This study is the first attempt to detect the aforementioned protozoan parasites in water samples from countries in SEA, using real-time polymerase chain reaction (qPCR) assays.Access to clean and safe drinking water that is free from pathogenic protozoan parasites, especially CA total of 221 water samples of 10 l each were collected between April and October 2013 from Malaysia (53), Thailand (120), the Philippines (33), and Vietnam (15). A physicochemical analysis was conducted. The water samples were processed in accordance with the US Environmental Protection Agency’s methods 1622/1623.1, microscopically observed and subsequently screened using qPCR assays.Cryptosporidium oocysts were detected in treated water samples from the Philippines (1/10), with a concentration of 0.06 ± 0.19 oocyst/L, and untreated water samples from Thailand (25/93), Malaysia (17/44), and the Philippines (11/23), with concentrations ranging from 0.13 ± 0.18 to 0.57 ± 1.41 oocyst/L. Giardia cysts were found in treated water samples from the Philippines (1/10), with a concentration of 0.02 ± 0.06 cyst/L, and in untreated water samples from Thailand (20/93), Vietnam (5/10), Malaysia (22/44), and the Philippines (16/23), with concentrations ranging from 0.12 ± 0.3 to 8.90 ± 19.65 cyst/L. The pathogens C. parvum and G. lamblia were detected using using qPCR assays by targeting the 138-bp fragment and the small subunit gene, respectively. C. parvum was detected in untreated water samples from the Philippines (1/23) and Malaysia (2/44), whilst, G. lamblia detected was detected in treated water samples from the Philippines (1/10) and in untreated water samples from Thailand (21/93), Malaysia (12/44), and the Philippines (17/23). Nitrate concentration was found to have a high positive correlation with (oo)cyst (0.993).C. parvum and G. lamblia in large water samples.The presence of (oo)cysts in the water samples means that there is potential risk for zoonotic disease transmission in the studied countries. Detection using qPCR is feasible for quantifying both pathogenic  Cryptosporidium parvum (C. parvum) and Giardia lamblia (G. lamblia) are protozoan parasites that can cause gastrointestinal illness in humans [Cryptosporidium oocyst and Giardia cyst stages. However, the aforementioned condition is highly unaffected in harsh water conditions or disrupted by conventional water disinfection treatments  due to its resistance [n humans . Both pan humans . The exisistance .Cryptosporidium spp. and Giardia spp. are two of the highest reported causative parasitic waterborne agents [All over the world, e agents , but thee agents  via conte agents , and viae agents ; infectiThe immunomagnetic separation (IMS) technique, which was developed by The United States Environmental Protection Agency (EPA) can be used to morphologically identify both protozoan parasites. However, identification at species-level can only be done using molecular techniques, such as those used in polymerase chain reaction (PCR)-restriction fragment length polymorphism , 9 and nG. lamblia and C. parvum was two cysts and one oocyst, respectively, in 20–1,500 l of spiked water samples [This study is the first attempt to detect the aforementioned protozoan parasites in water samples from countries in Southeast Asia (SEA), using real-time PCR (qPCR) assays. Since it was developed in 1992 by Higuchi and coworkers , qPCR as samples .C. parvum and G. lamblia in the water basins of these countries would indicate if there is potential risk for zoonotic disease transmission in the region.Because the data on water contamination with protozoan parasites is limited, this study aims to examine the current distribution of waterborne protozoan parasites in various types of water samples from four countries in SEA, namely Malaysia, Thailand, the Philippines, and Vietnam. The distribution patterns of A total of 221 water samples were collected between April and October 2013; 53 samples were collected from Malaysia, 120 samples from Thailand, 33 samples from the Philippines, and 15 samples from Vietnam. They comprised both treated and untreated water , as well tap water, which is supplied to buildings after treatment via coagulation, flocculation, sedimentation, filtration, and disinfection. Swimming pool water was also included in the treated water category because pools undergo a treatment process that includes purification, flocculation, filtration, and chemical disinfection . In the untreated water category, water samples were collected from rain, springs, wells, recreational water bodies , and effluent water sources.Physical parameters  were initially measured in situ using a multiprobe parameter  at the sampling sites. Meanwhile, chemical parameters  were measured using a colorimeter . Both physical and chemical  parameters were recorded as mean values.Each of water was transported to a laboratory within 24 h of collection and filtered through a flatbed membrane filtration machine , using a 142 mm diameter and a 1.2 μm pore-sized nitrocellulose membrane . The accumulated sediments were removed from the top of the membrane using a 0.1 % Tween 80 solution and collected in a Falcon™ 15 ml tube . The 10 ml eluate was then subjected to the IMS procedure.Cryptosporidium and Giardia (oo)cysts. The eluate was purified using the IMS technique . Magnetic beads with Cryptosporidium and Giardia-specific monoclonal antibodies were magnetized, separating the (oo)cyst-magnetic beads complex from the debris [The EPA-established method 1623.1  was emple debris . FinallyGiardia/Cryptosporidium monoclonal antibody reagent  and 4′, 6′-diamidino-2-phenylindole (DAPI) solution , before being fixed with methanol. The slides were then examined through an epifluorescence microscope , alongside positive  and negative (purified water) controls. Morphology was determined based on round oocysts (4–6 μm) for Cryptosporidium and oval cysts (9–12 μm) for Giardia. The numbers of Giardia cysts and Cryptosporidium oocysts were calculated as follows:Each microscopy well slide  was prepared with 5 μl of 1 N NaOH solution, and then 25 μL of the sample was spotted onto a well. The slide was then stained with fluorescein isothiocyanate-conjugated (FITC) The end product of the remaining samples were frozen in liquid nitrogen and thawed in a hot water bath at 56 °C five times before overnight incubation for 12 h to break the tough (oo)cyst cell wall. Then, they were subjected to the standard DNA extraction method . All extracted DNA were measured for purity and concentration using a fluorescence spectrophotometer . The DNA that did not fall within the purity range of 1.7–2.2 were re-concentrated and purified following the protocol for DNA cleanup using Genomic-tips .C. parvum  were used to amplify a 138-bp fragment inside the C. parvum specific 452-bp. For G. lamblia, the primers and probe sequences  were chosen to specifically detect a 62-bp fragment within the SSU RNA gene [The primers and probe sequences for RNA gene , 22.Singleplex qPCR assays were amplified in a concentration with a volume of 25 μL, consisting of: PCR premix of 2 x SensiFAST™ Probe No-ROX Kit , 3.75-pmol of each primer, 1.25-pmol of probe, and 2.5-μL of DNA template. Amplification was performed using the CFX96™ system , and consisted of five minutes at 95 °C, followed by 45 cycles of 10 s at 95 °C, 20 s at 55 °C, and 20 s at 72 °C. Fluorescence was measured during the annealing step of each cycle.G. lamblia axenic culture broth sample , or from the seeded Cryptosporidium oocyst samples that were prepared following the EasySeed™ Cryptosporidium only procedure . At the threshold peak, fluorescence emitted at 490 nm for G. lamblia and at 575 nm for C. parvum.The sample’s first signal threshold cycle (Ct) value was quantified by comparing it with the standard reference Ct value of the positive control. The positive control either originated from the Physicochemical data for both treated and untreated water samples were computed as mean ± standard deviation (SD), with 95 % confidence intervals (CIs), as shown in Table The highest ammonia reading was recorded in treated water samples from Malaysia, 2.07 ± 5.63 mg/L (CI: 3.90), whereas the highest reading of chlorine was recorded in treated water samples from the Philippines, 0.58 ± 0.74 mg/L (CI: 0.52). Two treated water samples from Thailand showed maximum levels of DO and nitrate, at 5.24 ± 6.18 mg/L (CI: 2.64) and 2.31 ± 8.03 mg/L (CI: 3.43), respectively, whereas untreated water samples from Thailand showed the highest average level of turbidity, at 62.51 ± 149.91 NTU (CI: 29.53). Untreated water samples from Vietnam showed maximum levels of TDS  and salinity , and treated water samples showed maximum levels of nitrite .The staining protocol used is based on the concept of conjugated monoclonal antibody with specific FITC dye by targeting the protein of the (oo)cyst cell wall. Meanwhile, DAPI permits the observation of the internal structure of both sporozoites and trophozoites, indicating the viability of individual (oo)cysts .Cryptosporidium oocysts were detected in both treated and untreated water samples, from the Philippines ; Malaysia ; and Thailand . Meanwhile, Giardia cysts were detected in both treated and untreated samples from all four countries: the Philippines ; Malaysia ; Vietnam ; and Thailand , as shown in Table Table C. parvum and G. lamblia, were computed as Ct values. Coefficient of variation (CV) was used to express consistency between cycles as follows:The number of cycles required to intersect the threshold level during amplification, which reflects the accumulation of desired fluorescent signals for both C. parvum was 26.40, compared to a value of 16.26 on the second consecutive amplification cycle and 38.54 on the tenth consecutive amplification cycle. Similarly, for G. lamblia, the optimum Ct value was 28.99, compared to a value of 20.15 on the second amplification cycle and 38.47 on the tenth cycle. The mean values of the 10 consecutive runs were 27.63 ± 7.63 for C. parvum and 29.14 ± 6.09 for G. lamblia, as shown in Table The optimum Ct value for Cryptosporidium oocysts were detected only in treated water samples from the Philippines; 10 % of the samples tested positive with a concentration of 0.06 ± 0.19 oocyst/L, as shown in Table C. parvum using the qPCR method, as shown in Table Cryptosporidium oocysts in untreated water samples from three countries, namely the Philippines (47.8 %), Malaysia (38.6 %), and Thailand (26.9 %). The highest oocyst concentration was recorded in water samples from Malaysia, with a concentration of 0.57 ± 1.41 oocyst/L, followed by samples from Thailand (0.22 ± 0.59 oocyst/L) and the Philippines (0.13 ± 0.18 oocyst/L).Table C. parvum was detected in 4.5 % of the samples from Malaysia  and 4.3 % of the samples from the Philippines (Ct: 36.53), as summarized in Table Using the qPCR method, C. parvum was found only in untreated water samples from Malaysia and the Philippines.A high presence of oocysts was detected in untreated water samples compared to treated water from the aforementioned countries. Giardia cysts were detected in one treated water sample from the Philippines (10 %), out of 10 total samples, with a concentration of 0.02 ± 0.06 cyst/L (see Table G. lamblia using the qPCR method (10 %), with a Ct value of 32.26 ± 0 (see Table Giardia cysts were detected in untreated water samples from the Philippines (8.90 ± 19.65 cyst/L), Malaysia (0.92 ± 1.74 cyst/L), Vietnam (0.51 ± 0.81 cyst/L), and Thailand (0.12 ± 0.30 cyst/L), as shown in Table G. lamblia (73.9 %), followed by Malaysia (27.3 %) and Thailand (22.6 %), with Ct values ranging from 30.49 to 34.41 and CVs ranging from 0.1 to 0.18 . Meanwhile, in untreated water samples, salinity had a positive correlation with ammonia (0.657) and TDS (0.708), whereas nitrite also had a high positive correlation with nitrate (0.851). Amongst the parameters, ammonia, nitrate, and nitrite (0.603–0.742) each had a positive correlation with In Thailand, the data from treated water samples revealed positive correlations between nitrate and salinity (0.627), nitrate and TDS (0.639), nitrate and nitrite (0.772), and nitrate and chlorine (0.735). Nitrite had a positive correlation with chlorine (0.518), salinity (0.535), TDS (0.55), while salinity showed a positive correlation with TDS (0.998). In untreated water samples, a positive correlation was observed between turbidity and salinity (0.577), and nitrite and nitrate (0.656).In the Philippines, a positive correlation was observed between nitrate and (oo)cyst concentration (0.993), salinity and (oo)cyst concentration (1), and TDS and (oo)cyst concentration (1) in treated water samples. Positive correlations were also observed between (oo)cyst and cyst concentrations (0.516), salinity and chlorine (0.527), nitrate and TDS (0.547), turbidity and chlorine (0.6), ammonia and nitrite (0.614), ammonia and turbidity (0.69), and nitrite and cyst concentration (0.720) in untreated water samples.In Vietnam, there was a positive correlation between DO and ammonia (0.642), salinity and TDS (0.96), chlorine and turbidity (0.996), and chlorine and nitrite (1) in the treated water samples. In untreated water samples, ammonia was positively correlated with cyst concentration and turbidity, with values of 0.648 and 0.73, respectively. There was a positive correlation between turbidity and both TDS and salinity (0.778), whereas ammonia had a positive correlation with nitrate, nitrite, and both TDS and salinity, with values of 0.847, 0.876, and 0.879, respectively. Nitrate also showed positive correlation with nitrite (0.882), both TDS and salinity (0.867), and turbidity (0.971), whereas salinity and TDS were both positively correlated with nitrite (0.999).Overall, positive correlations with the presence of parasites were obtained from the chemicals parameters of ammonia, nitrate, and nitrite from Malaysia, the Philippines, and Vietnam.In recent years, the SEA region has seen an exponential increase in the number of residents, with an estimated total population of 290.5 million in the four studied countries. The growing population has meant that there is an urgent need for clean water for daily usage, however, the ability to provide a potable water supply is rather limited in some countries due to the costs involved in building dams. Slow economic growth in some countries in SEA has been a limiting factor in preparing, treating, and supplying clean water to the inhabitants. Clean and treated water is readily available in urban areas but in rural areas, people still depend on natural water sources, such as rainwater, rivers, lakes, etc., and these may have become contaminated by waterborne protozoan parasites.Physicochemical data can be used to preliminary determine water quality, and play a vital role in reflecting nutrient availability which enables parasites to survive. For example, fluctuations in TDS can affect the pH level and DO of the water both directly and indirectly, depending on particle matters. The increase in TDS, and DO are mainly due to increased water usage and precipitation, and are unfavorable conditions for the survival of (oo)cysts, as shown by the negative correlations between (oo)cysts and these parameters in the present study. Previous studies have postulated to use the correlation of nitrate and nitrite concentrations to predict the presence of (oo)cysts in water . MoreoveCryptosporidium and Giardia. It has been proven that turbidity is positively correlated with a number of (oo)cysts due to run-off intensity and effluent discharge. However, in this study, a negative correlation between (oo)cysts and turbidity was observed. Supporting this, another study has demonstrated that the recovery rate of Cryptosporidium oocysts and Giardia cysts declined as the turbidity level in the water increased, regardless of the filtration method used [This study showed the existence of an interesting physicochemical relationship between the presence of hod used , 24.Cryptosporidium oocysts and Giardia cysts found in the selected treated water samples from Malaysia, Thailand, and Vietnam. This means that the process of treating water can effectively eliminate (oo)cysts.Mostly it was the untreated water samples in this study that were contaminated with parasites. The presence of protozoan parasites in waterfalls could explain how humans become directly exposed to parasitic infections . Water basins have also been polluted due to high water usage for domestic, fishing, and recreational purposes by local inhabitants living near rivers. Fortunately, there were no Giardia cysts (cyst/L) appeared to be a major contaminant, and were detected in untreated water samples at a rate of 11.4 times higher than Cryptosporidium oocysts (oocysts/L). This finding is confirmed by the common discovery of Giardia cysts in raw water [Giardia cysts  and its thickness (0.3 to 0.5 μm), compared to Cryptosporidium oocysts, as reported by the EPA [In this study, aw water . This is the EPA . Due to In this study, we chose to purify recovered (oo)cysts using filtration and concentrated them via centrifugation without using any chemicals that are potential PCR inhibitors , 28. TheCryptosporidium oocysts and Giardia cysts in environmental samples, even a small loss can have a significant influence on their detection via quantitative PCR [Although it has been widely proven that the IMS purification technique is considered to be an efficient step pre-PCR, the presence of foreign particles is inevitable  due to poor water quality (cloudy and turbid) . Disturbtive PCR .Not knowing the time when the (oo)cysts were released into water bodies, the extended time needed for processing samples in the lab until molecular analysis can begin, and the loss that occurs during the physical lysis technique and centrifugation steps might contribute to low recoveries during molecular analysis. These factors could also explain the low level or absence of intact (oo)cysts that are needed to maintain good DNA integral contents . FurtherC. parvum and G. lamblia due to a lack of antibody specificity.The IMS technique has been the subject of many studies globally and is considered the gold standard for the identification of (oo)cysts . HoweverReal-time PCR can provide comparable data to that acquired using the IMS technique without compromising the number of samples. The selection of primer-probe targets is based on the unique molecules of each organism, the biology of which are equally important. Detection of these parasites at a molecular level also takes into account the sensitivity and specificity of each primer-probe used in the PCR method , 36. It Some environmental factors can also be used to predict the incidence of (oo)cysts in water bodies. For example, meteorological data  can affect the leachate sediment process after downpour, thus influencing the turbidity and TDS levels of water bodies. Moderate to high correlations were observed between the eight physicochemical parameters employed in this study. Other studies have also pointed to significant interactions of waterborne parasites with other chemical and biological factors , 38.C. parvum oocysts and G. lamblia cysts purified from various types of water samples. The qPCR method can be used for large samples, and is useful for understanding the abundance and distribution of protozoan parasites across different geographic locations. The presence of C. parvum and G. lamblia in both raw and treated water samples can result in potential risk for zoonotic disease transmission in SEA. This requires the urgent attention of relevant policymakers who can implement the necessary preventive measures.Based on the results obtained, physicochemical parameters can be used to obtain baseline data on the deterioration of water quality and how this is correlated with parasite occurrence (i.e. (oo)cyst). Real-time PCR assay allows for feasible detection and identification of


Polygonatum odoratum. The optimal extraction parameters were determined as the following: NaOH concentration (A) 0.3 M, temperature (B) 80°C, ratio of NaOH to solid (C) 10-fold, and extraction time (D) 4 h, in which ratio of NaOH to solid was a key factor. The order of the factors was ratio of NaOH to solid  > extraction temperature  > NaOH concentration  > extraction time . The monosaccharide compositions of polysaccharides from P. odoratum were rhamnose, mannose, xylose, and arabinose with the molecular ratio of 31.78, 31.89, 11.11, and 1.00, respectively. The reducing power, the 1, 1-diphenyl-2-picryl-hydrazil (DPPH) radical scavenging rate, the hydroxyl radicals scavenging rate, and the inhibition rate to polyunsaturated fatty acid (PUFA) peroxidation of the alkaline soluble polysaccharides from P. odoratum at 1 mg/mL were 9.81%, 52.84%, 19.22%, and 19.42% of ascorbic acid at the same concentration, respectively. They also showed antimicrobial activity against pathogenic bacteria Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, and Escherichia coli.The present study is to explore the optimal extraction parameters, antioxidant activity, and antimicrobial activity of alkaline soluble polysaccharides from rhizome of  Polygonatum odoratum (Mill.) Druce is an edible herb mainly distributed in the Southern area of China and some other south east Asian countries [ P. odoratum is customarily called “Xiang Yuzu” by local people, and the dried rhizome is often used as a common Chinese traditional medicinal herb for removing dryness, promoting secretion, and quenching thirst [ P. odoratum is now attracting more and more attention for its healthy and medical value.ountries , and it ountries , 3. In pg thirst . It is ag thirst . People  P. odoratum such as mucous polysaccharides [ P. odoratum and considered as one of the important bioactive components with functions of antitumor, antidiabetes, and antioxidation. Extraction of polysaccharides from P. odoratum is an important approach to further research or scale application. Tomshich et al. [ P. odoratum, and several reports were documented on extraction and bioactivities of polysaccharides from P. odoratum in recent years [ P. odoratum, using hot water extraction or hot water extraction assisted by physical technologies such as microwave and ultrasound, to optimize the productivity of polysaccharides. Nevertheless, it should be noted that hot water extraction is often associated with lower leaching efficiency, higher extraction temperature, more time-consuming, and fewer monosaccharide compositions [ P. odoratum was only nearly 7%, and three kinds of monosaccharides were detected, suggesting that there were plenty of polysaccharides in extraction residues insoluble in hot water. Kim et al. [ Ganoderma lucidum was significantly higher compared with hot water extraction. But there were no reports published about the alkaline extraction of polysaccharides from P. odoratum until now.Many active compounds have been identified incharides , homoisocharides , azetidicharides , 8, sapocharides , and stecharides . Among th et al.  reportednt years , 12. Genositions . In our m et al.  reportedm et al.  reported P. odoratum through an orthogonal test, which explored the relationship between four selected variables and determined the optimal variable values to optimize the alkaline extraction conditions, and to further investigate the bioactivity of the alkaline soluble polysaccharides extracts.The objective of the work reported in this paper was to improve polysaccharides yield of alkaline soluble extraction fromEthyl mercaptan, trifluoroacetic acid (TFA), acetic anhydride, pyridine, and 1, 1-diphenyl-2-picryl-hydrazil (DPPH) were from Sigma Co. . Beef extraction peptone medium and LB medium were from Guangrui Biomart Co. . All the other chemicals and reagents used were of analytical grade. P. odoratum was collected in August, 2013, from a natural habitat in Hunan, China, and authenticated by associate Professor Zefa Liu, Loudi Agricultural Institute, Hunan, China. Fresh rhizome was washed, sliced to pieces, and dried at 50°C for 48 h. Then the pieces were ground to powder with a grinding machine. The powder was sieved with a nylon sieve of 0.18 mm diameter grids. 100 g of the sieved powder was mixed with 300 mL petroleum ether and heated at 55°C water bath for 4 h. After filtration, the sediment was dried at 80°C for 12 h, then mixed with 80% ethyl alcohol and heated at a 65°C water bath for 1 h. After filtration again, the sediment was dried at 65°C for 12 h to get powder of P. odoratum.Rhizome of9(34) array. After finishing all the tests, the solution was filtered by vacuum, and 2 M HCl was added to it to adjust pH value to 7.0 and then subjected to 100 mL 95% ethyl alcohol at room temperature for 12 h. After centrifugation at 6,000 ×g for 10 min, the sediment was dissolved in distilled water to determine the yield. The sediment was dried to get the crude polysaccharides and stored at room temperature until use.According to the previous study of single variable test, four control variables were selected as follows: NaOH concentration (M), extraction temperature (°C), ratio of NaOH to solid , and extraction time (hours). The variables of extraction optimization design were as shown in 0.5 g of crude polysaccharides was dissolved in 5 mL distilled water, added to 5 mL 10% trichloroacetic acid (TCA), and stored overnight at room temperature. After centrifugation at 8,500 ×g for 30 min, the supernatant was treated with 1/5 volume of Sevag reagent . The proPolysaccharide hydrolysis and derivatization were performed according to the method of Pitthard and Finch . 15 mg oμm film . The split-splitless injector temperature was 260°C. The chromatographic procedures were as follows: initial temperature 80°C for 2 min, up to 210°C at 1°C/min, then up to 280°C at 25°C/min, and kept at 280°C for 6 min. Helium (99.999%) was used as a carrier at 1 mL/min. Electron impact ionisation was 70 eV with 1.5 scans/s at range of m/z 35–600, and the source temperature was 220°C.GC-MS detection wascarried out according to Chunsriimyatav et al. , using a3Fe(CN)6 were added to a test tube and mixed with polysaccharides (1–10 mg/mL). The solution was incubated at 50°C for 20 min, then 1 mL 10% TCA was added, and centrifuged at 3,000 ×g for 10 min. 2 mL of the supernatant was mixed with 0.5 mL 0.3% FeCl3 to measure 700 nm absorbance after 10 min.The reducing power of the polysaccharides was determined according to the method of Juntachote and Berghofer . 0.5 mL Free radical scavenging rate of the polysaccharides was determined according to the method of Chen et al.  and Ulbi4, 0.2 mL 30% H2O2, 2.8 mL distilled water, and 1.0 mL different concentration of polysaccharides (1–10 mg/mL). The reaction mixture was stirred and immediately incubated at 37°C for 40 min. Absorbance was measured at 510 nm, and the hydroxyl radical scavenging rate was calculated as the following equation:Asample was absorbance of mixture containing polysaccharides and H2O2; Adamage was absorbance of mixture containing H2O2; Anondamage was absorbance of mixture containing polysaccharides.Hydroxyl radical scavenging rate of the polysaccharides was determined according to Li et al.  with min2 and 1.5 mL 0.1 M pH 7.4 phosphate buffer were added to the mixture and incubated at 37°C for 15 min with continuous vibration. And then 0.5 mL 20% TCA was added to the mixture and centrifuged at 5,000 ×g for 10 min. 2 mL of the supernatant was added with 1 mL 0.8% thiobarbituric acid, and the solution was incubated at 75°C for 10 min. After cooling to room temperature, absorbance was determined at 532 nm. The inhibition rate was calculated using the following equation:Acontrol was absorbance of mixture using the same volume of distilled water instead of polysaccharides; Asample was absorbance of mixture containing polysaccharides.Inhibition rate of polyunsaturated fatty acid (PUFA) peroxidation from lipoprotein was determined according to Zhang et al. . Yolk su Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, and Escherichia coli were individually incubated on LB medium at 37°C for 24 h, and bacterial plaques were selected and suspended to 9 mL sterile saline to prepare bacterial suspension at concentration of 1 × 108 cfu/mL. 0.2 mL bacterial suspension was uniformly coated on beef extraction peptone medium of the petri dishes. Plates of sterile filter paper, 6 mm in diameter each, were soaked in different concentration of polysaccharides (1–10 mg/mL) and dried and then were placed into the petri dishes to incubate at 37°C for 24 h. Result was calculated by measuring the zone of inhibition in millimeters.The antimicrobial activity was determined following the method of Lipipun et al.  with mod LSD test (P < 0.05).All experiments were repeated at least three times and the results were expressed as mean ± standard error. Statistical analyses were performed using SPSS13.0, and the data were analyzed using one-way ANOVA followed by9(34) array were performed, and factors and levels were depicted in R = 6.35) than the other factors, and the order of the factors was found to be ratio of NaOH to solid  > extraction temperature  > NaOH concentration  > extraction time . The optimal extraction parameters were A1, B3, C1, and D3, namely, NaOH concentration 0.3 M, extraction temperature 80°C, ratio of NaOH to solid 10-fold, and extraction time 4 h, respectively. Under the optimal condition, the extraction was repeated and the actual yield was 17.28% (n = 3). Jiang et al. [ P. odoratum was higher than that of hot water extraction. In addition, this method was much more time-saving because it was needed to be extracted only once.The factorial design of four factors and three levels was carried out to evaluate the extraction efficiency of polysaccharides. The four factors were NaOH concentration (A), extraction temperature (B), ratio of NaOH to solid (C), and extraction time (D). Therefore, nine experiments of Lg et al.  reportedTCA and Sevag reagent were employed to get rid of impurities such as proteins and nucleic acids before GC-MS analysis. As shown in  P. odoratum by GC, which were fucose, mannose, and galactose with the molecular ratio of 4.72, 3.90, and 1.00, respectively. In our experiment, monosaccharide compositions of alkaline soluble polysaccharides in P. odoratum were detected by GC-MS  was more significant (R = 6.35) than the other factors, and the order of the factors was found to be ratio of NaOH to solid  > extraction temperature  > NaOH concentration  > extraction time . The alkaline soluble polysaccharides consisted of four monosaccharide compositions, which were rhamnose, mannose, xylose, and arabinose with the molecular ratio of 31.78, 31.89, 11.11, and 1.00, respectively. They showed antioxidant activity such as DPPH radical scavenging ability, hydroxyl radical scavenging ability, hydroxyl radical scavenging ability, and PUFA protective ability, but the antioxidant activity was lower compared with the same concentration of ascorbic acid. Moreover, the alkaline soluble polysaccharides from P. odoratum showed antimicrobial activity against S. aureus, P. aeruginosa, B. subtilis, and E. coli.In the present study, an orthogonal test was carried out to optimize extraction of alkaline soluble polysaccharides from


Many strength and conditioning coaches utilize the good morning (GM) to strengthen the hamstrings and spinal erectors. However, little research exists on its electromyography (EMG) activity and kinematics, and how these variables change as a function of load. The purpose of this investigation was to examine how estimated hamstring length, integrated EMG (IEMG) activity of the hamstrings and spinal erectors, and kinematics of the lumbar spine, hip, knee, and ankle are affected by changes in load. Fifteen trained male participants  were recruited for this study. Participants performed five sets of the GM, utilizing 50, 60, 70, 80, and 90% of one-repetition maximum (1RM) in a randomized fashion. IEMG activity of hamstrings and spinal erectors tended to increase with load. Knee flexion increased with load on all trials. Estimated hamstring length decreased with load. However, lumbar flexion, hip flexion, and plantar flexion experienced no remarkable changes between trials. These data provide insight as to how changing the load of the GM affects EMG activity, kinematic variables, and estimated hamstring length. Implications for hamstring injury prevention are discussed. More research is needed for further insight as to how load affects EMG activity and kinematics of other exercises. Training specificity, defined as “how exercise programs must match functional activities to produce the greatest performance gains” , is a weKnowing how load affects movement kinematics and electromyography (EMG) activity allows the role of load in resistance training to not only control for intensity, but to also improve specificity, as certain loads may better mimic other movements, especially those in sport. The good morning (GM) is a popular exercise in the strength and conditioning community for increasing hamstring and spinal erector strength , but litThe biomechanics of a GM are similar to that of the stiff leg deadlift (SLDL). Both exercises are used to target the hamstrings muscle group  via an eccentric muscle action during hip flexion and a concentric muscle action during hip extension . During In addition to training for hamstring strengthening, the GM is utilized to target back extensor muscles . BurnettDespite the GM being a popular exercise in the strength training community, there is a paucity of data on its kinematics and EMG activity . The effThe purpose of this study was to examine the effects of load on movement kinematics and EMG activity during the GM. During the course of one session, participants warmed up, performed a submaximal 1RM estimation test, and performed one repetition of the GM with varying loads  in a randomized order, with two minutes rest between each repetition. We hypothesized that as load increases, medial and lateral hamstrings integrated EMG (IEMG) activity would increase, then plateau from 80 to 90%, similar to the findings of n = 15; age = 24.6 ± 5.3 years; body mass = 84.7 ± 11.3 kg; height = 180.9 ± 6.8 cm) with 8.6 ± 5.5 years of weightlifting experience, recruited by word of mouth, participated in this study. All participants had weight-trained consistently for at least two years prior to this investigation. All participants were experienced with the GM, having had performed it on a minimum of 12 different occasions over the 12 months prior to testing. All participants were healthy and denied the existence of any current musculoskeletal or neuromuscular injuries, pain, or illnesses; if one had been discovered during testing, that participant was excluded. All participants filled out an Informed Consent and Physical Activity Readiness Questionnaire (PAR-Q) before beginning. Any participant that answered “Yes” to any of the questions on the PAR-Q was excluded. Participants were advised to refrain from resistance training that targeted the lower body or back for 72 h prior to testing. Using only the barbell on the first warm-up set, participants’ form was evaluated to ensure the movement was comfortable and acceptable. If a participant reported pain, discomfort, or failed to perform the movement correctly, that participant was excluded. If, for any reason, a participant could not complete a trial, that trial was excluded; however, if the participant felt comfortable completing them, subsequent trials were not excluded. The study was approved by the Institutional Review Board at Arizona State University (IRB ID: STUDY00000284).Fifteen healthy men , with a diameter of 1 cm and an inter-electrode distance of 2 cm, were placed on the muscle bellies, parallel with muscle fibers of the right lateral hamstrings (LH), right medial hamstrings (MH), right lumbar erector spinae 3 cm lateral to spinous process L3 (LES), and right thoracic erector spinae 5 cm lateral to spinous process T9 (TES) .Lumbar flexion was calculated by placing 25.4 mm spherical markers on T11, L1, and S2, and a 14.0 mm marker on the ASIS . In ordeMVIC was taken for the erector spinae by performing a “superman”; in other words, having the participant lay prone and hyperextend their spine with their arms above their head. This position was chosen due to other data suggesting that a prone hyperextension elicits near-maximal erector spinae EMG , and pilThe barbell was placed on participants’ upper trapezius, slightly above the level of the acromion (high bar position); arms were abducted and placed on the bar so the participants’ elbows would not block the T11 marker upon descent; participants were instructed to stand how he normally would when performing the GM. Participants’ shoes were removed before beginning. Participants performed one repetition of the GM using 50, 60, 70, 80, and 90% of estimated 1RM in a randomized order and were allowed two minutes rest between each repetition. No directions for depth or tempo were provided, as to let the participant perform the GM as he normally would in training.EMG signals of the entire movement (eccentric and concentric), collected at 1,500 Hz, were analyzed using MyoResearch 3.4 . A 10–500 Hz bandpass filter was applied to all EMG data. Peak and mean EMG data were rectified, smoothed using root mean square with a 50 ms window, and normalized to peak mean of a 100 ms window from the MVIC trial. While peak allows for all near-instantaneous increases in muscle activation to be seen, mean is robust to both movement artifact and time, thus providing a reliable average of EMG activity over the entire movement . EMG datpost hoc tests were employed on any measure that achieved a main effect. Alpha was set to 0.05 for significance. Partial eta-squared effect sizes were calculated and reported, as were their 95% confidence intervals (95% CI). Group means for each trial were used to calculate hamstrings length, utilizing the coefficients presented by All data were normalized and entered into Stata 13 , wherein one-way analyses of variance (ANOVA) with repeated measures were performed on all EMG and kinematic variables, with load being the independent variable. The normalization process was that described by n = 15; age = 24.6 ± 5.3 years; body mass = 84.7 ± 11.3 kg; height = 180.9 ± 6.8 cm) had 8.6 ± 5.5 years of weightlifting experience. During submaximal testing, participants completed 8.7 ± 2.5 repetitions with 55.5 ± 25.4 kg, and mean estimated 1RM was 76.0 ± 24.9 kg. All but one participant were able to complete every trial; the participant that failed to complete a repetition of 90% 1RM opted not to complete his 80% 1RM trial thereafter; however, his other trials were included. One participant’s LH EMG was discarded due to abnormally high readings, indicative of an invalid signal, and one participant’s 80% kinematic data was not included due to corruption of the motion capture video file.Participants  = 11.77; partial η2 = 0.47 ), LH  = 12.57; partial η2 = 0.50 ), TES  = 10.20; partial η2 = 0.52 ), and LES  = 6.08; partial η2 = 0.31 ) mean EMG activity  = 3.34; partial η2 = 0.20 ) and TES  = 7.12; partial η2 = 0.35 ) peak EMG activity, but not LH  = 1.64; partial η2 = 0.12 ) or LES  = 0.61; partial η2 = 0.04 ) ) .p < 0.001; F = 6.31; partial η2 = 0.32 ), LH  = 11.99; partial η2 = 0.49 ), TES  = 8.36; partial η2 = 0.38 ), and LES  = 2.75; partial η2 = 0.17 ) IEMG activity  = 15.07; partial η2 = 0.53 ), but not lumbar  = 1.67; partial η2 = 0.11 ), hip  = 0.53; partial η2 = 0.04 ), or ankle  = 1.62; partial η2 = 0.11 ) kinematics  and semitendinosus (ST), respectively, are the two most commonly injured muscles of the hamstrings group , are subDuring the GM, the trunk is to be held stable as movement occurs about the hip joint, thus requiring an isometric action of the spinal erectors. However, these data, and others , show thAlthough statistical significance was found in these data, one cannot infer clinical significance, as what constitutes a clinically significant difference in EMG activity is not known. It is important to consider that the erector spinae MVIC position chosen in this investigation may not be a true MVIC position for everyone. Due to large inter-participant variability , it is cThis was the first study to examine both EMG and kinematic data as a function of load. In time, similar studies may show that certain loads may better mimic kinematic and EMG patterns in sport than other loads, or that certain loads emphasize some muscles more than others due to both changes in torque and EMG activity. It is recommended that future research examine these variables in addition to other kinetic variables, which will improve our understanding of the GM exercise and potentially allow coaches to improve program design strategies.The GM is a closed kinetic chain movement that is assumed to involve pure hip extension with a neutral spine. We have shown that the spine does not remain neutral during the GM exercise and in fact moves through flexion and extension. The hips move through a large degree of hip flexion, which requires a high degree of loaded stretch of the hamstring musculature. The hamstrings are of particular interest due to their propensity for injury , and due10.7717/peerj.708/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.708/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.708/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.708/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.708/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.708/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.708/supp-7Supplemental Information 7Click here for additional data file.


The aim of this study was to examine the physical demands imposed on professional soccer players during 11-a-side friendly matches in relation to their playing position, using global positioning system (GPS) technology. One hundred and eleven match performances of a Spanish “La Liga” team during the 2010–11 and 2011–12 pre-seasons were selected for analysis. The activities of the players were monitored using GPS technology with a sampling frequency of 1 Hz. Total distance covered, distance in different speed categories, accelerations, and heart rate responses were analyzed in relation to five different playing positions: central defenders (n=23), full-backs (n=20), central midfielders (n=22), wide midfielders (n=26), and forwards (n=20). Distance covered during a match averaged 10.8 km, with wide and central midfielders covering the greatest total distance. Specifically, wide midfielders covered the greatest distances by very high-intensity running (>19.8 km·h-1) and central midfielders by jogging and running (7.2–19.7 km·h-1). On the other hand, central defenders covered the least total distance and at high intensity, although carried out more (p<0.05–0.01) accelerations than forwards, wide midfielders, and fullbacks. The work rate profile of the players obtained with the GPS was very similar to that obtained with semi-automatic image technologies. However, when comparing results from this study with data available in the literature, important differences were detected in the amount of distance covered by sprinting, which suggests that caution should be taken when comparing data obtained with the GPS with other motion analysis systems, especially regarding high-intensity activities. Traditional time-motion analysis systems have been replaced during the last years by semi-automatic computerized player tracking technologies which are currently used at the elite European club level . The useThe physical demands imposed on top-class soccer players have been extensively documented during the last years . From alThe amount of high-intensity exercise performed by the players has been identified as the activity exceeding a certain speed threshold . HoweverThus, the aim of this study was to examine the physical demands imposed on professional soccer players during 11-a-side friendly matches in relation to their playing position using GPS technology. An additional objective was to determine the number of accelerations carried out by the players in the course of the matches.n=9) and 2012–13 (n=8) season. The soccer players  were classified according to their positional role in the playing formation of the team (n=23), full-backs , central midfielders , wide midfielders , and forwards . All the players were fully informed of all experimental procedures before giving their written informed consent to participate. The study was approved by the Ethics Committee of the University of Madrid and conformed to the Declaration of Helsinki.One hundred and eleven match performances of a Spanish “La Liga”  team were selected for analysis. All the measurements were carried out during 17 pre-season 90-min friendly matches prior to the commencement of the 2011–12  accelerometer which captured data at 100 Hz and allowed determining the changes of speed in the displacements of the players. In addition, the heart rate was monitored synchronously by means of short range telemetry in 1 s intervals during the matches . At the end of the matches, all data were downloaded to a laptop and treated using the software Team AMS V2.1 .The activities of the players were monitored using GPS technology  with a sampling frequency of 1 Hz. Before the beginning of the warm-up, the GPS units were switched on and placed on the upper back of the players, using a neoprene harness to avoid its movement during the exercise. The GPS unit integrated a triaxial −1), (ii) walking (0.7–7.1 km·h−1), (iii) jogging (7.2–14.3 km·h−1), (iv) running (14.4–19.7 km·h−1), (v) high-speed running (19.8–25.1 km·h−1), and (vi) sprinting (>25.1 km·h−1). High-intensity running consisted of running, high-speed running, and sprinting (running speed >14.4 km·h−1), whereas very high-intensity running represented the sum of high-speed running and sprinting (running speed >19.8 km·h−1). Maximal running speed was defined as the peak speed a player reached during the match. The frequency of accelerations was computed into the following categories  1.1–1.5 m·s−2, (iii) 1.6–2.0 m·s−2, (iv) 1.6–2.0 m·s−2, and (v) >2.5 m·s−2. After −2) were also included in the analysis. Finally, the heart rate was expressed in relation to the individual maximal heart rate (%HRmax) of the participants and classified into the following categories  61–70% HRmax, (iii) 71–80% HRmax, (iv) 81–90% HRmax, (v) 91–95% HRmax, and (vi) >95% HRmax. As players used heart rate monitors during matches, fitness tests, and training sessions, peak values reached in any of these conditions were determined as the individual HRmax.The activities carried out by the players were classified into categories according to the following speed thresholds after : (i) staes after : (i) <1.es after : (i) <60The validity and reliability of the GPS for match analysis have been reported in previous studies . Specifipost-hoc tests were used. The significance of the difference between two means was determined by the effect size, with values of 0.2, 0.5, and 0.8 representing small, medium, and large differences, respectively . The normality distribution of the data was checked using the Kolmogorov-Smirnov test and homogeneity of variance was assessed by the Hartley test. Differences between playing positions were tested with one-way analysis of variance (ANOVA). When a significant difference occurred, Tukey’s ectively . Statistp < 0.05) distance than CD . p < 0.05) than by FB (effect size: 1.26) and FW (effect size: 1.03). In addition, CM covered greater (p < 0.01) distances by running than FB (effect size: 1.57) and CD (effect size: 1.38).Total distance covered during a match averaged 10793 ± 1153 m, with WM covering more  than FW , central , and wide  midfielders. Full-backs covered 2364 ± 599 m by high-intensity running. p < 0.001; effect size: 1.80 and 1.62, respectively), FW , and FB .Distance covered running at speeds exceeding 14.4 km·h−1. Central midfielders achieved lower (p < 0.001) peak speeds (26.0 ± 2.1 km·h−1) than FB , WM , and FW . Maximal running speed achieved by CD averaged 27.7 ± 1.7 km·h−1 , WM , and FB . The average number of maximal accelerations per match was 10.1 ± 5.2, with no significant differences between playing positions . An increment in this rate would probably increase the accuracy of the measurements, especially regarding high-speed efforts . The devThe results of this study show that physical demands are influenced by the playing position of the players and should be used to prescribe specific training plans in accordance to them. Wide midfielders experienced the greatest physical requirements during the game, both in terms of total distance covered and by very high-intensity running. Sprinting was predominant in FW and FB, who showed moderate values of total distance covered during the games. The physical performance of CM was characterized by covering a high overall distance, especially at moderate to high speeds (jogging and running). Finally, CM covered the lowest total distance and by high-intensities. An additional finding from this study was the necessity to be cautious when comparing physical data obtained with different motion analysis systems as important differences were detected between our results and previous data calculated with semi automatic tracking systems in players of a similar standard of competition.


Renal impairment is known to be associated with atherosclerosis, which in turn is reported to be positively associated with hemoglobin levels. In addition, renal impairment is known to be associated with a form of anemia known as renal anemia.To clarify the associations between renal impairment and anemia, we conducted a cross-sectional study of 1,105 60 to 89-year-old men, who were not taking medication for anemia and were undergoing general health check-ups.2 was found to constitute a significant risk of anemia. However, we noted that this risk was lower for mild renal impairment (60 mL/min/1.73 m2 ≤ GFR <90 mL/min/1.73 m2). Compared with the non-CKD reference group, the classical cardiovascular risk factors adjusted odds ratio (OR) for anemia was 1.81 (1.23 to 2.68) and compared with the normal renal function (GFR ≥90 mL/min/1.73 m2) reference group, the ORs for mild renal impairment and CKD were 0.26 (0.15 to 0.47) and 0.60 (0.33 to 1.09).Compared with non-chronic kidney disease, chronic kidney disease (CKD) with a glomerular filtration rate (GFR) <60 mL/min/1.73 mIndependent from classical cardiovascular risk factors, CKD, which was identified during general health check-ups, appeared to constitute a significant risk of anemia for older Japanese men. For mild renal impairment, however, this association was a reduced risk of anemia and thus possibly a higher risk of atherosclerosis. A decline in the glomerular filtration rate (GFR) was identified as a marker of chronic kidney disease (CKD) that is known to be associated with a form of anemia known as renal anemia.In a previous study of ours, positive associations were detected between hemoglobin levels and increased arterial stiffness assessed with the cardio-ankle index (CAVI) . Since CTo investigate this potential association, we conducted a cross-sectional study of older Japanese men who participated in a general health check-up between 2005 and 2012.The survey population covered 1,175 men aged 60 to 89 years, who were all residents of the western Japan rural community of the Goto Islands and participated in this study between 2005 and 2012. We used data that were obtained the first time the subjects participated in this study. A total of 66 individuals with missing data and 4 individuals who were taking medication for anemia were excluded, leaving 1,105 men for enrollment in this study. The mean age of the study population was 70.2 years . Written consent forms were available in Japanese to ensure comprehensive understanding of the study objectives, and informed consent was signed by the participants. This study was approved by the Ethics Committee for Use of Humans of Nagasaki University (project registration number 0501120073).1c), were measured with standard laboratory procedures. The glomerular filtration rate (GFR) was estimated by using an established method with three variations recently proposed by a working group of the Japanese Chronic Kidney Disease Initiative [Systolic blood pressures were recorded at rest. Body weight and height were measured with an automatic body composition analyzer  at the same time blood was drawn. Trained interviewers obtained information on smoking status, drinking status, medical history, use of antihypertensive agents, and use of medication for diabetes mellitus. Fasting blood samples were obtained in an EDTA-2 K tube and were used for measuring hemoglobin with the sodium lauryl sulfate (SLS)-hemoglobin method. Serum concentrations of HDL cholesterol, triglycerides (TG), aspartate aminotransferase (AST), γ-glutamyltraspeptidase (γGTP), creatinine, and hemoglobin A1c  and/or initiation of glucose-lowering medication or insulin therapy [2, reduced renal function as GFR <90 mL/min/1.73 m2, mild renal impairment as GFR 60 to 89 mL/min/1.73 m2, and CKD as GFR <60 mL/min/1.73 m2.Anemia for men was defined as 13 g/dL > hemoglobin in accordance with the recommendation by the World Health Organization (WHO) Study Group . Diabete2), diabetes , history of cardiovascular disease , HDL cholesterol (mg/dL), TG (mg/dL), AST (IU/L), and γGTP (IU/L). All statistical analyses were performed with the SAS system for Windows . All P values for statistical tests were two-tailed, and values of <0.05 were regarded as statistically significant.Differences in age-adjusted mean values or prevalence of potential confounding factors in relation to hemoglobin levels were calculated using ANOVA or logistic regression models. Odds ratios (ORs) and 95% confidence intervals (CIs) for anemia associated with renal impairment  were calculated with the aid of logistic regression models. First, we adjusted only for age. Second, we included other potential confounding factors, that is, smoking status , alcohol consumption (non-drinker and current light to moderate drinker (1 to 6 times/week), current heavy drinker (every day)), systolic blood pressure (mmHg), antihypertensive medication use , body mass index  and a mild renal impairment group (60 < GFR ≤90 mL/min/1.73 m2), and used the normal renal function group for reference, mild renal impairment was found to be associated with a significantly lower risk of anemia. CKD was not associated with a higher risk of anemia.We found that compared with non-CKD, CKD constituted a significant risk factor for anemia Table . However2 for participants with non-anemic CKD and 44.8 mL/min/1.73 m2 for those with anemic CKD (P <0.001). For non-CKD participants, the corresponding values were 75.5 mL/min/1.73 m2 and 84.3 mL/min/1.73 m2 (P <0.001).Participants with CKD and anemia showed significantly lower GFR whereas their non-CKD counterparts showed significantly higher GFR. The age-adjusted values for GFR were 50.6 mL/min/1.73 m2) and in hemoglobin (1.3 g/dL) were 0.82  and 1.31 , respectively.As part of our study, we conducted a further investigation of 1,059 men for whom CAVI data were available. We found that both GFR and hemoglobin were significantly associated with atherosclerosis (CAVI ≥9.0). The age-adjusted odds ratios for risk of atherosclerosis of a one standard deviation (SD) increment in GFR  showed that the odds ratio for risk of anemia was significantly higher for a GFR of less than 90 mL/min/1.73 mThis study has certain potential limitations, which warrant consideration. One of these is that we could not establish any causal relationships because this study was cross-sectional. Since we consider the positive association between hemoglobin and renal impairment to be caused by active atherosclerosis progression, the atherosclerosis should also be classified into stable and progressive atherosclerosis. This indicates the need for a longitudinal study with a larger number of participants.In conclusion, CKD appeared to constitute a significant risk of anemia for older, rural Japanese men as determined during general health check-ups. For mild renal impairment, however, this association was the result of a reduction in the risk of anemia and thus possibly a higher risk of atherosclerosis.AST: Aspartate aminotransferase; CAVI: Cardio-ankle index; CI: Confidence intervals; CKD: Chronic kidney disease; GFR: Glomerular filtration rate; HbA1c: Hemoglobin A1c; NGSP: National Glycohemoglobin Standardization Program; ORs: Odds ratios; SLS: Sodium lauryl sulfate; TG: Triglycerides; WHO: World Health Organization; γGTP: γ-glutamyltraspeptidase.The authors have no financial or any other kinds of conflicts in connection with this paper.YS designed the study and performed the statistical analyses, interpreted the data, and drafted and revised the manuscript. MN, KK, SS and JK assisted with the design of the study, were involved in data collection, and checked the manuscript. HY, NT, MN, KA, MT, and YK participated in the study concept and checked the manuscript. TM was the general coordinator and also designed the study. All authors read and approved the final manuscript.


Stenotrophomonas sp. DDT-1 based on morphological characteristics, BIOLOG GN2 microplate profile, and 16S rDNA phylogeny. Genome sequencing and functional annotation of the isolate DDT-1 showed a 4,514,569 bp genome size, 66.92% GC content, 4,033 protein-coding genes, and 76 RNA genes including 8 rRNA genes. Totally, 2,807 protein-coding genes were assigned to Clusters of Orthologous Groups (COGs), and 1,601 protein-coding genes were mapped to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. The degradation half-lives of DDT increased with substrate concentration from 0.1 to 10.0 mg/l, whereas decreased with temperature from 15 °C to 35 °C. Neutral condition was the most favorable for DDT biodegradation. Based on genome annotation of DDT degradation genes and the metabolites detected by GC-MS, a mineralization pathway was proposed for DDT biodegradation in which it was orderly converted into DDE/DDD, DDMU, DDOH, and DDA via dechlorination, hydroxylation, and carboxylation, and ultimately mineralized to carbon dioxide. The results indicate that the isolate DDT-1 is a promising bacterial resource for the removal or detoxification of DDT residues in the environment.A novel bacterium capable of utilizing 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) as the sole carbon and energy source was isolated from a contaminated soil which was identified as  KK, Bacillus sp. BHD-4, Cladosporium sp. AJRPseudomonas sp. 12–3, Serratia marcescens DT-1P, Stenotrophomonas sp. 103–105, Sphingobacterium sp. D6, and white-rot fungi1011Sphingobacterium sp. D-6 could convert DDT into six metabolites  propionic acid) and then mineralize to carbon dioxide in mineral salts medium (MSM)Pseudomonas aeruginosa and Flavimonas oryzihabitans could transform DDT into DDE, DDMU and DDOH in broth mediumRhodococcus sp. IITR03 could degrade DDT to DDE, DDD, and DDMU in minimal mediumBiodegradation has been considered as a cost-effective, safe, and promising method for the removal or detoxification of DDT residues in the environment8In the present study, a bacterial strain DDT-1 capable of utilizing DDT as the sole source of carbon and energy was isolated, purified, and identified. The objectives of this study were: 1) to measure physiological characterization of the isolate DDT-1; 2) to conduct genome sequencing and analysis of the isolate DDT-1; 3) to examine the effect of substrate concentration, pH, and temperature on the degradation of DDT by the isolate DDT-1; and 4) to reveal the potential DDT biodegradation pathway by DDT degradation genes database (DDG) search and GC-MS analysis. The results will provide a new insight into the understanding of DDT biodegradation mechanisms.Stenotrophomonas with 0.74 similarity (24 h) using BIOLOG Microlog 4 database software. As shown in Stenotrophomonas. According to the above identifications, the strain was designated as Stenotrophomonas sp. DDT-1.A bacterial strain, designated as DDT-1, with the capability to utilize DDT as the sole carbon and energy source was isolated from the DDT-contaminated soil. Growth curve of the isolate DDT-1 in MSM (pH 7.0) containing 1.0 mg/l of DDT is shown in Genome properties of the isolate DDT-1 are shown in r = 0.94), suggesting that the degradation is subjected to pseudo first-order kinetics. As shown in Bacillus, Staphylococcus, and Stenotrophomonas for 31 days at 30 °CFlavimonas oryzihabitans and Pseudomonas aeruginosa at the biomass level of OD600 = 0.6 for 7 days at 30 °CStenotrophomonas maltophilia AJR5–106 cells/ml in basal salts medium containing 1.0 g/l of peptone resulted in a 35% decrease in DDT concentration after 28 daysPseudomonas fluorescens in a glass column packed with tezontle was as high as 55–99%The degradation effects of different concentrations of DDT by the isolate DDT-1 in MSM of pH 7.0 at 25 °C is shown in Stenotrophomonas sp. strain in M9 minimal medium increased with temperature from 28 °C to 35 °C.The effect of various temperatures on the degradation of 1.0 mg/l of DDT by the isolate DDT-1 in MSM at pH 7.0 is shown in p ≤ 0.05) shorter than those at pH 5.0 and 9.0. The results indicated that neutral condition was favorable for the degradation of DDT by the isolate DDT-1. A similar result was also reported by Fang et al.Sphingobacterium sp. D6 in MSM. However, Xie et al.Alcaligenes sp. KK at weak acidic condition (pH 6.0).The degradation of DDT (1.0 mg/l) by the isolate DDT-1 in MSM of pH 5.0, 7.0, and 9.0 is shown in dhc gene) by dehydrochlorination, DDT→DDD by dechlorination (rdh gene), DDMU→DDOH by hydrogenation and hydroxylation (sds and dhg genes), DDNU→DDOH by hydroxylation (hdt gene), DDA→DDM by decarboxylation (dcl gene), and OHCTT→CDP by carboxylation (hdl gene). As shown in meta-ring cleavage, and then into CDP by carboxylation, and ultimately mineralize to carbon dioxide. Similar biodegradation pathways of DDT using metagenomic analysis was also revealed by Fang et al.meta-ring cleavage reactions.Potential DDGs that hit the isolate DDT-1 genome are summarized in m/z of 320, 318, 266, 284, 282 in MSM by GC-MS  and mass spectra of DDT metabolites by the isolate DDT-1 in MSM are shown in by GC-MS . Referriet al.Pseudomonas putida T5 could transform DDT to DDE or DDD by eliminating either HCl or Cl-. Fang et al.Sphingobacterium sp. D-6 could dechlorinate DDT to DDD, DDE, DDMU, and DDNS, and then to DDA and DBP by hydroxylation, carboxylation, and decarboxylation, and finally mineralize to carbon dioxide. Xiao et al.Phlebia lindtneri GB-1027 and Phlebia brevispora TMIC34596 could metabolize DDT to DDD, DDA, and DBP. Additionally, a similar biodegradation pathway in coastal sediment was also reported by Yu et al.Based on these metabolites detected by GC-MS, a potential biodegradation pathway was proposed: DDT was initially converted into DDE and DDD by dechlorination at the trichloromethyl group, and then into DDMU, which was further transformed to DDOH and DDA by hydroxylation and carboxylation, and finally mineralized to carbon dioxide . SeveralThe shared degradation pathway of DDT by the isolate DDT-1 between genome annotation and GC-MS analysis is shown in p,p’-DDT (purity 99.5%), p,p’-DDD (purity 98.0%), p,p’-DDE (purity 98.5%), DDMU (purity 99.0%), DDOH (purity 99.5%), and DDA (purity 99.0%) were purchased from Dr. Ehrenstorfer GmbH . Sodium chloride, anhydrous sodium sulfate, and n-hexane of analytical grade were provided by Sinopharm Chemical Reagent Co., Shanghai, China. n-Hexane of chromatographic grade was provided by Tedia Co., USA.Standard samples of 4·7H2O, 0.40 g; FeSO4·7H2O, 0.002 g; K2HPO4, 0.20 g; (NH4)2SO4, 0.20 g; CaSO4, 0.08 g; H2O, 1000 ml; pH 7.0) supplemented with 1.0 mg/l of DDT as the sole source of carbon and energy. Each culture was incubated for 1 week on a rotary shaker at 30 °C and 150 rpm, and one loop of the culture was inoculated into 20 ml of MSM supplemented with 2.0 mg/l of DDT and incubated for another 1 week under the same conditions. The culture was repeatedly acclimated in fresh MSM with increasing concentration of DDT from 2.0 to 10.0 mg/l. Subsequently, one loop of the enrichment culture was streaked onto MSM agar plates containing 10.0 mg/l of DDT and incubated for 6 d at 30 °C, and individual pure colonies were then restreaked in fresh MSM agar plates and incubated for another 6 d. Once pure cultures were achieved, they were stored for further study.Soil samples were collected from the vegetable field located in Cixi, Zhejiang, China. These soil samples were homogenized and divided into three subsamples (10.0 g). Each subsample was mixed thoroughly with 100 ml of sterilized distilled water in 250-ml Erlenmeyer flask. Subsequently, an aliquot of 1.0 ml from soil suspension was inoculated into a 100-ml Erlenmeyer flask containing 20 ml of sterile mineral salts medium . The output data were recorded by the automatic threshold option using BIOLOG software (BIOLOG Gen III database v2.7).Carbon substrate utilization for the isolate DDT-1 was assessed using BIOLOG GN system . The isolate DDT-1 was incubated on BUG  at 30 °C for 24 h prior to assay. The fresh colonies were removed from BUG media with a long cotton swab and suspended in 15 ml of inoculation fluid with turbidity equivalent to 72% transmittance as measured by BIOLOG turbidimeter. Each well in the BIOLOG GN2 microplate2(15 mmol/l), 2 μl of dNTPs(2.5 mmol/l), 1 μl of primers , 0.25 μl of Taq DNA polymerase (5 U/μl), and 16.25 μl of ultrapure water. Amplification conditions were as follows: an initial denaturation step for 5 min at 94 °C, 34 cycles of denaturation for 30 s at 94 °C, annealing for 45 s at 58 °C, extension for 90 s at 72 °C, followed by a final extension for 5 min at 72 °C. The PCR products were purified and then sequenced using an ABI 3730 automated DNA sequencer . Sequences obtained were assembled and then compared with the NCBI GenBank sequences using online BLASTn search.DNA was extracted from 1 ml of suspension of the isolate DDT-1 using a QIAamp DNA mini kit  according to the manufacturer’s protocol. The concentration and quality of the extracted DNA were examined with spectrophotometry . The 16S rDNA was amplified using PCR with an Eppendorf Thermocycler  in a volume of 25 μl containing 1 μl of template DNA (20 ng/μl), 2.5 μl of 10×PCR buffer, 2 μl of MgClPrepared genomic DNA samples were sent to Novegene , and approximately 5 μg of DNA samples was used for shotgun library construction. DNA was first mechanically fragmented with an enrichment size of ~500 bp. The DNA fragments were recycled by gel purification and quality check, and were then used for shotgun library construction, which was finally used for genome sequencing using an Illumina MiSeq platform with the paired-end 250 bp sequencing strategy. Approximately 1.77 Gb of raw reads were generated for the isolate DDT-1.dhc, rrat, sds, cpo, doa, dcl, ort, hdl, ods, rdh, dhg, and hdt) was retrieved from the KEGG pathway maps (http://www.genome.jp/kegg/). DDGs database was deduplicated to obtain 982 non-redundant proteins (Protein database of DDT degradation genes (DDGs) for complete metabolic pathways containing 12 sub-databases  and alignment length (≥25 aa) by a self-written Python script.The flow chart of this study is shown in −10)http://stothard.afns.ualberta.ca/cgview_server/)The draft genome was annotated by GeneMarkS+ tool according to NCBI Prokaryotic Genome Annotation Pipeline (PGAP). The genomic functional annotation of the isolate DDT-1 was conducted by Reversed Position Specific BLAST (RPS BLAST) program search against the Clusters of Orthologous Groups (COGs) database with an E-value cutoff , and finally suspended in the same phosphate buffer as the seed. In order to study the degradation of DDT by the isolate DDT-1, the MSM was supplemented with DDT as the sole carbon and energy source. Each flask was inoculated with a suspension of the isolate DDT-1 at a biomass level of OD600 = 0.2. All flasks were incubated in a shaker at 150 rpm in the dark. The whole culture was sampled for the determination of DDT residues immediately after inoculation and after 3, 7, 14, and 21 days. Each treatment was performed in triplicate, and the control experiment without the isolate DDT-1 was performed under the same conditions. To determine the effect of DDT concentrations on its biodegradation, the medium (pH 7.0) was fortified with DDT at three levels of 0.1, 1.0, and 10.0 mg/l, respectively. To measure the effect of pH on the biodegradation, the medium was prepared with buffers at pH 5.0, 7.0, and 9.0, respectively , and then immediately washed 3 times with 20 mL of NaH600 = 0.4. The extraction methods of DDT and its metabolites in MSM were included in To reveal the degradation pathway of DDT by the isolate DDT-1, the experiment was carried out in a 100-ml Erlenmeyer flask containing 20 ml of MSM (pH 7.0) supplemented with 10.0 mg/l of DDT at an inoculation level of ODTo confirm the validity of the extraction method of DDT, recovery studies were carried out at three spiking levels of 0.1, 1.0, and 10.0 mg/l in MSM. The recovery of DDT ranged from 92.2% to 101.5% with relative standard deviation ≤ 6.9% in MSM. The limit of detection (LOD) and limit of quantitation (LOQ) of DDT were 0.1 and 0.5 μg/l, respectively. These data indicate that the extraction method is satisfactory for the analysis of DDT residues.The isolate DDT-1 has been submitted to CCTCC under M 2015153. The 16S rDNA sequence for the isolate DDT-1 has been deposited in the NCBI GenBank database under the accession number KP729429. The whole genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number LEKR00000000 .How to cite this article: Pan, X. et al. Biodegradation of DDT by Stenotrophomonas sp. DDT-1: Characterization and genome functional analysis. Sci. Rep.6, 21332; doi: 10.1038/srep21332 (2016).


Phasic alertness represents the ability to increase response readiness to a target following an external warning stimulus. Specific networks in the frontal and parietal regions appear to be involved in the alert state. In this study, we examined the role of the right dorsolateral prefrontal cortex (DLPFC) during the attentional processing of a stimulus using a cued double-choice reaction time task. The evaluation of these processes was conducted by means of Event-Related Potentials (ERPs), in particular by using the Contingent Negative Variation (CNV), and repetitive 1-Hz Transcranial Magnetic Stimulation (rTMS). Transient virtual inhibition of the right DLPFC induced by real 1-Hz rTMS stimulation led to a significant decrease in total CNV and W1-CNV areas if compared with the basal and post-sham rTMS conditions. Reaction times (RTs) did not decrease after inhibitory rTMS, but they did improve after sham stimulation. These results suggest that the right DLPFC plays a crucial role in the genesis and maintenance of the alerting state and learning processes. Alertness provides the capacity to increase vigilance to an impending stimulus and to achieve and maintain an alerting state . Two typA substantial body of research has investigated the brain areas involved in alertness, particularly the phasic one , 2. NeurThe DLPFC is involved in detecting salient events and preparing or inhibiting motor responses, moving the focus of attention, and engaging attention at the new target , 6. MoniOne valid approach to the study of alerting is the use of psychophysiological techniques, such as Event-Related Potentials (ERPs). ERPs include the Contingent Negative Variation (CNV), which was first described by Walter et al. . The CNVThe CNV is an electrophysiological signature of a task-specific preparatory state that facilitates the stimulus perception and the required responses. It reflects the activation of multiple areas, mainly involved in the actual processing of S2, which compose a specific sensorimotor neural set attentionally controlled by frontoparietal networks , 13. ThiThe aim of this study was to investigate, from a psychophysiological point of view, the role of the right DLPFC during attentional processing of the stimulus using a CNV paradigm in which the S2 is represented by a double-choice reaction time task. For this purpose, we delivered inhibitory rTMS stimulation to the right DLPFC in a sample of healthy subjects.Transcranial Magnetic Stimulation (TMS) can be used to map and study perceptual, motor, and cognitive functions in the human brain , 22. TMSWe hypothesize that a virtual dysfunction in the right DLPFC interferes with the CNV phenomenon given the role played by this area in phasic alertness.Twelve right-handed, healthy subjects  were enrolled from among the staff working in the Careggi Hospital of Florence to participate in a double-blind, sham-controlled crossover study. None of the subjects had a history of neurological or psychiatric disease or of head injury, and none reported consuming excessive amounts of alcohol or were taking any medication that affects the central nervous system. Written informed consent was obtained from all participants prior to the experiment and the study was approved by the local medical ethics committee.T0, T1, and T2) two of which followed a TMS session that lasted 30 minutes.The experiment consisted of a sequence of three CNV recordings, lasting about 25 minutes, performed in the same day at three consecutive time-points (https://www.random.org/), either real 1 Hz rTMS or sham 1 Hz rTMS was administered. Right after the TMS sessions, the CNV task was performed again so that for each subject a basal CNV recording, a post-real TMS CNV recording and a post-sham TMS CNV recording were obtained.First, the CNV task was recorded in basal condition. Then, according to a randomized order  . For rTMEach rTMS session consisted of 1800 pulses at a frequency of 1 Hz, at 80% of the individual Resting Motor Threshold (RMT), which was determined for each subject according to standardized criteria . On the Participants were told that they would receive rTMS on two occasions and that it would be administered at slightly different intensity levels. Neither the subjects nor the ERP investigators, with the exception of the investigator applying the rTMS, were aware of whether real or sham stimulation was being performed. The subjective sensations of coil-scalp contact and discharge noise during sham stimulation were similar to those of the real stimulation. Since the skin sensation induced by the TMS pulse is different between real and sham stimulation, a postexperiment interview was conducted and confirmed that the subjects did not perceive any significant physical differences between the two stimulation conditions. The setting falls well within the safety guidelines .Participants were seated in an anatomic chair in a partially soundproof, faradized, and light-attenuated room. The electrophysiological signals were recorded by means of a 21-channel carbon cap with active electrodes at the F3, Fz, F4, C3, Cz, C4, P3, Pz, and P4 sites, according to the International 10–20 System, referred to linked mastoids, and grounded at the forehead. The bipolar electrooculogram (EOG) was recorded from above and below the left eye. To reduce residual TMS-related artifacts, the impedance at each electrode was kept below 3 kΩ. EEG signals and EOG were filtered using a 0.01–30 Hz bandpass. A notch filter was also applied. The data were digitized with an analog/digital (A/D) converter at a sampling rate of 1024 Hz and stored on a hard disk. A Mizar Sirius EEG-EP multifunctional system was used.μs-1.5 J (S1) (delivered by a strobe lamp at a distance of 30 cm from the subject) and, 1750 ms later, of a sound (S2) at an intensity of 80 dB SPL, lasting 200 ms, which was randomly presented at 1000 Hz  or 2000 Hz . The subject was instructed to push a button, held in the right hand, as quickly as possible upon hearing the 2000 Hz sound, as requested by the double-choice reaction time task for paired stimuli. The intertrial interval varied randomly between 6 and 12 s. A total of 100 trials were acquired. The task lasted about 25 minutes.The CNV task consisted of a sequence of two paired stimuli: a first stimulus (warning: S1) that informed the subject to expect a second stimulus (imperative: S2). The warning and imperative stimuli consisted, respectively, of a flash of 100 μV in the eye channels were automatically rejected online, according to clinical guidelines [μV in any channel were also excluded. A further selection was performed in the offline analysis to reject other kinds of artifacts not detected by the automatic rejection procedure .Trials containing eye movements (including blinks) that exceeded ±100 idelines . Trials The ERPs were evaluated for each subject in each of the three experimental conditions: basal, post-sham rTMS, and post-real rTMS. The analysis epoch for each CNV was 5 s with a 500 ms prestimulus baseline before S1. The CNV amplitude was measured as the total area (negative shift between S1 and S2) and as two temporal windows of interest: the early orienting window: W1 (between 500 and 700 ms following S1), and the late window: W2 (200 ms preceding S2) compared with the prestimulus baseline , 19. AddData are expressed as the mean (±1 standard deviation) for continuous variables and as proportions for categorical variables. The Kolmogorov-Smirnov test was applied to assess the normal distribution of the data and to ensure that the assumption of normality was not violated for any of the data.T0), sham (T1), and real (T2); group 2: basal (T0), real (T1), and sham (T2)) as the between-subject factor.In order to avoid the order effect for EEG data, given the consecutive within-design at one day, CNV parameters  were analysed separately by means of a factorial repeated measures ANOVA, with the experimental “condition”  and the “electrode”  as the within-subject factors and the order in which subjects received rTMS  were analysed separately by means of a factorial repeated measures ANOVA, with the “condition” , the “electrode” , and the “stimulus”  as the within-subject factors, and the “group” factor as the between-subject factor.p values are reported; the original degrees of freedom are reported together with their correction factor epsilon.A post hoc correction according to Bonferroni was then applied. Degrees of freedom were adjusted, when necessary, using the Greenhouse-Geisser epsilon coefficient for possible violations of the sphericity assumption and corrected Owing to the effect exerted by task repetitions on behavioural performance (RT), the RT mean values were compared by using a factorial repeated measures ANOVA, with the “condition”  as the within-subject factor and the “group” factor as the between-subject factor. A post hoc correction according to Bonferroni was applied when necessary.p < 0.05 was considered statistically significant. All the analyses were performed using the SPSS statistical package (Version 20.0).The Pearson correlation test was performed to assess any correlations between CNV areas  and RT. A CNV was elicited in 100% of the subjects. In one subject, the electrophysiological data set for the post-sham CNV could not be evaluated owing to the high artifact rate. Figures F = 0.37, p = 0.56), W2  = 0.0003, p = 0.98), and total area  = 0.73, p = 0.42). A significant main effect of the “condition” factor was observed for total area and W1  = 11.21, p = 0.001; F = 11.64, p = 0.001, resp.). After Bonferroni correction for multiple comparisons, a reduction in amplitudes emerged only after real rTMS both for total area  – basal: −8514.8; sham: −9128.4; real: −5606.8) and for W1  – basal: −783.6; sham: −1045.2; real: −378.8). By contrast, ANOVA did not yield a significant main effect of the “condition” factor for W2  = 2.37, p = 0.13). A significant effect of the “electrode” factor was observed for each parameter  = 10.21, p = 0.002, ε = 0.24; W1: F = 8.22, p = 0.015, ε = 0.95; W2: F = 20.71, p < 0.001). After Bonferroni correction, a significant difference emerged between frontal and parietal electrodes, with lower amplitudes being observed in the latter, for total area, W1, and W2  = 1.80, p = 0.05); after Bonferroni correction, a significant difference emerged for each electrode between the real rTMS condition and basal condition as well as between the real rTMS condition and sham condition, while no difference emerged between the basal and sham conditions. The same findings emerged for W1  = 3.88, p = 0.016; ε = 0.240), though not for W2  = 1.14, p = 0.33).CNV Parameters: no significant difference emerged between groups  for both W1  = 0.07, p = 0.79; N1 latency: F = 0.12, p = 0.74; P3-like amplitude: F = 0.87, p = 0.39; P3-like latency: F = 0.67, p = 0.45). ANOVA did not reveal a main effect of the “condition” factor for either the N1 amplitude  = 0.23, p = 0.80) or the P3-like latency  = 0.53, p = 0.60) and amplitude  = 0.36, p = 0.70). ANOVA detected a main effect of the “condition” factor only for N1 latency  = 16.89, p < 0.001), with shorter latencies being detected in the real rTMS condition; the p values after Bonferroni correction were as follows: basal versus rTMS p = 0.001; basal versus sham p = 0.02; sham versus real p = 0.42.Post-S2 parameters: no significant difference emerged between groups for post-S2 parameters  = 0.48, p = 0.96; F = 6.52, p = 0.06; F = 0.72, p = 0.76, resp.) or amplitude  = 1.47, p = 0.18, ε = 0.18, F = 2.98, p = 0.10; F = 0.58, p = 0.89, resp.) or P3-like wave latency  = 0.85, p = 0.62; F = 0.37, p = 0.63; F = 0.56, p = 0.90, resp.) and amplitude  = 0.59, p = 0.88; F = 0.09, p = 0.91; F = 0.85, p = 0.62, resp.). Moreover, ANOVA did not reveal a main effect on N1 and P3 amplitudes of either the “stimulus” factor  = 0.03, p = 0.86; F = 0.02, p = 0.89, resp.) or the “stimulus” × “electrode” interaction  = 3.29, p = 0.08; F = 1.33, p = 0.25, resp.).There was no significant effect of the “condition” × “electrode”, of the “condition” × “stimulus,” or of the “condition” × “electrode” × “stimulus” interactions on either N1 latency .A negative correlation emerged between RT and CNV areas , with longer values after real rTMS (means RT and SD (ms)—basal: 264.8 ± 9.8; sham: 241.6 ± 7.0; rTMS: 267.5 ± 7.5); the p values after Bonferroni correction were as follows: basal versus real p = 1; basal versus sham p = 0.006; sham versus real p < 0.001. With regard to the between-subject factor, a difference bordering on significance emerged between groups (p = 0.056), with shorter RTs in group 1 (241.01 versus 274.08). No main effect emerged for the “condition” × “group” interaction  = 0.97; p = 0.39)  . CorrectThe aim of this study was to examine whether transient inhibition of the right DLPFC interferes with the CNV phenomenon and post-S2 activities.The following methodological issue should be taken into account before the possible implications of our results are discussed: on the basis of data that have emerged from behavioral TMS studies regarding the after-effects of a 1 Hz protocol, we chose a duration of stimulation that would ensure an inhibition effect throughout the subsequent CNV task . MoreoveAs hypothesized, the functional inhibition induced by low-frequency rTMS on the right DLPFC significantly reduced the overall area of the CNV waveforms in all the recording sites, particularly in the frontocentral areas. Moreover, the early CNV area was also reduced following inhibition of the right DLPFC.These electrophysiological changes point to the suppression of the cerebral activity believed to prepare the system for a rapid response after a warning signal  and suggThe reduction in CNV we observed confirms that the right DLPFC is involved in the functioning of alerting, as previous neuroimaging studies have also shown. Critchley et al.  suggesteOur experiment also revealed that the CNV phenomenon was reduced bilaterally, which indicates that the inhibitory cortical effect was widespread even though rTMS stimulation was unilateral. This widespread reduction in CNV yields further evidence of the capacity of the right DLPFC to achieve cognitive integration during sustained attentional processing of a stimulus, which may be ascribed to the activation of bilateral neural areas. The PFC is known to consist of a set of neocortical areas that have bidirectional links with several brain cortical regions, including the sensory and motor areas , and a wIt is noteworthy that the functional inhibition of the right DLPFC does not interfere with the late CNV area, which indicates that the electrical activity related to motor readiness is not directly modified by the virtual lesion in this area. We therefore believe that, during a motor task, the DLPFC sustains the activation of sensory and motor cortices by maintaining a state of alertness induced by a warning signal, without interfering with preprogramming and motor preparation.Interestingly, the post-S2 components did not change significantly after inhibitory rTMS stimulation. N1 latency became even shorter over repetitions, likely reflecting a growing confidence in stimulus discrimination , 46. TheThe relationship between the right DLPFC and motor performances also deserves consideration. It is known that the repetition of a CNV motor task like ours is associated with a reduction in RTs, which is probably due to the maintenance of high levels of attention displayed by high CNV amplitudes over sessions , 49. TheAlthough the results of our psychophysiological study should be considered as preliminary owing to the limited sample size and the nature of the procedure adopted, they do provide noteworthy findings on the anatomofunctional correlates of sustained phasic alertness. Further studies conducted on larger samples and using more sophisticated techniques are warranted to confirm these findings. Lastly, the results of our study strongly indicate that the right DLPFC plays a critical role in the genesis and maintenance of the alerting state.


Debate continues about the optimal anticoagulation level for elderly Japanese patients with non-valvular atrial fibrillation (NVAF) receiving warfarin. The Japanese Circulation Society guideline has recommended prothrombin time-international normalized ratios (PT-INR) of 1.6 – 2.6 for elderly patients and 2.0 – 3.0 for non-elderly patients, because previous observational studies indicated increased risk of bleeding when the ratio exceeded 2.6. We aimed to reappraise the relationship between PT-INR and the risk of major bleeding in elderly Japanese patients.From the electronic medical records, we selected a cohort of elderly (age ≥ 70 years) Japanese patients with NVAF who were prescribed warfarin for the prevention of thromboembolic diseases between November 2010 and March 2014 at Kanto Rosai Hospital. We identified those who developed major bleeding (cases). For each case, we randomly selected two matched controls by adopting a risk-set sampling method defined by calendar date, age, gender, length of warfarin administration, and the prescriber of warfarin. The risk of major bleeding in patients having PT-INR ≤ 1.49, 1.50 – 1.99, 2.00 – 2.49 (the reference), 2.50 – 2.99, and ≥ 3.00 were compared using the conditional logistic regression method. The study protocol was approved by the IRB before the study was begun.n = 20), 1.50 – 1.99 (n = 32), 2.00 – 2.49 (n = 18), 2.50 – 2.99 (n = 10), and ≥ 3.00 (n = 16) were 1.0 , 0.3 , 1.0 (reference), 1.2 , and 19.8 , respectively, with a significant difference between ≥ 3.00 and reference.Among the cohort of 806 elderly patients, we identified 32 cases and selected 64 matched controls. The overall incidence of major bleeding was 3.5 per 100 patient-years. The odds ratios  for the risk of developing major bleeding in patients with PT-INR ≤ 1.49 (Among elderly Japanese patients with NVAF, PT-INR 2.0 – 3.0 may be associated with a clinically permissible risk of major bleeding while PT-INR ≥ 3.00 a significant risk. Further studies are warranted to determine whether the risk of major bleeding is significantly lower for PT-INR 2.50 – 2.99 than for PT-INR ≥ 3.00. Non-valvular atrial fibrillation (NVAF) is the most prevalent arrhythmia in the elderly and poses substantial morbidity and mortality risks because of an increase in cardiogenic thromboembolic complications . Oral anOne of the reasons for recommending a lower PT-INR range (1.6 – 2.6) for elderly Japanese patients is that an observational study on approximately 200 Japanese NVAF patients of all ages for the secondary prevention of stroke demonstrated an increased risk of major bleeding for PT-INR ≥ 2.60 . UnfortuA case-control study design would complement prospective randomized or cohort studies in assessing the risk of outcomes with low event rates (such as major bleeding caused by warfarin). Indeed, an optimal PT-INR range of 2.0 – 3.0 for warfarin was first proposed from case-control studies conducted by Hyleck et al. , 16 in tThe present study was performed at the Japan Labour Health and Welfare Organization Kanto Rosai Hospital with 610 beds located in an urban area of Tokyo. Patients eligible for the present study were retrieved retrospectively from the electronic medical records between November 2010 and March 2014, utilizing as indices atrial fibrillation in the diagnosis  and age (≥70 years). We also extracted elderly (age ≥ 70 years) patients who were prescribed warfarin using records of the electronic prescribing system. Patient retrieved from both databases were combined. Then those who were prescribed warfarin for clinical indications other than prevention of thromboembolic events associated with NVAF were excluded to obtain the final cohort for the present case-control study.The electronic medical records were searched for occurrence of major bleeding events in the cohort using the following ICD-10 codes as event identifiers: D69 (purpura and other hemorrhagic conditions), I60 to 62 , K25.0, .2, .4, and .6 , K26.0, .2, .4, and .6 , K27.0, .2, .4, and .6 , K28.0, .2, .4, and .6 , K29.0 (acute hemorrhagic gastritis), K62.5 (hemorrhage of anus and rectum), K92.0, .1, and .2 , and R04 (hemorrhage from respiratory passages). Because no consensus has been reached regarding the criteria for major bleeding, we adopted the following composite criteria by modifying that used in the RE-LY study . SpecifiFor each case patient, we randomly assigned two control patients by adopting a risk-set sampling method defined by the calendar date of the bleeding event, age, gender, length of warfarin administration, and the prescriber of warfarin . In cont2 and HAS-BLED scores were extracted from the medical records of the retrieved patients [Age, gender, height, weight, estimated glomerular filtration rate (eGFR), PT-INR, warfarin doses, and other clinical data required to calculate CHADSpatients , 20. Forpatients . The metpatients . We exclThe present study was planned and conducted in compliance with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement and the Declaration of Helsinki. The protocol was approved by the institutional review board of Kanto Rosai Hospital .Comparisons of continuous variables were performed by paired t-test and those for categorical variables by McNemar’s test. We stratified the case and control patients into five categories according to their PT-INR values . The risk of major bleeding for each group was evaluated by conditional logistic regression analysis and expressed as odd ratio (OR) and 95 % confidence interval (CI) versus patients with PT-INR 2.00 – 2.49 (reference).We performed power calculation for the statistical analysis of a case-control study before we began the study, according to the method reported previously . We made2 and HAS-BLED scores were observed between the two groups. The CHADS2 and HAS-BLED scores  were identical in the two groups. In addition, there was no significant difference in the numbers of patients receiving CYP2C9 inhibitors or those augment anticoagulation effect of warfarin: 17 of 32 (53 %) cases and 35 of 64 (55 %) controls ingested at least one of the above drugs by McNemar’s test. The PT-INR  for the cases  was significantly (p < 0.05) greater than the ratio for the controls   greater than that of the reference , ORs for patients with PT-INRs 2.50 – 2.99, 1.50 – 1.99, and ≤ 1.49 were not significantly different from the reference, whereas OR for patients with PT-INR ≥ 3.00 was significantly  is required to obtain sufficient power for detecting a significant difference in the risk of bleeding between patients exposed to PT-INR ≥ 2.60 and those exposed to < 2.60.Five patients were considered to develop major bleeding solely by the criterion of clinical judgement of the necessity of admission. When the statistical analysis was performed by excluding these data, essentially similar results were obtained (data are not shown).2 score of 3 [To the best of our knowledge, this is the first nested case-control study carried out to assess the relationship between PT-INR and the risk of developing major hemorrhage in elderly Japanese patients with NVAF receiving warfarin. Our data show that patients having PT-INR ≥ 3.00 had approximately 20 times greater risk of developing major bleeding compared to patients having PT-INR 2.00 – 2.49 (reference), whereas those with PT-INR 2.50 – 2.99 appeared to have similar risk OR 1.23) compared to the reference. Our data support the notion that the upper limit of target PT-INR for elderly Japanese patients with NVAF may be extended to 3.00. Target PT-INR of 2.0 – 3.0 has been recommended for non-elderly Japanese patients. It is noteworthy that both case and control patients participating in the present study had a high risk of thromboembolic complications, because the mean age of both groups was 81 years and both had a median CHADSore of 3 . In addi.23 compaOur data in general agree with previous studies. A subgroup analysis of elderly (age ≥ 70 years) patients in the J-Rhythm registry showed that the hazard ratio (95 % CI) of major bleeding in patients with PT-INR 2.00 – 2.59, 2.60 – 2.99, and ≥ 3.00 increased gradually to 2.87 1.12 – 7.35), 3.99 (1.33 – 11.89), and 7.02 (2.23 – 22.13), respectively, against those who did not receive warfarin. Nevertheless, the 95 % CIs of these groups overlapped substantially  – 7.35, . Howevern = 8) were observed in our cohort. A recent study has reported that the risk of intracranial hemorrhage in elderly Japanese may increase approximately four-fold  at PT-INR 2.5 – 3.0 [The present study has several drawbacks. First, we were unable to analyze the risk of intracranial hemorrhage at different PT-INR levels separately, because only a small number of events (.5 – 3.0 . Ultimat.5 – 3.0 . They peThe present study demonstrates that PT-INR 2.0 – 3.0 may be associated with a clinically permissible risk of major bleeding while PT-INR ≥ 3.00 a significant risk in elderly Japanese patients with NVAF. The present study warrants further case-control studies with a greater number of patients to obtain conclusive evidence.