during traditional purposes, the animals are exposed to them continuously throughout the day for several months. Consequently, cows are also exposed to the bells during resting periods. The chime of a bell is characterised by high and varying amplitudes, varying frequencies and sounds arising intermittently, depending on the movement of the bell. These characteristics have been shown to be more aversive than uniform sounds of similar amplitudes in pigs , 12], g, g12], gIn ruminants, feeding and rumination are indispensable activities and provide useful information regarding health and welfare , 16]. R. R16]. RAlthough rumination can take place during standing, it mainly takes place while cows are lying . As feedTo our knowledge, the impact of wearing a bell on welfare-related indicators has not been investigated in dairy cows. We examined whether and how wearing a functional bell (exposure to weight and chime) and a bell that does not produce any noise (exposure to weight only) for 3 days affects behaviour as well as vagal activity of cows compared with not wearing a bell. Bells become audible when the animals move their heads. Thus, we were interested in whether the cows would move less to quieten the bell or to possibly reduce the burden of its weight. As a consequence, we expected feeding and ruminating durations to decrease because both behaviours include head movements. Moreover, we expected lying duration to increase and locomotion activity as well as head movements to decrease as an adaptation to wearing a functional bell. In addition, herd members may increase the distance to a cow that is wearing a functional bell to avoid the sound. Furthermore, wearing a bell may lead to a change in vago-sympathetic balance resulting in reduced heart rate variability. All these changes were expected to be more pronounced when cows were exposed to both the chime and the weight of the bell compared with only the weight. According to the finding that goats were habituating to once daily minute-long playbacks of bell sounds within 3 days , we expeTo test our hypotheses, dairy cows were equipped with no bell (control), with a bell with inactivated clapper (silent bell) or with a functional bell  for 3 days each in a balanced order. During the treatment period, lying, feeding and ruminating behaviours, leg and head movements, nearest neighbour distances and heart rate variability were recorded. The study was performed between June and November 2012 on a working farm near Zurich, Switzerland . The owner of the land gave permission to conduct the study on this site. Procedures used on the animals included fixing a belt for heart rate measurements, a halter and a pedometer. All cows used in this study had previously been habituated to wearing these devices and were used to human contact. Hence, no negative effect of either the equipment or the handling on the cows’ behaviour was expected. The study did not involve endangered or protected species. Ethical approval to conduct the study was obtained from the Zurich Cantonal Veterinary Office, Switzerland . Animal care and all experimental procedures were in accordance with the ARRIVE guidelines for animal research .In this study, we used 19 dry, multiparous Brown-Swiss cows with an average milk yield of 8,000 kg per 305 days of standard lactation, between 3 and 10 years of age. All cows had previous experience with wearing a bell, as all of them had been equipped with a bell on an Alp for 4–5 months when they were 1 year old. Because the experiment had to be conducted on pasture, its duration was limited to the grazing period (end of May–mid-November). During the experiment, the cows were managed under standard summer grazing conditions with 24 hours access to two flat lowland pastures of approximately 3.5 ha in total. Trees provided protection against rain, wind and sun. Hay was provided ad libitum in a round rack with headlocks, and the cows had access to a water trough 24 hours a day. The herd of dry cows was managed in that cows were added to the herd approximately 5 weeks before calving and were removed from the herd 3–5 days before the estimated calving date. Thus, the herd varied in size and consisted of both focal and non-focal cows . During The experiment was conducted during the grazing season (May to November) in six consecutive batches using one unique group of focal cows per batch. Each group of focal cows consisted of two to five individuals with groups being tested one after the other . The numIn the morning (between 0800 h and 0900 h) of the first day of the second and third treatments, the focal cows were again fitted with a halter, a 3D-accelerometer logger on the hind leg and a thorax belt. At this time on the first day of each bell treatment, cows were also fitted with a silent or a functioning bell . During The casted cow bells that were used in this study were bells made from a bronze containing about 23% tin known as bell metal, and all were similar in size and identical in weight  including the strap used to attach the bell to the neck of the cows . The belAmplitudes were measured in dB(A) by using a precision noise level measuring instrument with integrated long-term storage . The A-weighting scale assigns low weights to the low-frequency tones, to which the human ear and the ears of some animals are less sensitive, and high weights to the typically more audible high-frequency tones . Thus, tThe experimental design resulted in a sample size of 38 cow-days (i.e. 19 cows × 2 days) per treatment for lying behaviour, leg accelerations and nearest neighbour distance, and 57 cow-days (i.e. 19 cows × 3 days) per treatment for feeding and rumination behaviours and acceleration of head movements. Due to technical problems, data collected on five animals of the treatment ‘functional bell’ and on one animal of the treatment ‘silent bell’ could not be used for the analysis of lying behaviour and activity, and data collected on six animals of the treatment ‘functional bell’ and on five animals of the treatments ‘no bell’ and ‘silent bell’ could not be used for feeding and rumination analysis. One cow approached calving and had to be excluded from the treatment ‘functional bell’, and data collected on another cow had to be excluded from the treatment ‘silent bell’ because the strap of the bell was broken. Concerning lying behaviour and leg acceleration, this resulted in a remaining sample size of 34 cow-days for the treatment ‘functional bell’, 38 cow-days for ‘silent bell’ and 38 cow-days for ‘no bell’. For feeding and rumination behaviours, we had a remaining sample size of 46 cow-days for ‘functional bell’, 47 cow-days for ‘silent bell’ and 52 cow-days for ‘no bell’.g. The accelerometer was powered by a 260 mAh lithium-polymer battery, rechargeable via USB connection, which enabled measurements for several days. Data were recorded on an integrated memory chip with a capacity of 2 million data points, transferred to a computer via USB connection and stored in a Microsoft CSV-file. MSR PC-Software was used for data transfer and analysis as well as settings of the data loggers . M. Mg. The by each  was usedUsing the scan sampling method , the dis2 Pro, version 1.3.0.3, Polar Electro Oy, Kempele, Finland).Heart rate variability was recorded using Polar Equine , allowing a non-invasive measurement of heartbeats . To imprAll calculations were carried out using the programs Polar ProTrainer 5 Equine Edition  and R version 3.1.2 . The rooAutomatic correction of the tachograms was carried out using the correction routines included in the Polar Software . Data with an error rate of more than 5% were excluded from the analysis according to von Borell and colleagues . The expLinear mixed-effects models were performed for the different outcome variables defined above using the lmer method from the lme4 package  in R , experimental day , and their interaction. The minimal model considered was the model including treatment only. Maximum models were reduced in a step-wise backward procedure using  account . Post-hoz = −3.0, p = 0.008; z = −1.0, p = 0.55; functional bell vs. silent bell: z = 1.9, p = 0.14). The duration of rumination was reduced by 131 min in the functional bell treatment compared with the no bell treatment  and by 160 min in the silent bell treatment compared with the no bell treatment . The number of chews per cud numerically decreased from the no bell to the silent bell and the functional bell treatments, but this pattern did not reach significance . There was no difference in the summed changes in head acceleration between the silent bell and the functional bell . The variability of head accelerations showed a similar pattern in that there was a reduction in variability with the silent bell  compared with no bell. Leg acceleration during standing bouts was slightly but not significantly reduced with the silent bell and the functional bell . The proportion of scans with another cow in a distance of up to 2 m to the focal animal slightly decreased from the no bell to the silent bell and the functional bell treatments, but this pattern did not reach significance (p ≥ 0.6) or RMSSD/SDNN .The lying duration with the functional bell was reduced by 231 min on the third day of treatment compared with no bell on the first day  to other cows independent of wearing a bell or not, although the chime of the bells was reduced to levels below 80 dB only at distances > 10 m. We thus assume that the need for close spatial contact was stronger than the avoidance of the chime of the bell. Nevertheless, although these effects were not significant, the mean values for the time spent in close distance to another cow, leg acceleration changes and the number of rumination chews per cud tended to decrease when cows were wearing a bell, suggesting that the behaviour of the cows may have been restricted in this situation. By contrast, the physiological measure heart rate variability did not differ between any of the treatments. Although some studies found an increase in heart rate during noise exposure in pigs and cattle  m to oth. HoweverFinally, none of the eight behavioural measures indicated habituation by the cows although all cows had had some experience wearing a bell during one summer season as heifers. This finding contradicts those from studies on goats , pigs 22 and catAdded effects by the functional bell in comparison with the silent bell may help distinguish the effects of weight alone from further effects of the sound of the bell. The functional bell reduced lying duration on the third day of treatment more than the silent bell, indicating that the cows may have felt more uncomfortable through the sound than the weight alone. The reduced duration of rumination with a bell, independent of sound production, may be explained by the head position of the animal during rumination. Cows usually ruminate with an upright position of the head, often while lying. The weight of the bell may result in discomfort when the cows are holding the head in this position, and thus the animals reduced the duration of rumination accordingly. By contrast, the head is always lowered for feeding, i.e. grazing, probably allowing the weight of the bell to be less burdensome. Cows do not have upper incisors, and thus jerky head movements, which make the bell chime, are required to pick a cluster of grass with the tongue. Nevertheless, feeding duration was reduced by almost 2 hours with a silent bell and 40 min with a functional bell. More research is needed to completely disentangle the effects of weight and sound.A bell usually combines weight and sound, with heavy bells being louder than light bells. Bells need to produce sound of certain (high) amplitudes because otherwise they are useless. Thus, a cow has to cope with the combined effects of weight and sound on-farm.Wearing a bell for 3 days interfered with feeding, ruminating and lying behaviours as well as head movements of cows compared with not wearing a bell, but it did not affect heart rate variability. Cows did not habituate to the bells over the 3 days of observation. The observed behavioural changes might challenge welfare if they lasted for an extended time period, but long-term observations are necessary to quantify the effects of bells on welfare.S1 Dataset(TXT)Click here for additional data file.S2 Dataset(TXT)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S1 Sound File(WAV)Click here for additional data file.S1 Video(MP4)Click here for additional data file.


In the combined treatment of La3+ (0.08 mM) and AR, the cell membrane permeability and the peroxidation of cell membrane lipid of soybean roots increased, and the superoxide dismutase, catalase, peroxidase and reduced ascorbic acid served as scavengers of reactive oxygen species. In other combined treatments of La3+  and AR, the membrane permeability, malonyldialdehyde content, superoxide dismutase activity, peroxidase activity and reduced ascorbic acid content increased, while the catalase activity decreased. The increased superoxide dismutase activity, peroxidase activity and reduced ascorbic acid content were inadequate to scavenge the excess hydrogen peroxide and superoxide, leading to the damage of the cell membrane, which was aggravated with the increase in the concentration of La3+ and the level of AR. The deleterious effects of the combined treatment of La3+ and AR were stronger than those of the single treatment of La3+ or AR. Moreover, the activity of antioxidant enzyme system in the combined treatment group was affected directly and indirectly by mineral element content in soybean plants.Rare earth element pollution (REEs) and acid rain (AR) pollution simultaneously occur in many regions, which resulted in a new environmental issue, the combined pollution of REEs and AR. The effects of the combined pollution on the antioxidant enzyme system of plant roots have not been reported. Here, the combined effects of lanthanum ion (La Rare earth elements (REEs) exhibit useful physical and chemical properties that enable their wide applications in petroleum, metallurgy, textiles, ceramics, glassmaking, new materials  production, and medicines –3. At thCucumis sativus L.), muskmelon (Cucumis melo L.) and birch (Betula pendula R.) indicated that the effects of AR on the activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) depend on the plant species, the pH level of AR and the duration of treatments [3+ and AR on plants remains largely unclear although we have tried to clarify the mechanism from the view of photosynthesis in leaves [3+ and AR on root phenotype of soybean [Acid rain (AR) is a global environmental issue . When iteatments –24. The eatments , and it n leaves . Plant r soybean . In orde3+ and AR on antioxidant enzyme system, cell membrane and metal element contents in soybean roots were investigated. The results could provide some references for the scientifically evaluating the potential ecological risk of REEs and AR on plants.Lanthanum (La), the first lanthanide element in periodic table, is ubiquitous in soils . Soybean2+, Na+, K+, NH4+, Mg2+, SO42-, NO3-, F- and Cl- to deionised water, in which the Ca2+, Na+, K+, NH4+, Mg2+, SO42-, NO3-, F- and Cl- contents were 0.83 μM, 1.32 μM, 0.15 μM, 5.34 μM, 0.36 μM, 0.64 μM, 0.47 μM, 0.69 μM and 1.80 μM, respectively. The ionic composition was derived from precipitation data in the southeast of China [2SO4 and HNO3 in a ratio of 1.10:1  [The control rain at pH 7.0 was prepared by adding Caof China –29. The valents) –29.2PO4 in the Hoagland’s solution (pH 7.0) with 1 mM KCl to avoid precipitation of lanthanum phosphate.The-P nutrient solution was prepared by replacing 1 mM KH3+ solutions with different concentrations  were prepared by dissolving appropriate quantities of LaCl3  in-P nutrient solution.The La2 (0.1%) solution for 5 min, rinsed several times with distilled water and germinated in an incubator at 25 ± 1°C. Three uniform seedlings with a radicle length of approximately 0.5 cm were transplanted into each pot  containing the 1.0 kg air-dried substrate . The-P nutrient solution was added to maintain substrate water content of 60%. Pots were placed in a greenhouse at 25 ± 3°C, a day/night cycle of 16/8 h, and a relative humidity of 70 − 80%. Photosynthetic photon flux density provided by the incandescent lamps at the greenhouse was selected as 300 μmol m-2 s-1, based on the fact that the light saturation point of soybean plant is usually 250~500 μmol-2 s-1 [3+ and AR was approximately 310 μmol-2 s-1, measured with photometer . The-P nutrient solution was used to irrigate the plants and to maintain substrate water content of 60%, and 1 mM KH2PO4 was sprayed on the leaves every other day to apply the required inorganic phosphate of plants.Soybeans were cultured as described in our previous study , 27. Theol-2 s-1  and the 3+ and AR in four classes [3+ or AR as well as the combined treatment of La3+ and AR. First was the control treatment. Soybean plants were irrigated with the-P nutrient solution (pH 7.0) and sprayed with the control rain. The substrate water content was 60%. Second was the La3+ treatment. Soybean plants were irrigated with the La3+ solution  and then sprayed with the control rain. The substrate water content was 60%. Third was the AR treatment. Soybean plants were irrigated with the-P nutrient solution and sprayed with AR . The substrate water content was 60%. Fourth was the combined treatment of La3+ and AR. Soybeans were irrigated with the La3+ solution , and then sprayed with AR. The water content of substrate was 60%. Soybeans were sprayed with AR by a sprayer. The diameter of drop was approximately 0.5 mm, which caused the optimal detention time and distribution area of AR on the surface of leaves. For the 16 treatments of La3+ and AR, the amount of simulated AR or control rain was 300 mL per pots, which was calculated according to the precipitation and evaporation in the southeast of China. All treatments were replicated in five pots, and KH2PO4 solution was sprayed every other day to apply the required inorganic phosphate of plants. After the La3+ and AR treatment for 7 d, the roots were collected for the determination of the test indices.Filling-stage soybean plants were subjected to 16 treatments of La classes , e.g. thC1), 2 h (C2), and 2 h after a freeze-thaw treatment . The membrane permeability was expressed as %/h = 100 × (C2-C1)/.Membrane permeability measurements were based upon the previous method . Leaves -1 butylated hydroxytoluene (BHT) using the sonication. After centrifugation, supernatants were pooled and an aliquot of appropriately diluted sample was added to a test tube with an equal volume of either (1)–thiobarbituric acid (TBA) solution containing 20% (w/v) trichloroacetic acid and 0.01% (w/v) BHT, or (2) +TBA solution containing the above materials plus 0.65% TBA. Samples were heated at 95°C for 25 min and after cooling, the absorbance was read at 440 nm, 532 nm, and 600 nm, respectively. The content of MDA was expressed as equation: MDA (nmol/mL) = 106 × (A-B)/157000; A = Abs532+TBA-Abs600+TBA- (Abs532-TBA-Abs600-TBA); B = (Abs440+TBA-Abs600+TBA) × 0.0571. Here 157000 was the molar extinction coefficient for MDA. The molar absorbance of 1–10 mM sucrose at 532 nm and 440 nm was 8.4 and 147, respectively, giving a ratio of 0.0571.The level of lipid peroxidation was expressed as the content of MDA . Samples2O2 was calculated based on a standard curve.Hydrogen peroxide content was determined by the previous method with some modifications . Root ti2- was calculated based on a standard curve.Superoxide content was determined by a modified method according to Elstner and Heupel . Two graRoots (5 g) were homogenized in 50 mM potassium phosphate buffer (pH 7.8) including 5 mM ascorbic acid, 5 mM dithiothreitol, 5 mM ethylene diamine tetraacetic acid (EDTA), and 2% (v/v) polyvinylpyrrolidone. The homogenates were centrifuged at 15,000×g for 15 min and the supernatants were used for enzyme activity assaying.2CO3/NaHCO3 buffer, pH 10.2, 0.1 mM EDTA, 0.015 mM ferricytochrome c, and 0.05 mM xanthine. One unit of SOD was defined as the amount of enzyme that which caused 50% inhibition of the rate of ferricytochrome c reduction.The SOD activity was determined essentially as described by Spychalla and Desborough . Each 3 2O2 at 240 nm according to the literature [2O2.The CAT activity was determined by following the consumption of Hterature . Each 3 2O2 (10 mM), and crude peroxidase. Activity was determined by the increase per minute in the absorbance at 470 nm due to guaiacol oxidation (E = 26.6 mM cm-1).The POD activity was determined by the literature . The rea-1. The total run time was 15 min. Calibration was achieved using purified ascorbic acid as standard.The reduced AsA content was determined by high performance liquid chromatography (HPLC)  at 254 nm according to previous research . Plant t3 and 9 M H2O2, v/v) for 30 min at 2600 kPa (80 psi) in a MDS-2000 microwave oven . The samples were diluted with deionized water to a final volume of 25 mL for determination of La and mineral element contents. In addition, standard solutions were used for the calibration.The mineral element and La contents in soybean roots and leaves were determined by inductively coupled plasma mass spectrometry (ICP-MS)  –41. The 3+ and AR was analyzed by two-way ANOVA.Each treatment was replicated five times. All values were presented as the means ±SD. The significance of differences between different treatments was analyzed by one-way ANOVA using SPSS 17. The interaction between La3+ and AR on membrane permeability and MDA content of soybean roots. When soybean roots were treated with 0.08 mM La3+, the membrane permeability and MDA content of soybean roots were unchanged compared with those of the control (3+ increased to 0.40 (1.20) mM, the membrane permeability and MDA content of roots increased by 49.27% (58.19%) and 21.18% (40.51%), respectively, in comparison with the control   .3+ and AR on the SOD, CAT and POD activities as well as the reduced AsA content in soybean roots. When soybean roots were treated with 0.08 mM La3+, the SOD and CAT activities in soybean roots increased by 6.04% and 15.81%, respectively, while the POD activity and the reduced AsA content were unchanged, compared with the control. As the concentration of La3+ increased to 0.40 and 1.20 mM, the CAT activity was lower, and the SOD activity, POD activity and reduced AsA content significantly were higher than those of the control  negatively correlated with the CAT activity, and positively correlated with the SOD activity, POD activity and reduced AsA content (p<0.05) (When soybean roots were treated with 0.08 mM La control . In othe control . The resan roots . The H2O(p<0.05) .3+, the contents of K, Ca, Mg and Mn in the roots and leaves were decreased, compared with those of the control. This effect was more evident at higher concentration of La3+ (0.40 and 1.20 mM), excepted for Mg in leaves. The Cu, Fe and Zn contents in the roots and leaves treated with La3+ were increased compared with those of the control except that the Zn content in the leaves decreased at 1.20 mM of La3+. The increased effects rose as the level of La3+ increased. When soybean roots were treated with AR, the K and Mg contents in the roots and leaves were increased, the Ca content in these organs was unchanged, the Cu, Mn, Fe and Zn contents in the roots and leaves (excepted for Zn content in the roots) were increased, compared with those of the control.3+ and acid rain at pH 4.5, the K, Ca, Mg and Mn contents were decreased, but the Cu, Zn and Fe contents were increased, compared with those of the control. These effects were more evident in other combined treatments of La3+ and AR. The results of two-way ANOVA indicated that there was an obvious interaction between La3+ and AR that affected the contents of mineral elements in the roots and leaves  and acid rain at pH 4.5 (3.5 or 3.0), the La contents in the roots and leaves were increased, compared with that in the control and La3+ or AR single treatment (excepted for La in leaves). The results of two-way ANOVA indicated that there was an obvious interaction between La3+ and AR that affected the La contents in soybean roots and leaves , including H and ·OH . The bal and ·OH . Abiotic and ·OH . Excess  and ·OH , trigger and ·OH . The inj and ·OH –44. In p and ·OH , 45. AsA and ·OH . In the 3+ did not affect the membrane permeability and MDA content in roots. Thus the generation and the elimination of ROS in roots remained in dynamic equilibrium under this treatment  in roots, which induced the membrane lipid peroxidation and the increase in the membrane permeability (Tables Oryza sativa) treated with Ce3+ and wheat (Triticum aestivum) treated with La3+ [2O2 and O2- excessively accumulated and finally induced significant damage in soybean roots. The similar results were also observed in cucumber (Cucumis sativus) and tomato (Lycopersicon esculentum) [The treatment of 0.08 mM Lareatment . The trey Tables  and 2. Tith La3+ , 11, 47.ulentum) , 48.3+ and AR, the combined treatment of La3+ and AR showed the different effects on the antioxidant enzyme system, which depended on the concentration of La3+ and the pH of AR  and AR, the CAT activity was inhibited, on the contrary, the SOD activity, POD activity and the reduced AsA content were increased  still excessively accumulated in soybean roots  positively (or negatively) correlated with the activities of antioxidant enzyme system in the roots . These r3+ and AR increase the membrane permeability and the peroxidation of membrane lipid. The increases resulted from the excess accumulation of H2O2 and O2- together with the changes in the activities of the antioxidant enzymes and the content of antioxidant. Moreover, the changes in the contents of mineral elements in soybean plants directly and indirectly affected the activities of the antioxidant enzyme system in roots. These effects mentioned above were higher than those of the single treatment of La3+ or AR. Thus more attention should be paid on the potential threat of the combined pollution of La3+ and AR. Furthermore, in this study, the experimental design excluded soil and the effects from xenobiotics in soil . Therefore, the obtained results need to be confirmed by extending the investigation in real and natural soil-plant systems.The combined treatment of La


AR) revealed a novel A2933 insertion that alters the Tyr codon to a termination codon (Y857X), resulting in a truncated form of the receptor. Computer simulation revealed major conformational changes in the hydrophobic pocket that accommodates the hormone. An insA2933 results in a truncated receptor incapable of binding the ligand and is responsible for the clinical symptoms of CAIS in the patient. The levels of the AR transcript in peripheral blood leukocytes were higher in the patient than in her heterozygous mother and her heterozygous sister, as well as in the two healthy sisters. It is hypothesized that elevated levels of the AR transcript in the patient might be caused by the inability of the truncated receptor to react with IFI-16, which functions in complex with AR to inhibit the expression of the AR gene.A patient with a female phenotype, 46,XY karyotype, and a diagnosis of complete androgen insensitivity syndrome (CAIS) was examined. Her mother and three 46,XX sisters were also included in the study. Sequence analysis of the androgen receptor gene ( AR) is located on the X chromosome (Xq11-13) and contains eight exons  and dihydrotestosterone (DHT) affect the development and differentiation of the male external genitalia during embryonic development and are responsible for virilization of the pubertal male .These defects are transmitted in an X-linked recessive manner and have a prevalence of about 1 in 65,000 individuals. Three main phenotypic forms of AIS have been identified: a complete form (CAIS), a partial form (PAIS), and mild form (MAIS). All patients with CAIS have a 46,XY karyotype and possess a female phenotype with nearly normal female external genitalia and breast development. Instead of female internal genitalia, they have testes located in the abdomen or inguinal canals. Their pubic hair is scarce and axillary hair is absent. Testosterone levels are within the normal male range and estradiol is within the normal female range, while serum gonadotropin levels are normal or slightly elevated. The partial form (PAIS) is characterized by a wide spectrum of phenotypes, ranging from an incomplete female phenotype through various degrees of feminization of male external genitalia. In the mild form (MAIS), the phenotype is usually male and the symptoms of feminization can be very weak  genes belonging to the p200 family. Their protein products are known to inhibit cell cycle progression, modulate apoptosis, and act as negative regulators of gene expression, including the AR gene  using Taq Polymerase  and previously described primers  reverse transcriptase and random hexamers , according to the manufacturer’s instructions. AR and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAs were amplified (RT-qPCR [reverse transcription quantitative PCR]) with the use of specific primers  of two repeats from three or more separate experiments and were subjected to one-way analysis of variance (ANOVA). All results were also tested with the post-hoc Student–Newman–Keuls test. The significance of the differences were tested at the level of Diagnosis of the 2-year-old girl undergoing a procedure to correct inguinal hernia was based on the following clinical symptoms: unambiguous female external genitalia, the absence of internal female genitalia, male gonads found bilaterally in the inguinal canals, and 46,XY karyotype. Post-operative histological examination of the gonads was consistent with the diagnosis of CAIS, as no signs of Wolffian derivatives and male gonad texture were found.AR by MSSCP, followed by sequencing of the mutated fragment and structural analysis of the protein product of the gene in silico.Genetic investigation of the patient, her mother, and her three healthy sisters Fig. a involveAR have been described  was found, while in the patient, the AR transcript level was significantly higher (p < 0.001) than that seen in her mother and three sisters  than in her heterozygous mother (I-1), her heterozygous sister (II-3), and her two healthy sisters (II-4 and II-5) is that the truncated receptor, devoid of a large part of the LBD, is probably incapable of androgen binding and, hence, would be unable to stimulate IFI-16 expression, because this requires a ligand–receptor complex. Therefore, the protein product of the IFI-16 gene will not be formed and the IFI-16–androgen complex could not be formed and inhibit the expression of AR.We hypothesize that the reason for higher This hypothesis, however, should be substantiated by further research using a group of CAIS patients with mutations leading to truncation of the LBD of AR.


The Ar I wavenumbers can be corrected by a constant multiplicative correction of 0.999 999 933.Recent observations at the National Institute of Standards and Technology indicate that the Ar I wavenumbers reported by Whaling et al.  are systematically too large. To investigate the source of this problem, selected lines of Ar I and Ar II were remeasured in the same spectra used by Whaling et al. The measurements show that the Ar I wavenumbers of Whaling et al. are systematically shifted with respect to the Ar II wavenumbers previously reported by Whaling et al. [J. Quant. Spectrosc. Radiat. Transfer  In Ref. , Whaling−1 to 25315 cm−1 were observed in a Hg/Ar electrodeless discharge lamp (edl) with an FTS optimized for the ultraviolet   2] were −1. For 26 of the lines the difference is less than 0.000 05 cm−1, and the largest individual deviations are +0.000 19 cm−1 and −0.000 28 cm−1. These deviations are less than the reported uncertainty for all of the lines; thus the data are entirely consistent with a double correction of the scale.Our speculation is supported by a comparison of the deviations in the third columns of Based on all of the evidence, we propose that the results of  be corre


To report a novel single nucleotide insertion mutation, and present the clinical, genetic, biochemical findings in a patient with primary amenorrhea.Chromosomal analysis was performed by harvesting lymphocytes from peripheral blood sample. Hormonal analysis was performed from the serum. After genomic DNA extraction from peripheral blood leukocytes the coding regions and corresponding exon–intron boundaries of sex-determining region Y (SRY) gene and androgen receptor (AR) gene were amplified by PCR and subjected to direct sequencing.HM010955), resulting in amino acid interchange cysteine to tryptophan at codon 669 in exon 4 [Cys669Trp] (GenBank Protein_id ADF47187).In the patient with a karyotype 46,XX, we identified a novel single nucleotide insertion mutation of the nucleotide G at position 2369 (GenBank accession number We report a novel single nucleotide insertion mutation in exon 4 region of the AR gene. The nature of the mutation presented in the patient is in the ligand-binding domain (LBD) of the AR gene. This insertion mutation was predicted to produce frame shift mutation and resulted in truncated form of the AR protein, implicating it in the phenotype observed with primary amenorrhea. •AR is a member of the steroid receptor superfamily.•AR gene was screened in a patient with primary amenorrhea by direct PCR sequencing.•A novel insertion mutation  was identified.•This causes frame shift mutation resulting in truncated form of the AR protein.•The finding further enriched the AR mutation spectrum. The androgen receptor (AR) is a member of the steroid receptor superfamily and involved in the process of sex differentiation. The principle action of androgen is to regulate gene expression through the AR. Mutations in the AR have been correlated with a wide spectrum of androgen insensitivity syndromes (AIS), that span from mild androgen insensitivity syndrome (MAIS) and partial androgen insensitivity syndrome (PAIS) to complete androgen insensitivity syndrome (CAIS). The symptoms range from phenotypically normal male with impaired spermatogenesis to phenotypically normal women with primary amenorrhea.AR has a well characterized modulator structure and is organized into functional domains. The AR gene is located on chromosome Xq11-12. Spanning 90 kb, it comprises 8 exons with 2757 bp of open reading frame within a 10.5-kb messenger RNA (mRNA) encoding for a modular protein of 919 amino acid residues , with thThe N-terminal transactivation domain is the largest AR domain, but it contains less than 15% of AR mutations leading to androgen insensitive syndrome . The C-tWe present a case of young woman affected with primary amenorrhea. The sequencing of AR gene revealed the presence of a novel insertion mutation that created a subsequent frame-shift mutation and resulted in truncated form of AR.A 19-year-old girl was referred to our institute because of primary amenorrhea, poor breast development, tall stature and without history of previous illness. She is the first daughter born to nonconsanguineous healthy parents. Her mother's pregnancy and delivery were normal. There was no family history of delayed puberty or infertility. At this stage the girl was screened for the clinical, genetic, biochemical, and molecular investigations. The study was approved by the institutional ethics committee, and informed written consent was obtained from the subject.The girl was 175 cm tall and weighed 55 kg. She showed normal body mass index, normal IQ and absence of Turner's stigma. The physical examination showed poor pubertal development  with no virilization. The clinical examination of the genitals showed normal female genitalia, clitoris and partially fused labia. Abdominal and retroperitoneal ultrasonographic studies detected hypoplastic uterus and absence of ovaries. Genitogram studies revealed blind-ending vagina and absence of cervix.Serum testosterone (T), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) levels were measured by radioimmunoassay.Chromosome analysis was performed by lymphocyte culture obtained from the peripheral blood of the subject. Conventional staining and the standard GTG banding technique were applied . The karFor mutational analysis, Genomic DNA was extracted from peripheral blood samples of the subject . The SRYThe SRY gene PCR conditions were initial denaturation at 94 °C for 1 min followed by 30 cycles of 94 °C for 30 s, 65 °C for 30 s, 72 °C for 1 min, with final extension at 72 °C for 5 min. The PCR conditions for exon 4 of the AR gene consisted of initial denaturation at 94 °C for 12 min followed by 30 cycles of denaturation at 94 °C for 45 s, annealing at 58 °C for 45 s, and an extension at 72 °C for 1 min, with a final extension at 72 °C for 10 min. The DNA sequencing was performed with the PCR primers using the BigDye terminator v3.1 Cycle Sequencing Kit and DNA sequences run on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). All the remaining exons of the AR gene were directly sequenced to confirm the presence of any other mutation.HM010955). The mutation was identified in the exon 4 region of AR gene that changes the sense of codon 669 from TGT (Cysteine) to TGG (Tryptophan) (GenBank Protein_id ADF47187). This mutation results in a frameshift and created a termination codon in place of His681X that resulted in a truncated form of AR protein. The mutation was confirmed by bidirectional sequencing  insertion at nucleotide number 2369 , results in amino acid change Cys669Trp in the conserved LBD of AR. This mutation results in a frameshift and created a termination codon in place of His681X that resulted in a truncated form of the AR protein, critical for AR function. This might result in a decrease in or even abolish the ligand-dependent transactivation. Thus, the novel insertion mutation identified in the AR gene is responsible for the symptoms in the subject and this finding is consistent with the clinical diagnosis. To the best of our knowledge, the mutation described in our study is novel and previously unpublished.


With the aging population, strokes have become a leading cause of disability and cognitive impairment. On average, someone has a stroke every 40 s in the United States, resulting in approximately 800,000 strokes annually , 2. As aForty percent of stroke survivors experience moderate to severe impairments requiring specialized care , while aAlthough CSCs treat acute stroke patients more effectively , we feelThe ideal way to integrate neurorehabilitation services in CSCs is to base recommendations on quality improvement studies that have demonstrated how to manage referral protocols to optimize rehabilitation outcomes. For example, studies examining the impact of referring patients for rehabilitation during the first days of admission vs. those referred at discharge can be used to create quality standards for internal and external monitoring. Unfortunately, studies comparing rehabilitation outcomes with different rehabilitation care referral procedures are not yet available. CSCs are ideal settings for examining differences in outcome based on systems of care, and thus we urge stroke researchers to begin evaluating and comparing rehabilitation referral pathways. However, until evidence-based protocols for rehabilitation are available, true quality monitoring in the CSC setting needs to be based on the best-practice standards.Psychological care is an integral part of all neurorehabilitation programs, due to the fact that stroke patients are at high risk of depression , 9. InteThe presence of neurorehabilitation services in designated CSCs can allow for the continuity of care from physiatrists, neurologists, rehabilitation nurses, physical and occupational therapists, speech-language pathologists, dieticians, social workers, neuropsychologists, case managers, and recreational therapists as part of the experienced and specialized interdisciplinary team paying careful attention to neurorehabilitation. With a more systematically integrated assessment of progress, including attention to psychosocial issues and early comprehensive discharge planning, this model for stroke-care not only potentially improves patient outcomes, but also decreases the financial burden on the medical care system and improves hospital–home transitions.Rehabilitation should begin in the hospital, as soon as possible, following the stroke. Any rehabilitation program should aim to improve function by allowing stroke survivors to operate as independently as possible . Stroke Since peak neurological recovery occurs within the first 3 months of the initial insult  and largAnother major advantage of involving rehabilitation in CSCs is to initiate a robust rehabilitation care pathway that includes post-acute, home-based and chronic components, patient and family education, and lifestyle adjustments  is extremely important.Because current stroke-care frequently involves a delay in initiating intensive acute inpatient rehabilitation, starting these services within a CSC, to continue in another sub-acute setting, may be optimal for qualifying patients . As thesThe importance of evaluating disability outcome measures is well recognized in patients undergoing inpatient neurorehabilitation . Prior tWe propose that all stroke patients be initially evaluated by the functional independence measure (FIM), or its short version (AlphaFIM) that is created for acute care settings , just beAlthough many acute stroke survivors show some level of improvement regardless of treatment, we urge our colleagues to plan quality improvement and care feasibility studies to determine how co-treatment with rehabilitation can be most effectively managed in CSC settings.Since the inception of physician quality research, the Center for Medicare and Medicaid Services has evaluated referral to rehabilitation as a quality care measure in stroke and stroke rehabilitation, however, no requirements have been specified about the process of rehabilitation evaluation. It is inevitable that these specifics should be defined and appropriate care processes be implemented as part of best-practice stroke-care. At this time, we feel that involving highly qualified subspecialists in rehabilitation in CSC rehabilitation referrals is the best way to enforce a clinical practice standard. The United Council for Neurologic Subspecialties (UCNS) certifies neurologists and physiatrists in neurorehabilitation. Starting in 2014, the American Board of Physical Medicine and Rehabilitation (ABPMR) will certify neurologists, physiatrists, and family practice physicians in Brain Injury Medicine. These specialists are the most appropriate experts to manage rehabilitation referrals. We also feel they can assemble the guidelines for an effective post-stroke neurorehabilitation regime to be implemented across stroke-care settings. The potential increase in efficiency and improvement in access to a multifaceted care regimen in turn justifies the need for more CSCs that offer neurorehabilitation services.Some stroke survivors with severe disabilities, premorbid (or new) dementia, major illness, or unstable medical problems can have difficulty tolerating intensive exercise therapy. For these patients, modified inpatient rehabilitation care pathways can be used so as to focus on other areas of function  and, after acute care or inpatient rehabilitation, care can be continued in sub-acute facilities that provide daily nursing care in association with other services .Emerging treatments to support motor recovery include serotonergic antidepressants – in the FLAME study, empirical (prophylactic) treatment with fluoxetine may even promote recovery as compared to placebo . A recenPublished data pertaining to the cost-benefit analysis between neurorehabilitation services in CSCs vs. elsewhere are scarce. Most of the published studies are related to comparison between traditional inpatient rehabilitation and early supported (in-home rehabilitation) discharge –33. The Early mobilization and recovery acceleration are likely to reduce events such as falls, and reduce the incidence of hospitalization-associated delirium . TherefoComprehensive stroke centers should be the leading choice for treating stroke victims. Concomitant neurorehabilitation program should be an integral part of any CSC . A coordThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In weakly bound systems like liquids and clusters electronically excited states can relax in inter-particle reactions via the interplay of electronic and nuclear dynamics. Here we report on the identification of two prominent examples, interatomic Coulombic decay (ICD) and radiative charge transfer (RCT), which are induced in argon dimers by electron collisions. After initial ionization of one dimer constituent ICD and RCT lead to the ionization of its neighbour either by energy transfer to or by electron transfer from the neighbour, respectively. By full quintuple-coincidence measurements, we unambiguously identify ICD and RCT, and trace the relaxation dynamics as function of the collisional excited state energies. Such interatomic processes multiply the number of electrons and shift their energies down to the critical 1–10 eV range, which can efficiently cause chemical degradation of biomolecules. Therefore, the observed relaxation channels might contribute to cause efficient radiation damage in biological systems. Inter-particle reactions in weakly bound systems are often difficult to pinpoint by detecting exclusively the kinetic energy of the produced ions. Here the authors present a full-coincidence experiment, where inter-particle reaction channels are determined by the measurement of all outgoing particles. This relaxation results from the behaviour of nature to reach the lowest energetic state. For the transition from isolated atoms and molecules in the gas phase to the weakly bound systems , a wealth of new energy transfer phenomena emerges like the recently discovered interatomic Coulombic decay (ICD), which is enabled only by the presence of the environment1Experimentally ICD was confirmed for the first time in 2003 ref. . From th781011All previous experiments based on electron impact relied on the detection of two ions formed and on the determination of the kinetic energy release . However, in this case the assignment to ICD is not ambiguous, since other competitive but fundamental relaxation mechanisms like radiative charge transfer (RCT)13In the present electron ionization study of argon dimers we have solved this problem by measuring all five charged particles in the outgoing reaction channel, that is, detecting both resulting ions and all three electrons. This full-coincidence experiment allows an unambiguous identification of the relaxation process accompanied by the electron ionization process. We show that both relaxation mechanisms are present and elucidate the ratio between both mechanisms. Moreover, we find a markedly enhanced production of low-energy electrons in the relaxation of argon dimer. The results suggest that the observed decay processes may have a significant influence on the radiation damage of biological tissues like DNA15++Ar+ fragment ions measured at increasing projectile energies of E0=37, 43 and 67 eV. For the highest impact energy (67 eV) the overall shape of the ion yield showing two peaks at 3.8 and 5.2 eV is in good agreement with theoretical predictions10et al.et al.+*(3p−2nl)−Ar) with shallow potential minima (between 36 and 130 meV binding energy) close to the equilibrium internuclear distance (Req) of the neutral argon dimer at 3.8 Å. They will quickly decay at Req via ICD to the repulsive Ar+−Ar+ final state due to the short lifetime (∼100 fs) compared with the vibrational periods (∼1 ps) and form the low-KER peak at 3.8 eV; (2) slow ICD, where higher ionization satellites are populated, which have deeper potential wells (250–340 meV) being shifted to shorter R around 3 Å  RCT for population of the states (Ar++−Ar) above the double ionization threshold of the argon atom (43.4 eV) (ref +−Ar+ state by transferring an electron from the neighbour atom to Ar++ accompanied by photon emission. Also this radiative process is rather slow (approximately nanosecond regime)R, and thus contributes to the higher KER peak.A multi-particle coincidence spectrometer (reaction microscope) designed for electron impact experiments103 Å ref. . These h eV) ref . The sysE0=37 eV in Our observation of only one peak at 3.8 eV KER for E0=67 eV. From this we obtain a two-dimensional correlation map between the KER and the binding energy (EB), as shown in EB as the initial projectile energy E0 minus the sum energy of all three outgoing electrons (EB=E0−(E1+E2+E3)). EB is the energy of the state reached after both initially bound electrons are ejected, that is, for ICD the Ar+−Ar+ repulsive states and for RCT the intermediate state Ar++−Ar . In contrast, the region at higher EB is characterized by transitions to doubly ionized states coupled with a neutral argon atom Ar++−Ar, that is, these are precursor states that will lead via RCT to the Ar+−Ar+ state. The additional EB observed here is taken by the emitted photon. This result shows clearly that the RCT relaxation mechanism is present in the clusters once the energy of the electron is above the threshold since energetically only the Ar+−Ar+ ion ground state channel is open.To clearly identify and separate the underlying relaxation processes above the threshold of both processes we carried out the fully quintuple-coincidence experiment of argon dimer at  Ar++−Ar . AccordiEB region in +* with energy Ar+EICD+KER, where EICD is the ICD electron energy. Moreover low ICD electron energies are observed for the fast ICD process at KER=3.8 eV and higher ICD electron energies occur for slow process consistent with our discussion above. For the strongest line at EICD≈1.8 eV +*(3p−2 4p) and Ar+*(3p−2 3d) contribute. The second visible line at EICD≈3.2 eV +*(3p−2 3d) and Ar+*(3p−2 5s). For slow ICD channels a band of contributions is found at KER=5.2 eV and EICD=3.0–6.0 eV, which can be assigned to a series of about 20 terms of the configurations Ar+*(3p−2nl) with nl=4d, 4f, 5d and 6s  the dimer result (e−/Ar+/Ar+) shows clear enhancement in the low-energy region (<8 eV) due to the presence of the ICD processes. Thus, the pure ICD electron spectrum is obtained and presented in EICD) and KER we can determine the binding energy of the ICD initial state as given below:In our experiment, monomer ionization is simultaneously recorded with dimer ionization and fragmentation. Since the ICD processes are absent for monomers, the monomer data can be considered as a reference to determine the continuous electron energy distribution due to scattered projectiles and ejected electrons from the ionizing collision. Thus, this contribution can be subtracted from the electron spectra recorded for the dimers to reveal the ICD electrons solely present for dimers. As one example the ionized electron energy spectra from monomer and dimer are presented in +−Ar+ state at R close to infinity.where How to cite this article: Ren, X. et al. Direct evidence of two interatomic relaxation mechanisms in argon dimers ionized by electron impact. Nat. Commun. 7:11093 doi: 10.1038/ncomms11093 (2016).


Acknowledgement of COPD underdiagnosis and misdiagnosis in primary care can contribute to improved disease diagnosis. PUMA is an international primary care study in Argentina, Colombia, Venezuela and Uruguay.To assess COPD underdiagnosis and misdiagnosis in primary care and identify factors associated with COPD underdiagnosis in this setting.1/FVC) <0.70 and the lower limit of normal (LLN). Prior diagnosis was self-reported physician diagnosis of emphysema, chronic bronchitis, or COPD. Those patients with spirometric COPD were considered to have correct prior diagnosis, while those without spirometric criteria had misdiagnosis. Individuals with spirometric criteria without previous diagnosis were considered as underdiagnosed.COPD was defined as post-bronchodilator (post-BD) forced expiratory volume in 1 second/forced vital capacity (FEV1/FVC <0.70) and 226 (LLN) had COPD. Underdiagnosis using post-BD FEV1/FVC <0.70 was 77% and 73% by LLN. Overall, 102 patients had a prior COPD diagnosis, 71/102 patients (69.6%) had a prior correct diagnosis and 31/102 (30.4%) had a misdiagnosis defined by post-BD FEV1/FVC ≥0.70. Underdiagnosis was associated with higher body mass index (≥30 kg/m2), milder airway obstruction (GOLD I–II), black skin color, absence of dyspnea, wheezing, no history of exacerbations or hospitalizations in the past-year. Those not visiting a doctor in the last year or only visiting a GP had more risk of underdiagnosis. COPD underdiagnosis (65.8%) and misdiagnosis (26.4%) were less prevalent in those with previous spirometry.1,743 patients were interviewed, 1,540 completed spirometry, 309 (post-BD FEVCOPD underdiagnosis is a major problem in primary care. Availability of spirometry should be a priority in this setting. Chronic obstructive pulmonary disease (COPD) is a considerable cause of morbidity and mortality. Sustained disease prevalence is partly due to the increasing age of the world’s population, the continued smoking of tobacco and in low-income countries to biomass exposure. Knowledge of the burden of disease in all settings is important for health systems worldwide.1/FVC) <0.70 (fixed ratio) to define COPD  a. How many such episodes have you had in the past 12 months? b. For how many of these episodes did you need to see a doctor in the past 12 months? c. For how many of these episodes were you hospitalized in the past 12 months?) and hospitalizations were also obtained.Spirometry was undertaken using a portable, battery-operated ultrasonic spirometer . Spirometry tests were performed at baseline and 15 min after the administration of 400 μg salbutamol, according to the American Thoracic Society criteria of acceptability and reproducibility.1/FVC <0.70 (fixed ratio), and second, the same ratio under the lower limit of normality . The reference equations used to calculate the LLN were the PLATINO study reference equations [Two definitions of COPD were used: first, a ratio of the post-bronchodilator (post-BD) FEVquations ,10. Sevequations .Prior diagnostic label of COPD was determined using a self-reported physician diagnosis of emphysema, chronic bronchitis, or COPD.Those individuals with previous medical diagnosis and spirometric criteria of COPD were considered as correct prior medical diagnosis.Among those with previous medical diagnosis and not spirometric criteria of COPD, the misdiagnosis or incorrect prior medical diagnosis was attributed.1/FVC <0.70 or <LLN), but had no previous medical diagnosis of COPD.Underdiagnosis of COPD was considered when participants had spirometric criteria of COPD .1/FVC <0.70 and LLN, respectively. From the 309 patients with COPD by post-BD FEV1/FVC <0.70, 90 patients (29.1%) do not have COPD by the LLN.Detailed descriptions of participation rates have been published previously . In summ1/FVC<0.70 criteria), and the proportion of correct prior diagnosis and misdiagnosis among those patients with prior medical diagnosis of COPD by post-BD FEV1/FVC <0.70 criteria are presented in 1/FVC <0.70 (correct prior diagnosis), while 31 patients (30.4%) had post-BD FEV1/FVC ≥0.70 (misdiagnosis). From the 309 COPD subjects by post-BD FEV1/FVC <0.70 criteria 82 subjects (26.5%) have prior asthma medical diagnosis. This probably represents asthma misdiagnosis.The proportion of COPD underdiagnosis among those patients with spirometric diagnosis (post-BD FEV1/FVC <0.70 definition was 77% (ranging from 63% in Colombia to 90% in Venezuela), and using LLN was 73% (ranging from 61% in Colombia to 87% in Venezuela) (1/FVC <0.70 definition was 15.5% (238/1540 patients) and using the LLN definition was 10.7% (165/1540 patients) (The proportion of COPD underdiagnosis (according to the different criteria) overall and by country is shown in nezuela) . The popatients) .1/FVC <0.70 and LLN criteria), patients with correct prior medical diagnosis, and the underdiagnosis proportion (post-BD FEV1/FVC <0.70 and LLN criteria) are shown in 2), without wheezing, dyspnea and history of exacerbations or hospitalizations due to exacerbations in the past year (2), had more severe airway obstruction (GOLD III–IV), had respiratory symptoms, and history of exacerbations or hospitalizations due to exacerbations in the past year. 1/FVC <0.70 and LLN). The crude odds of COPD underdiagnosis were significantly higher among individuals who were black, had a higher BMI (≥30 kg/m2), without dyspnea and wheezing in past year, had mild to moderate airway obstruction (GOLD I–II) and no history of exacerbations and hospitalizations due to exacerbations in the past year, whereas the adjusted odds of COPD underdiagnosis were significantly higher only in those individuals who were black, had a higher BMI (≥30 kg/m2), and had mild to moderate airway obstruction (GOLD I–II).Description of the population’s characteristics according to selected variables, prevalence of COPD , those who did not visit a clinician in the last year or those who only attended the GP have more risk of underdiagnosis than those who attended “both” .1/FVC <0.70 criteria decreased (The proportion of COPD misdiagnosis (26.4%) and underdiagnosis (65.8%) by post-BD FEVecreased  among th1/FVC <0.70 definition of COPD, the prevalence of COPD underdiagnosis was 77% and using the LLN definition was 73%. Second, COPD underdiagnosis was associated with higher BMI (≥30 kg/m2), milder airway obstruction (GOLD I–II), black color, absence of dyspnea, wheezing, and no history of exacerbations and hospitalizations due to exacerbations in the past year. Third, assistance by a specialist was protective for COPD underdiagnosis. Fourth, having performed spirometry in past 12 months decreased COPD underdiagnosis and misdiagnosis.The most important findings of this study of COPD underdiagnosis in the Latin American primary care setting were: first, using the post-BD FEV1/FVC <LLN) were underdiagnosed; there was considerable variation across sites, and underdiagnosis was associated with male gender, younger age, never and current smoking, poor education, no previous spirometry and lower airflow limitation [Several studies have assessed the prevalence of COPD underdiagnosis in different settings ,5–7,12. 1/FVC <0.70 definition was higher (15.5%) compared to the LLN definition (10.7%). The wide variation in the prevalence of undiagnosed COPD across the regions studied is noteworthy , lower airway obstruction (GOLD I–II), black color, absence of dyspnea, wheezing, and no history of exacerbations and hospitalizations due to exacerbations in the past year. These results are in line with those reported in population based surveys, suggesting that patients with fewer respiratory symptoms and milder airway obstruction are less likely to receive a diagnosis.In population-based surveys, Lamprecht et al  found thDifferent studies have documented a widespread underuse of spirometry by general practitioners to establish COPD diagnosis –17. De MThere are some limitations to the present study that deserve to be discussed. It is possible that the results found in this study do not apply to all Latin American countries. As a result of resources available, the sample size varied among centers and countries (from 104 eligible patients in Uruguay to 726 in Venezuela). Due to the smaller sample size in Uruguay and Colombia we recalculated the margin of error for prevalence for these two sites and they are: Uruguay (7.8% percentage points) and Colombia (4.35% percentage points). Uruguay is the worst scenario . Nevertheless, even if a convenience sample was utilized in this study, the procedure used was the most reasonable in view of the operational possibilities in each country: to avoid selection bias, centers were selected on the basis of available lists of primary care physicians and the study patients were those who visited the center spontaneously. Another possible limitation is the fact that the study design did not include revision of the medical record, so the definition of prior diagnostic label of COPD was only determined using self-reported physician diagnosis of emphysema, chronic bronchitis, or COPD. This could overestimate the underdiagnosis rate of COPD; due to patients may unknown the exact diagnosis.In summary, this study shows that irrespective of the spirometric definition used, COPD underdiagnosis is a major health problem in the primary care setting in Latin America, and highlights the need to improve the diagnosis of COPD in this setting. It is necessary to insist on the greater use and availability of spirometry in the primary care setting to enhance COPD diagnosis and to reduce misdiagnosis. It is also essential to optimize coordination between different levels of care to improve the disease approach.S1 Fig(TIF)Click here for additional data file.S1 File(PDF)Click here for additional data file.


All synthesized compounds were screened for in vitro antimicrobial activity against various pathogenic bacterial and fungal strains and were found to possess good to moderate inhibitory potential against all tested strains. Antimicrobial results reveal that compounds bearing lipophilic electron withdrawing groups such as chloro and bromo displayed significant inhibitory potential against both bacterial and fungal strains. Particularly, compound 3c displayed significant inhibitory against bacterial strains and compound 3h exhibits significant inhibitory potential in comparison to standard drug fluconazole against fungal strain S. cerevisiae.Novel substituted 1,2,4-dithiazolylchromones  Fungal and bacterial infections are affecting millions of people worldwide, and are associated with high rates of mortality and morbidity . Resistachloro and fluoro linked with chromone ring showed more inhibitory potential against fungal strains than standard drug -dithiazol-3′-yl]-4H-chromen-4-one (3a–j). The crude mixtures were purified by column chromatography over silica gel (60–120 mesh) and eluted with 1% ethyl acetate in hexane. The purified products were characterized by spectroscopic techniques such as IR, 1H and 13C NMR, and mass and elemental analysis  assigned to C3′-H and another doublet at δ 7.75 (J = 0.9 Hz) attributed to C2-H of chromone moiety. These NMR spectral features are indicative of an intact chromone ring. 13C NMR spectrum showed resonances at δ 174.8 (C=O), 156.2 (C3′), 140.3 (C3), and at 82.8 corresponding to (C5′), besides the other anticipated carbon resonances of chromone as well as dithiazole ring of compound 3c. Its IR spectrum showed a characteristic band at 1645.7 cm−1 attributed to (C=O) group and band at 771.4 cm−1 corresponding to the disulphide linkage (-S-S-). Mass spectrum revealed a molecular ion peak at m/z 361.5(M+) corresponding to molecular mass 361.5 of compound 3c and its elemental composition is also corroborated by the microanalytical data.In vitro antibacterial activity of compounds (3a–j) were established against five human pathogenic bacteria: Staphylococcus aureus (MTCC96), Bacillus subtilis (MTCC 2451), Escherichia coli (MTCC 82), Pseudomonas aeruginosa (MTCC 2642), and Salmonella typhimurium (MTCC 1251). The antibacterial activity of compounds was determined, with comparison to standard antibiotic discs of amoxicillin and gentamycin. Inhibitory potential of the synthesized compounds was determined in terms of minimum inhibitory concentration (MIC) in μg/mL (in μg/mL  by using3a–j) were found to be active against all pathogenic bacterial strains. Compound 3c showed excellent inhibitory activity against B. subtilis with MIC 0.78, which is comparable with positive control gentamycin, followed by compound 3h with MIC 1.56, wherein compound 3f showed inhibitory activity with MIC 3.12. Compounds 3a, 3c, and 3e displayed promising activity against S. aureus with MIC 6.25. Compound 3h exhibited great inhibitory activity against E. coli with MIC 1.56 followed by compounds 3c and 3g with MIC 3.12, wherein compounds 3f and 3i with MIC 6.25. Compounds 3h and 3c showed good inhibitory activity against P. aeruginosa and S. typhi with MIC 6.25 and 12.5, respectively.All synthesized compounds  was carried out against three fungal strains, that is, Saccharomyces cerevisiae (MTCC 172), Candida albicans (MTCC 3018), and Cryptococcus gastricus (MTCC 1715). Fluconazole was used as a positive control. Inhibitory potential of the synthesized compounds was determined in terms of minimum inhibitory concentration (MIC) in μg/mL using turbidity method  with standard drug fluconazole, followed by 3c with MIC 12.5, respectively. The structure activity relationship of all the synthesized compounds was developed on the basis of obtained in vitro antimicrobial results. The substitution of -chloro group at para position of phenyl ring of the 1,2,4-dithiazole enhanced the inhibitory activity against various bacterial and fungal strains comparison to our previously synthesized compounds -dithiazol-3′-yl]-4H-chromen-4-one (3a–j) were synthesized and characterized by rigorous spectroscopic techniques such as 1H and 13C NMR, IR, and mass and elemental analysis. All synthesized compounds (3a–j) were screened for in vitro antimicrobial activity against various pathogenic bacterial and fungal strains. Compound 3c exhibited excellent inhibitory activity against B. subtilis with MIC 0.78 followed by compound 3h which displayed promising inhibitory activity against B. subtilis and E. coli with MIC 1.56. Compound 3h showed excellent inhibitory activity against S. cerevisiae with MIC 0.78 as compared to standard drug fluconazole and compound 3c showed promising inhibitory activity against C. albicans with MIC 3.12. These compounds can be taken as “lead” to develop more potent antimicrobial agents.Various substituted 3-[5-(1H and 13C NMR (75 MHz) spectra and chemical shifts (δ) are reported as downfield displacements from tetramethylsilane (TMS) used as internal standard and coupling constants (j) are reported in Hz. IR spectra were recorded with Shimadzu FT-IR-8400S on KBr pellets. Mass spectra (ESI method) were recorded on Bruker Daltonics esquire 300 mass spectrometer and HR-MS (ESI-TOF). Elemental analyses were carried out on a Thermoelectron EA-112 elemental analyser and reported in percent abundance.Starting materials and reagents were purchased and used after further purification . JEOL AL-300FT (300 MHz) NMR spectrometer was used to record p-chlorothiobenzamide  in a 100 mL round bottom flask using dry xylene (10 mL) as a solvent at a temperature of 200°C. The heating duration was standardized by monitoring the progress of reaction by TLC. After completion of the reaction, the contents were allowed to cool and the solvent (xylene) was evaporated under reduced pressure [1H and 13C NMR, and mass and elemental analysis.The reaction were carried out by refluxing substituted 3-formylchromones  with pressure . The cru IR (CHCl3) νmax (cm−1): 1647, 1465, 761; 1H NMR : δ 8.31 , 8.27 , 7.90 , 7.74  7.45 , 7.25 , 7.21–7.05 ; 13C NMR : δ 174.8 (C=O), 156.5 (C5), 154.2 (q-arom), 152.7 (C2), 138.9 (q-arom), 134.1 (q-arom), 130.6 (Ar-CH), 129.4 (Ar-CH), 126.4 (Ar-CH), 125.4 (q-arom), 123.8 (Ar-CH), 122.9 (Ar-CH), 118.4 (Ar-CH), 83.8 (C3′); MS (ESI) m/z 395.5 (M+); analysis: Calcad for (C17H10ClNO2S2): C 56.74 H 2.80 N 3.89%; found C 56.70 H 2.75 N 3.80%.Yellow crystalline solid, ; mp: 145–148°C; IR (CHCl3) νmax (cm−1): 1643, 1483, 775; 1H NMR : δ 8.30 , 8.15 , 8.03 , 7.89 , 7.71 , 7.48–7.35 , 1.78 ; 13C NMR : δ 174.8 (C=O), 156.5 (C5′), 154.7 (q-arom), 152.6 (C2), 135.4 (q-arom), 134.1 (Ar-CH), 131.6 (Ar-CH), 129.1 (Ar-CH), 128.8 (Ar-CH), 125.1 (q-arom), 124.8 (q-arom), 122.4 (q-arom), 118.4 (Ar-CH), 83.8 (C3′), 22.4 (-CH3); MS (ESI): m/z 359.5 (M+); analysis: Calcad for (C18H12ClNO2S2): C 57.82 H 3.24 N 3.75%; found C 57.75 H 3.16 N 3.70%.Yellow crystalline solid, ; mp: 155–158°C; IR (CHCl3) νmax (cm−1): 1645, 1475, 773; 1H NMR : δ 8.30 , 8.21 , 7.98 , 7.98 , 7.91–7.87 , 7.75 ; 13C NMR : δ 174.8 (C=O), 156.2 (C5′), 153.6 (C2), 140.3 (q-arom), 138.3 (q-arom), 135.2 (q-arom), 130.5 (Ar-CH), 129.5 (Ar-CH), 128.4 (Ar-CH), 127.5 (Ar-CH), 124.6 (q-arom), 123.4 (q-arom), 118.3 (Ar-CH), 82.8 (C3′); MS (ESI): m/z 361.5 (M+); analysis: Calcad for (C17H9ClFNO2S2): C 57.82 H 2.40 N 3.71%; found C 57.73 H 2.32 N 3.65%.Yellow crystalline solid, ; mp: 181–183°C;IR (CHCl3) νmax (cm−1): 1647, 1425, 771; 1H NMR : δ 8.24 , 7.89 , 7.88 , 7.73 , 7.49–7.41 ; 13C NMR : δ 174.8 (C=O), 155.3 (C5′), 153.2 (q-arom), 152.5 (C2), 139.6 (q-arom), 135.4 (Ar-CH), 131.8 (q-arom), 130.5 (Ar-CH), 130.1 (q-arom), 129.7 (Ar-CH), 125.8 (Ar-CH), 123.4 (q-arom), 119.8 (Ar-CH), 82.8 (C3′); HRMS (ESI-TOF): Calcad for (C17H9ClBrNO2S2): m/z 437.2050; found 437.2050 (M+); analysis: Calcad for (C17H9ClBrNO2S2): C 46.54 H 2.07 N 3.19%; found C 46.46 H 2.02 N 3.12%.Orange crystalline solid, ; mp: 180–182°C; IR (CHCl3) νmax (cm−1): 1685, 1442, 771; 1H NMR : δ 8.13 , 8.10 , 7.92 , 7.64 , 7.42–7.31 ; 13C NMR : δ 174.8 (C=O), 156.6 (C5′), 152.6 (C2), 140.3 (q-arom), 138.8 (q-arom), 130.5 (Ar-CH), 130.1 (q-arom), 129.3 (Ar-CH), 127.6 (Ar-CH), 126.4 (Ar-CH), 123.2 (q-arom), 122.3 (q-arom), 118.3 (Ar-CH), 82.8 (C3′); HRMS (ESI-TOF): Calcad for (C17H9Cl2NO2S2) m/z 399.9694; found 399.9694 (M+); analysis: Calcad for (C17H9Cl2NO2S2): C 51.78 H 2.30 N 3.55%; found C 51.70 H 2.30 N 3.55%.Light orange crystalline solid, ; mp: 140-143°C;  IR (CHCl3) νmax (cm−1): 1649, 1461, 771; 1H NMR : δ 8.16 , 7.92 , 7.83 , 7.65 , 7.56 , 7.40 , 7.35 ; 13C NMR : δ 174.8 (C=O), 156.8 (C5′), 152.6 (C2), 139.1 (q-arom), 134.3 (Ar-CH), 133.8 (q-arom),131.7 (q-arom), 130.5 (Ar-CH), 129.8 (q-arom), 129.3 (C5), 125.7 (Ar-CH), 124.8 (q-arom), 119.9 (Ar-CH), 82.8 (C3′); HRMS (ESI-TOF): Calcad for (C17H9Cl2NO2S2) m/z 415.2220; found 415.2220 (M + Na+); analysis: Calcad for (C17H9Cl2NO2S2): C 51.78 H 2.30 N 3.55%; found C 51.70 H 2.30 N 3.55%.Light yellow crystalline solid, , mp: 170–175°C; IR (CHCl3) νmax (cm−1): 1635, 1458, 775; 1H NMR : δ 8.13 , 7.89 , 7.69 , 7.66 , 7.56 , 7.45 , 7.40 ; 13C NMR : δ 174.8 (C=O), 156.3 (q-arom), 153.5 (C2), 152.5 (C5′), 139.7 (q-arom), 135.6 (Ar-CH), 131.8 (q-arom), 130.5 (Ar-CH), 130.3 (q-arom), 129.3 (Ar-CH), 125.8 (Ar-CH), 123.4 (q-arom), 118.8 (Ar-CH), 82.8 (C3′); HRMS (ESI-TOF): Calcad for (C17H9ClBrNO2S2) m/z 461.8813; found 461.8813 (M + Na+); analysis: Calcad for (C17H9ClBrNO2S2): C 46.54 H 2.07 N 3.19%; found C 46.46 H 2.02 N 3.12%.Orange crystalline solid, , mp: 183–185°C; IR (CHCl3) νmax (cm−1): 1635, 1458, 775; 1H NMR : δ 8.30 , 7.90 , 7.71 , 7.40 , 7.20 , 7.16–7.08 ; 13C NMR : δ 174.8 (C=O), 156.2 (C5′), 153.6 (C2), 140.3 (q-arom), 138.2 (q-arom), 135.4 (q-arom), 130.5 (Ar-CH), 129.3 (Ar-CH), 128.4 (Ar-CH), 127.5 (Ar-CH), 124.7 (q-arom), 123.4 (q-arom), 118.3 (Ar-CH), 82.8 (C3′); HRMS (ESI-TOF): Calcad for (C17H9ClFNO2S2) m/z 413.2697; found 413.2697 (M + Na+); analysis: Calcad for (C17H9ClFNO2S2): C 57.82 H 2.40 N 3.71%; found C 57.73 H 2.32 N 3.65%.Yellow crystalline solid, ; mp: 165–169°C; IR (CHCl3) νmax (cm−1): 1647, 1442.7, 771; 1H NMR : δ 8.39 , 8.14 , 8.07 , 8.04 , 7.97–7.26 ;13C NMR : δ 174.8 (C=O), 156.8 (C5′), 156.2 (C2), 140.3 (q-arom), 138.5 (q-arom), 130.5 (Ar-CH), 130.1 (q-arom), 129.8 (q-arom), 128.3 (Ar-CH), 126.4 (Ar-CH), 123.5 (q-arom), 122.5 (q-arom), 118.3 (Ar-CH), 82.8 (C3′); analysis; Calcad for (C17H8Cl3NO2S2): C 47.62 H 1.88 N 3.27%; found C 47.52 H 1.70 N 3.19%; Calcad for (C17H9Cl2NO2S2): C 51.78 H 2.30 N 3.55%; found C 51.70 H 2.30 N 3.55%.Orange crystalline solid, ; mp: 175–178°C; IR (CHCl3) νmax (cm−1): 1647, 1456, 771; 1H NMR : δ 8.19 , 7.90 , 7.87 , 7.75 , 7.48 , 7.43 , 7.41-7.36 ; 13C NMR : δ 174.8 (C=O), 156.8 (C5′), 156.2 (C2), 139.5 (q-arom), 135.6 (q-arom), 133.6 (q-arom), 131.4 (q-arom), 130.5 (Ar-CH), 129.6 (q-arom), 129.3 (Ar-CH), 125.7 (Ar-CH), 124.8 (q-arom), 119.5 (Ar-CH), 82.8 (C3′); analysis: Calcad for (C17H9Cl2NO2S2): C 51.78 H 2.30 N 3.55%; found C 51.70 H 2.30 N 3.55%.Yellow crystalline solid, ; mp: 171–175°C;Antibacterial and antifungal activities of synthesized compounds were evaluated using the broth macro dilution method to determine the minimum inhibitory concentration (MIC) , 14.Staphylococcus aureus (MTCC 96), Bacillus subtilis (MTCC2451), Escherichia coli (MTCC 82), Pseudomonas aeruginosa (MTCC 2642), and Salmonella typhimurium (MTCC 1251) from Microbial Type Culture Collection, IMTECH Chandigarh, India. Bacteria were cultivated at 37°C in Mueller-Hinton agar medium. The compounds (3a–j) were dissolved in methanol and applied in different concentrations. Methanol was used as negative control and amoxicillin and gentamycin were used as positive controls.The bacterial strains used were μL) was added to obtain a final concentration. A growth control tube and sterility control tube were used in each test. After overnight incubation at 37°C, the MIC was determined by measuring optical density at 600 nm as the lowest concentration that inhibits growth, evidenced by the absence of turbidity.The broth dilution test was performed in test tubes. In twofold serial dilutions of the compounds, a standardised suspension (McFarland turbidity standard) of test bacteria (100 Saccharomyces cerevisiae (MTCC 172), Candida albicans (MTCC 3018) and Cryptococcus gastricus (MTCC 1715). Fungi were cultivated at 26°C in Sabouraud Dextrose Broth (SBD). The compounds (119a–j) were dissolved in methanol and applied in different concentrations. Methanol was used as negative control and fluconazole as positive control.The fungal strains used were The broth dilution test was performed in test tubes. The conidial suspension gave the final concentration. A growth tube and sterility control tube were used in each test. After 48 h incubation at 26°C, the MIC was determined visually as the lowest concentration that inhibits growth, evidenced by the absence of turbidity.


Zingiber officinale (ginger), Curcuma longa (turmeric) Allium sativum (garlic) and Trigonella foenum graecum (fenugreek). Molecular docking was performed for lead identification and molecular dynamics simulations were performed to study the dynamic behaviour of these protein-ligand interactions. Gingerenones A, B and C, lariciresinol, quercetin and calebin A from these spices exhibited high docking score, binding affinity and sustained protein-ligand interactions. Rescoring of protein ligand interactions at the end of MD simulations produced binding scores that were better than the initially docked conformations. Docking results, ligand interactions and ADMET properties of these molecules were significantly better than commercially available AR inhibitors like epalrestat, sorbinil and ranirestat. Thus, these natural molecules could be potent AR inhibitors.Hyperglycemia in diabetic patients results in a diverse range of complications such as diabetic retinopathy, neuropathy, nephropathy and cardiovascular diseases. The role of aldose reductase (AR), the key enzyme in the polyol pathway, in these complications is well established. Due to notable side-effects of several drugs, phytochemicals as an alternative has gained considerable importance for the treatment of several ailments. In order to evaluate the inhibitory effects of dietary spices on AR, a collection of phytochemicals were identified from  This stBased on the NADPH cofactor binding location, the active site of the AR is located at carboxyl terminal end of the beta barrel . NonetheFor predicting the binding orientation, affinity and activity of ligand molecules with the targets, molecular docking was employed . Grid baAfter docking, top ranked compounds were arranged based on the GlideScore . Lower GIn order to explore the stability and variability of top ranking docked complexes, MD simulations were performed . Two top2 on the solute atoms. Four short MD simulations were then performed: (i) 12 ps MD simulation in NVT  ensemble at 10 K with solute heavy atoms restrained with a force constant of 50 kcal/mol/Å2; (ii) 12 ps MD simulation in NPT  ensemble at 10 K with the same restraint; (iii) 12 ps MD simulation in NPT ensemble at 300 K with the same restraint; (iv) 24 ps MD simulation in NPT ensemble at 300 K without any restaints. Unrestrained production simulations were then performed in the NPT ensemble for 100 ns at 300 K temperature and 1.01325 bar pressure. For this, the Nosé-Hoover chain thermostat [The default six-stage system relaxation protocol was employed before starting production runs. This consisted of a series of minimization and equilibration simulations to slowly relax the system while not significantly deviating from the initial conformation. The first two stages consisted of 2000 steps of steepest descent minimization with and without a restraint of 50 kcal/mol/Åermostat  was usedermostat  was usedermostat . The RESermostat  was usedDrug likeliness and ADME studies of ligands was performed to eliminate unfavourable compounds in the early stage of drug development process. Schrodinger Qikprop  was usedOne of the most widely used methods for validating a docking protocol is the re-docking of co-crystalized ligand to the target protein . In thisTo confirm the validity of the results, docking was performed with three structures of AR . All ligand conformers were flexibly docked into the selected AR X-ray crystal structures. Initially, SP docking method was employed and the highest scoring compounds were subjected to XP docking. After XP analysis, the best interacting compounds were ranked based on GlideScore and the best pose of the ligand was chosen. The top ranked compounds that docked to AR are shown in ingiber officinalae and the basic structure consist of two aromatic rings connected by a heptane chain [From Tables ne chain . Only thne chain . Due to ne chain . In agrene chain . In the In the case of 4GCA-gingerenone A complex Figs  and 4A, In 4GCA-gingerenone B complex Figs  and 4B, Curcuma longa produced interactions with structures 4GCA  were calculated. To study the dynamic interaction of AR with the docked phytochemicals, molecular dynamics (MD) simulations of the AR structure (PDB ID: 4GCA) complexed with gingerenones A and B were performed using Desmond. 100 ns simulations of these structures were performed to observe how the binding site adapts to the docked ligand. RMSD of the protein Cα atoms with respect to the initial structure in these simulations stabilized to under 2 Å indicating a stable conformation of the protein  Figs. ThThe binding site is a dynamic region and the simulations indicate that the protein structure is adapting around the docked ligand . GingereRescoring of the protein-ligand interactions was done using Glide after extracting frames every 5 ns from the equilibrium MD simulation. Ligand interaction diagrams depicting the protein-ligand interactions in the highest and the lowest scoring frames are shown in in vitro and in vivo experimental studies are being considered for the validation of the results. Thus, this study sheds light on a mechanism through which these spices may have a positive effect for diabetics. The results further our understanding about how these natural molecules, present in our diet, may interact with proteins in our body.Aldose reductase, the major rate limiting enzyme in the polyol pathway, plays a critical role in diabetic complications. Molecular docking revealed that natural compounds such as gingerenone A, gingerenone B, gingerenone C, quercetin, lariciresinol and calebin A from spices exhibited much better binding score and binding energy than commercially available drugs. MD simulations were performed to study how protein-ligand interactions evolved on a temporal scale. At the end of 100 ns simulations, rescoring of the protein-ligand interactions produced an improvement in the docking score due to novel interactions with proteins and water molecules. Thus, MD simulations could be an effective tool for refining protein-ligand interactions and obtaining a more accurate indication of the dynamic evolution of the binding site interactions. Molecular interactions and pharmacokinetic properties of these compounds are extremely favourable for developing new therapeutic strategies. Further S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Fig(A) AR-gingerenone A complex (B) AR-gingerenone B complex (C) AR-gingerenone C complex (D) AR-calebin A complex (E) AR-lariciresinol complex (F) AR-quercetin complex. Protein is shown in grey cartoon representation, amino acid side chains are shown in stick representation and the docked ligand is in orange. Hydrogen bonds are shown as black dotted lines and π–π interactions are shown as blue lines.(TIF)Click here for additional data file.S2 Fig(A) AR-gingerenone A complex (B) AR-gingerenone B complex (C) AR-gingerenone C complex (D) AR-calebin A complex (E) AR-lariciresinol complex (F) AR-quercetin complex. Protein is shown in grey cartoon representation, amino acid side chains are shown in stick representation and the docked ligand is in orange. Hydrogen bonds are shown as black dotted lines and π–π interactions are shown as blue lines.(TIF)Click here for additional data file.S3 Fig(A) AR-epalrestat complex (B) AR-ranirestat complex (C) AR-sorbinil complex. Protein is shown in grey cartoon representation, amino acid side chains are shown in stick representation and the docked ligand is in orange. Hydrogen bonds are shown as black dotted lines and π–π interactions are shown as blue lines.(TIF)Click here for additional data file.S4 Fig(A) AR-gingerenone A complex (B) AR-gingerenone B complex (C) AR-gingerenone C complex (D) AR-calebin A complex (E) AR-lariciresinol complex (F) AR-quercetin complex. Colored circles indicate amino acids that interact with the bound ligand. Negatively charged amino acids are represented with red circles, positively charged amino acids are represented with dark blue circles, polar amino acids are represented with light blue circles and hydrophobic amino acids are represented with green circles. Hydrogen bonds are represented with purple arrows–dashed arrows for hydrogen bonds involving amino acid side chain and regular arrows for hydrogen bonds involving amino acid backbone. π–π interactions are shown with green lines.(TIF)Click here for additional data file.S5 Fig(A) AR-gingerenone A complex (B) AR-gingerenone B complex (C) AR-gingerenone C complex (D) AR-calebin A complex (E) AR-lariciresinol complex (F) AR-quercetin complex. Colored circles indicate amino acids that interact with the bound ligand. Negatively charged amino acids are represented with red circles, positively charged amino acids are represented with dark blue circles, polar amino acids are represented with light blue circles and hydrophobic amino acids are represented with green circles. Hydrogen bonds are represented with purple arrows–dashed arrows for hydrogen bonds involving amino acid side chain and regular arrows for hydrogen bonds involving amino acid backbone. π–π interactions are shown with green lines.(TIF)Click here for additional data file.S6 Fig(A) RMSD of Cα atoms of AR with respect to the initial structure during the course of the simulation. Simulation reaches equilibrium in the first few nanoseconds as indicated by the plateauing of the RMSD plot. (B) RMSF of Cα atoms of AR indicating backbone regions with major motions. Significant movement is observed in the loop region between residues 217–223.(TIF)Click here for additional data file.S7 Fig(A) RMSD of Cα atoms of AR with respect to the initial structure during the course of the simulation. Simulation reaches equilibrium in the first few nanoseconds as indicated by the plateauing of the RMSD plot. (B) RMSF of Cα atoms of AR indicating backbone regions with major motions. Significant movement is observed in the loop region between residues 217–223.(TIF)Click here for additional data file.S8 FigThe top 3 highest and lowest scoring frames are shown along with the corresponding rescored GlideScore. Colored circles indicate amino acids that interact with the bound ligand. Negatively charged amino acids are represented with red circles, positively charged amino acids are represented with dark blue circles, polar amino acids are represented with light blue circles and hydrophobic amino acids are represented with green circles. Water molecules are represented with gray circles. Hydrogen bonds are represented with purple arrows–dashed arrows for hydrogen bonds involving amino acid side chain and regular arrows for hydrogen bonds involving amino acid backbone. π–π interactions are shown with green lines.(TIF)Click here for additional data file.S9 FigThe top 3 highest and lowest scoring frames are shown along with the corresponding rescored GlideScore. Colored circles indicate amino acids that interact with the bound ligand. Negatively charged amino acids are represented with red circles, positively charged amino acids are represented with dark blue circles, polar amino acids are represented with light blue circles and hydrophobic amino acids are represented with green circles. Water molecules are represented with gray circles. Hydrogen bonds are represented with purple arrows–dashed arrows for hydrogen bonds involving amino acid side chain and regular arrows for hydrogen bonds involving amino acid backbone. π–π interactions are shown with green lines.(TIF)Click here for additional data file.


PMEPA1 silencing on CaP biology. PMEPA1 knockdown accelerated the growth of CaP tumor cells in athymic nude mice. In cell culture models knockdown of PMEPA1 resulted in resistance to AR inhibitors enzalutamide and bicalutamide. While, AR protein down regulation by NEDD4 was PMEPA1 dependent, we also noted a PMEPA1 independent downregulation of PTEN by NEDD4. In a subset of human CaP, decreased PMEPA1 mRNA expression significantly correlated with increased levels of AR transcription target PSA, as a surrogate for elevated AR. This study highlights that silencing of PMEPA1 accelerates the growth of CaP cells through AR, NEDD4 and PTEN. Thus, the therapeutic restoration of PMEPA1 represents a promising complementary strategy correcting for AR and PTEN defects in CaP. Statement of significance: Here we define that silencing of PMEPA1 facilitates the growth of CaP cells and modulates AR through NEDD4 and PTEN. The restoration of PMEPA1 represents a promising complementary therapeutic strategy correcting for AR and PTEN defects.Androgen Receptor (AR) is the male hormone receptor and a nuclear transcription factor which plays a central role in the growth of normal and malignant prostate gland. Our earlier studies defined a mechanistic model for male hormone dependent regulation of AR protein levels in prostate cancer (CaP) cells through a negative feed-back loop between AR and PMEPA1, an androgen induced NEDD4 E3 ubiquitin ligase binding protein. This report focuses on the impact of  SPOP noted in 5–10% of CaPs impair the SPOP mediated AR degradation [AR is a nuclear transcription factor that executes key function in normal and malignant growth of the prostate gland –5. The tradation , 23.PMEPA1 was originally identified by our laboratory as a prostate abundant, highly androgen induced gene that mapped to chromosome 20q13.31-q13.33 [PMEPA1 is a direct transcriptional target of AR in CaP cells [PMEPA1 mRNA expression in matched normal and prostate tumor specimens suggested decreased expression of PMEPA1 in two-third of CaP patients [PMEPA1 has been noted in multiple solid tumors [PMEPA1 expression by transforming growth factor-β (TGF-β) that was associated with colonocyte terminal differentiation [PMEPA1 expression in breast and lung cancer may lead to inhibition of TGF-β signaling [PMEPA1 expression may promote tumorigenesis through distinct cell signaling pathways in a given cellular background.1-q13.33 . Human P1-q13.33 , 24. PMEaP cells . FurtheraP cells . MembersaP cells , 26. Inipatients . In contd tumors , 28. Stuntiation . Subsequntiation . Increasignaling , 35. TakPMEPA1 expression. Loss or decreased PMEPA1 expression in CaP contributes to accelerated cell growth through increased AR and NEDD4, decreased PTEN levels and confers resistance to AR inhibitors used in androgen ablation therapy.In this report, we present new findings on the cancer biologic properties of decreased PMEPA1 depletion on tumor growth in vivo. We have characterized PMEPA1shRNA-LNCaP and PMEPA1shRNA-VCaP stable transfectants to assess the growth of prostate tumor cells in vitro. We observed enhanced cancer cell biologic features by monitoring cell growth, BrdU incorporation, cell plating efficiency, soft agar colony formation, growth response to androgen and cell cycle    in PMEPA1shRNA harboring xenografts (increased by 304%), in contrast to the control group (increased by 69%) (p < 0.05)  Figure . At 9 we) Figure . Taken tPMEPA1 levels in enhancing tumor cell growth by gain of AR function, dose and time kinetic response to the AR inhibitors, enzalutamide and bicalutamide were assessed in cell growth assays. PMEPA1 depletion conferred resistance to AR inhibitors in both LNCaP and VCaP cells  of the AR mediated signaling, play critical roles in the development and progression of CaP. While AR has been extensively studied for alterations of its structure, expression and transcriptional functions in CaP, defects of the AR protein degradation in CaP warrant careful evaluations. Better understanding of mechanisms controlling AR protein levels may also help in developing new therapeutic strategies focusing on AR degradation in a majority of metastatic CaP associating with the gain of AR function.PMEPA1 expression in CaP cells with increased AR due to impaired AR degradation [PMEPA1 expression in the interface of NEDD4, AR and PTEN in CaP biology and the effects on therapeutic AR inhibitors has not been examined before. Three major consequences of loss of PMEPA1 in CaP are summarized in the working model .LNCaP and VCaP cells  were maintained in RPMI 1640 and DMEM growth medium, respectively, supplemented with 10% fetal bovine serum (FBS). For the androgen-depletion experiments, cells were grown in 10% charcoal stripped FBS supplemented cell growth medium for 5 days. Subsequently, cells were treated with various dosages  of synthetic androgen R1881  for indicated time. Hexamethrine bromide (Cat#H9268–5G) and puromycin (Cat#A1113802) used for selection PMEPA1, pCMV-AR (wild-type) and pCMV-AR (mutant T877A) were described before ; pCMV-XL4  and pCMV-XL4-NEDD4 (Cat#SC107811) were purchased from OriGene . The siRNAs for various genes were all purchased from Dharmacon . Each gene of interest was targeted with two different siRNAs  was also purchased from Dharmacon . The Mission shRNA lentiviral transduction particles  were purchased from Sigma-Aldrich . The targeting sequence of PMEPA1 shRNA1 was 5′-GAGCAAAGAGAAGGATAAACA-3′, and PMEPA1 shRNA2 was 5′-GAGTTTGTTCAGATCATCATC-3′. The transfection of plasmids or small interfering RNA were performed following the standard protocol of lipofectamine 2000 . The infection of mission shRNA lentiviruses transduction particles and selection of positive colonies were performed as recommended by the supplier.The mammalian plasmids used were as follows: pcDNA3.1 as vehicle control, pcDNA3.1-t siRNAs  using op5 cells/dish. Cells were grown at 37°C, 5% CO2 for 2 days followed by transfection with plasmids  or siRNA  using lipofectamine 2000 procedure. The AR inhibitors treatment experiments were conducted with PMEPA1 shRNA1 harboring LNCaP and VCaP cells. The transfected cells or the stable transfectants were harvested at indicated time points post-transfection with 0.25% trypsin plus EDTA . Cells were re-suspended into 10 ml regular medium, and 10 μl of single cell suspension was applied to hemocytometer for cell counting with trypan blue .The CaP cells (LNCaP and VCaP cells) were seeded in 6 cm culture dishes at the density of 2 × 10PMEPA1 shRNA transfectant LNCaP or VCaP cells were harvested by centrifugation at 180 × g for 5 min. Cells were resuspended in 300 μl PBS (pH 7.4), and 5 ml methanol was added drop-wise and vortexed at low speed. The fixed cells were incubated in methanol at −20°C for 30 min. After removing the methanol cells were stained by propidium iodide dye  at final concentration of 60 μg/ml containing 50 μg/ml RNaseA in PBS at room temperature (RT) for 30 min and were processed for fluorescence-activated cell sorting (FACS) analysis.Ten million cells of The cells were harvested with M-PER mammalian protein extraction reagent  supplemented with protease inhibitor mixture  and phosphatase inhibitor mixture . Cell lysates were analyzed using Western blot procedures described in details in the Supplementary Methods.Antibodies used for Western blot assay were as follows: anti-AR rabbit polyclonal antibody ; anti-PMEPA1 mouse monoclonal antibody ; anti-NEDD4 rabbit polyclonal antibody ; anti-PTEN mouse monoclonal antibody , anti-PSA rabbit polyclonal antibody ; anti-GAPDH polyclonal antibody ; and horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse TrueBlot antibodies . Antibodies for immunohistochemistry (IHC) assay were: anti-PMEPA1 antibody mentioned above, anti-AR mouse monoclonal antibody , anti-PSA rabbit polyclonal antibody , biotinylated horse anti-mouse antibody , biotinylated goat anti-rabbit antibody  and VIP peroxidase (HRP) substrate kit .PMEPA1 shRNA LNCaP and control-shRNA LNCaP cells. Mice were maintained under pathogen-free conditions in accordance with established NIH guideline under the animal protocol approved by Uniformed Services of University of the Health Sciences, Institutional Animal Use and Care Committee. A total of 40 athymic nude mice were subdivided into two groups: control-shRNA LNCaP and PMEPA1-shRNA LNCaP with 20 mice in each group. Transfectant cells were resuspended in RPMI1640 medium mixed with 50% matrigel . Each mouse received 4 × 106 cells in 200 μl medium by subcutaneous injection to right frank side. Injected mice were monitored for tumor formation twice per week post injection. The end point of the experiment represented tumor diameter of 1.5 cm or 6 weeks after xenograft tumor formation. The excised tumor tissue was fixed in 10% formalin, paraffin-embedded and sectioned for 5 μm slides for Hematoxylin and Eosin stain and immunohistochemistry (IHC) staining for PMEPA1, AR and PSA. Total 10 foci with 20X magnification were randomly examined from each IHC staining slides for quantitative analysis with ImageJ software . The IHC procedures were described in details in the Supplementary Methods.Ten weeks old athymic Ncr-nu/nu mice  were evaluated in tumor xenografts derived from et al [PMEPA1, 5′-CATGATCCCCGAGCTGCT-3′ (forward), 5′-TGATCTGAACAAACTCCAGCTCC-3′ (reverse), and 6FAM-5′-AGGCGGACAGTCTCCTGCGAAACC-3′-TAMRA (probe); PSA/KLK3, 5′-CCCACTG CATCAGGAACAAA-3′ (forward), 5′-GAGCGGGTGTG GGAAGCT-3′ (reverse), and 6FAM-5′-ACACAGGCCAG GTATTTCAGGTCAGCC-3′-TAMRA (probe). The expression of GAPDH served as an endogenous control measured in the same tube as the target gene . Target gene expression in each sample was normalized to GAPDH. Results were plotted as average Ct (cycle threshold) values of duplicate samples. Relative gene expression level was presented as dCt in tumor versus matched benign cells, where dCt represents normalized Ct value of target genes to GAPDH. Statistical analysis of quantitative expression of PMEPA1 and PSA mRNA was evaluated in 82 patients . The sample size (N = 82) was determined by statistical power calculation . Distributions of other clinico-pathological variables were examined by using Chi-square or Fisher exact tests.Quantitative RT-PCR was performed using RNA laser capture microdissected matched normal and prostate tumor specimens as described in Sterbis et al . PCR prit-test. Data are calculated as mean ±SEM or +SEM.If not otherwise stated, significant was calculated utilizing an unpaired