Michael Valenzuela and colleagues systematically review and meta-analyze the evidence that computerized cognitive training improves cognitive skills in older adults with normal cognition.Please see later in the article for the Editors' Summary New effective interventions to attenuate age-related cognitive decline are a global priority. Computerized cognitive training (CCT) is believed to be safe and can be inexpensive, but neither its efficacy in enhancing cognitive performance in healthy older adults nor the impact of design factors on such efficacy has been systematically analyzed. Our aim therefore was to quantitatively assess whether CCT programs can enhance cognition in healthy older adults, discriminate responsive from nonresponsive cognitive domains, and identify the most salient design factors.I2 = 29.92%). There was no systematic evidence of publication bias. The overall effect size  for CCT versus control was small and statistically significant, g = 0.22 (95% CI 0.15 to 0.29). Small to moderate effect sizes were found for nonverbal memory, g = 0.24 (95% CI 0.09 to 0.38); verbal memory, g = 0.08 (95% CI 0.01 to 0.15); working memory (WM), g = 0.22 (95% CI 0.09 to 0.35); processing speed, g = 0.31 (95% CI 0.11 to 0.50); and visuospatial skills, g = 0.30 (95% CI 0.07 to 0.54). No significant effects were found for executive functions and attention. Moderator analyses revealed that home-based administration was ineffective compared to group-based training, and that more than three training sessions per week was ineffective versus three or fewer. There was no evidence for the effectiveness of WM training, and only weak evidence for sessions less than 30 min. These results are limited to healthy older adults, and do not address the durability of training effects.We systematically searched Medline, Embase, and PsycINFO for relevant studies from the databases' inception to 9 July 2014. Eligible studies were randomized controlled trials investigating the effects of ≥4 h of CCT on performance in neuropsychological tests in older adults without dementia or other cognitive impairment. Fifty-two studies encompassing 4,885 participants were eligible. Intervention designs varied considerably, but after removal of one outlier, heterogeneity across studies was small  packages, which provide standardized, cognitively challenging tasks designed to “exercise” various cognitive functions, is a lucrative and expanding business. But does CCT work? Given the rising global incidence of dementia, effective interventions that attenuate age-related cognitive decline are urgently needed. However, the impact of CCT on cognitive performance in older adults is unclear, and little is known about what makes a good CCT package. In this systematic review and meta-analysis, the researchers assess whether CCT programs improve cognitive test performance in cognitively healthy older adults and identify the aspects of cognition (cognitive domains) that are responsive to CCT, and the CCT design features that are most important in improving cognitive performance. A systematic review uses pre-defined criteria to identify all the research on a given topic; meta-analysis uses statistical methods to combine the results of several studies.The researchers identified 51 trials that investigated the effects of more than four hours of CCT on nearly 5,000 cognitively healthy older adults by measuring several cognitive functions before and after CCT. Meta-analysis of these studies indicated that the overall effect size for CCT  was small but statistically significant. An effect size quantifies the difference between two groups; a statistically significant result is a result that is unlikely to have occurred by chance. So, the meta-analysis suggests that CCT slightly increased overall cognitive function. Notably, CCT also had small to moderate significant effects on individual cognitive functions. For example, some CCT slightly improved nonverbal memory  and working memory . However, CCT had no significant effect on executive functions (cognitive processes involved in planning and judgment) or attention (selective concentration on one aspect of the environment). The design of CCT used in the different studies varied considerably, and “moderator” analyses revealed that home-based CCT was not effective, whereas center-based CCT was effective, and that training sessions undertaken more than three times a week were not effective. There was also some weak evidence suggesting that CCT sessions lasting less than 30 minutes may be ineffective. Finally, there was no evidence for the effectiveness of working memory training by itself .These findings suggest that CCT produces small improvements in cognitive performance in cognitively healthy older adults but that the efficacy of CCT varies across cognitive domains and is largely determined by design aspects of CCT. The most important result was that “do-it-yourself” CCT at home did not produce improvements. Rather, the small improvements seen were in individuals supervised by a trainer in a center and undergoing sessions 1–3 times a week. Because only cognitively healthy older adults were enrolled in the studies considered in this systematic review and meta-analysis, these findings do not necessarily apply to cognitively impaired individuals. Moreover, because all the included studies measured cognitive function immediately after CCT, these findings provide no information about the durability of the effects of CCT or about how the effects of CCT on cognitive function translate into real-life outcomes for individuals such as independence and the long-term risk of dementia. The researchers call, therefore, for additional research into CCT, an intervention that might help to attenuate age-related cognitive decline and improve the quality of life for older individuals.http://dx.doi.org/10.1371/journal.pmed.1001756.Please access these websites via the online version of this summary at PLOS MedicinePerspective by Druin BurchThis study is further discussed in a age-related forgetfulness, about memory and cognitive health, and about dementia (in English and Spanish)The US National Institute on Aging provides information for patients and carers about dementia and about memory lossThe UK National Health Service Choices website also provides information about memory, mild cognitive impairment, and dementia (in English and Spanish)MedlinePlus provides links to additional resources about  Cognitive decline and impairment are amongst the most feared and costly aspects of aging Several studies have now established strong and independent links between engagement in cognitively stimulating activities throughout the life span and enhanced late-life cognition, compression of cognitive burden, and reduced risk of cognitive impairment and dementia Prior systematic reviews of generic cognitive interventions in healthy older adults The effectiveness of CCT in enhancing cognitive performance in healthy older adults is therefore currently unclear, and the impact of design and implementation factors on efficacy has yet to be systematically analyzed. Using data from RCTs of narrowly defined CCT, we aimed to quantitatively evaluate the efficacy of CCT with respect to multiple cognitive outcomes in healthy older adults. Furthermore, we aimed to test the moderating effect of several key study features in order to better inform future CCT trial design and clinical implementation.This work fully complies with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines Eligible studies were published, peer-reviewed articles reporting results from RCTs of the effects of CCT on one or more cognitive outcomes in healthy older adults.Eligible studies had mean participant age ≥60 y and participants who lacked any major cognitive, neurological, psychiatric, and/or sensory impairments. Studies with MCI as an inclusion criterion were excluded, as cognitive performance in this population may vary substantially, particularly with respect to variability in the diagnostic criteria of MCI Eligible trials compared the effects of ≥4 h of practice on standardized computerized tasks or video games with clear cognitive rationale, administered on personal computers, mobile devices, or gaming consoles, versus an active or passive control condition. Lab-specific interventions that did not involve interaction with a computer were excluded.Outcomes included performance on one or more cognitive tests that were not included in the training program , administered both before and after training. This review is limited to change in performance from baseline to immediately post-training on tests of global cognition, verbal memory, nonverbal memory, working memory (WM), processing speed, attention, language, visuospatial skills, and executive functions. Both primary and secondary outcomes were included. Long-term outcomes, subjective measures , noncognitive outcomes , imaging data, and activities of daily living outcome measures were excluded from the analysis.We searched Medline, Embase, and PsycINFO using the search terms “cognitive training” OR “brain training” OR “memory training” OR “attention training” OR “reasoning training” OR “computerized training” OR “computer training” OR “video game” OR “computer game”, and by scanning reference lists of previous reviews. No limits were applied for publication dates, and non-English papers were translated. The first search was conducted on 2 December 2013. An updated search was conducted on 9 July 2014.Two reviewers (A. L. and H. H.) independently screened search results for initial eligibility based on title and abstract. Full-text versions of potentially eligible studies and those whose eligibility was unclear based on title and abstract were assessed by A. L. and H. H., who also contacted authors when eligibility was unclear based on the full report. Disagreements regarding study eligibility were resolved by consulting with M. V., who approved the final list of included studies.Coding of outcome measures into cognitive domains was done by two reviewers (A. L. and H. H.) based on accepted neuropsychological categorization CCT programs were divided into five content types: speed of processing (SOP) training, WM training, attention training, multidomain training, and video games. Video games were defined as computer programs that were distributed for entertainment purposes before they were tried as cognitive interventions When studies presented data for both active and passive control groups, only the active control group was used as a comparison to the CCT group. When studies presented data from both young and older adults, only data from the older group were analyzed.Risk of bias in individual studies was assessed using the items recommended in the Cochrane's Collaboration's risk of bias tool In addition, we used the Physiotherapy Evidence Database (PEDro) scale to assess study quality. The PEDro scale is a 11-item scale designed to assess the methodological quality and reporting of RCTs, and is reliable for rating trials of non-pharmacological interventions g) of post-training change between CCT and control groups. Analyses were conducted for all cognitive results combined, as well as for each of the following cognitive domains: verbal memory, nonverbal memory, WM, processing speed, attention, visuospatial skills, and executive functions . Precision of the SMD was calculated for each trial by the 95% CI. A positive SMD implies better therapeutic effects over time in the CCT group compared to the control group.The primary outcome was standardized mean difference  intervals I2 values of 25%, 50%, and 75% imply small, moderate, and large heterogeneity, respectively Because we expected studies to report multiple cognitive outcomes and display methodological variability Q statistic p<0.05 level. The following moderating factors were included in our analysis plan: type of CCT program , delivery format (group or home-based training), session length, session frequency, total duration of the program (dose), control condition (active or passive control), and risk of bias (high or low risk of bias as defined above).In the third stage, subgroup analyses were based on a mixed-effects model, which uses a random-effects model to generate within-subgroup variance and a fixed-effects model to compare effects between subgroups p<0.1), we report primary outcomes based on a fixed-effects model along with a random-effects model, as the former gives more weight to larger trials and helps to counterbalance a possible inflation of therapeutic effect In order to assess risk of publication bias, funnel plots for overall outcomes as well as for each cognitive domain were inspected for asymmetry  k = 3), and use of a fixed-effects model instead of a random-effects model. These results are reported in For the main analysis , we tested the robustness of our results to parametric variation of the following assumptions: test–retest correlation (set at 0.6 and tested from 0.5 to 0.7), within-study multiple outcome intercorrelation (set at 0.7 and tested from 0.6 to 0.8), inclusion of passive controls instead of active controls in studies with multiple controls  = −4.324, p<0.001).An active control group was used in 26 studies (50%), and assessor blinding was confirmed in 24 (46.2%) of studies. The average PEDro score was 6.2/9 (SD = 1.35), and 35 (66.6%) studies were found to have a high risk of bias . As expeType of CCT varied considerably across studies . Twenty-g = 0.28, 95% CI 0.18 to 0.39, p<0.001). Heterogeneity across studies was moderate . The forest plot revealed one conspicuous outlier g>3.0; see g = 0.22, 95% CI 0.15 to 0.29, p<0.001; I2 = 29.92%, 95% CI 0.63% to 50.57%; p = 0.12; The overall effect of CCT on cognition was small and statistically significant . The Funnel plot showed potential asymmetry , and the funnel plot did not show evidence of asymmetry . The funnel plot did not show significant asymmetry . We detected evidence of unusual funnel plot asymmetry, whereby larger studies reported larger effect sizes . The funnel plot suggested larger effect sizes in smaller studies . The funnel plot did not display notable asymmetry . The funnel plot revealed potential asymmetry, suggesting a greater effect in smaller studies  compared to home-based administration . Study-to-study heterogeneity within the group-based training studies was low  or 2–3 sessions per week  but not in studies that trained their participants more than three times per week . Within-subgroup heterogeneity was low for training either once per week  or 2–3 times per week . The intersection of these two moderators (group- versus home-based administration and number of sessions per week), i.e., group-based CCT studies that administered 2–3 sessions per week, comprised a subset of k = 25 studies and produced a similar effect estimate: g = 0.29, 95% CI 0.18 to 0.39, p<0.001; Q statistic for within-subgroup heterogeneity = 30.84, df = 24, p = 0.16; I2 = 22.18%, CI 0% to 52.44%.In order to examine the relationship between CCT design choices and training outcomes, we evaluated efficacy in predefined subgroups . Based oA similar sequence of moderator analyses for each cognitive domain can be found in CCT research involving healthy older participants has now matured to a substantial literature, encompassing 51 RCTs of reasonable quality. When examined en masse, CCT is effective at enhancing cognitive function in healthy older adults, but small effect sizes are to be expected. By definition this result pertains to the theoretical “average” older person—it is currently not possible to predict whether a given individual's cognitive abilities will improve beyond normal practice effects. More importantly, the efficacy of CCT depends on particular design choices as well as the cognitive outcome of interest. Moderator analyses revealed the inefficacy of home-based training compared to group-based training, as well as training more than three times a week. Domain-specific analyses found evidence of efficacy for nonverbal memory, processing speed, WM, and visuospatial outcomes, but not for attention and executive functions. Equally important, we found consistent evidence for the likely inefficacy of WM training and the use of brief training sessions.g = 0.08, 95% CI 0.01 to 0.15). Somewhat atypically, the funnel plot for SOP outcomes found that the largest trials tended to find the largest effect sizes. Given that more than half of all participants in this systematic review undertook speed-based training Evidence of possible publication bias was found only for reports of verbal memory outcomes. In this case a more conservative fixed-effects model was used and found that CCT efficacy in this domain is weak at best  revealed a significant interaction between delivery setting and therapeutic outcome, whereby group-based delivery was effective  and home-based delivery was not . A high degree of consistency amongst group-based training studies suggests that this conclusion is robust (One of the attractions of home-based (often Internet-delivered) CCT is the ability to administer a customized and adaptive intervention in the individual's home, with potential for decreased implementation cost s robust . If tranWe also found interesting evidence for the importance of correct CCT dose. The results suggested that short sessions of less than 30 min may be ineffective, possibly because synaptic plasticity is more likely after 30–60 min of stimulation k = 51), the typical trial was modest in size (median N = 45). Future studies incorporating supervised group-based delivery and a session frequency of 2–3 sessions per week can anticipate an approximate effect size of g = 0.29, suggesting that a sample of 87 is sufficient to designate power at 0.8 and allow for 15% attrition.To our knowledge, this is the first quantitative meta-analysis of RCTs in the defined field of CCT in cognitively healthy older adults. As opposed to previous reviews that included various cognitive interventions and research designs Discussion of CCT tends to focus on whether it “works” rather than on what factors may contribute to efficacy and inefficacy Figure S1Funnel plots. (A) Verbal memory, (B) nonverbal memory, (C) WM, (D) processing speed, (E) executive functions, (F) attention, and (G) visuospatial skills.(TIF)Click here for additional data file.Figure S2Moderators of efficacy of CCT for verbal memory.aQ-test for between-group heterogeneity, fixed-effects model. bTotal number of training hours.(TIF)Click here for additional data file.Figure S3Moderators of efficacy of CCT for nonverbal memory.aQ-test for between-group heterogeneity, mixed-effects model. bTotal number of training hours.(TIF)Click here for additional data file.Figure S4Moderators of efficacy of CCT for working memory.aQ-test for between-group heterogeneity, mixed-effects model. bOne study that combined data from both home- and group-based training cTotal number of training hours.(TIF)Click here for additional data file.Figure S5Moderators of efficacy of CCT for processing speed.aQ-test for between-group heterogeneity, mixed-effects model. bOne study that combined data from both home- and group-based training cTotal number of training hours. dSession length could not be determined for one study (TIF)Click here for additional data file.Figure S6Moderators of efficacy of CCT for executive function.aQ-test for between-group heterogeneity, mixed-effects model. bTotal number of training hours. cSession length could not be determined for one study (TIF)Click here for additional data file.Figure S7Moderators of efficacy of CCT for attention.aQ-test for between-group heterogeneity, mixed-effects model. bTotal number of training hours.(TIF)Click here for additional data file.Figure S8Moderators of efficacy of CCT for visuospatial skills.aQ-test for between-group heterogeneity, mixed-effects model. bTotal number of training hours.(TIF)Click here for additional data file.Table S1Classification of neuropsychological outcomes.(DOCX)Click here for additional data file.Table S2Group data extraction and results of individual studies.(DOCX)Click here for additional data file.Table S3Data provided by primary authors.(DOCX)Click here for additional data file.Table S4Risk of bias within studies.(DOCX)Click here for additional data file.Table S5Results of sensitivity analyses.(DOCX)Click here for additional data file.Checklist S1PRISMA checklist.(DOC)Click here for additional data file.Dataset S1Raw effect size and moderator data for overall (combined) and domain-specific results.(XLSX)Click here for additional data file.Protocol S1Study protocol.(DOCX)Click here for additional data file.


To report the thickness of the retina, retinal ganglion cell (RGC)-related layers, and choroid in healthy subjects using swept source optical coherence tomography (SS-OCT).One hundred and forty-six healthy volunteers were consecutively recruited for this prospective observational study. Thickness of retina, RGC-related layers, and choroid in the standard early treatment of diabetic retinopathy study (ETDRS) grid were automatically measured using one SS-OCT . The IOL Master  was used to measure axial length (AL).p = 0.000; r = 0.813, p = 0.000, respectively) and with similar topographic distributions within the retina. The thicknesses of retina and GCC/GCIPL in the inner sectors were significantly higher than in the outer sectors of the EDTRS area, while in the same region of the macula, the choroid exhibited completely different patterns of topographic variation. Men had 7.8 um thicker retina and 34.9 um thicker choroid than women after adjustment for age and AL . Age and AL could significantly influence the choroidal thickness but not the retina .Thicknesses of the average macular ganglion cell complex (GCC) and ganglion cell-inner plexiform layer (GCIPL) were 105.3 ± 9.7 and 78.5 ± 6.2 um respectively. Neither of them was significantly related with sex, age, or AL. Both showed strong correlations with retinal thickness  can be significantly influenced by age and AL.The online version of this article (doi:10.1186/s12886-015-0110-3) contains supplementary material, which is available to authorized users. This high resolution has enabled clinicians to accurately measure the thickness of local RGC-related layers, including ganglion cell complex (GCC) [Various retinal and choroidal pathologies, including age-related macular degeneration (AMD) and choroidal neovascularization (CNV), are among the most common reasons for severe visual impairment and blindness. For this reason, qualitative and quantitative analyses of retinal and choroidal structures are critical for the diagnosis and treatment of vitreo-retinal and choroidal diseases. Optical coherence tomography (OCT), as an essential tool in ophthalmology, can noninvasively capture detailed ex (GCC) , ganglioex (GCC) , retina ex (GCC) .Three inner retinal layers, including the nerve fiber, ganglion cell, and inner plexiform layer, are collectively known as the GCC , 5. The The Early Treatment of Diabetic Retinopathy Study (ETDRS) chart is considered to be the gold standard in the evaluation of retinal and choroidal structures in the posterior pole, and is widely used in clinical application of ophthalmology. Many studies have reported the normal distribution of data for retinal and choroidal thickness in the ETDRS area , 10–13. Cumulative evidence supports the view that choroidal thickness is clearly influenced by sex, age, and axial length (AL) in healthy subjects , 14–16. The transversal study included 146 healthy Chinese volunteers, recruited from our hospital staff and the students of Sun Yat-sen University, from January 2014 to June 2014. All participants underwent a complete ophthalmic evaluation in the clinical research center at Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China, and gave written informed consent after study approval by the Ethical Review Committee of Zhongshan Ophthalmic Center. The study adhered to the provisions of the Declaration of Helsinki for research involving human subjects Additional file All the study participants were healthy individuals with no history of ocular disease or visual symptoms; aged at least18 years; intraocular pressure (IOP) <21 mmHg; normal appearance of optic nerve head; normal anterior chamber angles; and a best-corrected visual acuity (BCVA) of 1.0 or better. Exclusion criteria included IOP > 21 mmHg; history of intraocular surgery or ocular trauma in the study eye; high myopia or hyperopia ; retinal or choroidal abnormality detected by SS-OCT; poor image quality due to severe cataract or unstable fixation; or severe systemic diseases such as diabetes mellitus, rheumatism, or malignant tumors.The comprehensive ophthalmical evaluation included: measurement of visual acuity and BCVA; IOP measurement using Goldmann applanation tonometry; slit lamp examination; fundus examination with a 90D lens; measurement of axial ocular dimension using ocular biometry ; and autorefractometry examinations. Refraction data were converted to spherical equivalents, counted as spherical diopters plus one half of the cylindrical dioptric power. Mean thicknesses of retina, GCC/GCIPL, and choroid were automatically acquired through one SS-OCT scan .After pupil dilation with 0.5 % tropicamide and 0.5 % phenylephrine, a 3D raster scan protocol with 3 um axial resolution and a speed of 100,000 A-scans per second was performed to acquire the retinal and choroidal thickness map in the macular region (12 × 9 mm) with a central fixation. Next, the thickness maps were overlapped to the EDTRS grid (6 × 6 mm) to obtain the values for each sector . Built-iAL was measured by IOL Master, which has been extensively used and is reported to be very precise , 18. Twot-test was used to compare two different groups where data were normally distributed; a Mann-Whitney U test was used when the data showed a non-parametric distribution. Pearson or Spearman correlation coefficients were used for evaluation of bivariate correlations. For comparison of the thickness of different layers at different subgroups according to age and AL, analysis of variance (ANOVA) or a Kruskal-Wallis test was performed. The relationship between macular retinal and choroidal thickness and sex, age and AL was investigated by linear and stepwise multiple regression analysis.All analyses used SPSS software version 19.0 . Normally distributed data were expressed as mean ± standard deviation (SD). An independent sample The current study included 146 randomly selected eyes from 146 healthy volunteers . Seventy-nine left eyes and 67 right eyes were analyzed. The mean AL was 23.5 ± 1.1 mm (range 20.9–26.9 mm).P < 0.0001). The thickness in the inner sectors was significantly thicker than in the outer sectors. In the inner sectors, the retina was thickest in the inner superior (309.7 um) and was significantly thicker than the other three sectors (P < 0.0001). In the outer sectors, the thinnest area was the inferior (267.1 um), while the nasal retina was the thickest (281.6 um), and with significantly differences between the four different regions (P < 0.0001).Table p = 0.000; r = 0.813, p = 0.000, respectively) and with similar topographic distributions within the retina in the EDTRS area .The average macular GCC and GCIPL thickness were 105.3 ± 9.7 um and 78.5 ± 6.2 um in the EDTRS area. Both of these showed strong correlations with retinal thickness  in the EDTRS region as automatically measured by SS-OCT. Unlike the retina and GCC/GCIPL, the choroid exhibited a completely different distribution pattern , but it seems that GCIPL thickness does not decrease with age .Table age Fig. . Mean thp < 0.001). There seemed to be no significant difference in retinal thickness, including GCC and GCIPL, between different AL subgroups  uses a longer wavelength-sweeping laser of 1050 nm, which allows for a much higher image acquisition speed and much deeper penetration of the ocular tissue. It enables automatic measurement of retina, local RGC-related layers (GCC and GCIPL), and choroid, and many clinical studies have confirmed its reliability in reproducing ocular tissue measurements, especially for assessment of the choroid –24. SS-OBy comparison with total retinal macular thickness, assessment of RGC-related layers provides higher diagnostic power for differentiating between healthy and pathologic eyes. GCC/GCIPL analysis maybe an early and susceptible structural marker for neuronal loss, and estimated macular GCC/GCIPL thickness is considered to be better a diagnostic measure of retinal nerve fiber layer in differentiating preperimetric and perimetric glaucomatous eyes from healthy eyes , 25–26. 2 elliptical annulus centered on the fovea. However, in the present study, the modified EDTRS grid (113.04 mm2) was used for measurement. In addition, SS-OCT as used here provided significantly better identification of the GCC/GCIPL layer in healthy eyes and reduced extraneous human factors as much as possible. The greater number of women and the differing ethnicity of enrolled volunteers may also partly account for the different results.Many studies have demonstrated an independent association between age, sex, AL, and GCC/GCIPL thickness, although the magnitude of this effect is not significant. Older age, female sex, and longer AL are associated with thinner GCC/GCIPL thickness –31. In tAs expected, inter-sex differences and age-related changes were also found in retinal and choroidal thickness. Men had a thicker retina and choroid, and after adjusting for age and AL, men still had 7.8 um greater retinal thickness and 34.9 um greater choroidal thickness than women, which is consistent with the previous results . The maiThe main strength of the present study  is the application of available built-in software, enabling automated segmentation and thickness measurements of retina, GCC/GCIPL, and choroid. Of course, this study, like others, also had some limitations. One of these was the fact that only healthy Chinese subjects were enrolled. However, our primary purpose was to evaluate the baseline thickness of GCC/GCIPL in the ETDRS grid of such subjects. A further limitation was that the numbers of men and women were not equivalent, as more women were recruited for this study.In conclusion, the thickness of GCC/GCIPL in healthy Chinese individuals is similar across different genders, ages, and AL groups in terms of the ETDRS chart. Men’s eyes were found to have thicker retinal and choroid structures. Age and AL also significantly influence the choroidal structure, but not the retina. These data will be of value in diagnosing and monitoring diseases of the ocular fundus and will provide a useful reference for measurements across different races.


Salmonella Typhi vaccine strain CVD 915 on the development of liver metastases in a mouse model of breast cancer. To this end, one group of BALB/c mice was orogastrically immunized with CVD 915, while another received PBS as a control. After 24 h, mice were injected with LM3 mammary adenocarcinoma cells into the spleen and subjected to splenectomy. This oral Salmonella-based vaccine produced an antitumor effect, leading to a decrease in the number and volume of liver metastases. Immunization with Salmonella induced an early cellular immune response in mice. This innate stimulation rendered a large production of IFN-γ by intrahepatic immune cells (IHIC) detected within 24 h. An antitumor adaptive immunity was found in the liver and celiac and portal lymph nodes (LDLN) 21 days after oral bacterial inoculation. The antitumor immune response inside the liver was associated with increased CD4+ and dendritic cell populations as well as with an inflammatory infiltrate located around liver metastatic nodules. Enlarged levels of inflammatory cytokines (IFN-γ and TNF) were also detected in IHIC. Furthermore, a tumor-specific production of IFN-γ and TNF as well as tumor-specific IFN-γ-producing CD8 T cells (CD8+IFN-γ+) were found in the celiac and portal lymph nodes of Salmonella-treated mice. This study provides first evidence for the involvement of LDLN in the development of an efficient cellular immune response against hepatic tumors, which resulted in the elimination of liver metastases after oral Salmonella-based vaccination.Primary tumor excision is one of the most widely used therapies of cancer. However, the risk of metastases development still exists following tumor resection. The liver is a common site of metastatic disease for numerous cancers. Breast cancer is one of the most frequent sources of metastases to the liver. The aim of this work was to evaluate the efficacy of the orally administered  The development of novel cancer treatments is a major subject of study around the globe. More than 90% of cancer-related deaths are due to metastatic disease and not from the primary tumors from which they arise . ExcisioSalmonella enterica serovar Typhi (S. Typhi)-attenuated vaccine strain CVD 915 as an immunotherapeutic agent in breast cancer  inoculated CVD 915 proves to be a good adjuvant in a prophylactic cancer vaccine against a mouse T-cell lymphoma . Based oS. Typhi CVD 915 promotes an efficient antitumor immune response that results in a decrease in hepatic metastases. Moreover, we found that the recently identified main liver-draining celiac and portal lymph nodes .Intrahepatic immune cells, mesenteric lymph nodes (MLN), and liver-draining lymph nodes (LDLN) cells obtained from BALB/c mice were cultured at 37°C in a humidified 5% COSalmonella Typhi CVD 915 was kindly provided by Dr. Myron Levine . This vaccine strain , Facultad de Medicina, UBA] approved the research protocol. Distress and suffering were avoided as much as possible. All procedures were carried out by a veterinarian.9 CFU) or PBS alone as a control, also via o.g. Twenty-four hours later, animals were challenged with LM3 tumor cells, using the liver metastases tumor model designed by Lafreniere and Rosenberg  and bled by heart puncture with a heparinized syringe. Livers were perfused with cold PBS via the portal vein and then removed, weighted, and processed for the isolation of IHIC. Spleen, MLN, and/or LDLN  were removed and disaggregated into a single-cell suspension. Celiac and portal lymph nodes were identified anatomically as described by Zheng et al.  and sampV = π/6 × f × (length × width)3/2 (Hepatic metastases were measured and quantified using a stereo zoom microscope (loupe) (4×) . The hepidth)3/2 .The liver metastases model used has an effectiveness of ~88% and does not allow discrimination between mice that failed to develop metastases from those eventually cured by the treatment. Thus, only those animals showing macroscopically visible metastases 20 days after the challenge were included in further analysis.Balb/c mice were treated with CVD 915 via o.g. route, as described previously. One, four, eight, or twenty-four hours after the bacterial inoculation, mice were euthanized and livers aseptically removed and homogenized in distilled water. Samples of homogenized livers were seeded in agar plates for CFU determination, as previously described .g for 1 min at room temperature without brake. The resulting supernatant was centrifuged at 480 × g with brake for 8 min at room temperature. The pellet obtained was resuspended in 5 ml of Percoll 33% in PBS with heparin (100 U/ml) and centrifuged at 850 × g for an additional 30 min at room temperature without brake. The resulting pellet, containing the IHIC and contaminant erythrocytes, was subjected to hypoosmotic lysis and then washed with complete medium. Cells were then resuspended in complete medium for cell surface phenotyping by flow cytometry or functional analysis.Intrahepatic immune cells were isolated by mechanical disruption followed by Percoll density separation as described by Blom et al. , with mieBioscience, San Diego, CA, USA). APC- or PE-conjugated streptavidin and isotype-matched controls were used as required. Immunofluorescence surface staining and flow cytometric analysis were performed, as previously described , and biotin anti-MHC II , and then labeled with fluorescent antibodies, as previously described. Samples were processed using a BD FACSCalibur or a BD Accuri C6 flow cytometer . Multicolor flow cytometric analysis was performed using the Flowing Software 2.5.1 .IFN-γ-production by tumor-specific CD8escribed . IHIC an6 and 1 × 106/well, respectively) obtained from treated and control mice were cultured separately in 96-well plates in the presence or absence of LM3 tumor cells at a 10:1 ratio. Supernatants were collected 72- or 96-h later, and cytokines  were measured by ELISA (eBioscience) following the manufacturer’s instructions. The detection limits were 30, 4, and 4 pg/ml, respectively.Intrahepatic immune cells and LDLN cells  staining of paraffin-embedded sections was performed, as previously described .All data are shown as mean ± SEM. For comparisons between groups, unpaired Student’s Salmonella Typhimurium has been shown to infect mouse liver  and neutrophils (Gr1+), were detected in the spleen  was also increased in treated mice as compared with control animals  and local (in liver and LDLN) innate immune response.Salmonella or PBS. Twenty-four hours later, LM3 tumor cells were inoculated using the liver metastases mouse model explained above. The prophylactic efficacy of bacterial administration on the development of liver metastases was evaluated 20 days later, by counting and measuring liver tumor nodules. A significant decrease in the hepatic tumor burden was found in Salmonella-treated mice  , indicating that oral Salmonella promotes tumor mass reduction. These results show a strong therapeutic efficacy of this bacteria-based vaccine in preventing the development of liver metastases.To assess the incidence of liver metastases following oral vaccination with CVD 915, mice were orally immunized with Salmonella, tumor-challenged and naive animals were weighted, before and after treatment, and side effects were monitored. Mice from all groups gained almost 2 g in 3 weeks. There was no disparity in body weights between control and treated mice, either inoculated or not with LM3 cells , as shown in Figure Salmonella-based treatment. We next examined whether the liver and LDLN are involved in the immune mechanism underlying the antitumoral effects of this oral vaccine. An increased frequency of CD4+ T cells at the expense of decreased B cells (B220+) was detected in CVD 915-treated mice when compared to controls  from treated-mice expressed higher levels of MHC II molecules than DC from controls  to LM3 cells. Then, levels of IL-2, TNF, and IFN-γ were determined in culture supernatants. In cultures unexposed to tumor cells, enhanced levels of these three cytokines were found in IHIC and LDLN cultures from  Figures A–C. When Figures A–C. When Figures A. These Salmonella-treated mice with (or without) LM3 tumor cells, immune cells were immunocharacterized and stained for intracytoplasmic IFN-γ. An enlarged percentage of CD8+IFN-γ+ T-cells was found in LDLN from bacteria-treated mice that had been cultured with tumor cells, as compared with non-treated controls . The liver is a common target of cancer metastases. The fact that Salmonella-based vaccines prove to be useful against breast and lymphoma metastases implies that they might be suitable to treat metastases from other types of cancer. Our findings correlate with other studies in which orally attenuated Salmonella Typhimurium was tested in a mouse model of liver metastases from colon adenocarcinoma  both in LDLN and liver from treated mice. These results are consistent with a predominant T-cell-mediated immune response located at the tumor site and LDLN. Besides, it is broadly known that IFN-γ is a critical component of the immune system in antitumor immune responses  cells, such as activated tumor-specific CD4- and CD8-T lymphocytes  in the cellular immune response against hepatic tumors, which resulted in the elimination of liver metastases in LM3 tumor-bearing mice after oral r et al. . These aS. Typhi vaccine strain can elicit a successful immune response, which is capable of preventing the development of liver metastases. The early type-1 innate immunity activated by a single oral dose of a S. Typhi is sufficient to break not only liver-inherent immune tolerance but also tumor-induced immunosuppression. The proinflammatory microenvironment elicited in the liver by oral Salmonella triggers a tumor-specific Th1-type immune response, which efficiently reaches the liver and its recently identified draining lymph nodes rendering a drastic reduction of hepatic metastases. The proven antitumor efficacy of oral S. Typhi vaccination encourages future clinical evaluation of this simple and inexpensive immunotherapy as a neoadjuvant and/or adjuvant treatment for liver metastatic cancer.In conclusion, this study demonstrates that an oral live attenuated Conception and design: AV and CW. Execution of experiments: AV and AT. Acquisition of data: AV, MG, AT, and AC. Analysis and interpretation of data: AV, CM, MG, JG, and CW. Drafting of the manuscript: AV and CW. Critical revision of the manuscript: MG, AC, JG, and CM. Obtained funding: AV, CM, and CW. Study supervision: CW. Final approval of the version to be published: AV, CM, MG, AT, AC, JG, and CW. Agreement to be accountable for all aspects of the work: AV, CM, MG, AT, AC, JG, and CW.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Staphylococcus aureus, and gram-negative Escherichia coli and Pseudomonas aeruginosa as bacteria models. Testing results revealed that, in the absence of any other antibacterial agents, the rPDA coatings exhibited remarkably enhanced antibacterial activities. In addition, such enhanced antibacterial activities of the rPDA coatings were found to be unimpaired by steam sterilization treatments.Strong antibacterial polydopamine (PDA) coatings prepared by a facile shaking-assisted method is reported for the first time. It was found that a minor modification made to the conventional synthesis procedure of PDA coatings, viz. replacing the static solution condition with a shaking solution condition by using a mechanical shaker, can produce the roughened polydopamine (rPDA) coatings at different substrates, e.g., glass, stainless steel, plastic, and gauze. The resulting rPDA coatings were characterized with Raman spectrum, zeta-potential analysis and contact angle measurement. The antibacterial activity of the rPDA coatings was evaluated by a shake flask test with gram-positive  Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) do not develop noticeable resistance against these coatings161718191617Nowadays interest in implantation of medical devices has continued to accelerate. However, bacterial infection at the site of implanted medical devices has meanwhile become a serious ongoing problem12356781011121314Autoxidation of dopamine forming polydopamine (PDA) coatings has recently emerged as an extremely attractive approach for single-step surface functionalization of almost all kinds of materials. PDA also allows easily functionalization through reactions with amino- or mercapto-nucleophiles. In addition, PDA possesses biocompatible property. Therefore, PDA coatings currently have become a hot field of scientific research and technological innovation22232425122627282812262729313233S. aureus, and gram-negative E. coli and Pseudomonas aeruginosa (P. aeruginosa). In addition, such strong antibacterial performances are unimpaired by steam sterilization treatment that is a necessary procedure for implantation.Herein, we report the shaking-assisted one-step preparation of strong contact-active antibacterial PDA coatings without using any other antibacterial reagents for the first time. It has been known that dopamine self-polymerization in a static solution can produce relatively smooth PDA coatings2 as oxidant)The preparation of the rPDA coatings follows the conventional synthesis approach of PDA coatings , Ko and ~82o, respectively. After sPDA coating, the static contact angles changed to ~59o (a2) and ~60o (b2). The converged contact angles were consistent with the published values of 50–60o for sPDA coatingso , indicating more hydrophilic surfaces. Evidently, the rough surface morphology of the formed rPDA coatings strongly affected the apparent contact angle. The low values of the apparent contact angles may be associated with the tendency of water droplets to immerse through the gaps between PDA particles and aggregates. This phenomenon is somewhat akin to that observed at the nanostructured coatings, for instance, the surface nanostructures of the ZnO nanorods-based films can also exert a remarkable influence on the wettabilityOn the other hand, contact angle measurement revealed an abnormal phenomenon that the rPDA coatings could exhibit obviously different wettability from the sPDA coatings. As shown in E. coli and S. aureus. The bactericidal ratios are shown in 5 colony-forming unit (CFU) mL−1, after 18–24 hour incubation, the bactericidal ratio of the sPDA coatings toward E. coli and S. aureus, respectively, was less than 5%. Due to that the colonies on the agar plate corresponding to antibacterial activities of the sPDA coatings and bare substrates were too dense to be exactly counted, the agar plate testing results were directly showed in the form of photographs P. aeruginosa was also evaluated. From P. aeruginosa was low. Typically, at a bacteria concentration of 105 CFU mL−1, after 18–24 hour incubation, the bactericidal ratio of the sPDA coatings toward P. aeruginosa was less than 5%.The antibacterial properties of the rPDA coatings were evaluated by a shake flask test according to ASTM E2149–01, a quantitative antibacterial test method for determining the antibacterial activity of non-leaching antibacterial agents under dynamic contact conditions2830tographs . As reve5 CFU mL−1, comparable with the classical QACs and Ag+. Even at the used highest bacteria concentration, i.e., 107 CFU mL−1, after 18–24 hour incubation, the bactericidal ratios of the rPDA coatings toward three kinds of bacteria still reached more than 90%. The bactericidal results were also directly visualized from the photographs of the agar plates  dehydration and air-drying. As revealed by SEM observations , indicating possible damage to cell membrane.The mechanism of cell death caused by contact-active antibacterial coatings is commonly associated with the damage of cell surfaces1643rvations , on the E. coli and S. aureus was measured respectively. Steam sterilization and subsequent incubation process were repeated twice. Antibacterial results are summarized in In addition, it is well-known that all materials implanted within the body or placed in contact with corporeal fluids must be sterilized, and the preferred sterilization process is high temperature steam sterilizationFurthermore, the biocompatibility of the rPDA coatings was evaluated by examining the viability of the adherent mammalian cancer cell line, HeLa cells, incubated with the rPDA coatings. No qualitative differences in the attached cells were observed by microscopy, as shown in S. aureus, and gram-negative E. coli and P. aeruginosa. The damage of the cell membranes of bacteria incubated with the rPDA coatings supported the contact-kill mechanism. In addition, the antibacterial property of the rPDA coatings was found to be unimpaired by steam sterilization treatments. The rPDA coatings thus represent an excellent approach for substrate material-independent, single-step, green, antibacterial modification to fabricate contact-active and biocompatible antibacterial polymer coatings.This study has demonstrated that a shaking condition applied to the self-polymerization of dopamine in alkaline solution can facilitate the formation of the rPDA coatings. The rPDA coatings exhibited remarkably enhanced antibacterial activities against gram-positive S. aureus (ATCC 29213) and E. coli (ATCC 8739) were obtained from China General Microbiological Culture Collection Center . Human cervix carcinoma cells (HeLa) were obtained from China Infrastructure of Cell Line Resources . Aqueous solutions were prepared using deionized water (Milli-Q system).Dopamine hydrochloride was purchased from Sigma-Aldrich. Tri(hydroxymethyl) amino methane (Tris)-HCl, tryptone, yeast extract, agar powder and other chemicals of at least analytical reagent were obtained from Sinopharm Chemical Reagent Co., Ltd. . −1 dopamine, 25 mM Tris-HCl buffer solution, pH 8.5) for 24 h at 37 °C and various shaking rates using an incubator shaker . Then, the substrates were taken out, rinsed using deionized water, and dried in air.Substrate materials including glass, stainless steel, plastic, and gauze (ca. 2.5 cm ×1 cm) were immersed in the freshly prepared dopamine solution . The culture was incubated at 37 °C and 200 rpm for 18–20 h. Then, the incubated test culture was diluted using a sterilized phosphate buffer (pH 7.0) to the desired concentration. Antibacterial activity of the PDA-modified substrate materials and blank control substrate materials (2.5 × 2.0 cm2) were evaluated according to the ASTM E2149–01, which is a quantitative antibacterial test method for determining the antibacterial activity of immobilized antibacterial agents under dynamic contact conditions. For testing the effect of steam sterilization on the antibacterial activity of the rPDA coatings, the rPDA coatings on glass slides were treated with high-pressure steam sterilizer at 121 °C for 15 min. These materials were respectively incubated with 4 mL of cell suspension in a 5 mL conical tube at 37 °C and 200 rpm. Samples were taken after 24 h, and plated in nutrient agar. The inoculated plates were incubated at 37 °C for 24 h, and the surviving cells were counted. The experiments were performed in triplicate. Antibacterial testings towards P. aeruginosa (ATCC 27853) were performed in the Antimicrobial Material Testing Center of Technical Institute of Physics and Chemistry, Chinese Academy of Sciences . The antibacterial activity was expressed as R = % reduction of the organism after contact with the test substrate materials compared to the number of bacterial cells surviving after contact with the control.S. aureus and E. coli, respectively, at 37 °C and 200 rpm. Blank stainless steel slides were used as the control. The slides were taken out after 5 h. The bacteria were fixed in 2.5% glutaraldehyde for 2 h and rinsed carefully with distilled water. Then, the bacteria-modified slides were dehydrated with ethanol (20–100%), air-dried, and sputter-coated with carbon for SEM observation.For SEM observation, the rPDA-coated stainless steel slides were incubated with the cell suspensions of 2. The cells were harvested using 0.25% trypsin, counted, and resuspended in the cell culture media at a concentration of 5 × 104 cells mL−1. 2 mL of this suspension was seeded in the wells of a 6-well plate containing the rPDA-coated and blank substrates, respectively, and incubated for 24 h at 37 °C in a humidified atmosphere of 5% CO2. The amount of cells attached to the substrates was evaluated by optical microscopy .HeLa cells were maintained in Dulbecco’s modified Eagle media (DMEM) containing 10% (v/v) fetal bovine serum (FBS) at 37 °C in a humidified atmosphere of 5% CORaman spectra were obtained at room temperature on a Raman system (JY-HR 800). Surface morphologies of samples were characterized with a ZEISS Supra 55 FE-SEM. Contact angles were measured at room temperature on an OCA20 system . The average contact angle values were obtained by measuring at five different positions on the same sample. Zeta-potential measurement was conducted on a Malvern Zetasizer Nano ZS90.How to cite this article: Su, L. et al. Strong Antibacterial Polydopamine Coatings Prepared by a Shaking-assisted Method. Sci. Rep.6, 24420; doi: 10.1038/srep24420 (2016).


Sleep fragmentation in OSA is independently associated with hyper-LDL cholesterolemia, which may predispose patients with OSA to a higher risk of cardiovascular disease.Obstructive sleep apnea (OSA) is independently associated with dyslipidemia. Previous studies have demonstrated that sleep fragmentation can impair lipid metabolism. The present study aimed to identify whether sleep fragmentation is independently associated with dyslipidemia, in a large-scale, clinic-based consecutive OSA sample. This cross-sectional study was conducted among 2,686 patients who underwent polysomnography (PSG) for suspicion of OSA from January 2008 to January 2013 at the sleep laboratory. Multivariate regression analyses were performed to evaluate the independent associations between the microarousal index (MAI) and lipid profiles adjusting for potential confounders, including metabolic syndrome components and nocturnal intermittent hypoxia. The adjusted odds ratios (ORs) for various types of dyslipidemia according to MAI quartiles, as determined by logistic regression were also evaluated. MAI was found positively associated with low-density lipoprotein cholesterol (LDL-c) but not with total cholesterol (TC), triglyceride (TG) or high-density lipoprotein cholesterol (HDL-c). Furthermore, the adjusted ORs  for hyper-LDL cholesterolemia increased across MAI quartiles, as follows: 1 (reference), 1.3 (1.1–1.7), 1.6 (1.2–2.0), and 1.6 (1.2–2.1) ( Obstructive sleep apnea (OSA) is a highly prevalent chronic disease affecting approximately 4–14% of men and 2–5% of women with 30–70 years of age in the past few decades1345Previous studies have attributed the pathophysiology of lipid metabolism disorders in OSA mainly to chronic nocturnal intermittent hypoxia781011In addition to these previous reports on the association between sleep fragmentation and dyslipidemia11p < 0.001)  values were more likely to be male, current smokers, current alcohol consumers and to have hypertension, diabetes, and a higher body mass index (BMI) and waist circumference (WC) . A posit< 0.001) .p < 0.05); however, a linear trend for HDL-c among the MAI quartiles was not found (p = 0.286)  but was not associated with the HDL-c level in model 1. Linear trends for the TC, LDL-c, and TG levels among the ODI quartiles were also significant in model 1  . The ODI model 1 .p < 0.001) (p < 0.001)  (p < 0.001)  . Similar< 0.001) , whereas< 0.001) . The losctively) . However< 0.001) , and the< 0.001) , suggestWhen obesity, hypertension and diabetes were used as the categorized covariates in the linear regression analyses in models 3 and 4, the association between the MAI and the lipid levels was similar in models 1 and 3, with similar results obtained for models 2 and 4  and S2.2 change in the MAI was 0.007 in model 2 and 0.008 in model 4 (both p < 0.001), which indicated a significant association between the MAI and the LDL-c level independent of the other covariates included in those models.The hierarchical regression analysis showed that the rp < 0.05). In model 2, only the OR (95% CI) for hyper-LDL cholesterolemia remained significant in the linear trend test OR (95% CI) = 1 (reference); 1.3 (1.1–1.7), 1.6 (1.2–2.0), and 1.6 (1.2–2.1)  when the ODI was further taken into consideration. For models 3 and 4, the ORs and linear trends of dyslipidemia across the MAI quartiles were similar between models 1 and 3 and between models 2 and 4  were referred to the sleep laboratory at the Shanghai Jiao Tong University Affiliated Sixth People’s Hospital for suspected OSA. Among these, 538 participants were excluded for the following reasons: 1) previous treatment for OSA (n = 106), 2) treatment with lipid-lowing medications before the study (n = 92), and 3) missing data (n = 340). Finally, 2,686 patients were included in this study. The study was approved by the Internal Review Board of the Institutional Ethics Committee of Shanghai Jiao Tong University Affiliated Sixth Hospital, and was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants.2 < 90% (CT90) was also calculated.To obtain objective sleep parameters, overnight polysomnography  was performed under attended conditions in the laboratory. The following channels were employed: bilateral central referential electroencephalogram (EEG) channels (C3-M2 and C4-M1), bilateral electroculograms (EOG), a bipolar chin electromyogram (EMG), airflow , finger pulse oximetry, chest and abdominal wall motion (piezo electrodes), electrocardiogram, and body position. Sleep stages, respiratory events, and microarousals were scored automatically using a computer software and were subsequently checked manually by a skilled technician, following the American Academic Sleep Medicine (AASM) criteriaFor each participant, a fasting blood sample was collected from the antecubital vein the morning after PSG evaluation. The following lipid parameters were tested in the hospital laboratory using routine procedures: total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-c), and low-density lipoprotein cholesterol (LDL-c). According to the US National Cholesterol Education Program Adult Treatment Panel III (NCEPIII) diagnostic criteria, dyslipidemia in terms of TC, LDL-c, HDL-c and TG were defined as TC levels >200 mg/dL, LDL-c levels >130 mg/dL, HDL-c levels < 39.8 mg/dL, and TG levels >150 mg/dL, separately2 and abdominal obesity as a WC ≥ 85 cm for males and ≥80 cm for females based on the recommendations of the Working Group on Obesity in ChinaAnthropometric variables including weight, height and waist circumference (WC) were measured with the participant in light weight clothing and bare feet using standard methods. Body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared. Waist circumference (WC) was measured horizontally, midway between the lowest rib margin and the iliac crest. Obesity was defined as a BMI ≥ 28 kg/mSmoking status and alcohol consumption were ascertained by means of a self- reported questionnaire completed on the night of each patient’s in-laboratory PSG recording. Smoking status was classified as current or non-current smoker. Patients were also stratified according to their reported alcohol consumption into 2 groups: current or non-current drinker. The specific questions asked in the questionnaire and the cut-off values are provided in the Daytime blood pressure (BP) was measured after at least 5 min of rest in a sitting position using a mercury sphygmomanometer, following the American Society of Hypertension Guidelines, and the mean of three measurements was recorded. Fasting serum glucose levels were measured in the hospital laboratory using an auto analyzer . An immunoradiological method was used to measure the fasting serum insulin level. Mean arterial pressure was calculated as (systolic BP+ 2* diastolic BP)/3. Insulin resistance was estimated using the previously described homeostasis model of assessment (HOMA) methodP-values for linear trends across the four MAI groups were calculated using the polynomial linear trend test for continuous variables and using the linear-by-linear association test for dichotomous variables.Descriptive statistics of the study population are displayed according to MAI quartiles using the mean and standard deviation (SD) for continuous variables and percentages for categorical data. To understand the relationship between sleep fragmentation and lipid profiles including TC, LDL-c, HDL-c, and TG, stepwise multiple regression analyses were performed. Fasting lipid levels were used as dependent variables, while MAI was used as a predictor. All the regression analyses were adjusted for potential confounding factors in four models.p-value for linear trends was determined by examining the median MAI value for each quartile and assessing the overall F test for the median MAI variable.Dichotomous logistic regressions were used to model the associations between MAI quartiles and the ORs  for hypercholesterolemia, hyper-LDL cholesterolemia, low HDL cholesterolemia, and hypertriglyceridemia, after adjusting for confounding factors in models 1–4. The The statistical analysis was preceded by the use of collinearity diagnostics to eliminate possible multicollinearity among variables. The 2 steps of the collinearity analyses were: (1) a preliminary analysis using Spearman’s correlation; and (2) collinearity diagnostics to determine the selected covariates in the multivariate regression analyses. Based on the collinearity diagnosis, in model 1 we adjusted for age, BMI, fasting glucose level, HOMA- IR, and mean arterial pressure as continuous variables, and sex, smoking status, alcohol consumption as dichotomous variables. As nocturnal intermittent hypoxia was considered a potential confounder for the association between the MAI and outcomes in patients with OSA, we also accounted for the oxygen desaturation index (ODI) in the second multivariate model. Model 3 included the following covariates: age as a continuous variable; and sex, obesity, abdominal obesity, hypertension, diabetes, smoking status, and alcohol consumption as dichotomous variables. Model 4 further added the ODI to model 3. For detailed results of the collinearity diagnoses, see 2 at each step of the analysis.Hierarchical regression was used to test the independent influence of the MAI in models 2 and 4. The method analyzes the effect of a predictor variable after controlling for other variables, achieved by calculating the change in the rp-value < 0.05 was considered statistically significant37Of note, ODI was examined as both a classified variable according to its quartiles as well as a continuous one. These yielded similar conclusions, and we presented only the results using the continuous variables in linear regression analyses and the quartile variables in logistic regression analyses for simplicity. All statistical analyses were conducted using SPSS software . A bilateral How to cite this article: Qian, Y. et al. Independent Association between Sleep Fragmentation and Dyslipidemia in Patients with Obstructive Sleep Apnea. Sci. Rep.6, 26089; doi: 10.1038/srep26089 (2016).


Amphipterygium adstringens is a plant widely used in Mexican traditional medicine for its known anti-inflammatory and antiulcer properties. In this work, we evaluated the in vitro antimicrobial and antiproliferative activities of the methanolic extract of A. adstringens against oral pathogens such as Streptococcus mutans, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Candida albicans, and Candida dubliniensis, using microdilution (MIC) and agar diffusion methods (MBC), and the antiproliferative activity evaluating total growth inhibition (TGI) by staining the protein content with sulforhodamine B (SRB), using nine human cancer cell lines. Crude extract (CE) of A. adstringens showed some degree of activity against one or more of the strains with a MIC from 0.125 mg/mL to 63 mg/mL and MBC from 1.6 to 6.3 mg/mL and cytotoxic activity, particularly against NCI-ADR/RES, an ovarian cell line expressing multiple resistance drugs phenotype. The CE is a complex mixture of possible multitarget metabolites that could be responsible for both antimicrobial and antiproliferative activities, and further investigation is required to elucidate the identity of active compounds. Nevertheless the CE itself is useful in the development of new antimicrobial treatment based on natural products to prevent oral diseases and as alternative natural source for cancer treatment and prevention. Natural molecules and products reemerge as promising sources of complex multitarget mixtures that are used as alternative therapeutic agents for various conditions, including infections and chronic diseases , 2, suchAmphipterygium adstringens Schiede ex Schlecht (Julianaceae) is an endemic species in Mexico commonly known as “cuachalalate.” A. adstringens is a dioecious tree of approximately 5–10 meters in height and 40 cm in diameter, with the twisted trunk, gray bark with large scales, and rounded lenticels exposed and oblong leaves from 6 to 15 cm  with the formation of large amounts of organic acids that demineralize tooth enamel, and to adapt and tolerate environmental stresses, particularly low pH (aciduricity) [uricity) .Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans are bacteria often isolated from the subgingival biofilm [ biofilm , 31 and  biofilm . Dental  biofilm  and it h biofilm , cardiov biofilm , preterm biofilm , and pan biofilm . Additio biofilm  with chr Candida, is a local infection commonly seen in infants, older adults who wear dentures, patients treated with antibiotics, chemotherapy, or radiation therapy on head and neck, and those with cellular immune deficiency states, such as HIV infection [ Candida species are the fourth leading cause of healthcare-associated infections, accounting for approximately 11% of all infections; Candida spp. are also responsible for nearly 12% of all central line-associated bloodstream infections, preceded only by Staphylococcus aureus and Enterococcus species [ Candida albicans is often guilty in oral candidiasis and has been implicated in persistent apical periodontitis [ Candida dubliniensis was involved with denture stomatitis and oropharyngeal candidiasis in immunocompromised subject [Oropharyngeal candidiasis, caused bynfection . Moreove species . Candidadontitis . On the  subject , 42 and  subject . TreatmeThe increasing cancer incidence is closely related to the raise in life expectancy and longevity that consequently results in a longer exposure to carcinogenic agents such as pollution, UV radiation, and microorganisms . The redMoreover, there has been a decline in research, development, and approvals of new antimicrobial agents. For example, from 1983 to 1987 compared to from 1998 to 2002, there was a 56% decrease in new antibacterial agents approved by the FDA (Food and Drugs Administration). Among other reasons, one main cause is the high cost with a low rate of return compared to drugs used to treat chronic conditions, limitations on the use of newly approved antibacterial agents, and trial design and regulatory issues . This en A. adstringens against microorganisms associated with oral infections, and the possible antiproliferative effect against a well-defined panel of human cancer cells lines. This is, undoubtedly, a significant contribution in the field of alternative medicine towards the elaboration of novel antimicrobial agents to prevent and treat oral infections and to reduce the potential risk of systemic chronic diseases and cancer.With this approach, we studied the antimicrobial activity of CE ofAmphipterygium adstringens bark was obtained by Dr. Julia Verde from the Department of Chemistry, Faculty of Biological Sciences, Autonomous University of Nuevo Leon (UANL). The taxonomic identification was made by Dr. Mauricio Gonzalez, following the last revision of the genus Amphipterygium (Julianaceae) [anaceae) . A vouch A. adstringens was air-dried at 45°C and ground in a Wiley type mill to a fine powder. About 390 g of powder was macerated with 500 mL methanol by shaking at 100 rpm for 72 hours. The extract was filtered with filter paper (Whatman Grade 1) to remove debris and filter-sterilized with 0.20 μm syringe filter . Extract was concentrated using a rotary evaporator (Yamato RE801), under reduced pressure at 30°C, providing 52.7 g of crude extract. The crude extract was stored at 4°C until its use.The bark of A. adstringens was determined in a preliminary study using the Kirby-Bauer disk diffusion method [ A. adstringens crude extract against oral pathogens.The antimicrobial effect of the crude extract ofn method  against  S. mutans (ATCC 700610) was cultured on BHI and 5% CO2. P. gingivalis (ATCC 33277) and A. actinomycetemcomitans (ATCC 43718) was cultured on TSB (Tryptic Soy Broth) and carried out in anaerobic conditions. Escherichia coli (ATCC 43895) and Escherichia coli (ATCC 0157-H7) were cultured on TSB in aerobic atmosphere and were used as biological control to validate microbicidal activity assay due to their multidrug resistance. Candida albicans (CBS-562) and Candida dubliniensis (CBS-7987) were cultured on Sabouraud dextrose agar in aerobic conditions [ Candida because it is frequently used in patients who suffer oropharyngeal and esophageal candidiasis.Strains were cultured following their specific requirements.nditions . All str5 colony forming units (CFU)/mL. The CE was dissolved in 1% DMSO followed by dilution in tryptic soy broth, brain heart infusion, or Sabouraud (depending on the strain), and, then, serial twofold to threefold dilutions were made in a concentration ranged from 78 to 0.01 mg/mL, in sterile water. 96-well plates were incubated at 35°C/24 hours aerobically and anaerobically. MIC was determined as the lowest concentration of CE that inhibited microorganism growth. The experiment was performed by duplicate in three independent tests.The minimum inhibitory concentrations (MIC) were determined by microdilution broth method according to the Clinical and Laboratory Standards Institute , using 9The MBC or MFC was determined removing the samples from the wells of the microplate with no visible bacterial growth and subcultured in agar , depending on the strain. The lowest concentration of the subculture with no growth was considered the minimum bactericidal concentration (MBC) or the minimum fungicidal concentration (MFC).P ≤ 0.05 were considered to be significant.Results were expressed as mean ± SD for analysis performed in triplicate. Statistical analysis of the data was performed by Analysis of Variance (ANOVA) followed by Duncan test using SPSS 17.0 software. Probability values of 3 flasks cell cultures (Nunc) with 5 mL of RPMI-1640  supplemented with 5% fetal bovine serum  plus penicillin/streptomycin (1000 μg/mL : 1000 IU/mL) and incubated at 37°C in humid atmosphere with 5% CO2. The cell line has less than 20 passages as laboratory good practices.Human tumor cell lines U251 (CNS), UACC-62 (skin), and OVCAR (ovary); NCI-ADR/RES (ovary multidrug resistant phenotype); 786-O (kidney); NCI-H460 ; PC-3 (prostate); HT-29 (colon); and K562 (bone marrow) were kindly provided by M. A. Frederick . Stock culture was grown in 25 cmIn vitro antiproliferative activity was performed using preincubated (24 hours at 37°C) cell lines into 96-well plates (Nunc), which were exposed at 0.25, 2.5, 25, and 250 μg/mL of A. adstringens CE for 48 hours. Cells were fixed with 50% trichloroacetic acid (30 min at 4°C) and cell proliferation was determined by spectrophotometric quantification (540 nm) of cellular protein content using sulforhodamine B assay. Using the concentration-response curve for each cell line, total growth inhibition (TGI) was defined as the concentration value that produces total growth inhibition and was determined by nonlinear regression analysis using the software Origin 7.5 . This value corresponded to the test extract concentration necessary to inhibit proliferation of the cells. Doxorubicin was used as positive control. The assay was performed by triplicate per concentration of CE in three independent tests. A. adstringens used in this work showed antimicrobial activity against all microbial strains tested with concentrations between 0.125 and 63 mg/mL  showed a MIC of 0.04 mg/mL and a MBC of 2.4 mg/mL, and 0.06 mg/mL and 1.2 mg/mL for Fluconazole.Methanolic extract of63 mg/mL . The mos in vitro cytotoxicity determination of the CE of A. adstringens demonstrated total inhibition of tumor cell growth at concentrations ranging from 7.30 to 27.50 μg/mL (μg/mL).In this model,50 μg/mL  and showThere exists an increasing prevalence of infections worldwide, due to, among other reasons, the growing resistance of pathogens to antibiotics and antifungals as well as alterations in the autoimmune system. Diseases as caries, periodontitis and gingivitis, oropharyngeal candidiasis, and cancer have had profound effects on human health. Microbial species in oral cavity are commensals under normal physiological conditions. The host immune response, supplemented by excellent oral hygiene, is sufficient to maintain healthy tissues. However, failures in immune defense or plaque control result in development of gingival inflammation –27. A. adstringens, against oral pathogens and human cancer cell lines and, as expected, we unambiguously demonstrated that the methanolic extract exerts significant antimicrobial and antiproliferative activities. Additionally our results provide novel evidence that A. adstringens also exerts growth control against Candida, and these results represent the first report for this plant.In this investigation, we tested the crude extract of S. mutans, our results showed that inhibitory concentrations of A. adstringens CE reached 0.125 mg/mL (MIC) and 0.31 mg/mL (MBC), while chlorhexidine had MIC and MBC values of 0.4 and 2.4 mg/mL, respectively. On the other hand, biocidal activity against periodontopathogens showed that the crude extract of A. adstringens had a MIC and MBC of 37 mg/mL for P. gingivalis and 63 mg/mL for A. actinomycetemcomitans. These results are consistent with previous work demonstrating higher sensitivity of S. mutans than periodontopathogens to medicinal plant components [In regard of activity shown againstmponents , 50, 51. A. adstringens, enhance proposals to include natural remedies as components of mouthwash, toothpaste, and delivery systems [Chlorhexidine is the most common mouthwash product used for caries and periodontal diseases and kills bacteria by causing a precipitation or coagulation of the cytoplasmic content. It is an effective antiplaque agent developed to date, but it has been proven that prolonged use causes several undesirable side effects; besides many clinical trials have shown that taste of chlorhexidine is not well accepted by children . Our res systems  to contr A. adstringens CE against yeasts, our findings indicated a strong antifungal activity showing MIC and MBC values of 0.4 mg/mL and MFC of 1.6 mg/mL. Those are the lowest among all MIC values shown in our data, suggesting that A. adstringens components exert fungicidal activity against candidiasis causal agents. By far, this represents our most original and promising evidence, considering the continuous quest for new and improved products, with emphasis placed on natural/nature identical compounds with the fewest side effects. Fluconazole is commonly used as therapy for candidiasis in HIV and cancer patients, but the rise in prevalence of fungal infections and drug resistance has exacerbated the need of novel antifungal compounds. Our results provide evidence of potential antifungal application of A. adstringens offering an alternative therapy to fluconazole.As for activity ofAs mentioned before, oral microbiota diversity is closely related to health conditions and systemic diseases such as cardiovascular and inflammatory diseases and cancer. This is why we designed experiments to holistically test both biological activity against oral pathogens and antiproliferative activity on human cancer cell lines. A. adstringens extract determining total growth inhibition (TGI) [The model used in this work to evaluate the antiproliferative activity was the assay with Sulforhodamine B (an anionic dye) that allowed the evaluation of antitumor activity through exposure of selected human tumor cell lines during exponential growth phase, at different concentrations of theon (TGI) .μg/mL, followed by melanoma UACC-62 and colon adenocarcinoma II HT-29 with TGI = 7.3 and 7.9 μg/mL, respectively. These are significant activities at low concentrations considering that CE is a mixture of nonpurified compounds, such as those found in actual traditional use of A. adstringens. Consistent with our results, other authors reported A. adstringens activity with TGI = 20.4 μg/mL to 48.2 μg/mL and 27.4 μg/mL to 38.3 μg/mL in the cell lines U251 and PC-3, respectively [Antiproliferative activity of the CE showed lowest TGI results on ovarian cancer OVCAR-3 adenocarcinoma with a TGI = 4.4 ectively . Similarμg/mL), whereas doxorubicin showed almost no effect at concentrations of 25 μg/mL.Interestingly, NCI/ADR-RES ovarian tumor cell line, expressing high levels of MDR1 and P-glycoprotein, which are related to multidrug resistance , 56, shoThis data confirms that complex mixtures of plants, essential oils, and natural alternative therapeutics that have been widely used in traditional medicine represent an effective, safe, and evidence based treatment for acute and chronic infections as well as chronic health conditions. Preparations and formulations of this CE can be improved and applied for daily use in dental products to prevent caries and periodontal diseases and concomitantly weaken the risk of cancer. A. adstringens, where the synergy of all components could be acting against all the strains tested, especially against S. mutans and Candida spp. A. adstringens has shown selective antiproliferative activity against multidrug resistant cell line NCI/ADR-RES. These effective antimicrobial and antiproliferative activities make A. adstringens a good candidate for further development of antimicrobial products for oral infectious therapy and cancer prevention and treatment. However, further research should be made to test whether secondary metabolites from A. adstringens maintain reported bioactivity when separated or act as a synergistic complex mixture of phytochemicals.The findings of the present study demonstrated a broad potential using the heterogeneous mixture of CE of


Glomus mosseae/Medicago sativa mycorrhiza on atrazine degradation was investigated. It was observed that the atrazine degradation rates with any addition level in mycorrhizal treatments were all significantly higher than those in non- mycorrhizal treatments. When atrazine was applied at 20 mg kg−1, the removal efficiency was up to 74.65%. Therefore, G. mosseae can be considered as ideal inhabitants of technical installations to facilitate phytoremediation. Furthermore, a total of 10.4 Gb was used for de novo transcriptome assembly, resulting in a comprehensive data set for the identification of genes corresponding to atrazine stress in the AM association. After comparative analysis with edgeR, a total of 2,060 differential expressed genes were identified, including 570 up-regulated genes and 1490 down-regulated genes. After excluding ‘function unknown’ and ‘general function predictions only’ genes, 172 up-regulated genes were obtained. The differentially expressed genes in AM association with and without atrazine stress were associated with molecular processes/other proteins, zinc finger protein, intracellular/extracellular enzymes, structural proteins, anti-stress/anti-disease protein, electron transport-related protein, and plant growth associated protein. Our results not only prove AMF has important ecological significance on atrazine degradation but also provide evidence for the molecular mechanisms of atrazine degradation by AMF.Arbuscular mycorrhizal fungi (AMF) protect host plants against diverse biotic and abiotic stresses, and promote biodegradation of various contaminants. In this study effect of  Zea mays and Triticum aestivum production, with 70,000–90,000 tons applied annually in the worldAtrazine is frequently used alone or in combination with other herbicides for combating grassy and broadleaf weeds in in situ of contaminated sites6et al. observed that atrazine dissipation in soil was enhanced by AM association9Atrazine degradation efficiency and growth characteristics of a wide range of bacteria have been reported worldwideGlomus mosseae, the dominant species in Heilongjiang Province of China, associated with Sorghum bicolor could reduce a maximum 91.6% of atrazine significantlyG. mosseae/Medicago sativa, one of atrazine-sensitive plant mycorrhiza with and without atrazine stress was investigated. A total of 10.4 Gb was used for de novo transcriptome assembly, resulting in a comprehensive data set for the identification of genes corresponding to atrazine stress in the AM association. The objective of this work was to prove AMF has important ecological significance on atrazine degradation91027In our previous studies, we observed that M. sativa were extensively colonized by G. mosseae. No mycorrhizal colonization was observed in control plants. Excellent symbiotic relationship between G. mosseae and M. sativa was established with the formation of vesicular structures and AMF hypha (Roots of inoculated MF hypha . The colMF hypha .P < 0.05). The dissipation of atrazine in non-mycorrhizal treatments was the results of host plants uptake and other environmental degradations. The differences decrease of atrazine between the mycorrhizal and control treatments attributes to AM in any atrazine addition level. When the atrazine was applied at 20 mg kg−1, degradation rate and its contributed by AM were both significantly higher than those in other addition levels.Compared with the initial concentrations, the atrazine concentration of Compartment A in inoculated treatments decreased from 51.29% to 74.65% , those 1−1 atrazine and without atrazine) and then used for Illumina sequencing. Using 101 bp pair-end sequencing based on Illumina sequencing approach, a total of 103,134,502 reads were obtained and then do novo assembled using Trinity Software. The longest assembled sequences containing blocks of unknown bases (Ns) were called contigs. A total of 75,957 contigs from 351 to 14643 bp were assembled with a mean size of 1132.66 bp  database, UniProtKB/Swiss-Prot (SwissProt), Gene Ontology (GO), Clusters of Orthologous Groups (COG) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. GO assignments were utilized to classify the predicted functions of l groups . In eachl groups .−1 atrazine addition and without treatments respectively. After comparative analysis with edgeR, a total of 2,060 differential expressed genes were identified, including 570 up-regulated genes and 1490 down-regulated genes . Excluding ‘function unknown’ and ‘general function predictions only’ genes, 172 functionally annotated genes were found in 570 up-regulated genes. We divide these genes into 7 categories according to their functions  zinc finger genes were found to be unevenly distributed on eight chromosomes and displayed different expression levels in response to various stress conditionsRhizoctonia solani, Puccinia striiformis f. sp. tritici and Magnaporthe oryzae2324G. mosseae and atrazine. The main function of the Zinc finger proteins is to trigger the expression of down-relgulated genes in response to stress. High level expression of zinc finger proteins in symbiotic roots under the stress of atrazine not only increases the resistance of adversity, but might be directly involved in atrazine degradation.Increasing evidences have revealed that zinc finger proteins (ZFPs), a well conserved and large family in many plant species participated in the regulation of plant growth, developmental processes and resistance mechanism for various biotic and abiotic stresses19et al. found that NAC transcription factors were involved in biotic and abiotic stress response in plantHigh level expression genes in 39 anti-stress/anti-disease proteins, such as NAC transcription factors and ethylene response transcription factor, might also play an important role in atrazine resistance. Wang et al. suggested that atrazine entered the chloroplasts depending on its liposolubility and directly attacked on the electron transport chain, especially PSII, contributing to reactive oxygen species (ROS) burstM. sativa inoculated has no symptoms such as growth arrest and leaf chlorosis after roots exposed to atrazine. It was suggested that the high expression of electron transport related genes may help to alleviate the detrimental effect of atrazine on electron transport system. Results of these investigations also support the role of atrazine in affecting mitochondrial electron transport and oxidative stress.Bai, X. We had two hypotheses about the molecular mechanisms of atrazine degradation in soil by AM. One was the secretion of atrazine-degrading enzyme under the stress of atrazine, causing degradation of atrazine in soil; the other was the absorption and degradation of atrazine in the plant. It could be possible that these two mechanisms of atrazine degradation work simultaneously. In agreement with the first hypothesis, our result could be explained by the high expression of laccase. Many studies have shown the laccase has degradation effect on atrazine and many other organic pollutantsM. sativa seeds were scarified with 20 °C to 30 °C in warm water, surface-sterilized with 10% (v/v) solution of hydrogen peroxide for 10 min, then rinsed with sterile distilled water. Seeds were then germinated in the dark on moist filter paper for 24 h. Well germinated seeds were selected for potting experiment.G. mosseae was propagated on sorghum and harvested after 40 days, preserved in the Ecology Laboratory of Heilongjiang University. The inoculum is a mixture of spores, mycelium, sand and root fragments. The inoculum contained about 25 spores per gram.The inoculum of The compartmented cultivation system partial referred−1). The experiment was conducted in a controlled-environment growth chamber that maintained a daily 16-h light period. The day/night temperature regime was 25/18 oC. The plants grew for 8 weeks. Deionized water was added as required to maintain soil moisture content at 40% WHC, because semi-arid condition was beneficial to form symbiosis between M. sativa and G. mosseae.A 5:2:3 (v/v/v) mixture of peat soil, sand and vermiculite was used as growth medium. The medium was autoclaved at 121 °C for 1.5 h, and air dried to moisture content to 20% of water holding capacity (WHC). In the growth compartment, 1.5 kg medium was thoroughly mixed with 30 g inoculum for mycorrhizal treatments and with the same amount of sterilized inoculum (30 g) for non-mycorrhizal treatments. In the side-arm compartments, 200 g atrazine-free medium was added to compartment B, and compartment A was filled with 200 g atrazine-contaminated medium with different addition levels  (20 mL /8g soil) using a ultrasonic apparatus for 5 min and centrifuged for 5 min at 2500 rpm. Finally supernatant was air-dried using nitrogen gas. To insure full recovery, soil samples were extracted multiple times. Final product was diluted in 2 mL of methanol. Atrazine content was analyzed by HPLC , using C18 alkyl column . The mobile phase was methanol / water . Flow rate was 0.8 mL/min.TM RNA sample prep kit, following the manufacturer’s instructions. The isolation of mRNA, fragment interruption, cDNA synthesis, addition of adapters, PCR amplification and RNASeq were performed by staff at Shanghai Majorbio Bio-Pharm Technology . Poly-A mRNA was isolated using Magnetic Oligo (dT) Beads, and then broken into small pieces using divalent cations under an elevated temperature. Then the double-stranded cDNA was synthesized using the SuperScript Double-Stranded cDNA Synthesis kit  with random hexamer primers (Illumina). The synthesized cDNA was subjected to end-repair and phosphorylation using T4 DNA polymerase, Klenow DNA polymerase and T4 PNK. These repaired cDNA fragments were 3′ adenylated using Klenow Exo- . Illumina Paired-end adapters were ligated to the ends of these 3′-adenylated cDNA fragments. Fragments (200 bp ± 25 bp) were then separated by agarose gel electrophoresis and selected for PCR amplification as sequencing templates. Finally, the mRNA-seq library was constructed for sequencing on the Illumina HiSeqTM 2500 sequencing platform. cDNA library construction process was shown in Total RNA of roots with and without atrazine were extracted using the TRIzol Reagent (Invitrogen) and treated with DNase I according to manufacturer’s instructions. RNA quality was examined using 2% agarose gel and the concentration was determined using a Nanodrap spectrophotometer . Then, preparation of cDNA libraries for Illumina HiSeq 2500 sequencing was done using the TruseqTranscriptome de novo assembly was carried out using Trinity Software. In order to reduce redundancy and chimeras in the Trinity pipeline, we used CAP3 tomerge and combine highly similarly assembled sequences into unisequences.http://www.geneontology.org/). The remaining sequences that putatively encoded proteins were searched against the SwissProt protein database (http://www.expasy.ch/sprot), the KEGG pathway database, and the COG database (http://www.ncbi.nlm.nih.gov/COG) by applying a typical E-value threshold of less than 10−5. Moreover, the edgeR (http://www.bioconductor.org/packages/release/bioc/html/edgeR.html) tool was applied in order to calculate the different expression and the significant differences.Sequences were annotated using a series of sequential BLAST searches designed to find the most descriptive annotation for each sequence. Assembled unique transcripts were compared with sequences in Nr using the BLAST algorithm, the GI accessions of best hits were retrieved, and the GO accessions were mapped to GO terms according to molecular function, biological process, and cellular component ontologies .