At the end of the first, fourth, and eighth month of follow-up, 91.1, 73.5, and 76.5% of participants respectively were e-cig users . They showed no significant variation in plasma cotinine and trans-3’-hydroxycotinine with respect to the start of the study when they smoked only tobacco cigarettes, but a significant reduction in breath CO. The proposed medically assisted training program of e-cig use led to a successful nicotine intake, lack of typical cigarette craving and overdosage symptoms and a significant decrease in the biomarker of cigarette combustion products.The electronic cigarette (e-cig) has gained popularity as an aid in smoking cessation programs mainly because it maintains the gestures and rituals of tobacco smoking. However, it has been shown in inexperienced e-cig users that ineffective nicotine delivery can cause tobacco craving that could be responsible for unsuccessful smoking reduction/cessation. Moreover, the incorrect use of an e-cig could also led to potential nicotine overdosage and intoxication. Medically assisted training on the proper use of an e-cig plus behavioral support for tobacco dependence could be a pivotal step in avoiding both issues. We performed an eight-month pilot study of adult smokers who started e-cig use after receiving a multi-component medically assisted training program with monitoring of nicotine intake as a biomarker of correct e-cig use. Participants were tested during follow-up for breath carbon monoxide (CO), plasma cotinine and  During the past few years electronic cigarettes (e-cigs) have gained popularity, primarily among smokers who want to reduce the risks of tobacco smoking. The popularity of e-cigs is linked to their perceived safety since they completely avoid the combustion of organic material (tobacco), and hence of the toxic and carcinogenic chemicals that are present in cigarette smoke; however long-term safety is currently unknown, and concerns have been raised about the potential risks of nicotine poisoning .trans-3’-hydroxycotinine (3-HCOT), as biomarkers of nicotine intake. Several studies have evaluated relationships between e-cig use and smoking reduction and cessation, but the results are still controversial ,3,4. Thei.e., relax or stress circumstances). Classical smoking cessation treatments  cannot replace the rituals associated with the act of smoking, and when the smoker stops smoking the need for the ritual sometimes still exists and can cause relapses; so classical treatments are generally strongly refused by smokers who do not want to stop smoking tobacco cigarettes [Adult smokers unwilling to quit smoking tobacco cigarettes and who have never tried a quit smoking protocol and/or have refused any smoking cessation treatment were recruited by word of mouth for the uncontrolled pre-post pilot study, carried out at the anti-smoking detoxification Centre at San Giovanni Bosco Hospital  that supplies individualized smoking cessation treatments since 2000 to an average of 300 smokers per year. The study was approved by the Istituto Superiore di Sanità ethical committee (CE 14/418) and carried out in compliance with the Helsinki Declaration. After a first meeting to inform about the study design, written informed consent for the involvement in the study was obtained from all participants. At the baseline, participants were investigated about their age, weight, sport habit, physical status, social class, education and smoking history; this has included years as a smoker, daily number of cigarettes, and cigarette brand. Additionally, participants were scored for their level of nicotine dependence by the Fagestrom Test of Nicotine Dependence (FTND). Levels of carbon monoxide (CO) in exhaled breath as biomarker of absorption of cigarette combustion products were measured using the MicroMedical micro Co monitor  and blood samples were collected to determine principal nicotine metabolites: COT and 3-HCOT. Participants were given a couple of commercially available e-cigs , two different chargers for each e-cig and PUFFIT e-liquids with nicotine content matching the individual nicotine daily intake and tobacco and/or other flavours freely chosen by each participant. Information about general working principles, safety and risks of e-cigs were given to the participants by Dr. Beatrice, the chief of the Detoxification Centre and his medical and nursing staff, together with medically assisted face-to-face training on how to correctly use the device to absorb nicotine vapor. This medical assisted training  has been developed by the same chief of the Detoxification Centre at San Giovanni Bosco Hospital and colleagues as a translational program from tobacco cigarette to e-cig for smokers unwilling to quit smoking especially because of the gestures and rituals of cigarette smoking, known to be linked with specific feelings  at any time of the day by Dr. Beatrice, and his medical and nursing staff, with any possible problem a related to e-cigs, both through established follow-up group sessions, and also through an instant messaging application for smartphones (Whatsapp Inc.) which included all the participants and the trainers in a chat group. Although participants were encouraged to use the e-cig, they were also permitted to smoke their favorite brand of tobacco cigarettes during this study. At the first, fourth, and eighth month follow-up visits, participants were tested again for CO levels in exhaled breath, and blood samples were collected to determine COT and 3-HCOT concentration. Finally, participants were asked to report the daily number of cigarettes smoked at the time of the visit and any adverse events experienced. The data were analysed using 2-tailed statistical tests, and trans-3’-hydroxycotinine (3-HCOT): 10 μL of internal standard  and 2 mL of a chloroform/isopropyl alcohol solution were added to 500 μL of each plasma sample. After centrifugation at 3000 rpm for 3 min, the organic phase was collected and evaporated under a nitrogen stream at ambient temperature. Samples were redissolved in 100 μL LC mobile phase . A 20 μL volume was injected into the LC/MS/MS system applying a methodology reported elsewhere [Blood samples underwent a liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis of principal nicotine metabolites: cotinine (COT) and lsewhere .Thirty-four participants  unwilling to quit smoking were recruited for the study. Participants were aged 18–63 years old , smoked ≥ 10 cigarettes per day  for at least 4 years  and the FTND indicated a moderate-strong nicotine dependence. The majority of the participants had an average education level  and a profession in the semi-skilled category (61.8%) .At the end of the first month of follow-up (T1), participants could be classified in three categories; only e-cig users, dual users, and only cigarette users ..A summary of their characteristics such as the values of breath CO, plasma COT and 3-HCOT during the study is given in At the end of the fourth month of follow up (T4) 40% (n = 10) of only e-cig users resumed cigarette smoking, increasing the percentage of dual and only cigarette users to 23.5% and 26.5%, respectively . HoweverAt the end of the eighth month follow up (T8) there were no major changes in the only e-cig, dual, and only cigarettes groups . As descThe use of e-cigs as an aid to smoking reduction/cessation is still controversial ,3,4. OneFurthermore, a recent systematic review on sixteen clinical studies on e-cigs highlights that even if second generation e-cigs are capable of delivering a similar amount of nicotine as traditional tobacco cigarettes, inexperienced users are not able to achieve systemic NIC and/or COT concentrations similar to those produced from traditional cigarettes . et al. showed that giving clear instructions on the use of a second generation e-cig in a group of smokers, unwilling to quit and who had never tried e-cigs, led to an immediate and strong craving reduction with only little withdrawal symptoms after e-cig abstinence for four hours. Accordingly, saliva COT levels did not show any difference between only e-cig users, dual smokers and only tobacco smokers and exhaled breath CO levels decreased in all the three groups of smokers following the reduction/abstinence from tobacco cigarettes [Conversely, Adriaens garettes .These data are in agreement with the results of the present study. Both e-cig users and dual users showed no significant variations in COT and 3-HCOT plasma concentrations at first, fourth and eighth month follow ups, meaning that they were obtaining a similar nicotine intake as when smoking only tobacco cigarettes, and a significant reduction in the absorption of cigarette combustion products was demonstrated by a significant decrease in breath CO. At the end of the T4 follow-up, the reintroduction of tobacco cigarettes was reported by 40% of only e-cig users at T1; however the number of smoked tobacco cigarettes was so low that it did not increase breath CO values. Data at the end of T8 follow up showed only a slight variation in the number of participants in the only e-cig and only cig groups. A significant reduction in the breath CO compared to T0 was still present, even when the 37.5% of only cigarette users reported an increased number of daily cigarettes compared to the fourth month of follow up. Plasma concentration of COT and 3-HCOT did not show significant variation in all three groups from the start of the study, consolidating our previous data. It should be noted that at the end of T8 follow-up about 53% of participants were still abstinent from tobacco cigarettes (only e-cig users); this cessation rate is higher than the 21% obtained by Adriaens and colleagues at the same time interval . In lighA criticism that could be raised for the present study is the absence of a control group of smokers not undergoing medically assisted training program before starting e-cigs in order to demonstrate the efficacy of the training. However, the group of smokers who tried and immediately refused to start the e-cig can be considered a “control” group.Although this study presents the important limitation of small number of participants, the promising results suggest that the proposed transitional program from tobacco cigarette by a patented multicomponent medically assisted training program on e-cig use assists the reduction of cigarette consumption in smokers not intending to quit, by assuring successful nicotine intake, avoiding nicotine overdosage and intoxication and absorption of cigarette combustion products, while considering the behavioural components of tobacco dependence. However, a more extended fully-powered randomized controlled trial with more participants and a control group not following the transitional program is necessary to strengthen these data.


To describe changes over time in extent of idiopathic pulmonary fibrosis (IPF) at multidetector computed tomography (MDCT) assessed by semi-quantitative visual scores (VSs) and fully automatic histogram-based quantitative evaluation and to test the relationship between these two methods of quantification.th-90th percentile in 5th percentile steps). Longitudinal changes in VSs and in the percentiles of attenuation histogram were obtained in 20 untreated patients and 20 patients treated with pirfenidone. Pearson correlation analysis was used to test the relationship between VSs and selected percentiles.Forty IPF patients  that underwent 2 MDCT at different time points with a median interval of 13 months (interquartile: 10-17 months) were retrospectively evaluated. In-house software YACTA quantified automatically lung density histogram  as compared to Δ 80th percentile ; closer correlation was found between Δ ground-glass extent and Δ 40th percentile  as compared to Δ 80th percentile , while the Δ reticulations correlated better with the Δ 80th percentile  in comparison to Δ 40th percentile .In follow-up MDCT, visual overall extent of parenchymal abnormalities (OE) increased in median by 5 %/year . Substantial difference was found between treated and untreated patients in HU changes of the 40th and of the 80th percentiles of density histogram. Correlation analysis between VSs and selected percentiles showed higher correlation between the changes (Δ) in OE and Δ 40th percentile might reflect the change in overall extent of lung abnormalities, notably of ground-glass pattern; furthermore Δ 80th percentile might reveal the course of reticular opacities.There is a relevant and fully automatically measurable difference at MDCT in VSs and in histogram analysis at one year follow-up of IPF patients, whether treated or untreated: Δ 40 Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible interstitial lung disease (ILD) of unknown etiology with a heterogeneous clinical course . NotablyAlthough predictive markers of response to therapy are still missing, pulmonary impairment, symptoms, and serial imaging changes are widely used in monitoring patients. Forced vital capacity (FVC) and diffusing capacity of the lung for carbon monoxide (DLCO) are both recommended in monitoring IPF . Howeverth percentile of lung density histogram is regarded as a useful tool in detecting patients with emphysema [In contrast, multidetector computed tomography (MDCT) is a potential alternative to assess “in vivo” the progression of the disease . Despitemphysema . It is tThe purposes of this study were to describe the changes over time in the extent of parenchymal abnormalities assessed on chest MDCT by both semi-quantitative visual scores (VSs) and automatic histogram-based quantitative analysis and to test the relationship between these two methods of quantification of lung abnormalities in patients suffering from IPF.All procedures were in accordance with the declaration of Helsinki. MDCTs were performed on clinical indication. Informed written consent for examination was obtained from all patients. Clinical records of 256 patients suffering from IPF admitted to our center for interstitial and rare lung disease between August 2006 and December 2013 were reviewed retrospectively. The retrospective analysis was approved by the ethics committee of the medical school of the University of Heidelberg . All patient records were anonymized and de-identified prior to analysis.The diagnosis of IPF was confirmed in each patient by a multidisciplinary team discussion meeting  and was based on a review of clinical data, chest MDCT, and histological assessment of lung biopsies. IPF was diagnosed according to the current consensus statement of the American thoracic society/European respiratory society/Japanese respiratory society/Latin American Thoracic Association (ATS/ERS/JRS/ALAT) for patients with first diagnosis after 2011, whereas prior cases were diagnosed following the previous guidelines . IndividDemographic and clinical data of the study population are shown in In all IPF patients, thin-section MDCT of the entire chest was routinely performed at inspiratory breath-hold in supine position using spiral mode scanning. Prior to the examination, all patients were trained to achieve a full end-inspiratory breath-hold after automatic patient instruction. Twenty-nine patients underwent the pair of baseline and follow-up MDCT scans on a 4-slice scanner  and 11 patients on a 64-slice scanner . The technical parameters for the 4-slice scanner were as follows: tube voltage 120 kV, effective tube current-time product of 70 mAs, collimation of 4x1.25 mm, rotation time 0.5 sec, and pitch of 2 . The acquisition parameters for the 64-slice scanner were as follows: tube voltage 120 kV, median effective tube current-time product of 67 mAs (range 48–151), collimation of 64x0.6 mm, rotation time of 0.33 sec, and pitch of 1.5 . Two patients were scanned both at baseline examination and at follow-up with contrast enhanced MDCT on the 4-slice scanner with identical administration . Baseline and follow-up scans were started automatically by bolus triggering with a region-of-interest (ROI) located in the pulmonary trunk, and scanning protocol as mentioned above.Image reconstruction was performed with a slice thickness of 1.25 and 1 mm increment. Since medium-soft kernels are more accurate and less noisy than sharp kernels for computer based quantitative analysis, either B40f kernel (4-slice scanner) or iterative I40f algorithm (64-slice scanner) were used . The scaMDCT images for each patient were reviewed by a trainee (D.C.) with 2 year of experience in thoracic imaging, who was blinded to individual therapy, clinical findings and histological data, after 6 months of focused training for ILD under interactive guidance of experienced chest radiologists (C.P.H and J.D.). For each patient, all the MDCT scans were scored on a certified PACS workstation simultaneously, using a window setting for lung parenchyma .MDCT scans were visually evaluated at six axial levels selected by the reader as follows: 1) the aortic arch, 2) the carina, 3) the pulmonary venous confluence, 4) the midpoint between level 3 and level 5, 5) 1 cm above the dome of the right hemi-diaphragm and 6) 2 cm below the dome of the right hemi-diaphragm . MDCT fith-90th percentile in 5th percentiles steps was derived from the histogram recording the densities of all lung voxels for each MDCT.The lung density histogram of each CT was obtained using the in-house YACTA software, which was primarily developed for the emphysema quantification . The staBody plethysmography  was performed according to the guidelines of the ERS and the standards of the ATS . Pulmonath percentile included in the range of 10th-90th percentile of lung density histogram (Δ PERC HU/y), and in PFTs (Δ PFTs %/y) were computed as the difference between the initial and follow-up MDCTs results divided by the interval. In order to identify a measurable difference in MDCT density, the percentiles of the attenuation histogram characterized by the largest difference and the lowest overlap in longitudinal HU changes between treated and untreated patients were identified. Pearson correlation analysis was used to evaluate the relationship between VSs, selected PERC, and PFTs%. All data were recorded using a dedicated database  and analyzed using SPSS version 20 . A p value less than 0.05 was considered significant.Individual longitudinal changes per year in VSs (Δ VSs %/y), in every 5th percentile included in the range 10th-90th percentile of lung density histogram for both treated and untreated patients. The largest difference with the lowest overlap between treated and untreated patients in longitudinal HU changes of the attenuation histogram was detected in the 40th and in the 80th percentiles. The initial median density of the 40th percentile was -822 HU , while the 80th percentile showed a median density of -595 HU ; at follow-up MDCT, the 40th percentile demonstrated a median increase of 22 HU  and the density of the 80th percentile increased of 35 HU . In particular, the median HU change of the 40th percentile was of 12 HU  in patients with the anti-fibrotic treatment and equal to 26 HU  in patients without specific anti-fibrotic therapy , while the reduction of DLCO% was 2%/y . Due to worsening of the respiratory symptoms, the follow-up VC% was not obtained in 2/40 patients, whereas follow-up DLCO% measurement was not performed in 7/40 patients for the same reason.th PERC . Additionally, the Δ 40th PERC correlated more tightly with the Δ GGO , whereas no significant correlation was observed between the same pattern and the variation of lung density in the 80th percentile.The Pearson correlation coefficient between the selected percentiles of lung density histogram and VSs are given in r = -0.63, p < 0.001). However, both the Δ GGO  and Δ RET  extent demonstrated a significant but weak negative relationship with the Δ VC%. Changes in HC extent was not significantly correlated with Δ VC%. The Δ VC% demonstrated a similar negative correlation with both the Δ 40th PERC  and the Δ 80th PERC . The Δ VSs and Δ DLCO% were not significantly correlated, while was found a significant, although weak, relationship between Δ DLCO % and Δ 40th PERC  as well as with Δ 80th PERC .The correlation coefficients obtained between the selected percentiles of lung density histogram, VSs, and PFT% are listed in th and the 80th percentile of the MDCT attenuation frequency histogram were identified as promising parameters for monitoring the disease extent. The correlation analysis between changes after 1 year in VSs and in selected percentiles suggested that the 40th percentile of lung density histogram, representing the low-density parenchymal areas , might reflect the overall extension of the abnormalities, including the GGO, which hypothetically represents potentially reversible acute inflammation [th percentile of attenuation histogram, representing the high-density parenchymal areas , might reveal the progression of the reticular opacities.This study describes the intra-individual changes over 1 year in a visual and a fully automatic histogram-based quantitative evaluation of MDCT in patients suffering from IPF. The visual assessment of the follow-up MDCT demonstrates an increase in the overall extent of the disease with a predominant increment of the fibrotic abnormalities. Changes in the density values of the 40ammation , 26. Addth percentile of frequency histogram was of about 30–40 HU, in contrast to an increment of 10–20 HU detected in the 40th percentile. Especially changes in the 80th percentile are of a significant amount, which is crucial in a high-contrast organ such as the lung, whose density is also influenced by different respiratory levels. In the majority of cases, the MDCT abnormalities detected in IPF patients consisted of mixed ground-glass opacities, reticulations and honeycombing that resulted in a wide range of attenuation values for the respective pattern. For instance, an increase of the gross cystic changes results in a decrease of the average regional HU values, possibly due to the air inside the cysts, in contrast to an increment of reticular opacities and ground-glass opacities extents that causes an increase of the MDCT attenuation [th percentile lung density, which represented the reticular opacities progression, was higher as compared to the increment observed in the 40th percentile, which probably reflected a combination of a predominant ground-glass pattern mixed with reticular opacities and honeycombing. Similarly to the findings obtained by Yoon et al., in this study a relatively good correlation was demonstrated between the automatic and the visual quantification of the lung abnormalities extent, though higher correlation was seen between the visual score of reticulations and correspondent automatic quantification  between the manual and the fully automatic segmentation volume. This difference might be regarded marginal considering that might be nearly constant in the longitudinal MDCTs during the course of the disease. Fifth, the CT measurements were not corrected for eventually different lung volumes. This would affect density measures significantly. Neither the visual scores, nor the attenuation percentiles were normalized for lung volumes. This should not affect the correlations results obtained in the present study between such methods of quantification. However, for future studies lung volume normalization is required, in order to eliminate the well-known limitation of sub-optimal inspiration during MDCT scan [There are several limitations in this study besides those already described above. First, it is a retrospective data analysis on a small number of patients from a single center. However, the presence of patients from only one center reduced the inter-scanner variability, which is a well-known drawback and limitation of any quantitative analysis and especially the automatic evaluation . Second,DCT scan . Sixth, DCT scan . HoweverDCT scan , 34. Howth percentile might reflect the overall extension of the lung abnormalities, in particular of the ground-glass pattern, which could represent potentially reversible acute inflammation; in addition, the change in the 80th percentile might reveal the progression of the irreversible fibrotic reticular opacities. Future studies with a larger number of patients might focus upon these values.Visual quantification of parenchymal abnormalities at multidetector computed tomography over 1 year in IPF patients showed an increase in the overall extent of the disease, with a predominant increment of the fibrotic abnormalities. The density changes in the 40


Various researchers have theorized that bereaved adults who report non-secure attachment are at higher risk of pathological grief. Yet past findings on avoidant attachment representations and grief have yielded limited and contradictory outcomes. Little research has been conducted with older adults to identify the psychological processes that mediate between self-reported attachment representations and the patterns of grief.To examine the impacts of avoidant attachment and anxious attachment dimensions on emotion and non-acceptance, in response to the loss of a conjugal partner, and the mediating effect of yearning thoughts.N = 21) and women (N = 68) aged 60 years and above who had lost a partner within the last 12 to 72 months were invited to participate. Participants rated their levels of yearning thoughts about the deceased, emotions and non-acceptance on the Texas Revised Inventory of Grief (TRIG-Present), and their type and level of general romantic attachment on the Experiences In Close Relationship questionnaire (ECR).Men  indicated that individuals who reported higher levels of avoidant attachment reported less emotional responses and less non-acceptance. SEM also showed that individuals who reported higher levels of anxious attachment reported greater emotional responses and greater non-acceptance. SEM further indicated that these relationships were mediated by yearning thoughts.People adopt different grief coping patterns according to their self-reported attachment representations, with the nature of their yearning thoughts influencing the process. Grief therapy may be organized according to individual differences in attachment representations. In 1990, 11% of the Australian population were aged 65 years or older. This figure rose to 14% in 2010 [Researchers, beginning with Bowlby , suggestconscious regard for interpersonal closeness or as fearful-avoidant whereby one prefers autonomy out of fear of being let down in relationships [Attachment representations have been conceptualized as degrees of avoidant attachment and anxious attachment. Adults who self-report as higher on avoidant attachment in romantic relationships typically strive for independence and emotional distance from their partner , 5. Someionships . A seconionships , 7. Baseionships . Securelionships .implicit cognitive processing revealed that beneath their minimalist concern with attachment needs, avoidantly attached adults indeed experience heightened yearning thoughts for their attachment figures during attachment threats but usually only when under high cognitive load imposed by having to simultaneously process multiple stimuli [both implicit and explicit cognitive hypervigilance to attachment-related negative memories, cues and threats, and higher attachment-related distress [Several studies indicate that avoidantly attached adults and anxiously attached adults vary in the nature and treatment of their attachment-related thoughts, leading to differences in how they cope with and express their distress over an attachment loss, such as the death of a loved one. Relative to other adults, avoidantly attached adults are less attentive to attachment-related concerns , 9, 10,  stimuli , 11. The stimuli . Bereave stimuli . These p stimuli , 14 thatdistress , 10, 12.The existing data on the differential patterns of attachment-related thoughts and affect regulation among avoidantly attached adults and anxiously attached adults begs the question of whether and how bereaved older adults may grieve differently due to their varying patterns of attachment. While past studies have either found contradictory or no links between avoidant attachment representations and grief, they have unequivocally posited that anxiously attached bereaved adults experience greater grief , 15, 16.full mediator model is illustrated in The current study aimed to test a mediator model of grief on a sample of older adults. The higher levels of self-reported romantic avoidant attachment will report fewer yearning thoughts about the deceased, leading to self-reports of lower levels of emotional responses and lower levels of non-acceptance about the loss.H1: Individuals with higher levels of self-reported romantic anxious attachment will report more yearning thoughts about the deceased, leading to self-reports of higher levels of emotional responses and higher levels of non-acceptance.H2: Individuals with Participants were recruited from senior centres and newspapers. The inclusion criteria were: Aged sixty years or older, the death of a spouse/long-term partner within the last 1 to 6 years, a relationship with the deceased for five years or more prior to the bereavement, English as a first/native language or possess sufficient English proficiency to read a standard English newspaper, and not presently suffering from depression or dementia. One hundred and twelve individuals who are residents of Australia volunteered to participate in the study. Three did not complete both questionnaires, nineteen did not return the questionnaires, and one had been bereaved for more than 6 years and was excluded from the study.M = 73.94 years, SD = 8.36). 61 (69%) are women and 28 are (31%) men. Of the 73 participants who had specified the length of their relationship with the deceased, the relationship ranged from 5 to 64 years . The remaining 16 participants had self-reported that the duration of their relationship had been at least 5 years but did not specify the exact duration. Of the 76 participants who had specified time since loss, time ranged from 12 to 72 months . The remaining 13 participants had self-reported that they had been bereaved for a minimum of 12 months but not more than 72 months, without indicating the exact length of time.The final sample comprised 89 older adults, ranging from 60 to 92 years (Experiences in Close Relationship (ECR)  is a 36-Each participant rated how well each of the thirty-six statements described his/her feelings towards intimate partners in general. Akin to past reviews on the ECR , the ECRThe Texas Revised Inventory of Grief-Present scale (TRIG-Present)  is a 13-Ethics approval was granted by the Curtin University Human Research Ethics Committee. Written informed consent for participation in the study and for publication of de-identified group data were obtained from the participants. Each participant was provided with an explanatory letter, consent form, background information checklist, TRIG-Present form and ECR form. These were returned to the researchers by email, mail or in person. The researchers also carried out follow-ups through phone calls three days after receiving the completed questionnaires, to ensure participants’ emotional well-being. Coordinators for each of the centres were provided with information sheets about behavioural symptoms of re-activated grief and free counselling helplines.Structural equation modelling (SEM) was conducted using LISREL software (Version 8.54). SEM is used to test the viability of causal models. Unlike traditional path models, which only incorporate observed variables measured with error, SEMs include both observed and latent variables. By including both observed and latent variables in the modeling process, SEM is able to account for measurement error in the observed variables .The basic analytical strategy involved comparing a saturated model  to a comThe following fit indices were used to evaluate model fit: the Comparative Fit Index (CFI), the Non-Normed Fit Index (NNFI), the Standardised Root Mean Square Residual (SRMR), and the Root Mean Square Error of Approximation (RMSEA). The fit statistics for the two models are reported in Individual participants’ mean scores for Avoidant Attachment, Anxious Attachment, Thoughts, Emotions, and Non-Acceptance are reported in Fit statistics for the two models are reported in 2/df, CFI, NNFI, and RMSEA favour the mediator model  indicating that the χ2 value for the saturated model was not significantly smaller than the χ2 value for the mediator model. The weight of the evidence indicates that the mediator model is at least as good as the saturated model at explaining the data. Scientific parsimony therefore dictates that we opt for the less complex mediator model.The χ2/df < 3 , CFI > .2/df < 3 , NNFI > 2/df < 3 , and RMS2/df < 3 . The medp-values for the indirect pathways in the mediator model were subsequently examined to confirm that Thoughts was mediating the relationships between the exogenous variables  and the endogenous variables (NA and Emotion). An initial requirement that must be met in order for Thoughts to be a mediator is the significance of each of the four component pathways that comprise the mediation effects. overall indirect effects. The strength of each of the four indirect effects is given by the product of its two component path coefficients. These effects are reported in The All indirect effects are significant.higher levels of self-reported romantic avoidant attachment tended to report fewer yearning thoughts about the deceased, and individuals who reported fewer yearning thoughts tended to report lower levels of emotional responses and lower levels of non-acceptance about the loss. H1 is therefore supported. The path coefficients also indicated that individuals with higher levels of self-reported romantic anxious attachment tended to report more yearning thoughts about the deceased, and individuals who reported more yearning thoughts tended to report higher levels of emotional responses and higher levels of non-acceptance about the loss. H2 is therefore supported.Each of the four pathways in the mediator model was significant. The relative directions of the path coefficients (positive or negative) indicated that individuals with This study examined the impact of older adults’ self-reported non-secure attachment representations on their yearning thoughts, emotions and non-acceptance in response to conjugal bereavement. The result supported the hypothesis that participants who self-report higher scores on the ECR avoidant attachment dimension would experience less emotional distress and less non-acceptance, modulated by their fewer yearning thoughts about the deceased, as self-reported by the TRIG-Present.This is a first study that empirically demonstrated the cognitive link between individuals’ avoidant attachment representation and grief. Consistent with the position that avoidantly attached adults tend to minimize attachment-related thoughts and that a reduction in these thoughts leads to less emotional distress , 13, 15,dismissing-avoidant attachment representation ocessing , 11, 12,The result offers support for the hypothesis that participants who self-report higher scores on the ECR anxious attachment dimension would experience more pining thoughts for the deceased, leading to greater distress and greater non-acceptance about the death, as self-reported by TRIG-Present. The influence of yearning thoughts is likewise indicated by the significant mediator pathways in Figs  and 3 inThe current data echo the unequivocal past findings , 15, 16 The statistical significance and direction of the anxious attachment mediator path model is consistent with existing literature that anxiously attached adults adopt cognitive hypervigilance to negative attachment stimuli, threats and memories , 5, 10 aOverall, the mediator pathways present important implications for grief therapy. The findings suggest that participants adopt different grieving patterns according to their attachment representations. These attachment representations form the basis for their differential nature of yearning thoughts, with these thoughts critically influencing their conscious self-reported levels of emotions and acceptance about the loss. The importance of attunement between one’s cognitive and affective states for emotional well-being is supported by various therapeutic approaches for pathological grief and other emotional difficulties. These include cognitive behavioural interventions , 31, 32,Several limitations exist in the study. Somatic symptoms and implicit processes of grief were not assessed, comparisons with a younger sample could have been incorporated, and results from this sample of participants may not readily generalize to the wider population of grieving older adults in Australia. In addition, the study was cross-sectional in design. Cross-sectionality reduces the degree to which causal inferences can be made. Future researchers may address the problem of cross-sectionality by using longitudinal data in which the attachment dimensions are measured at Time 1, thoughts at Time 2, and non-acceptance and emotions at Time 3.The current research provides a springboard for a richer investigation into the role of thoughts in grieving adults with different attachment representations; in particular, for the self-reported avoidantly attached older adults. We suggest future researchers consider using a self-report measure that assesses the dismissing-avoidant and fearful-avoidant attachment subtypes respectively, as well as an assessment of implicit cognitive processing and emotions of grieving. Finally, future researchers who aim to replicate this study could recruit a larger, more diverse and more representative sample of bereaved older adults in Australia.S1 Fig(SAV)Click here for additional data file.


Cardioprotective properties of volatile agents and of remote ischemic preconditioning have survival effects in patients undergoing cardiac surgery. We performed a Bayesian network meta-analysis to confirm the beneficial effects of these strategies on survival in cardiac surgery, to evaluate which is the best strategy and if these strategies have additive or competitive effects.Pertinent studies were independently searched in BioMedCentral, MEDLINE/PubMed, Embase, and the Cochrane Central Register (updated November 2013). A Bayesian network meta-analysis was performed. Four groups of patients were compared: total intravenous anesthesia (with or without remote ischemic preconditioning) and an anesthesia plan including volatile agents (with or without remote ischemic preconditioning). Mortality was the main investigated outcome.We identified 55 randomized trials published between 1991 and 2013 and including 6,921 patients undergoing cardiac surgery. The use of volatile agents  and the combination of volatile agents with remote preconditioning  were associated with a reduction in mortality when compared to total intravenous anesthesia. Posterior distribution of the probability of each treatment to be the best one, showed that the association of volatile anesthetic and remote ischemic preconditioning is the best treatment to improve short- and long-term survival after cardiac surgery, suggesting an additive effect of these two strategies.In patients undergoing cardiac surgery, the use of volatile anesthetics and the combination of volatile agents with remote preconditioning reduce mortality when compared to TIVA and have additive effects. It is necessary to confirm these results with large, multicenter, randomized, double-blinded trials comparing these different strategies in cardiac and non-cardiac surgery, to establish which volatile agent is more protective than the others and how to best apply remote ischemic preconditioning. Acute cardiac damage in cardiac surgery is associated with increased morbidity and mortality and the identification of cardioprotective strategies might influence patients’ outcome.  CardioprIschemic preconditioning is a response at cellular level to brief sub-lethal episodes of ischemia leading to a major protection against subsequent lethal ischemia. Remote ischemic preconditioning consists in the stimulation of brief episodes of ischemia and reperfusion in a tissue or in an organ different from the heart (e.g. limb ischemia), inducing myocardial protection from ischemic injury.  Recent mVolatile agents are among the few interventions that might reduce perioperative mortality, ,7 probabTo address the question whether the choice of anesthetic regimen and the application of remote ischemic preconditioning have beneficial additive effects on patients’ survival after cardiac surgery, we carried out a Bayesian network meta-analysis, a valid and robust statistical technique that compares indirectly various treatments.We performed a network Bayesian meta-analysis in order to evaluate the effects on mortality of remote preconditioning, volatile agents and total intravenous anesthesia (TIVA). Our aim was to assess whether remote preconditioning and the choice of anesthetic regimen can influence survival on patients after cardiac surgery.st 2013. The full PubMed search strategy is available in the The search for relevant studies was carried out independently in BioMedCentral, Embase, MEDLINE/PubMed and in the Cochrane Central Register of clinical trials by two experienced investigators. Literature searches were last updated on November 1All references obtained from literature searches with the abovementioned methods were independently evaluated at a title/abstract level by two investigators and then, if potentially reasonable, assessed as full-text articles. Non-English articles were translated in order not to exclude them in the analyses. Inclusion criteria were random allocation to treatment and comparison between a total intravenous anesthesia (TIVA) and a combined plan including administration of a volatile agent ; comparison between the use of remote ischemic preconditioning and not; cardiac surgery setting. There was no restriction in dose and time of anesthetic administration or methods of remote ischemic preconditioning. The exclusion criteria were missing outcome data, experimental studies on animal subjects and duplicate publications. In case of duplicate publications we included the article reporting the longest follow-up. We also included in the analysis studies in which epidural anesthesia or analgesia was given to all subjects.Two investigators were independently tasked with verifying study compatibility with selection criteria. Any potential divergences were sorted out by agreement.Two trained investigators independently obtained baseline, procedural and outcome data with divergences resolved by consensus .Specifically, we extracted survival, study design , clinical setting, population, and anesthetic comparators. “TIVA” was defined as a group not receiving desflurane, isoflurane or sevoflurane. “Volatile” was defined as a group receiving at least 5 minutes of a volatile agent (even if added over a TIVA regimen). “Remote” was defined as a group receiving remote ischemic preconditioning.The endpoint of the present review was to identify the effects on mortality at the longest follow-up available of remote ischemic preconditioning, volatile agents and TIVA using a network Bayesian meta-analysis. If the study had missing or incomplete data on survival we contacted all authors by e-mail, letter or both.The internal validity of each trial included in this review was carefully evaluated for bias in accordance with The Cochrane Collaboration guidelines.  We assesThe posterior probability check method  was usedFor each trial, the number of reported deaths and number of patients included were expressed as the logarithm of odds ratio (OR) and of its standard error (SE). If available, we used data referred to the intention-to-treat population.The primary treatment strategies of interest in this network meta-analysis were 1) TIVA, 2) volatile, 3) remote-TIVA and 4) remote-volatile. A graphical representation of the network was used to show the pairwise association between each treatment. In this diagram each node represents a single anesthetic agent and each edge connects treatments that have been directly compared in one or more RCTs. The font size of each anesthetic agent node is proportional to the number of randomized patients per group and the edge size is proportional to the number of studies per comparisons. The dashed line indicates the comparison derived from a multi-arm trial.Q statistic and the I-square statistic (I-square >25% was used as a threshold indicating significant heterogeneity).To analyze the binary outcome, among pairwise comparisons and through standard meta-analysis, we pooled the study-specific estimates using the inverse-variance method and a fixed-effect model.  To assesres) and the Deviance Information Criterion (DIC) statistics. We selected the random effect model because it is usually the most conservative option.We performed the network analysis modeling the binary outcome for mortality with a Bayesian hierarchical model  using the Markov chain Monte Carlo (MCMC) estimating algorithm. We used non-informative priors to produce the posterior distributions for the treatment effect in reference group (TIVA) and the treatment difference effects . To overcome the zero-cell count problem, we ran the random effect model with a more informative prior (Inverse-Gamma distribution) on the variance parameter. ,14 ModelPooled ORs, comparing different anesthetic agents, were estimated from the mean of the posterior distribution obtained with the Bayesian approach. After confirming the consistency hypothesis, the indirect estimate was calculated as difference from the appropriate direct estimates; the corresponding 95% credibility intervals (CrI) were obtained by normal approximation. We assumed that the between-trial variance is homogeneous across treatment contrast and we took into account the correlation between the treatment difference effect for each group of multi-arm trials. ,16 We chWe performed two meta-regressions to evaluate the statistical influence on treatment effects. We included in the model (i) the length of follow-up and (ii) the study-specific precision (measured as inverse of sample size) to detect the potential publication bias.Sensitivity meta-analyses were implemented analyzing data from studies with low risk of bias, with trials with more than 80 patients and removing the largest study of Hong DM et al. The statistical analysis will be performed by winBUGS  and STATA . Statistical significance was set at the two-tailed 0.05 level for hypothesis testing. Unadjusted P values are reported throughout.This study was performed in compliance with The Cochrane Collaboration and Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines . 10,18],1810,18]The process of selection of randomized controlled trials is shown in We finally spotted 55 eligible randomized clinical trials, ,17,19–69The 55 included trials randomized 6,921 patients including 2,704 (39% in 50 studies) receiving volatile agents, 2,564 (37% in 41 studies) receiving TIVA, 902 (13% in 7 studies) receiving remote-TIVA, and 751 11% in 15 studies) receiving remote-volatile. The most common pairwise comparison was volatile agents versus TIVA (34 [62%] studies) followed by volatile agents versus remote-volatile (14 [25%] studies) and TIVA versus remote-TIVA (5 [9.1%] studies). We included one three-arm study  studies  The dateStudy quality appraisal indicated that studies were of medium/high quality  and thatThe network configuration of each contrast analyzed by a Bayesian network meta-analysis is reported in When comparing TIVA, volatile, remote-TIVA, remote-volatile through simple direct comparisons we found the following differences in mortality: 1) volatile agents (27/1997 [1.4%]) vs. TIVA (41/1683 [2.4%]), OR = 0.56, 95% IC 0.36–0.88, p for effect = 0.01, I-square = 0% with 36 studied included ; 2) volares = 159.6 and DIC = 188.7) and fixed (Dres = 160.6 and DIC = 188.5) effects models had no differences in model fit. To estimate the treatment effect, we selected the first that is the most conservative method. The WinBUGS code and the data entry, used to fit the random effect model, are available in the The random  and remote-volatile  were associated with decreased mortality when compared to TIVA at the longest follow-up available. Besides, we observed that the use of remote-volatile was as well associated with a reduction in mortality when compared to remote-TIVA .Different length of follow-up did not affect our findings as documented by the ORs that were constant over time: posterior mean of slope coefficient = 0.00003, 95% CrI -0.004 to 0.005. Furthermore, the study precision was not associated with the risk of mortality .When repeating the analysis using low-risk-of-bias studies, we confirmed the significant reduction in mortality using halogenated  and remote-volatile  instead of TIVA. Moreover, when the largest study  was remoThe overall results were confirmed when we performed the network meta-analysis using trials with more than 80 patients.This Bayesian network meta-analysis demonstrates that in patients undergoing cardiac surgery the use of volatile agents  and the combination of volatile agents with remote preconditioning  are associated with a reduction in mortality at the longest follow-up available when compared to TIVA. This result is confirmed by repeating the analysis with low-risk-of-bias trials, removing the largest study,  and inclPosterior distribution of the probability of each treatment to be the best one shows that the association volatile-remote is the best treatment to improve short- and long-term survival after cardiac surgery , S1 Fig The effects of halogenated anesthetics in improving survival in cardiac surgery are not a new finding. It was first suggested in 2007  and thenRemote preconditioning is a simple and non-invasive strategy that can protect myocardial tissue: a brief previous ischemic period can reduce tissue infarct size when a sustained and more intense ischemic insult is applied. In most trials included in our meta-analysis preconditioning was achieved through various cycles of mechanical limb ischemia with a pressure cuff or a tourniquet for some minutes.A recent meta-analysis  suggesteAnother recent meta-analysis  of 19 raOur findings suggest that volatile agents in the cardiac surgery setting are associated with a reduction in mortality and that the combination of volatile agents with remote ischemic preconditioning gives further protection.Bayesian network meta-analysis is a research method that directly and indirectly compares different groups, with an established role in clinical research. We have included in our meta-analysis all randomized trials ever done in adult cardiac setting that compare volatile agents, TIVA and remote ischemic preconditioning. On the basis of these findings, we strongly encourage the use of volatile agents alone or associated with remote ischemic preconditioning in patients undergoing cardiac surgery.Ischemic preconditioning was described for the first time by Murry et al  in 1986:On the basis of our findings, general anesthesia in adult cardiac surgery should be maintained with volatile agents. In fact there is no evidence of any beneficial properties of propofol when compared to volatile agents on clinically relevant outcomes. Heusch et al recently discovered that protection by remote ischemic preconditioning is associated with the activation of signal transducer and activator of transcription 5 in left ventricular biopsy samples of patients undergoing coronary artery bypass grafting during isoflurane anesthesia.  In theirApplication of mechanical ischemic preconditioning through a pressure cuff or tourniquet in patients under general anesthesia undergoing cardiac surgery is an economical and simple strategy that could change short and long term outcome, especially in high risk patients.This meta-analysis presents some limitations: most of the included studies are small, single-center and not double blind. In some of them, authors do not clarify if patients that were taking drugs such as sulfonylurea, theophylline or allopurinol have been excluded; in studies in which patients were under these drugs, they do not specify the timing of preoperative withdrawal. These drugs, in fact, seem to interfere with preconditioning mechanism. Also data on intraoperative amount of opioids have not been clarified at all. Opioids reduce cardiovascular stress and could influence cardio-protective effects of volatile agents. Furthermore, the length of follow-up was not identical in the different RCTs included in the meta-analysis; nonetheless a metaregression suggested that the beneficial effect of volatile agents and remote ischemic preconditioning did not vary over timeLarge, multicenter, randomized, double-blinded trials comparing these different strategies  in cardiac surgery are necessary to confirm our results. It is also necessary to establish which is the most protective volatile agent and in what manner remote ischemic preconditioning should be applied. These findings could be of great interest also in non-cardiac setting, especially in high risk patients, since major non cardiac surgery is often associated with myocardial damage, leading to prolonged hospital stay and increasing perioperative morbidity and mortality.The limitations of Bayesian network meta-analyses are well known.  TraditioIn patients undergoing cardiac surgery the use of volatile agents and the combination of volatile agents with remote preconditioning are associated with a reduction in mortality when compared to TIVA at the longest follow-up available. On the basis of our findings, general anesthesia in cardiac surgery should be performed with volatile agents because there is no evidence of any beneficial properties of total intravenous anesthesia alone compared with volatile agents. Furthermore, application of mechanical remote ischemic preconditioning is a simple strategy that could change short- and long-term outcome. It is necessary to confirm these results with large, multicenter, randomized, double-blinded trials comparing these different strategy  in cardiac and also in non-cardiac surgery, and to establish which volatile agent is more protective than the others and how to applied remote ischemic precondition.S1 Checklist(PDF)Click here for additional data file.S1 Fig(PNG)Click here for additional data file.S2 Fig(PNG)Click here for additional data file.S3 Fig(PNG)Click here for additional data file.S4 Fig(PNG)Click here for additional data file.S5 Fig(PNG)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Pseudomonas putida KT2440, Rup4959, a multidomain response regulator with diguanylate cyclase activity, when overexpressed causes an increment in the intracellular levels of c-di-GMP that gives rise to a pleiotropic phenotype consisting of increased biofilm formation and crinkly colony morphology. In a broad genomic screen we have isolated mutant derivatives that lose the crinkly morphology, designed as cfc (crinkle free colony). A total of 19 different genes have been identified as being related with the emergence of the cfc phenotype either because the expression or functionality of Rup4959 is compromised, or due to a lack of transduction of the c-di-GMP signal to downstream elements involved in the acquisition of the phenotype. Discernment between these possibilities was investigated by using a c-di-GMP biosensor and by HPLC-MS quantification of the second messenger. Interestingly five of the identified genes encode proteins with AAA+ ATPase domain. Among the bacterial determinants found in this screen are the global transcriptional regulators GacA, AlgU and FleQ and two enzymes involved in the arginine biosynthesis pathway. We present evidences that this pathway seems to be an important element to both the availability of the free pool of the second messenger c-di-GMP and to its further transduction as a signal for biosynthesis of biopolimers. In addition we have identified an uncharacterized hybrid sensor histidine kinase whose phosphoaceptor conserved histidine residue has been shown in this work to be required for in vivo activation of the orphan response regulator Rup4959, which suggests these two elements constitute a two-component phosphorelay system.Most bacteria grow in nature forming multicellular structures named biofilms. The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) is a key player in the regulation of the transition from planktonic to sessile lifestyles and this regulation is crucial in the development of biofilms. In  In recent years cyclic diguanosine monophosphate (c-di-GMP) has emerged as an important second messenger in prokaryotic cells, where its role in defining the bacterial mode of life has been established. Changes in the levels of this signaling molecule are associated with phenotypic modifications related to virulence, motility, production of exopolysaccharides (EPSs), colony morphology, and expression of functions involved in biofilm development . High inThe turnover of this second messenger is carried out by proteins containing the domains GGDEF or EAL/HD-GYP, which are responsible for the synthesis and hydrolysis of c-di-GMP through their diguanylate cyclase (DGC) and phosphodiesterase activities (PDE), respectively. Proteins containing one or both of these domains have been described in many bacteria. Often multiple of these proteins are found in the same organism and therefore there appears to be some redundancy .Pseudomonas putida KT2440 by transcriptomic analysis of genes preferentially expressed in the rhizosphere of corn plants. This plant-beneficial bacterium colonizes the root surface and surrounding soil area (the rhizosphere) of different plants, a process that is accompanied by a global reprogramming of gene expression  and the P. putida-specific EPS Pea. Additionally this phenotype is dependent on the stationary phase sigma factor RpoS . Interestingly, the appearance of phenotypes like flocculation and increased biofilm formation can be accelerated by the addition of spent supernatants of KT2440 cultures  medium : ampicillin, 100 (E. coli); chloramphenicol, 30; gentamicin, 10 (E. coli), and 50 or 100 as indicated (P. putida); kanamycin 25; nitrofurantoine (NF), 100; piperacillin 30, routinely, or 50 (for counter selection of P. putida); rifampin, 10; streptomycin, 50 (E. coli), and 100 (P. putida); tetracycline, 7.5. To induce gene expression in P. putida from expression vectors IPTG 0.1 mM was used.) medium  or M9 mi) medium  supplemeeviously . Glucose5[Km1], or mini-Tn5[ΩTc], two mini-Tn5 derivatives carrying respectively a kanamycin or tetracycline resistance marker (P. putida KT2440) and donor , were grown overnight in LB with the appropriate antibiotics. A volume of 0.75 ml of each strain was mixed and cells were collected by centrifugation, then rinsed and resuspended in 50 μl of fresh LB, and finally spotted on a filter of 0.45 μm of pore diameter. Up to 10 independent conjugations were performed in parallel to maximize heterogeneity and minimize siblings in the mutant library. After overnight incubation at 30°C, cells were scraped off the mating filter and resuspended in 10 ml of M9 buffer, and serial dilutions were plated on selective LB medium supplied with Km or Tc plus NF to counterselect the E. coli donor strain. Two independent mutant libraries were generated, one based on mini-Tn5[Km1] and the other based on mini-Tn5[ΩTc].Transposon mutagenesis was performed by mating with mini-Tne marker . The rec5 derivatives using specific probes against Km or Tc resistance-encoding genes. (Primers used in this work are listed in Supplementary Table S1).All selected mutants were verified by Southern-blot to have single insertions of the mini-Tn® genomic DNA purification kit (Promega Cat. No. A1120). Plasmid DNA was isolated using the Quantum Prep Plasmid Miniprep Kit (Biorad Cat. No. 732-6100). For DNA digestion, the manufacturer’s instructions were followed (Roche and New England Biolabs). Separated DNA fragments were recovered from agarose using the high pure PCR cleanup micro kit (Roche Cat No. 04983955001). Alkaline phosphatase, ligation reactions, Southern blots and colony hybridization were performed by standard protocols  as a helper. Filter matings were used as described in the previous section.Transfer of pMAMV1 or pMIR178 from Phenotypes of the exconjugants  were analyzed as specified below.Mutants of interest were cured of their plasmid by removing the selective antibiotic, Gm or Km, from the culture media followed by screening for antibiotic sensitive colonies. After plasmid extraction antibiotic sensitive colonies were confirmed to be free of plasmids.P. putida cells caused rearrangement within these plasmids, which was not observed when they were transferred by conjugation. At least 50 exconjugant colonies were tested per strain to confirm their phenotype.Once strains were cured of plasmid, pMAMV1 or pMIR178 were newly transferred to the transposon insertional mutant strains by triparental mating as described above. These plasmids were transferred by conjugation because electrotransformation of For Mini-Tn5[Km1] insertional mutants, arbitrary PCR was used following the previously described method . ArbitraP. putida KT2440 , to check the absence of mutations.Plasmid pMIR203 was generated by cloning a 650 bp fragment containing the GTCGACCTGGTGTTGATGCTG GTA) and pMAMV1-MfeIRev (CACAACGTCGAGCAATTGGG). Restriction sites are underlined. The codon for A65 is underlined and italicized and the base cytosine mutated to attain this residue is in bold. A further amplicon of 436 bp was obtained which was cloned in pGEM-T generating pMIR189. The wild type SalI/MfeI fragment in pMAMV1 was replaced with that of pMIR189 to generate pMIR190. The replacement of the wild type sequence GAT with GCT in pMIR189, as well as the absence of further mutations, were confirmed by sequencing.The aspartic acid residue present in Rup4959 at position 65 (D65) was replaced with alanine (A65). For the mutation of the codon encoding D65 in pMAMV1, one amplicon was obtained by Expand High Fidelity PCR system (Roche) with the pair of primers pMAMV1-SalI . Upstream and downstream amplicons were obtained with the pairs of primers CfcA-OutF 5′GAGAGTGCCCGTTATCGCCAG3′/CfcA-AlaR 5′GC GCAGCTCGCAGGACATGTTG3′ and CfcA-AlaF 5′GCCAAC ATGTCCGCT GAGCTGC3′/CfcA-OutR 5′GCTGAACACGCT GCCTTGC3′, respectively. These oligonucleotides were designed to replace the codon CAT (His496) with the codon GCT (Ala496). Oligo CfcA-SbfIF 5′GCCTGCAGGAAGTGCTGGC3′, which anneals internal to the upstream amplicon, and oligo CfcA-SalIR 5′CTGCTGTCGACATTGATCTGGC3′, which anneals internal to the downstream amplicon, were further used for the amplification of 910 bp containing the mutated codon. This PCR amplicon was cloned at the EcoRV site of circular pCR2.1-TOPO generating pMIR229. Restriction sites are underlined. The codon for A496 is underlined and italicized and the bases GC mutated to attain this residue are in bold. The wild type SbflI/SalI fragment in pMIR231 was replaced with that of pMIR229 and pMIR232 was generated. The replacement of the wild type sequence CAT with GCT in pMIR232, as well as the absence of further mutations, were confirmed by sequencing.The histidine residue present in PP_3761 at position 496 (H496) was replaced with alanine (A496). An intermediate construction, plasmid pMIR231, was first generated by removing from pMIR205 the genes downstream from the Pictures of the bacterial streaks were taken after 24 or 48 h incubation as indicated. Pictures of flocculation were taken after incubation for 16 h under orbital shaking (200 rpm) followed by static incubation on the bench for 30 min.Biofilm formation assays were performed in borosilicate tubes that were incubated in an orbital shaker (40 rpm) with bacterial cultures grown in LB medium or glucose-M9 medium supplemented with Casaminoacids 0.5% (w/v) as described before . The starup4959 expression. Cells corresponding to 7 ml of culture were then lysed by heat and the nucleotides extracted twice, as previously described, with 65% ethanol . Growth and fluorescence (excited at 485 nm and read at 535 nm) were monitored in an Infinite 200 Tecan fluorometer. The experiments were conducted in triplicate over 24 h, taking measures every 30 min.Indirect detection of c-di-GMP contents was carried out by microtiter plate-based assays of the strains with the reporter pCdrA::rup4959 of Pseudomonas putida (Table 1). Mini-Tn5[Km1] was used for random mutagenesis of KT2440. The resulting KmR clones were pooled and en-masse mating experiments were used to transfer pMAMV1 to the mutants. About fifteen thousand ex-conjugant clones were then streaked onto LB-agar plates and their morphology visually analyzed. Approximately eighty clones were initially isolated as having lost the crinkly colony morphology despite carrying extra copies of rup4959. These mutants were termed cfcK (for crinkle-free colony/KmR). Selected mutants were cured of plasmid pMAMV1 and the same plasmid was newly transferred into them by conjugation in order to confirm their cfc phenotype and to analyze the incidence of this phenotype in a greater number of isolates of the mutant strain populations. In all cases the presence of the intact pMAMV1 plasmid was confirmed, ensuring that the loss of the phenotype was not due to plasmid deletions or rearrangements. Results are summarized in Figure 1. In addition to morphology, we also analyzed culture flocculation  and biofilm formation in all the cfcK mutants (Table 2). The gene disrupted in each case was identified by arbitrary PCR from the genome as described in Section “Materials and Methods”. Several of the cfcK mutants turned out to be siblings. In particular, it is noticeable that nine clones harbored the transposon insertion in the last codon of the algU gene, just before the stop codon, although they were in two different positions (termed cfcK-16 in Table 2). When re-transformed by conjugation with pMAMV1 as reported above, only a subpopulation of cfcK-16 carrying pMAMV1 showed the cfc phenotype. The other subpopulation was given the name cfcK-16F (pMAMV1) . By contrast, an independent transposon insertional inactivation located in the core of the algU gene did not cause any mutant phenotype (not shown). This result was consistent in all the clones and indicates that the loss of functionality of algU was probably not the reason for the crinkle-free phenotype of the variant cfcK-16 (pMAMV1). Two other transcriptional regulators, PP_4630 and FleQ (cfcK-77), were also identified in this screen. The later has been previously reported to function in association with c-di-GMP in P. aeruginosa (argG (cfcK-66) and argH (cfck-74), which encode enzymes acting sequentially in the arginine biosynthetic pathway . Interestingly these mutants still retained the flocculation phenotype and were only partially hampered in biofilm formation capacity when carrying pMAMV1. Several other genes involved in metabolism were also identified in this screen, i.e., gpsA (encoding a glycerol-3-P dehydrogenase) and betA (encoding a choline dehydrogenase). When examined under the stereomicroscope only mutants in the glycerol-3-P dehydrogenase and the chaperone protein DnaK kept partial crinkle phenotype .A genetic screen was designed to identify, by visual inspection, mutants that did not form crinkly streaks after introducing the medium-copy number plasmid pMAMV1 harboring the gene ruginosa . The binruginosa . In addi5[ΩTc] was also generated and analyzed by en-masse transfer of plasmid pMIR178. This plasmid confers kanamycin resistance but otherwise carries the same insert as pMAMV1 encoding Rup4959. Mutants in gacA, flhF, argH and in one of the seven copies for RNA23S present in P. putida KT2440 were isolated as crinkle-free colonies when carrying pMIR178 ; these strains were designated cfcT . It was not possible to discern which of three possible RNA23S was affected in cfcT-45, and this mutant was not studied further. Like FleQ, the flagellar biosynthetic protein FlhF also contains a Walker A motif. The two component system GacS/GacA is considered a master regulator of Pseudomonas secondary metabolism .To increase mutants heterogeneity a smaller gene library based on mini-Tntabolism  and regutabolism , which ipression . It shouTable 2). To ensure the consistency of these phenotypes, cfcK mutants were cured of plasmid pMAMV1, and then the plasmid was reintroduced and the phenotypes reassessed in a minimum of 50 exconjugants.None of the cfc mutants were hampered in growth, either in rich LB or defined media, except for cfcT-5 and cfcK-16, which exhibited a reduced lag phase in all media tested, whereas this stage was slightly longer in cfcK-54. Most of the crinkle-free colony mutants did not show any flocculation, while a few kept somewhat increased biofilm formation as compared to the wild type parent strain (free of plasmid pMAMV1 or pMIR178) which was negative for the three phenotypes (gacA), cfcK-16 , and cfcK-77 (fleQ), which allowed to investigate the significance of the transcriptional regulators on rup4959 expression; cfcK-66 (argG) and cfcK-74 (argH), both hampered in the arginine biosynthesis pathway; and cfcK-54 (PP_3761), the unique mutant inactivated in a HK, which together with a response regulator are the elements of a two-component phosphorelay system. For the complementation assays, each plasmid/cosmid was introduced into the corresponding cfc strain by conjugation. Once the presence of these plasmids, and that of pMIR178 or pMAMV1, was confirmed then colony morphology, flocculation and increased biofilm formation were re-analyzed. The results from these studies are summarized in Table 4 and shown in Figure 2. Complementation was observed for cfcK-54, cfck-74, and cfcK-77, although in the later two the crinkle phenotype did not reach the border of the streak. For mutant cfcT-5 crinkly morphology was not restored although biofilm formation was partially and flocculation completely restored. After receiving their wild type alleles mutants cfcK-16 and cfcK-66 both showed subpopulations with nearly full complementation and others presented the non-crinkly morphology . Interestingly complementation of cfcK-54 could be observed after 24 h incubation whereas crinkly morphology was better observed in the wild type only after incubation for 48 h .To check whether the incorporation of the intact gene restored the phenotypes associated with high c-di-GMP in crinkle-free colony mutants, two approaches were used: either, using a recombinant cosmid isolated from a gene library of KT2440 carrying the corresponding wild type allele, or by PCR amplification of the knocked-out gene and subsequent cloning in an expression vector. A selection of mutants were chosen for the complementation analyses: cfcT-5 . As previously reported, specific levels of the second messenger were under the detection limit in P. putida KT2440 and those strains harboring expression vectors  were due to a loss of rup4959 expression . In addition, it is tempting to speculate that the HK encoded by PP_3761 might be required to phosphorylate and activate the REC domain of Rup4959. Thus a strain with a mutation in this kinase (PP_3761) carrying pMAMV1 would present an increased level of an unphosphorylated and thus inactive form of Rup4959, which explains why the plasmid did not augment the levels of c-di-GMP in this particular genetic background. We have confirmed that mutants argG and argH are both auxotrophic for arginine . However, the mutant argH, unlike argG, presented detectable c-di-GMP levels although they were significantly lower than those of the wild type when both strains carried pMAMV1 . The altered second messenger content in these mutants was not due to a modification in the rup4959 expression pattern . Besides c-di-GMP values of fleQ when harboring pMAMV1 presented 40% reduction compared to the wild type. This decrease can be explained by the reduced expression of rup4959 found in a fleQ background . Interestingly, a reduction of rup4959 expression was also detected in the cfcK-16 genetic background  indicating that FleQ and AlgU/MucA are in the regulation cascade of rup4959. These two elements have been postulated to be part of the GacS/GacA regulatory cascade in P. fluorescens F113 ; interestingly, the non-flocculating variant strain showed the highest level of c-di-GMP which cannot be explained by the negative effect of cfcK-16 upon rup4959 expression. Furthermore this latter strain showed slight reduced motility (data not shown), a state that usually corresponds with increased c-di-GMP , total c-di-GMP intracellular levels were measured  we used an alternative technique to indirectly detect free second messenger with the biosensor based on the promoter of cdrA fused to gfpC . In addition, the higher intensity of fluorescence observed for argH compared to argG  was in accordance with the c-di-GMP levels measured for these strains using the analytical method . Interestingly, the non-flocculating variant of cfcK-16 harboring pMAMV1 showed somewhat comparable fluorescence intensity to the flocculating variant, in spite of the noticeable differences in morphology observed in the streaks , which is in accordance with the second messenger being detectable by the analytical method in both strains . Strain cfcK-16 cured of plasmid pMAMV1 presented the highest c-di-GMP levels among all the plasmid-cured strains tested  and as we have shown above this cannot be explained as a consequence of an increase of rup4959 expression in this genetic background.Given the difficulty in detecting c-di-GMP in several strains using our standard analytic method . To unequivocally demonstrate that this phosphorylation is required for Rup4959 to be an active DGC, the D65 was replaced with A65 by directed mutagenesis of plasmid pMAMV1 as described in Section “Materials and Methods”. It was confirmed that unlike pMAMV1, the plasmid pMIR190 encoding the mutant Rup4959-Ala65 did not confer increased biofilm or crinkle morphology  typical of high c-di-GMP levels to KT2440. The c-di-GMP content of KT2440 overexpressing Rup4959-Ala65 detected by the biosensor was comparable to that of the wild type containing physiological content of Rup4959 . Thus the accumulation of Rup4959-Ala65 was not enough for this protein to exhibit DGC activity indicating that the response regulator Rup4959 requires phosphorylation for its functionality.Unlike what was observed with the wild type, we have shown above that the global levels of c-di-GMP were undetectable in the mutant cfcK-54 carrying pMAMV1 and also that this mutant is hampered in a HK. An “Table 2). This orf encodes a hybrid sensor HK, whose phosphoacceptor domain presents a conserved histidine residue (H496) which is usually trans-autophosphorylated by the catalytic domain present in the HK . To unequivocally demonstrate that this residue was required for Rup4959 to catalyze the transfer of the phosphoryl group to an aspartate residue on its receiver domain, the H496 was replaced with A496 by directed mutagenesis of plasmid pMIR231 as described in Section “Materials and Methods”. It was confirmed that unlike pMIR231 encoding the wild type HK (PP_3761-H496), the plasmid pMIR232 encoding the mutant PP_3761-A496 did not restore any of the multiple phenotypes  typical of high c-di-GMP levels to KT2440 under Rup4949 overexpression conditions . Thus the conserved autophosphorylation histidine residue (H496) of PP_3761 is essential for Rup4959 to exhibit DGC activity.As we have shown above, mutant cfcK-54 is knocked out in PP_3761 . In addition, they showed a characteristic “whiter and drier” phenotype  and this characteristic was independent of carrying any plasmid. Auxotrophy was overcome by supplementing M9-glucose with arginine (1 mM) . Remarkably, an intact argG allele supplied via pME1088 only complemented the Arg auxotrophy of cfcK-66 when this strain harbored pMAMV1, even though to our knowledge there is no previous evidence that arginine biosynthesis is regulated by c-di-GMP. The presence of pMAMV1 was not a requirement in order to complement the auxotrophy of cfcK-74 with an intact argH allele provided via pME184. The cfc phenotype of mutants argG and argH when carrying pMAMV1 was also complemented by adding arginine, but this complementation required higher concentration of the amino acid .It has been shown above that mutants cfcK-66 and cfcK-74 had a non-functional arginine biosynthesis pathway. As expected these mutants were confirmed to be auxotrophs for this amino acid , a characteristic that was prevented by supplementing LB with Arg . This color development was not observed for cfcK-66 , initially suggesting that the accumulation of L-arginosuccinate might be involved in the color formation observed for cfcK-74. However, when cfcK-66 harbored pMAMV1, the development of a similar color was also observed . An intact argG almost completely prevented the color development in cfcK-66 (pMAMV1) . Similarly an intact argH allele abolished color development in cfcK-74  and cfcK-74 (pMAMV1) . Thus plasmids pME1088 and pME184 were able to complement all phenotypes observed in these strains, including, the cfc phenotype, and the two other phenotypes associated with argG and argH mutations, namely, auxotrophy and brownish color development.Curiously, LB-agar plates of cfcK-74 showed a brownish pigment , aspartic acid had the opposite effect , as an indication that the expected accumulation of the latter amino acid in the mutant cfcK-66 (argG)  might be the reason for the lower c-di-GMP content observed for this strain compared to cfcK-74 .Dissimilarities described above between cfcK-66 and cfcK-74 stimulated our interest to examine the effect that metabolic intermediates of the arginine biosynthetic pathway might have on the levels of c-di-GMP in the wild type P. putida. The variety of functions identified in this work, using mutants that completely or partially lost a crinkle morphology characteristic of the increased c-di-GMP levels gives an idea of the global influence of this compound as intracellular second messenger in this bacterium. The fact that none of the genetic determinants identified here were among those previously found to be required by P. putida to attain a pleiotropic phenotype, including crinkle morphology . In this bacterium, four gene clusters code for the production of cellulose, alginate and two species-specific EPSs named Pea and Peb (pea has been recently documented (our unpublished results). In addition, the transcription of lapA was compromised in a fleQ mutant , it is tempting to speculate that the transposon insertion at the end of algU in cfcK-16 had caused a polar effect upon the expression of mucA, which encodes the AlgU anti-sigma factor MucA . At the beginning of this work it was unknown whether such post-translational modification was a requisite for the activity exhibited by Rup4959. Given that the D65 and other conserved residues at its receiver domain suggested so, we decided to substitute D65 with A65 and have concluded that an aspartic acid residue at this position is required for the functionality of this response regulator as a DGC. Interestingly a sensor HK was identified in this work as being essential for crinkle morphology. The locus encoding this HK is the first orf of a cluster of five genes that we propose to name cfcABCDE. The genes cfcC and cfcB encode one of the three chemosensing systems CheB/CheR present in P. putida, although the role of CfcB and CfcC in chemotaxis has been ruled out . Thus we propose CfcA as the HK most likely transferring the phosphoryl group to the D65 residue on the receiver domain of Rup4959. In support of this, a point mutation in the autophosphorylation H496 of CfcA caused loss of activity in Rup4959. Furthermore, we identified that the REC domain present in CfcD showed the highest score (corresponding to 48% identity and 66% similarity) found in KT2440 to the REC domain of Rup4959 , suggesting that both domains are candidates to be phosphorylated by the same HK, namely CfcA. Genes encoding sensor kinase/response regulator systems often appear clustered in the genome. Remarkably genes orthologous to cfcA and rup4959 come together in the chromosome of P. resinovorans NBRC 106553 , in spite of being auxotrophic for arginine, suggesting that auxotrophy for this amino acid may not be the only or ultimate cause for the crinkle-free morphology exhibited by argG and argH mutants. At this point the connection between this metabolic pathway and c-di-GMP in Pseudomonas is unclear. However, it is worth mentioning that previous reports indicate a connection between amino acids and bacterial social behaviors. D-amino acids have been shown to promote dispersal of Bacillus subtilis biofilms , although cfcR transcription was not altered in these mutants (our unpublished results). In fact if, as it is presumable, accumulation of aspartic acid takes place in a mutant argG, we hypothesized that this amino acid might exert a negative effect upon the free content of the second messenger in the bacteria cell. We could confirm this hypothesis  although the underlying mechanism remains unknown. Given that species specific EPS Pea and LPS were known to be essential for P. putida to acquire the crinkle phenotype (argH mutant. However, the argG and argH mutant strains showed increased biofilm formation capacity and a full flocculation phenotype under cfcR overexpression conditions, indicating that the adhesins LapA and LapF, the major determinants of biofilm formation (fleQ mutant, in which the transcription of lapA was compromised (Another block of mutants identified are related to metabolic processes. We have focused our attention particularly on two genes in the arginine biosynthetic pathway, biofilms . On the swarming . Althougg domain . Based og domain , our dathenotype , it is pormation  and flocpromised .P. putida there is no clear equivalent of the above mentioned DGC STM1987 containing a periplasmic Arg-sensing domain, what emphasizes the interest in unveiling the mechanism by which arginine may control c-di-GMP levels in this bacterium and related species. We are currently investigating the specific role of CfcA/CfcR in the modulation of c-di-GMP content by metabolites of the arginine biosynthesis pathway. In addition we are examining the different steps of this pathway to determine their influence on c-di-GMP global cell content and their concomitant effect on signal transduction.In MIR-G and ME-U conceived the study. MIR-G designed the research. MLT and MIS performed molecular work. MIR-G, MLT and LB-M took pictures. MAM organized and performed the sampling and analyzed the data for c-di-GMP quantification. OH-R performed experiments with TECAN. OH-R, LB-M, and VGT performed biofilm experiments and helped with data interpretation. MIR-G and ME-U wrote the manuscript and discussed the results and all the authors specially MAM commented on the manuscript.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.The handling Editor declared a shared affiliation, though no other collaboration with all the authors and states that the process nevertheless met the standards of a fair and objective review.


In this research, we develop a micro-engineered conductive elastomeric electrode for measurements of human bio-potentials with the absence of conductive pastes. Mixing the biocompatible polydimethylsiloxane (PDMS) silicone with other biocompatible conductive nano-particles further provides the material with an electrical conductivity. We apply micro-replica mold casting for the micro-structures, which are arrays of micro-pillars embedded between two bulk conductive-PDMS layers. These micro-structures can reduce the micro-structural deformations along the direction of signal transmission; therefore the corresponding electrical impedance under the physical stretch by the movement of the human body can be maintained. Additionally, we conduct experiments to compare the electrical properties between the bulk conductive-PDMS material and the microengineered electrodes under stretch. We also demonstrate the working performance of these micro-engineered electrodes in the acquisition of the 12-lead electrocardiographs (ECG) of a healthy subject. Together, the presented gel-less microengineered electrodes can provide a more convenient and stable bio-potential measurement platform, making tele-medical care more achievable with reduced technical barriers for instrument installation performed by patients/users themselves. Conductive materials are often applied as electrodes for measurements of biopotential, which involves the voltage variation generated by muscle contraction/relaxation . Furtheri.e., no additional gels are required [et al., reported a dry electrode composed of iridium oxide (IrO) [i.e., the micro-needles pinned into the epidermis) in order to increase the contact area of measurement. Arrays of carbon nanotubes were also applied as the invasive needle-type electrodes, which penetrated into the outer layers of the skin and the stratum corneum to improve the electrical contact [et al. reported a biopotential electrode using conductive polyethylenedioxythiophene doped with polystyrenesulfonate and demonstrated its great potentials in electrophysiological measurements such as EOG, ECG and EEG [et al., reported the implementation of long-term electrophysiological measurements using capacitive epidermal electronics [Researchers have been working on development of the ‘dry’ biopotential electrodes (required ). For inde (IrO) . Needle- contact . Neverth contact ,16. Rece contact ,18,19. L and EEG ,20. Joenctronics . These cIn this paper, we report the development of dry microengineered conductive elastomeric biopotential electrodes for ECG measurement with higher electrical consistence. We chose the PDMS elastomer as the structural material because it is biocompatible, chemically inert, gas/moisture-permeable, and highly deformable. With these material properties, PDMS has become one of the most widely used materials in many biological or biomedical applications ,23. MixiAs the conductive-PDMS is highly flexible, the physical conformity on human skin can be achieved by the material flexibility, rather than having sharp nano-needles penetrate the skin’s surface. However, body movements of the user can significantly deform the bulk material and subsequently cause variations in its electrical properties. Therefore, we have designed and micro-fabricated the conductive-PDMS material as shown in w/w ratio of 10:1). The mixture was stirred for three to five minutes, degassed and poured onto the silicon master (with microstructures) and baked at 80 °C overnight for thorough cure (Step 1). After cutting and peeling the PDMS substrate off from the silicon master (Step 2), this PDMS negative-mold was silanized overnight with trichloro  silane. The PDMS pre-polymer was prepared again by mixing the monomer and curing agent with the w/w ratio of 10:1, and then mixed with selected conductive nanoparticles using a defined w/w ratio. The conductive-PDMS mixture was then poured onto the pre-fabricated negative pure-PDMS mold (Step 3). In order to remove the gaps and air trapped in the conductive material substrate, the conductive-PDMS and the mold were degassed and compacted using two PMMA boards (Step 4). The substrate was then placed into an oven and baked at 80 °C overnight for thorough curing. Meanwhile, a flat conductive-PDMS layer was prepared with the silicon master (only with boss) using mixed PDMS with the same nanoparticles. Before baking this flat substrate overnight, an electrical wire was inserted into the conductive-PDMS pre-polymer for the signal transmission purpose. To finish the fabrication, both the flat and micro-pillar conductive-PDMS substrates were peeled off and chopped. Oxygen plasma treatment was applied on their surfaces to covalently bond them together (Steps 5–6).We fabricated the conductive elastomeric electrode a by two C of each specimen was calculated by C = L/(RA), where R is measured sample resistance, A is cross-sectional area and L is sample length. −1 with a weight concentration of ~24%, whereas graphite-PDMS can induce a conductivity of only ~2 Sm−1 with a high weight concentration of ~60%. Thus, we have chosen 20% (w/w) of acetylene black in PDMS as the target substrate material for the microengineered electrode development. Our results indicate that acetylene black can induce higher conductivity to the PDMS substrate. In the electrode fabrication, we chose the weight ratio of acetylene black in PDMS to be 20% in order to retain sufficient material strength. Importantly, there are also medical grade versions of the selected materials available, e.g., Silastic biomedical grade ETR elastomers  [In order to optimize the composite candidates as bio-potential electrodes, we performed a series of experiments on conductive-PDMS specimens with the same size and shape but different nanoparticle types and concentrations. We considered three nanoparticle candidates: acetylene black, carbon black, and graphite. We measured the electrical resistances of these specimens by clamping the two ends of each sample using crocodile clips connecting to an LCR meter (VICTOR VC9808+). The electrical conductivity MI, USA)  for siliMI, USA) . Further3, Poisson’s ratio: 0.45; relative permittivity: ~2.8, and electrical conductivity: 23.7 Sm−1) were determined by our tests on the bulk material (~20% weight ratio of acetylene black in PDMS). We applied the Neo-Hookean hyper-elastic model to describe the material deformation because the composite material contained mainly the elastomeric PDMS. We considered the lower half of one micro-pillar region as our simulation model, due to the repeating micro-pillar structures in the electrode, and the symmetry between the upper and lower halves of one micro-pillar region. The model is a cylinder  fixed on top of a square block . On the other hand, we also simulated the resistance of a bulk conductive-PDMS with dimensions equivalent to a pillar region with a pillar height 400 µm, i.e., 1.5 mm (w) × 1.5 mm (l) × 500 µm (h). We then imposed a range of defined lateral strains (0%–30%) on region boundaries of both the micro-pillar model and the bulk PDMS model, and further examined for their strain profiles and the electrical resistance along the “height” direction of the models. We set the voltage difference between the top and bottom surfaces of the model to be 1 V and computed for the current density , which were normalized as the ratio to the unstretched status. We have performed measurements to characterize both the bulk and microengineered conductive PDMS electrodes (1 cm (W) × 1 cm (L) × 1 mm (H)). We selected the composite of 20% acetylene black in PDMS as the substrates for the bulk and microengineered materials. The electrode contact impedances  oFurther, we investigated the contact impedance variations under different uniaxial tensile strain levels of the conductive-PDMS electrodes. We applied stretches on the conductive-PDMS specimens  using an electromechanical material testing system  at a rate of 1 mm/min for a total distance of 1 mm (5% strain), and we simultaneously measured the conductivity along the direction of substrate thickness. Considering that the metal fixtures of the mechanical tester are conductive, we isolated both ends of the mounted samples by polymeric sealing films . The electrical resistances of the conductive-PDMS at different strains were measured at different distances of the stretching. The corresponding equivalent uniaxial elastic moduli b of the Additionally, we have applied the microengineered conductive-PDMS dry electrodes to implement the clinically recognized 12-lead ECG acquisition by developing a fully functionally ECG vest a and theDetection of biopotential from human skin should involve the potential signals in the range of <1 mV, which requires relatively sensitive electrodes with good conductivity, a high quality signal amplifier, and a signal filter. In the portable ECG acquisition device mentioned previously, we have included a biopotential amplifying and filtering module for signal acquisition of each ECG electrode. For each lead of the measurement, an individual circuitry was integrated from multiple operational amplifiers (UA741CN) as shown in The following amplifier circuit was a simple non-inverting amplifier (Gain: 11) that amplifies the output from the low-pass filter. Next, a twin-T filter and a voltage follower composed as a Notch filter to eliminate any possible noise caused by the 50 Hz alternating current signals from other appliance driven by the building power system.i.e., with waist muscular movements and physical vibration generated). The ECG measured with the microengineered electrodes , two on the two sides of the waist (LL and RL), and six on the rib cage across the chest (V1–V6). Only bandages were required to tighten the forearms (LA and RA) and waist (LL and RL) for the skin-electrode contact. A zipper was added in the front of the vest so that the wearer could put on the vest more easily. We also added a side waist bag a for carectrodes b should To further improve the captured signal quality, we implemented additional digital time-invariant band-pass linear filtering on each lead-signal of the measured ECG, by including the corresponding resolved difference equation in the GUI. In this stage, we chose the lower and higher cut-off frequencies to be 1 Hz and 100 Hz, respectively. i.e., the subject lifted his left and right legs alternatively) and (2) running, and the representative ECGs are shown in Furthermore, we have simultaneously measured the ECGs using both the conventional wet electrodes and the microengineered dry electrodes connected to our ECG acquisition system, in order to compare the measurement performance between the two electrode types. The wet electrodes and microengineered electrodes were placed next to each other to measure the same subject’s ECG. Considering that our ECG acquisition system had only limited number of input channels, 6-lead ECG   were adoIn this paper, we report a microengineered conductive-PDMS electrode for human electrophysiological measurement without the addition of gels. To reduce the changes in the electrical conductivity caused by mechanical stretching of the electrode during body movements of the user, we designed and configured the conductive-PDMS as an array of micro-pillars embedded between two layers. The electrode has been fabricated by micro-replica molding of conductive-PDMS. Based on our characterization of the composites of different nanoparticles  in PDMS, we chose acetylene black with a 20% weight ratio in PDMS as the substrate material for the sufficiently low skin contact impedance (17.5 kΩ) and rigidity (4.45 MPa). Altogether, the reported microengineered conductive-PDMS electrodes have a great potential for extended biomedical applications such as the long-term electrophysiological diagnosis , and electrooculography (EOG)) and remote medical care or healthcare of general users at home, due to the measurement consistency and simple electrode placement scheme resultant from embedding the electrodes into clothing and other gadgets.