My health for life program.Chronic disease is the leading cause of premature death globally, and many of these deaths are preventable by modifying some key behavioural and metabolic risk factors. This study examines changes in health behaviours among men and women at risk of diabetes or cardiovascular disease (CVD) who participated in a 6-month lifestyle intervention called the My health for life program is a Queensland Government-funded multi-component program designed to reduce chronic disease risk factors amongst at-risk adults in Queensland, Australia. The intervention comprises six sessions over a 6-month period, delivered by a trained facilitator or telephone health coach. The analysis presented in this paper stems from 9,372 participants who participated in the program between July 2017 and December 2019. Primary outcomes included fruit and vegetable intake, consumption of sugar-sweetened drinks and take-away, alcohol consumption, tobacco smoking, and physical activity. Variables were summed to form a single Healthy Lifestyle Index (HLI) ranging from 0 to 13, with higher scores denoting healthier behaviours. Longitudinal associations between lifestyle indices, program characteristics and socio-demographic characteristics were assessed using Gaussian Generalized Estimating Equations (GEE) models with an identity link and robust standard errors.The p < .001 for all) but not risky alcohol intake. Modelling showed higher average HLI among those aged 45 or older  with vocational educational qualifications  while being male, Aboriginal or Torres Strait Islander background, or not currently working conferred lower average HLI scores .Improvements in HLI scores were noted between baseline  and 26-weeks  which corresponded with increases in fruit and vegetable consumption and decreases in takeaway frequency (While participants showed improvements in dietary indicators, changes in alcohol consumption and physical activity were less amenable to the program. Additional research is needed to help understand the multi-level barriers and facilitators of behaviour change in this context to further tailor the intervention for priority groups.The online version contains supplementary material available at 10.1186/s12889-022-14056-1. Chronic disease poses the greatest threat to global health, with a higher morbidity and mortality rate, than do all other causes contributing to around 41 million deaths each year . The ramThe development of chronic disease is underpinned by varying risk factors, including both non-modifiable  and modifiable health behaviours –3, 5 witAddressing chronic disease is important for the 2030 Agenda for Sustainable Development, specifically Sustainable Development Goal (SDG) target 3.4 calls for a one-third reduction in premature mortality from chronic disease by 2030 . To accehttps://www.lifeprogram.org.au/), the Queensland Government invested in a large public health program, My health for life. The program, aims to reduce the risk of cardiovascular disease and diabetes in priority groups, such as those at high risk of developing chronic disease, Aboriginal and Torres Strait Islander People and culturally and linguistically diverse people, through supporting individuals to make changes to their health behaviours. The program targets multiple modifiable health behaviours associated with increased chronic disease risk, therefore to assess the joint association of the multiple modifiable health behaviours targeted by the program, a healthy lifestyle index (HLI) score was created by combining dietary, alcohol and tobacco smoking, and physical activity indices to form a composite score. To understand the effectiveness of the program, the purpose of this paper is to examine changes in the primary outcome  in participants of the My health for life program.Modifiable health behaviours including diet, alcohol, tobacco smoking, and physical activity are linked with physical and psychological symptoms including pain, fatigue and depressive symptoms . Among pMy health for life program is a novel, multi-component program that aimed to reduce chronic disease risk factors among adults at risk of diabetes or CVD in the state of Queensland, Australia. The Queensland Government funded program was developed by an alliance of health organisations led by Diabetes Queensland including Stroke Foundation, Heart Foundation, Queensland Primary Health Networks, Ethnic Communities Council of Queensland and Queensland Aboriginal and Islander Health Council . Details about the program are here https://www.myhealthforlife.com.au/.The https://www.myhealthforlife.com.au/risk-assessment). A range of marketing and communication activities were undertaken to lead potential participants to the online health check including television advertisements on regional and metropolitan television channels, outdoor advertising on billboards and bus shelters, Facebook advertising and print advertisements in local newspapers and a motoring club magazine. Eligible participants were identified using adapted risk assessment tools  [To recruit people at-risk of developing chronic disease, Stroke Foundation staff undertook health checks at community events and workplaces across the state of Queensland, health clinicians undertook health checks in clinical settings  and potential participants undertook a health check online via the website (USDRISK)  or AbsolUSDRISK) ). The prUSDRISK) . High rin = 245, 63.0%), with a small number contracted to a Pharmacy . In total, 264 facilitators (face-to-face or THC) delivered at least one program and had a variety of backgrounds in Allied Health (Dietetics or Exercise Physiology), Nursing, Pharmacy, Health Promotion, Counselling, Aboriginal Health Work or Multicultural Health Work. Retention of facilitators that delivered at least one program was 61.7% (n = 163). Retention of provider organisations that delivered at least one program was 81.8% (n = 112). All provider organisations received financial payment to deliver the face-to-face program, paid on a per participant basis.The program offered is either the face-to-face group-based program (GBP) or one-on-one telephone health coaching (THC), with participants choosing the most suitable option for themselves. The GBP consists of small groups of 6–8 participants, delivered by a trained facilitator in a community setting running for approximately two hours and the THC offering is delivered one-on-one via telephone in house by the lead organisation, Diabetes Queensland with a trained facilitator  running for approximately one hour. Potential provider organisations for the face-to-face program responded to a call for expressions of interest to deliver the program. The organisations nominated qualified and experienced health professionals for training by the program implementation team as a facilitator for the delivery of the face-to-face program with 136 approved providers engaged to deliver the program. Diabetes Queensland recruited the telephone health coaches to work in house through a standard employment recruitment process of qualified and experienced health professionals or through identification of appropriately qualified and experienced existing health professional staff. In total, 408 health professionals attended facilitator training with 403 completing training to become a certified facilitator. Of these, 389 were trained and certified to deliver face-to-face groups, and 14 were trained and certified to deliver the THC program. Training of facilitators included completion of prior reading, attendance at a two day face-to-face training course and successful completion of all assessment. All facilitators are required to maintain accreditation through participation in professional development activities on an annual basis. Training and certification of facilitators was conducted be the My health for life program implementation team. Most facilitators were contracted to an Allied Health Service  with session 6 (related to maintenance) occurring at around 24 weeks. The program, underpinned by the Health Action Process Approach (HAPA), aimed to develop knowledge, skills, and strategies to adopt positive lifestyle behaviours, while educating participants on different risk factors, including healthy eating, alcohol, tobacco use, and physical activity. Session activities target modifiable health behaviours using behaviour change techniques  as outli–5 with sMy Health for Life program between July 2017 and December 2019 who contributed weight, diet, alcohol, smoking and physical activity data towards the composite healthy lifestyle index (HLI). Participants consented to participate upon commencement in the program and completion of the first survey. Telephone health coaches or program facilitators assisted participants to enrol in the program. Ethical approval was granted from the Darling Downs Health Human Research Ethics Committee (HREA/2021/QTDD/72406) and Griffith University (GU Ref No: 2021/143) before accessing de-identified, secondary data.This paper draws upon survey data from 9,372 participants of the This study uses a pragmatic non-randomised, time–series analysis adopting observational, goal-based and pretest–posttest design for the program evaluation  and vegetables , intake of sugar-sweetened drinks  and take-away consumption  . The dieThe alcohol and tobacco index, based on the health guidelines for drinking alcohol , comprisFor the physical activity component, a single indicator was used. The variable, derived from the Active Australia Survey, was collapsed to form a single trichotomous variable indicating being sedentary (no points), insufficiently active (1 point), and sufficiently active for health (2 points) .Details of the scoring for each indicator is in Supplementary Table My health for life program. However, while excess weight was a primary outcome for the study, it was not included in the healthy lifestyle index as it could have been an intermediate factor between modifiable health behaviour and health outcomes [2 representing normal weight, 25–29.9 kg/m2 representing overweight, 30–39.9 kg/m2 representing obesity and >  = 40 kg/m2 representing extreme obesity. Sex-specific waist circumference was grouped according to increased risk (94-101 cm in men and 80-87 cm in women) and greater increased risk (> 102 cm in men and > 88 cm in women). Both measures were included in this analysis to adequately capture adiposity. BMI is an adequate measure of adiposity for clinical purposes [Overweight and obesity are associated with around 8% of Australia’s burden of disease  and was outcomes . Neverthoutcomes  with a Bpurposes .Adjustment was made for other covariates including socio-demographic characteristics , Culturally or Linguistically Diverse (CALD) background, and employment ), relati2) tests and ANOVA with statistical significance set at α = 0.01 and clinical significance achieved with percentage differences greater than 10% [Statistical analyses were performed using SPSS  version 23  and STATthan 10% .Before undertaking multivariate analysis, the patterns of missing data were examined. For the primary outcomes, the amount of missing data at Session 1 varied from < 1% on dietary and alcohol indicators   = 285.15, p < 0.01), sex  = 22.34, p < 0.001), retirement (χ2(16) = 328.41, p < 0.001), higher educational attainment (χ2(16) = 79.10, p < 0.001), and greater relative advantage  = 124.93, p < 0.001). Socio-demographic characteristics by HLI quintile are further outlined in Table Baseline bivariate comparisons of the healthy lifestyle index showed that healthy lifestyle was associated with age  = 16.41, p < 0.01) aged 45 years or less (χ2(2) = 67.36, p < 0.01) with primary or secondary school education (χ2(8) = 16.93, p = 0.03).Overall, three-quarters of participants were female, most were aged 45 years or older (> 80%), around two-thirds reported a secondary school or certificate/diploma level education, and half were employed outside the home. Some modest but statistically significant differences were noted with attrition highest in men (p < 0.001) and vegetables increased over time  while the frequency takeaways decreased. Risky alcohol intake . Finalles < 10%  increaseHowever, while there were general trends towards healthy lifestyle behaviours over the program period, attrition might have influenced prevalence and therefore data were imputed. To assess the robustness of imputation, the original and imputed healthy lifestyle indices summary statistics are provided. Point estimates for the HLI (range 0—13) did not change at each time point with the average HLI at Session 1 being 8.6 (SD = 2.1), 9.6 (SD = 1.9) at Session 5 and 9.9 (SD = 1.9) at Session 6.p < 0.001; Model 2: β = 0.96, 95% CI = 0.89, 1.03, p < 0.001; Model 3: β = 0.98, 95% CI = 0.91, 1.05, p < 0.001) and this was sustained at Session 6 .The results of Gaussian Generalized Estimating Equations which incrementally adjusted for program characteristics (Model 2) and personal background (Model 3) are shown in Table p < 0.001) and program mode  significantly influenced HLI scores though following adjustment for background socio-demographic factors (Model 3) mode was no longer significant (p = 0.076). Findings showed that being retired , aged 45 or older , and having a certificate or diploma  or bachelor’s degree or higher  conferred a higher average HLI while being male, Aboriginal or Torres Strait Islander background, or not currently working conferred lower average HLI scores .Model 2 examined the additive effect of program characteristics. In Model 2, number of sessions attended  though around one-third (37%) of participants consumed an average of 5 or more alcoholic drinks in one session and this was largely unchanged over the program period. Finally, at baseline 19% of participants reported being sedentary and 46% were insufficiently active for health. Over the program period, the proportion of people meeting physical activity guidelines increased, though at Session 6, only 53% reported being sufficiently active for health. Percentage changes using predicted probabilities in individual health behaviours from Session 1 to Session 6 are illustrated in Fig. % though My health for life program, which aimed to reduce the risk factors of chronic diseases among at-risk populations. When compared with the lifestyle indicators of Queenslanders more generally, My health for life participants reported lower compliance with recommended daily fruit consumption, higher baseline average single occasion risky drinking, and low physical activity levels that were sufficient for health [My health for life program period, the proportion of participants meeting recommended health behaviour guidelines , in some instances, was greater than is reported by Queensland adults [This paper explores changes in primary health outcomes of participants from the r health . Notablyd adults .2) in ‘at-risk’ populations, is associated with a significant reduction in disease burden [My health for life program participants has the potential to have a significant impact on the burden of chronic disease. The reduction in BMI for program participants is similar to previous literature which demonstrates the potential for programs targeting multiple health behaviours to contribute to reduction in BMI and waist circumference [During the intervention, the proportion of participants in the extremely obese, obese, categories decreased from Session 1 to Session 6 while those in the normal weight range increased from 9 to 13%. Overweight and obesity is the fourth highest risk factor for burden of disease in Australia. A large proportion of total disease burden can be prevented avoiding or reducing exposure to risk factors including tobacco use, overweight (including obesity), dietary risks, and alcohol use. Overweight including obesity accounts for 8.4% of the burden of disease in Australia . Obesitymference . This shmference .Dietary indicators improved over time, with many participants increasingly likely to meet recommended fruit and vegetable intake at Session 5. In this study 73.5% of participants were meeting dietary guidelines for fruit consumption, whereas in Queensland, it is estimated that around 2.1 million (53%) adults were meeting recommendations for fruit consumption. Around one quarter (25.7%) of participants in this study were meeting the recommendations for vegetable consumption, compared to only 320,000 (8.0%) of Queensland adults meeting recommendations for vegetable consumption . ImportaIn 2019 in Australia there were 11.6% of the adult population who smoke tobacco daily , compareWhile smoking and alcohol consumption rates in this group were lower than the Australian population at baseline, there were improvements across the life of the program.There were general improvements in participants’ physical activity behaviour between Session 1 (34.1%) and 5 (58%), though only around half of participants were sufficiently active for health at program completion, returning to lower levels (53.3%). At baseline for this study there was a considerably smaller percentage of participants who were sufficiently active for health (34.1%), than that previously reported for Queensland adults aged 18–75 years  and Australian adults more broadly (45%) , 52. ThiThe results from this study are consistent with the growing body of research showing the effectiveness of lifestyle interventions on improving modifiable health behaviours. Key components of these interventions include the use of motivational interviews to allow participants to set their own intentions and goals , 54, strThe intervention was associated with improvements in healthy lifestyle indicators which contributes new information to this research area. While participants showed improvements in dietary and smoking indicators, changes in alcohol consumption, and physical activity were less amenable to the program. Conducting additional research will help understand the multi-level barriers and facilitators of behaviour change in this context to further tailor the intervention for priority groups.This paper has several limitations to acknowledge. First, a pragmatic non-randomised, time–series analysis adopting observational, goal-based, and pretest–posttest design features underpins the program evaluation. The absence of a control group, which would be extremely challenging to establish given the size and intent of the program, could impact on the ability to make causal inferences about the impact of the intervention of health indicators, however, where possible program results are compared to Queensland and Australian rates of behaviour. Participants enrolled in a lifestyle modification program, are potentially highly motivated to improve their health regardless of the intervention which may impact on the positive outcomes of the program. Second, akin with many longitudinal studies, missing data were present in around 42% of participants at Session 5 and 58% of participants at Session 6 and this might have impacted the magnitude of outcomes and the representativeness of the sample . To accoAdditional file 1: Supplementary Table 1. Healthy lifestyleindex construction. Supplementary Table 2. Sensitivity analysisof the longitudinal modelling of a HLI using Gaussian Generalized EstimatingEquations with an exchangeable structure and robust standard errors (with andwithout adjustment for baseline excess weight). Supplementary Table 3. Associations between participant characteristics and missing data at baseline. SupplementaryTable 4. Proportion of missing data from indicators at sessions 1-6.


We recently reported the correlation of gut bacterial diversity with heart failure using a mouse model of heart failure due to pressure overload induced by transverse aortic constriction (TAC). We found that gut the bacterial diversity is significantly altered and is directly correlated to the severity of heart failure . In addition, stool samples that were collected for the gut microbial diversity analysis, we dissected ileum from the mice after 42 days of TAC. The total DNA was extracted to identify the bacterial diversity resided in ileum using 16S rRNA gene amplicon shotgun sequencing and downstream bioinformatics analysis to determine if it is correlated to the heart failure. We found that gut the bacterial diversity is significantly altered and is directly correlated to the severity of heart failure  were used for the present study. Mice were acclimatized to the conditions in our animal facility for 2 weeks before start of the experiments. All mice were fed a standard rodent chow diet and kept under 12-h light and dark cycle.2.2Mice underwent TAC (or sham) surgeries as described earlier 2.3https://www.ebi.ac.uk/ena/browser/view/PRJEB45533?show=reads).DNA extraction, PCR and 16S rRNA gene sequencing was carried out as described earlier 2.4Analysis of the 16S rRNA gene sequences was carried out as previously described 2.5Bacterial composition was analyzed as described earlier 2.6Statistical significance for bacterial composition was analyzed by non-parametric Wilcoxon's test followed by significant grouping using PERMANOVA. Whereas, the statistical significance for microbial abundances in two groups was calculated by Wilcoxon test and the P-values were adjusted using FDR. Mice echocardiography results were analyzed by one-way ANOVA  followed by Student–Newman–Keuls post-hoc test. P-values ≤ 0.05 were considered statistically significant.All animal experiments complied with the ARRIVE guidelines and were carried out in accordance with the EU Directive 2010/63/EU for animal experiments. The ethics protocol was approved by the Ministry of Energy Transition, Agriculture, Environment, Nature and Digitalization (MELUND) of the state of Schleswig-Holstein, Germany with the reference number V241–60,965/2017 (129–10/17).Martina E. Spehlmann: Conceptualization, Resources, Formal analysis, Writing – original draft, Writing – review & editing. Dhiraj P. Dhotre: Software, Formal analysis, Data curation, Supervision. Nesrin Schmiedel: Methodology, Formal analysis. Nikita Chavan: Methodology, Software, Formal analysis, Visualization. Corinna Bang: Methodology, Resources. Ashraf Y. Rangrez: Conceptualization, Writing – original draft, Writing – review & editing, Visualization, Supervision.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


The results identified shorter fever and rash resolution time in Obs compared with Con. The posttreatment CRP, PCT, IL-6, and CD8+ and the incidence of adverse events reduced notably in Obs and were lower than Con, while CD3+, CD4+, and CD4+/CD8+ elevated statistically and were higher than that of Con. Our results indicate that GC plus IVIG can significantly promote symptom resolution, alleviate inflammatory response, and improve immune function in children with Ig-insensitivity KD, with favorable safety and clinical promotion value.This study mainly analyzes the clinical effect of glucocorticoid (GC) plus intravenous immunoglobulin (IVIG) in treating children with immunoglobulin (Ig)-insensitive Kawasaki disease (KD). From September 2013 to November 2021, 86 Ig-insensitive KD children were selected, including 46 children  treated with GC plus IVIG, and 40 children  treated with IVIG. The symptom (fever and fever) resolution time, inflammatory factors , immune indicators (CD3 Kawasaki disease (KD), a mucocutaneous lymph node syndrome named after its discoverer (Kawasaki), is an acute febrile inflammatory disease that tends to occur among infants and children , 2. KD iImmunoglobulin (Ig), a regulator of the inflammatory process and immune response, is also vital in the effective connection between adaptability and the innate immune system . IntraveGiven the current lack of research on the combined use of GC and IVIG in children with Ig-insensitive KD, we conduct related research to provide new clues for the treatment of this type of patient population.n = 46) and the control group  based on different therapies . The male-to-female and the mean age in Obs were 32 : 14 and (6.18 ± 1.12) years, respectively, while the data in Con were 28 : 12 and (5.85 ± 1.03) years. The two cohorts of patients exhibited no evident difference in baseline data like gender and age , and the dose was reduced to 3–5 mg/kg per day when fever subsided. On this basis, Con was given IVIG therapy, 1 g/kg per day for 15 days. Obs was additionally treated with GC based on the control group. Methylprednisolone sodium succinate (3–5 mg/kg/d) was injected intravenously for 30 min each time, once a day. After three consecutive days of treatment, it was changed to oral administration. The course of treatment was 15 days.Symptom resolution time: we mainly recorded the fever and rash resolution time of the two groups of children.Inflammatory factors: we collected 5 mL of fasting cubital venous blood from both groups the next morning before and after treatment. After serum separation, enzyme-linked immunosorbent assay (ELISA)  was used+, CD4+, CD8+, and CD4+/CD8+) of children were detected by flow cytometry, and the experimental operation strictly followed the instrument instructions.Immune indices: T lymphocytes (CD3Incidence of adverse reactions (ARs): we mainly observed, recorded, and statistically analyzed the number of cases of adverse events such as nausea and vomiting, diarrhea, rash, dizziness and headache, renal function injury, and thromboembolic diseases in the two groups.χ2) was utilized for comparisons of categorical data expressed as several cases/percentages (n/%) in the two groups. The intergroup and intragroup (before and after treatment) comparisons of continuous data represented by mean ± SEM were made using the independent samples t-test and paired t-test, respectively. P values less than 0.05 were considered statistically significant.We used SPSS software  for statistical analysis and exported the images through the GraphPad Prism software (version 6.0). The chi-square test (p > 0.05). The results were as follows: significant differences were absent between the groups in terms of sex, average age, time from disease onset to hospital admission, duration of fever, coronary artery disease, acute fever, rash, mucosal congestion, cardiovascular system damage, and family history (p > 0.05), as given in We analyzed children's general data and determined that the two arms were comparable (Fever and rash resolution time in Obs was observed as (1.22 ± 0.19) d and (2.53 ± 0.51) d, respectively. While, the data in Con was found to be (5.42 ± 1.24) d and (6.93 ± 1.51) d. Fever and rash resolution time was shorter in Obs than that of Con .p > 0.05). While, the posttreatment levels of the above inflammatory factors decreased in both groups (p < 0.05), with notably lower parameters in Obs (p < 0.05)  .p > 0.05). After treatment, CD3+, CD4+, and CD4+/CD8+ increased (p < 0.05), while CD8+ decreased (p < 0.05), with significant differences in T lymphocytes between the groups (p < 0.05)  .p < 0.05)  .KD is a self-limiting systemic vasculitis that can cause multiorgan arteritis, resulting in impaired multiple organ function . Its incIn this study, we included 86 Ig-insensitive KD children and intervened with either the combination therapy of GC and IVIG (Obs) or IVIG monotherapy (Con). Our research results showed that the combined treatment had a remarkable effect on resolving the symptoms of fever and rash in children, suggesting that the combined intervention is more conducive to the clinical symptoms of children. IVIG preparations are composed of polyclonal plasma-derived IgG from thousands of donors, which secrete a large number of antibodies that are specific and conducive to play an anti-inflammatory role –21. In KThis study has confirmed the efficacy and safety of GC plus IVIG in treating children with Ig-insensitive KD, which can significantly resolve clinical symptoms, alleviate inflammation, and improve the immune function of children. However, this research still has potential limitations, which need to be gradually addressed in future research. First of all, the sample size of the study is small, and increasing the sample size will be more beneficial for the accuracy of the results. Second, if follow-up can be supplemented for prognostic analysis, the impact of the combined treatment on the prognosis of children with Ig-insensitive KD can be further evaluated. Finally, the underlying mechanism of their therapeutic effect can be further elucidated if the relevant basic research of combination therapy can be supplemented and their therapeutic mechanism in Ig-insensitive KD can be explored.Here, we demonstrated the effect of the combined therapy of GC and IVIG in children with Ig-insensitive KD, and the results revealed that the combined therapy of GC and IVIG exhibited a remarkable effect for resolving the clinical symptoms of fever and rash in children. Additionally, in terms of inflammation, combination therapy significantly inhibited inflammatory indexes such as CRP, PCT, and IL-6, with more obvious inhibition than monotherapy. The combined treatment of GC and IVIG validly improve the immune function of children by causing significant improvement in T lymphocytes. Hence, this study suggests that GC plus IVIG has satisfactory clinical effects in treating children with Ig-insensitive KD, which can resolve the clinical symptoms, inflammatory reaction, and immune function of children to varying degrees, with certain safety, providing a new understanding for the treatment of such children.


Essential oils are a category of agro-based industrial products experiencing increasing demand. In this research, three essential oils obtained by steam distillation from lavender, sage and basil plants cultivated in temperate continental conditions of Transylvania were investigated for chemical composition, physical characteristics and biological activity . The number of identified compounds varied: 38 for lavender, 29 for sage essential oil and 41 for basil. The volatile profile was dominated by terpenes and terpenoids (>80%). Major components were beta-linalool and linalool acetate in lavender essential oil; thujones and camphor in sage essential oil; beta-linalool, thujone, camphor and eucalyptol in basil essential oil. Refractive index of the essential oils was lowest for lavender and highest for sage. Antibacterial activity was strongest for basil, moderate for lavender and weakest for sage essential oil. The most active on both colon adenocarcinoma (Caco-2) and ovary carcinoma (A2780) was sage essential oil. Essential oils (EOs) represent an important group of agro-based industrial products with wide utilization in food flavorings, pharmaceutical, agriculture, wellness, fragrance and cosmetics industries ,2,3,4. EEOs consist of a complex and diverse mixture of chemical constituents with low molecular weight. The aroma profile of EO can be given by a dominant constituent or a mixture of constituents depending on botanical identity . Among tThere is a multitude of factors influencing EO characteristics, such as age of the plant , genotypThe timeline of EOs from production to commercialization is a temporal sequence of interconnected links that concerns particularities related to cultivation, harvesting or extraction. These are potential contributors impressing an influence on quality and properties of EOs, as proposed in Due to increasing demand, there is a need for transparency across the production chain timeline of EOs as a measure of green responsibility that can build robust knowledge bridging EO use with quality parameters. To date, there is stringent need to complete the knowledge base at an international level on the quality parameters of EOs in traceability systems. This requires that compelling information should be available on the identity of vegetal material sourced from given regions and obtained under known cultivation conditions, in the research publications. This could help researchers and industries to recognize and explain patterns of variation regarding certain physical–chemical as well as bioactivity parameters of EOs, that due to scarce or incomplete data cannot be achieved. Considering the lack of reports on quality of EOs from aromatic crops of Romania, this paper provides information on quality parameters for EOs, that could serve as reference for some physicochemical parameters and biological activity of EOs obtained in this region as well as a comparison base for the parameters of EOs from other regions.The family Lamiaceae comprises herbs and shrubs, many having high economic importance ,13,14,15evaluation of chemical composition of EOs,description of some physical parameters,evaluation of microbiological activity,evaluation of inhibitory activity on cancer cell lines.The aim of this research was to investigate some quality parameters of EOs for three frequently cultivated Lamiaceae species in Romania , that coThe research was conducted in order to assess the potential of some medical and paramedical uses of EOs obtained locally, and determine the relationship between their composition and the bioactivity.The experimental field was located within Agro-Botanical Garden UASVM Cluj-Napoca, located at latitude 46°45′36″ N; longitude 23°34′24″ E; elevation (AMSL) 380–430 m. Mean annual temperature is 8.1 °C, and multiannual average sum of precipitation is 635 mm . The locLavandula angustifolia cv. Sevstopolis), sage  and sweet basil (Ocimum basilicum). Vouchers were deposited at the Herbarium of Agro-Botanical Garden of UASVM Cluj-Napoca (CLA), numbers 30302–30304. This research focused on three aromatic species  from the family Lamiaceae: lavender . The fresh plant material was placed in the distillation unit, and water was added at the base. When the heated water temperature reached 80–85 °C, the steam rose through the plant material, mobilizing the essential oil from the plant tissue that was separated into a collector. The entire process for one lot of plant material lasted about 3 h, and during this time the temperature was maintained constantly at 85 °C. The extracted essential oil was packed in brown glass vials of 10 mL, sealed and stored in a dark and cool place.The volatile fingerprint of the essential oil samples was assessed by the gas chromatography coupled with mass spectrometry technique (GC-MS) using a GC-MS  equipped with a Combi-PAL AOC-5000 autosampler . An aliquot from each essential oil sample was diluted in hexane and 1 μL injected in the GC-MS at a split ratio of 1:100. The separation of the volatile constituents was performed on a capillary column  using the following temperature program: from 50 °C (maintained for 2 min) the temperature was increased to 160 °C at a rate of 4°/min and then to 250 °C with 15°/min and held for 10 min. The carrier gas was helium at a constant flow of 1 mL/min. The temperature for injector, ionic source and interface was set at 250 °C. The detection was performed on a quadrupole mass spectrometer operating in full scan (40–500 m/z), with electron impact (EI) as ion source at an ionization energy of 70 eV. The tentative identification of the volatile compounds was achieved by comparing their recorded mass spectra and the fragmentation patterns with those from the software’s NIST27 and NIST147 mass spectra libraries (considering a minimum similarity of 85%). The relative percentage of each compound was estimated as a fraction of its integrated ion area from the total ion chromatogram (TIC) area (100%).−1 and resolution 4.0 cm−1, with accumulation of 256. Raw spectra received five corrections for CO2 and five corrections for H2O using Spectra Manager software. Then, spectra were exported to Origin software for peak readings. Interpretation was based on tentative assignment to bands according to data from the literature.Fourier transform infrared spectroscopy (FT-IR) analysis of the EO samples was conducted at the spectroscopy laboratory of the Life Sciences Institute “King Michael I of Romania” in Cluj-Napoca. Pellets were obtained from 200 mg calcined KBr using a Specac hydraulic press at a pressure of 10 tons for two minutes. The resulting vitrified pellets were transparent. A quantity of 2 μL EOs was used per pellet . ImmediaRefractive index analysis for the EOs of the three aromatic species was conducted at the Biophysics Laboratory of UASVM Cluj-Napoca, using an Optika refractometer with horizontal prism. Refractive index of the EOs as well as changes in refractive index for essential oils following mixing into a vegetable oil were investigated. The vegetable oil used was commercial grade canola oil (CO), produced locally . Readings were performed for the following concentrations: 100% EO, 50% EO + 50% CO, 40% EO + 60% CO, 30% EO + 70% CO, 20% EO + 80% CO, 10% EO + 90% CO, 100% CO. Sample drops were placed with a pipette abundantly on the horizontal prism, then prisms were locked and the reading was performed according to the instructions manual.Staphylococcus aureus ATCC 6538P, Escherichia coli ATCC 25922, Salmonella enteritidis ATCC 13076 and Listeria monocytogenes ATCC 19114. Each strain was grown in a test tube containing 10 mL sterile nutrient broth  at 37 °C for 24 h. The purity of the inoculum was confirmed by microscopic examination of the Gram-stained smear. A loopful of inoculum was transferred to selective medium: Baird-Parker agar base supplemented with egg yolk tellurite emulsion for S. aureus, TBX agar for E. coli, XLD agar for Salmonella enteritidis  and Palcam agar  for Listeria monocytogenes. Plates were incubated for 24 h at 37 °C. Bacterial morphology was confirmed by optical microscopy. Several colonies were transferred to sterile saline solution (8.5 g/L), and adjusted to match the turbidity of McFarland 0.5 standard (1.5 × 108 CFU/mL) [The following microorganisms were tested:  CFU/mL) ,37.8 CFU/mL) was added. Gentamicin  was used as a positive control. For the negative control, a mixture with one part of the saline solution, eight parts of 50% ethanol and one part of Tween 80 was used. Microplates were incubated for 20–22 h at 37 °C. To each well, 20 µL resazurin aqueous solution (0.2 mg/mL) was added. Microplates were incubated for 2 h at 37 °C. The concentration that completely inhibited bacterial growth (MIC) was the concentration at which the blue color did not change into pink. Three replicates were run for each sample.The MIC was determined using the resazurin microtiter plate-based antibacterial assay. Stock solutions of the essential oils were prepared in eight parts 50% ethanol by mixing with one part Tween 80 . Into thMBC was determined by plating a 10 μL aliquot on solid culture Mueller–Hinton medium  from the last 4 wells that showed inhibition of bacterial growth in the MIC testing. The plates were incubated for 24 h at 37 °C. The lowest concentration that prevented the growth of bacteria (no colonies on the plate) was considered the MBC. Three different biological replicates were performed for each plate .v/v) NEAA, supplemented with 10% (v/v) fetal bovine serum (FBS) and without antibiotics, while A2780 was cultured in RPMI-1640 supplemented with 2 mM L-glutamine, 10% FBS and without antibiotics, in an atmosphere of 5% CO2 in air, 95% relative humidity and 37 °C . At approx. 80% confluence, cell lines were detached using 0.25% (w/v) trypsin–0.53 mM EDTA solution and were seeded in 96-well microplates at a concentration of 5 × 104 cells per well in 200 μL culture medium. After 24 h, 0.001, 0.002, 0.004, 0.008, 0.016, 0.032 and 0.064% (v/v) for each EO  were added to the culture medium and cells were incubated for the next 24 h under the same conditions. Tween 20 was used as a solvent for EO (10%) and the cytotoxic activity of Tween 20 was also tested at the maximum concentration of 0.0064%. Working dilutions were freshly prepared on the day of testing. At the end of incubation time, cells of each well were examined in contrast phase microscopy (Olympus IX51). The cytotoxicity assay was assessed by using 3--2,5-diphenyl-2H-terazolium bromide reagent (MTT). After a PBS washing step, cells were incubated with 150 μL/well MTT solution (5 mg/mL) for 1 h at 37 °C. The resulting formazan crystals were dissolved in 150 μL/well dimethyl sulfoxide (DMSO). The absorbance values were measured using wavelengths of 550 nm and 630 nm with an HT BioTek Synergy microplate reader . Cell viability was expressed as percentage of control . All experiments were conducted in triplicate.The experiments were carried out on two human tumor cell lines: Caco-2  and A2780 . Caco-2 was cultured in Eagle’s minimum essential medium (MEM) containing 2 mM L-glutamine, 1 mM sodium pyruvate, 1%  for triplicate determinations. The ANOVA analysis of variance was used to compare the average mean values, using SPSS 19.0 for statistical analysis  and Tukey’s honestly significant difference (HSD) test with a confidence interval of 95% or 99%. A For the lavender essential oil, 40 compounds were separated and of these 38 identified based on their mass spectra . The terpenes and terpenoids represented 94.41% of the volatile profile. The major volatile compounds were beta-linalool (30.91%) and linalool acetate (28.75%), followed by caryophyllene (5.66%) and beta-farnesene (6.45%) while camphor was identified only in a very small percentage (0.18%). Beta-linalool and linalool acetate were also the main compounds identified in the lavender essential oil from Middle-Friuli Venezia Giulia  togetherSalvia officinalis essential oil, with thujone concentration up to half of the total and camphor not less than 20%. Caution is required on the part of the public when using EOs. According to Cvetkovikj et al. [Salvia sp. EO is related to the toxicity of cis- and beta-thujone and camphor  and camphor (16.17%)  being thj et al. , an impoterature ,43 with In the case of basil essential oil, there were 53 separated compounds of which 41 were identified. The terpenic compounds represented >80% of the total volatile profile with beta-linalool (26.27%), thujone (14.84%), camphor (5.39%) and eucalyptol (5.22%) as the main representatives . There i−1, where most particularities were observed, and a second range between 2000–4000 cm−1 where the only differences between spectra were due to differences in band absorbance.Two spectral ranges were identified and examined. First, between 400–2000 cm−1, that could be assigned to the C–H molecular bond vibrations, as attributed in previous studies on basil and lavender EOs [−1 the sage oil presented several peaks of low intensity compared to lavender and basil that presented just one distinctive peak  [−1 that could be assigned to terpenes with tertiary and secondary alcoholic functions [−1, respectively, that can be assigned to C–O stretching vibrations [−1, respectively, that was not present in sage. Sage EO displayed a distinctive peak of low intensity at 1079 cm−1 that was not observed in the other two spectra. A study from France on lavender and lavandin EOs assigned the bands at 1079–1082 cm−1 to camphor [−1 could be attributed to camphor. Study on EOs of several Lamiaceae species indicated that the area between 1100 and 1300 cm−1 corresponds to stretching vibrations of the C–O group, while the O–C–O band originating from primary alcohols appears in the region from 1020 and 1100 cm−1 [At around 1000 cmranched) . Sage EOunctions . Lavendebrations . This pe camphor . Conside100 cm−1 .−1, all three EOs presented a peak  [−1, attributed to alkane asymmetrical and symmetrical C-H stretches [From 2800–3000 cmve peaks b, situatretching . FT-IR sH3, CH2) . FT-IR stretches .−1 in Lamiaceae family volatile extracts was attributed to the content of phenolics (and flavonoids), that are more abundant in this medicinal plant family compared to others such as Asteraceae [The broad band at 3400–3500 cmteraceae . The sigLavender EO presented the lowest refractive index out of the three species (1.4565 ± 0.0005 SD), and sage EO the highest (1.4712 ± 0.0003 SD). Canola oil presented a refractive index of 1.4690, which was the closest to the refractive index of basil EO (1.4692 ± 0.0003 SD). With incremental dilution with the vegetable oil, the optical properties changed and the refractive index shifted , with a Staphylococcus aureus ATCC 6538P. The bacterial strains used in the present study are more or less susceptible to each essential oil. The results related that the tested essential oils were characterized by varied antimicrobial activity. Gram-negative bacteria are more resistant to sage than to lavender and basil essential oils; however, sage EO manifests similar behaviors against Escherichia coli ATCC 25922, followed by Salmonella enteritidis ATCC 13076 and by Staphylococcus aureus ATCC 6538P. This result is in accordance with that found by Gaio et al. [Escherichia coli ATCC 25922, followed by Listeria monocytogenes ATCC 19114; lavender EO manifests similar behaviors against Listeria monocytogenes ATCC 19114. The results confirm that the antibacterial activity of basil EO is strong, that of lavender EO is moderate and that of sage EO is weak.The results obtained show that basil EO is the most effective against o et al. . The EO The cytotoxic effect of the lavender, sage and basil essential oils was evaluated using the MTT assay and the results were expressed as percentage of the control. The results of cytotoxicity on the Caco-2 cell line are shown in Essential oils (EOs) are recognized for their health-promoting properties and having diverse applications in various industries and agriculture. EOs can vary strongly depending on geographical origin of the plant material , and refDue to the high demand for and increasing use of EOs, regulations with the purpose to maintain a high standard of quality and safety remain of central importance. In the European Union, regulation of commercialized EOs falls under the incidence of Regulation EC Number 1907/2006 EC on “Registration, Evaluation, Authorization and Restriction of Chemicals” (REACH) currently in force , as wellAlthough authentication of EOs can be obtained by employing a variety of techniques such as sensory analysis and screening various chemical and physical parameters against standards , the aduFollowing the results of this study, it was determined that the three essential oils obtained from local crops reached high standards with regard to their composition, while the biological activity showed effectiveness with some variability between species attributed to their composition.−1 and the broad one at 3400–3500 cm−1 [−1 [Physical parameters can also be powerful tools in the characterization of EOs. IR spectral particularities could be used for defining fingerprint regions for botanical identification or potential authentication. Investigation of 30 essential oils from several botanical families by ATR-FTIR revealed, based on PCA clustering, that the strongest influences on the principal components in the Lamiaceae family group EOs are the bands at around 842, 1375, 1450 cm500 cm−1 . Researc500 cm−1 . Dilutiocm−1 [−1 . OpticalL. monocytogenes [Listeria monocytogenes is an opportunistic foodborne pathogen which often shows resistance against antimicrobials and lavender EO. Aelenei et al. [Staphylococcus aureus, Staphylococcus epidermidis) and Gram-negative bacteria (Escherichia coli) using broth microdilution assays. Adaszyńska-Skwirzyńska and Szczerbińska [Staphylococcus aureus) bacteria were the most vulnerable to the lavender essential oil. Additionally, the growth of strains such as S. aureus; S. aureus MRSA/ORSA; C. albicans; E. coli; P. aeruginosa; S. pullorum; S. enteritidis; S. typhimurium was inhibited by linalool. Relevant studies have reported that lavender essential oil of Moldovan origin showed good antibacterial activity against both non-pathogenic Gram-positive and Gram-negative bacteria (B. subtilis and P. fluorescens) [Staphylococcus aureus, but a low activity against Pseudomonas aeruginosa [Ocimum basilicum var. purpureum) EO extracted from air-dried plant material  by hydro-distillation was quite active against B. subtilis [S. aureus, L. monocytogenes, E. coli and S. enteritidis. The tested sage essential oil was active against the S. aureus strain with an MIC value of 3.795 µL/mL. Similar results were obtained by Sienkiewicz et al. [S. aureus clinical strains with MIC values ranging from 3.75 µL/mL to 5.25 µL/mL. According to the results obtained by Ghavam et al. [Both lavender and basil EOs from this study presented as major components beta-linalool and linalool acetate. Linalool  is an acyclic monoterpene tertiary alcohol with volatile flavors, that was extracted from more than 200 plants worldwide ,60. Linai et al.  studied erbińska  demonstrrescens) . Similarrescens) . Althougrescens) . The basruginosa . The preruginosa ,65,66. Tz et al.  for clarm et al. , the antm et al. .L. angustifolia) on human skin cells  at a 0.25% concentration. Kozics et al. [L. angustifolia EO the value of IC50 was lower (0.20%) than that of the in-house one (0.36%). In another study conducted by Niksic et al. [L. angustifolia EO on three cancer cell lines, MOLT-4 (acute lymphoblastic leukemia), MCF-7 (mammary adenocarcinoma) and NCI-H460 (pulmonary carcinoma), was reported. In the research of Zhao et al. [L. angustifolia EO depended on the type of prostatic cell line: 0.037% for PC-3 and 0.199% for DU145.Biological activity of different types of lavender EOs was tested in vitro on a large number of cell types. A notable result was achieved by Prashar et al.  regardins et al.  determins et al.  too, obsc et al. , a signio et al. , the IC5L. atriplicifolia on LoVo  and HepG2 (hepatocellular) cell lines also had cytotoxic and antiproliferative effects at a concentration of 10 µg/mL [L. angustifolia EO.Forty-eight-hour treatments with EO from aerial parts of 10 µg/mL . Even if10 µg/mL  studied 10 µg/mL  exploredS. officinalis EO on different cell types [In vitro studies concerning anti-inflammatory and immunomodulatory effects have also been explored in the case of sage EO ,79. Howell types ,82,83,84S. officinalis EO was tested on the RAW264.7 (murine macrophage) and HCT-116 (colon cancer) lines [S. sclarea exhibited stronger cytotoxic action against NALM-6 (lymphoblastic cell line) in comparison with the essential oil from in vivo plants.The cytotoxic effect of different fractions of r) lines , and ther) lines  observedr) lines  points oOcimum sp. genotypes, including O. basilicum. Based on sixteen published studies, basil demonstrated important anticancer activities in in vivo and in vitro models, suggesting that it could act as a potential anticancer agent. The EO isolated from O. basilicum L. was analyzed by Mahmoud [O. basilicum leaf extract have significant anticancer activity on the U-87 MG (glioblastoma), MDA-MB-231 (breast cancer) and MCF-7cancer cell lines. Kathirvel and Ravi [O. basilicum EOs obtained from plants collected from the Western Ghats area may belong to the methyl cinnamate and linalool chemotype and were shown to be active against the HeLa, HEp-2 and NIH 3T3 cancer cell lines. On the other hand, the study of Taie et al. [O. basilicum plant extract.In an article published in 2022, Perna et al.  reviewed Mahmoud  in order Mahmoud  concludeand Ravi  observede et al.  showed aAll these studies demonstrate that in recent years there has been an intense preoccupation and concern regarding the most accurate identification of biological activity of EOs. Particularly, there is a growing interest in the effects on different normal or tumor cell types, that could be fundamental for not only external medical uses but also internal uses. The results obtained to date confirm a large number of factors that influence the cytotoxicity of these products, such as genus and species, method of extract action, chemical composition  and also the effect of cellular type upon their action. The activity against pathogenic bacteria suggests that effectiveness depends on the synergistic effect of the specific composition found in a given EO, with inhibitory activity dependent on botanical identity, and therefore on the specific composition of the EO.This study investigated the physical and chemical composition as well as the preliminary biological activity of essential oils from three commonly cultivated species of the family Lamiaceae: lavender, sage and sweet basil. The EOs were obtained by steam distillation from plant material cultivated in climatic conditions of Transylvania, Romania.Results showed that in all three species, terpenes and terpenoids dominated the volatile profile: 94.41% in lavender EO, 95.35% in sage EO and 81.31% in basil EO, with over 20 terpenes and terpenoids identified in each case. Based on the literature, the EOs obtained are of high quality.Investigation of the physical properties of EOs through FT-IR identified signals of the main functional groups from the structure of various compounds of the EOs studied, suggesting that FT-IR spectra particularities might be used for defining fingerprint regions for botanical identification. Refractive index was lowest for lavender EO (1.4565), and highest for sage (1.4712), and changes occurred following dilution with a vegetable oil. Therefore, refractive index could be optimized to serve for the qualitative diagnosis in the authentication of EOs. Investigation of antibacterial activity showed that the effect of basil EO was strong, that of lavender EO was moderate and that of sage EO was weak.Cytotoxicity of EOs on cancer cell lines showed that the most active on both colon adenocarcinoma (Caco-2) and ovary carcinoma (A2780) was sage EO, while less cytotoxic activity was exhibited by basil EO on Caco-2 and lavender EO on A2780 cell culture.


The main aim was a systematic evaluation of the current evidence on outcomes for patients undergoing right ventricular assist device (RVAD) implantation following left ventricular assist device (LVAD) implantation.This systematic review was registered on PROSPERO (CRD42019130131). Reports evaluating in-hospital as well as follow-up outcome in LVAD and LVAD/RVAD implantation were identified through Ovid Medline, Web of Science and EMBASE. The primary endpoint was mortality at the hospital stay and at follow-up. Pooled incidence of defined endpoints was calculated by using random effects models.A total of 35 retrospective studies that included 3260 patients were analyzed. 30 days mortality was in favour of isolated LVAD implantation 6.74% (1.98–11.5%) versus 31.9% (19.78–44.02%) p = 0.001 in LVAD with temporary need for RVAD. During the hospital stay the incidence of major bleeding was 18.7% (18.2–19.4%) versus 40.0% (36.3–48.8%) and stroke rate was 5.6% (5.4–5.8%) versus 20.9% (16.8–28.3%) and was in favour of isolated LVAD implantation. Mortality reported at short-term as well at long-term was 19.66% (CI 15.73–23.59%) and 33.90% (CI 8.84–59.96%) in LVAD respectively versus 45.35% (CI 35.31–55.4%) p ⩽ 0.001 and 48.23% (CI 16.01–80.45%) p = 0.686 in LVAD/RVAD group respectively.Implantation of a temporary RVAD is allied with a worse outcome during the primary hospitalization and at follow-up. Compared to isolated LVAD support, biventricular mechanical circulatory support leads to an elevated mortality and higher incidence of adverse events such as bleeding and stroke. Results of clinical practice have led to an expanded role of LVAD in clinical use.24 LVAD implantation improves exercise tolerance and end-organ dysfunction and can improve hemodynamics.5Implantable mechanical circulatory devices, such as left ventricular assist device (LVAD), have emerged as a relevant option for improving quality of life and survival in patients with end-stage heart failure and are commonly utilized. The most common indications include bridge to transplant, bridge to candidacy, destination therapy and bridge to recovery.6 The incidence of patients with right ventricular failure following LVAD implantation ranges between 9 and 44%7 and about 10%–15% of these patients may require subsequent implantation of a separate right ventricular assist device (RVAD), associated with a higher mortality.9However, despite the excellent results, early right heart failure (RHF) or a progressive decline of right ventricular function remain major problems. Right heart failure may lead to impaired LVAD flow, difficulty in weaning from cardio-pulmonary bypass (CPB), decreased tissue perfusion, prolonged inotropic support and multi-organ failure, all associated with increased morbidity and mortality.1Patients requiring RVAD support represent a heterogeneous group ranging from patients in cardiogenic shock after a myocardial infarction to those with a known history of chronic heart failure. Additionally, it seems that timing of RVAD implantation may play a crucial role in in-hospital as well in short-term survival.The main aim of this systematic review was to synthesize all available evidence on the implantation of a RVAD in a LVAD setting and contribute to a better understanding of long-term outcomes following this surgical intervention. A systematic review summarizes the research evidence on a subject of interest and provides a more objective insight at the research level, compared to other study methods such as narrative reviews and expert commentaries.10 It was prospectively registered on PROSPERO (CRD42019130131).This systematic review follows the recommendations for preferred reporting items in systematic reviews and the meta-analyses (PRISMA) statement on conducting systematic reviews of RCTs.Appendix 1 . Search results were exported to EndNote X9 and deduplicated. Articles in English, German and French were considered for analysis by two reviewers (C.M. and G.R.) who screened the titles and abstracts of identified records and subsequently, the full texts of selected abstracts. An arbitrary reviewer (D.B.) assessed whether inclusion and exclusion were performed correctly. In cases of disagreement, an agreement was negotiated. References of selected articles were crosschecked for other relevant studies. Authors were contacted when a publication could not be obtained or not all required information could be retrieved from the publication.An information specialist (C.A.-H.) developed the search strategy, which was internally peer-reviewed by a second information specialist. Text words (synonyms and word variations) and database-specific subject headings for ventricular assist devices, right ventricular failure and ‘left and right’ were used. The detailed search strategies for Ovid Medline, Web of Science and EMBASE can be found in n ⩾ 10. In a case of multiple publications on the same patient cohort, the most recent publication was included in the analysis.The analysis did not include laboratory experimental reports, animal studies, studies on a paediatric population, in vitro studies, editorials, letters and case reports, as well reports on other clinical results. Criteria for inclusion were reports on adults, articles reporting on mortality/survival and/or morbidity after LVAD and/or after a LVAD/RVAD procedure, minimal duration of follow-up ⩾1 year, completeness of follow-up 90%  and study size 11The internal validity of each study was assessed using the Scottish Intercollegiate Guidelines Network (SIGN) Methodology checklist for RCTs by two independent reviewers (C.M. and R.G.).Quality was rated as follows: ‘High (+ +)’ denoted that most of the criteria were fulfilled. If not fulfilled, the conclusions of the study are very unlikely to alter. ‘Moderate (+)’ denoted that some criteria were fulfilled. Criteria not adequately described are unlikely to alter the conclusions. ‘Low (−)’ denoted that few or no criteria were fulfilled and the conclusions are likely to alter.12Microsoft Excel and Review Manager version 4.2 for Windows  were used for data extraction. To control for potential heterogeneity caused by the procedure, publications were allocated to the following categories: (1) series on LVAD and; (2) series on LVAD and RVAD, respectively. Study design was documented in each paper entering inclusion criteria. Data with regard to the authorship, date of publication, cohort quantity , genesis of heart failure, study design, follow-up duration as well the defined endpoint were extracted. Outcome events were registered according to the 2008 American Association for Thoracic Surgery/Society of Thoracic Surgeons/European Association for Cardiothoracic Surgery guidelines.Primary endpoints were defined as: in-hospital mortality (30 days), mortality/survival at 1 year (short-term), 3 years (mid-term) and 5 years (long-term) of follow-up. Secondary endpoints were: stroke, thromboembolic event, bleeding and transplantation rate.We calculated median and IQR of reported patient characteristics at baseline per treatment group (LVAD vs LVAD/RVAD), to approximately address the question of homogeneous indication criteria for either treatment. In the case of papers not reporting the mean, we included the median as an approximation. If standard deviation was not reported, we replaced it by the mean of all reported standard deviations. We calculated pooled Kaplan–Meier estimates of survival at 30 days, 1 year, 3 and 5 years using random effects models. Corresponding confidence intervals were calculated using Wilsons method based on number of patients at risk if reported. In studies that did not report patients at risk but only Kaplan–Meier estimates, we imputed the mean standard error to approximate confidence intervals. We assumed no patients lost to follow-up within the first 30 days. We used meta-regression with random effects to address heterogeneity, including time of publication as covariate in addition to treatment, first as before or during 2014 versus later, then as year of publication.We calculated pooled rates per 100 patient years and standard errors for transplantation by using random effects models on log transformed rates per year. We back-transformed the rates and corresponding limits and derived rates per 100 patient-years. If cumulated years of follow-up was not reported, we approximated it by multiplying the number of patients by the mean follow-up time. All other secondary outcomes were reported less exhaustively in the original publications. Therefore, we refrained from calculating pooled rates of complications, but summarized these variables as medians and IQRs for LVAD procedure and LVAD/RVAD procedure separately. Analyses were performed using Stata version 16 .n = 21), double publication (n = 3), case report (n = 1), surgical technique (n = 3), follow-up not completed (n = 56), studies under defined cut-off (n = 90) and other (n = 68)  .I2.Finally, 25 retrospective studies including 3260 patients published between 1991 and 2020 met our inclusion criteria. No additional relevant records were identified in the reference lists of these articles. Overall, 10 comparator (two-arm) studies were analysed, along with a further nine studies dealing with the LVAD procedure and six studies with the LVAD/RVAD procedure. The meta regressions showed a high heterogeneity among studies with respect to 1-year survival, with a high Supplemental material Table 1 provides an overview of the publications considered for analysis, 10 reports compared both surgical procedures, with n = 1962 patients in LVAD and with n = 469 patients in the LVAD/RVAD group, nine series evaluated the outcome only on the LVAD procedure (n = 829 patients) and five reports were on LVAD and permanent RVAD procedure (n = 182 patients).Median patient number per study arm was 98 (IQR 56–241) for LVAD and 27 (IQR 16–37.5) for the LVAD/RVAD procedure. supplementary material available online with this article) was used to assess risk of bias within studies. The study quality was good in all studies.The SIGN checklist  in LVAD versus 31.9% (95% CI 19.78–44.02) p = 0.001 in the LVAD/RVAD cohort . One-yeaN = 409) with a pooled mortality estimate of 33.90% (95% CI 8.84–58.96%) and two studies were on the LVAD/RVAD procedure (N = 52) with a pooled mortality estimate of 48.23% (95% CI 16.01–80.45%) p = 0.686. As CIs were very large in all studies, due to small numbers of patients at risk, heterogeneity I2 was small  was 69% for studies before/during 2014; and 86% or 70%, respectively, if >2014 or as year.The meta regressions showed that time of publication did not explain heterogeneity among studies with respect to 1-year survival, as inclusion of time did not decrease n = 3 patients at risk) and two on the LVAD/RVAD procedure (n = 15 patients at risk). After 5 years, the median proportion of patients with a bleeding complication in LVAD was inferior to the proportion in LVAD/RVAD at 7% versus 28.6% (IQR 23.8–33.3%). The proportion of patients that had stroke was 12.2% (IQR 11.1–13.3%) in LVAD versus 21.4% (IQR 5.9–33.3%) in LVAD/RVAD. Transplantation rate was reported in 14 studies, eight were on the LVAD procedure (n = 911 patients at risk) and six in the LVAD/RVAD procedure (n = 215 patients at risk)  .I2 = 97%) and LVAD&RVAD studies (I2 = 89%). This may emphasize again different indications and approaches at different sites. After LVAD implant, transplantation rate was 31 per 100 patient years (95% CI 20–48) which was slightly higher than after LVAD&RVAD, where the rate was 11 per 100 patient years (CI 4–29), p = 0.055. After excluding the study of Shah 2018 from the LVAD&RVAD group, heterogeneity became small (I2 < 1%) and the rate dropped to 7 per 100 patient years (CI 5–10), p < 0.001.Transplantation rates showed large heterogeneity among LVAD studies . Application of permanent biventricular mechanical support (BiVAD) is effective, but is associated with lower survival and reduced quality of life, when compared to isolated LVAD support. Furthermore, the postoperative course is complicated by an elevated incidence of major adverse events. It is however a necessity when conservative treatment by pulmonary vasodilators and inotropes is insufficient.1417 Similar conclusions regarding the mid-term survival can be drawn, where the implementation of temporary RVAD at index intervention was associated with reduced survival.1821 Nevertheless, it seems that the implementation of temporary RVAD at the time of LVAD implantation is associated with a worse outcome.The main finding of our analysis is that temporary RVAD implantation following LVAD is associated with decreased in-hospital, as well as short-term survival as compared to isolated LVAD implantation  and 3.1,1 We found a significantly higher number of patients to require preoperative circulatory support by IABP and ECMO in the LVAD/RVAD group.Our findings conclude that patients requiring temporary RVAD support are more ill at the time of LVAD implantation, compared to those who do not need the support. Indeed, there is evidence that INTERMACS patient profiles were found to be independently associated with temporary RVAD use. Class 1 and 2 were associated with 2–3 times the risk of RVAD compared with profiles > Class 3.2224 Additionally, the ratio between RAP and PAPP was identified as a risk factor for RVAD.25 Although the individual variables of the pulmonary artery pulsatility index (RAP and PAPP ratio) were predictors in the INTERMACS model, the index itself did not add incremental value to risk prediction.26 Similarly, the RAP/pulmonary capillary wedge pressure (PCWP) ratio, where the index had a significant univariate association with RVAD use which is in contrast to RAP alone, was not an independent predictor.26Besides a ‘general frailty’ of the individual, the hemodynamic performance of the right ventricle at the time of the LVAD implantation as a predictive element for subsequent RHF plays a crucial role. Different metric methods have been developed to assess the risk for RHF. Narrow pulmonary artery pulse pressure (PAPP) is correlated with impaired RV function and is an independent predictive element for RVAD implantation and similar evidence exists for elevated right atrial pressure (RAP).2731 The same may be stated for the central venous pressure, coupled with a lower pulmonary artery pressure.143035 The relation between RAP and PAPP (and/or relation to the PCWP) was not evaluated in any of the reports examined. Tricuspid valve insufficiency (TVI) has also been associated with worse outcomes among patients undergoing mechanical circulatory support including RHF.37 The severity of the TVI is a variable strongly dependent on hemodynamic status and volume load. It may be present in the absence of annular dilatation,38 a fact that complicates the liaison between severity of the TVI and RV function. In our analysis, severe TVI was higher in the LVAD/RVAD pooled cohort.35 The annulus size of the tricuspid valve, an element that seems to be a more precise indicator of the RV dysfunction and provides an accurate prediction of RHF after LVAD implantation,39 was not evaluated in the included reports.Regarding hemodynamic variables evaluating pre LVAD right ventricular performance, we found the RAP in the LVAD/RVAD cohort, to be higher when compared to the LVAD cohort.1827324043 It therefore seems that ischaemic cardiomyopathy is associated with an elevated risk for RVAD implantation. This may be linked to the history of coronary bypass graft surgery, namely it is likely that past cardiac surgery increases the risk of RV injury, prolonged cardiopulmonary bypass time and perioperative bleeding. Elements that may be due to an elevated transfusion rate precipitate the RHF by RV distension and increased pulmonary resistance.Ischaemic cardiomyopathy in our pooled cohorts was higher in LVAD/RVAD as compared to the isolated LVAD group.1442 A similar conclusion may be drawn for the short- and mid-term period, where the high mortality rate in the temporary RVAD group may in part be explained by an elevated incidence of thromboembolic events, stroke rate and higher bleeding rate in the post discharge period respectively.42Beside the previously mentioned ‘general frailty’ and worse RV performance at the time of the LVAD implantation, the elevated mortality in the temporary RVAD cohort, may in part be explained by an elevated stroke risk and the need for re-exploration due to bleeding during the hospital stay.supplementary Table 1 . Significant differences were found only in the preoperative variables age and Bilirubin serum levels, whereby patients in the LVAD only group were seen to be younger and have lower serum levels, respectively. In-hospital, as well as short-term mortality were significantly higher in temporary RVAD groups. The hemodynamic status of the right ventricle at the time of implantation, augmented biomarkers of end organs as a sign of congestion, as well as a combination of adverse events such as bleeding or thromboembolic events were identified as predictive factors for in-hospital and short-term mortality. In the mid- and long-term mortality analysis, there were very limited studies reporting data. This can be in part due to short follow-up periods in the publications themselves, as well as a non-uniform method of reporting transplantation as an outcome. Furthermore, the studies included were from a variety of healthcare systems.The literature on mid- and/or long-term survival analysing the outcome of patients requiring a temporary RVAD after LVAD implantation versus isolated LVAD implantations in one single cohort is very limited. We summarized population characteristics of the few studies reporting on both treatments in supplementary Table 3. We aimed to calculate event rates per 100 patients per year of observation time with standard error within studies, and then derive pooled estimates and corresponding limits of the 95% CI of all primary and secondary outcomes. This was not possible due to incomplete reporting and very small groups. Furthermore, single-arm LVAD studies could not be comprehensively identified. This is due to the search strategy chosen. Other possible limitations include a morbidity bias in the LVAD/RVAD cohorts, as these are inherently prone to be a sicker population associated with a higher mortality.Timing of RVAD implantation following LVAD was not reported as was not the time frame of the RV failure. Furthermore, the device modalities used as RV circulatory support were not distinguished. The reported devices used varied between nearly all studies and are summarized in Implantation of a temporary RVAD is allied with a worse outcome during the primary hospitalization and at follow up. There is a higher incidence of adverse events such as bleeding and stroke, that may be a functional expression of a dysbalanced hemodynamic status.Click here for additional data file.Supplemental material, sj-pdf-1-prf-10.1177_02676591211024817 for Outcome of right ventricular assist device implantation following left ventricular assist device implantation: Systematic review and meta-analysis by Gregory Reid, Constantin Mork, Brigita Gahl, Christian Appenzeller-Herzog, Ludwig K von Segesser, Friedrich Eckstein and Denis A Berdajs in PerfusionClick here for additional data file.Supplemental material, sj-pdf-2-prf-10.1177_02676591211024817 for Outcome of right ventricular assist device implantation following left ventricular assist device implantation: Systematic review and meta-analysis by Gregory Reid, Constantin Mork, Brigita Gahl, Christian Appenzeller-Herzog, Ludwig K von Segesser, Friedrich Eckstein and Denis A Berdajs in PerfusionClick here for additional data file.Supplemental material, sj-pdf-3-prf-10.1177_02676591211024817 for Outcome of right ventricular assist device implantation following left ventricular assist device implantation: Systematic review and meta-analysis by Gregory Reid, Constantin Mork, Brigita Gahl, Christian Appenzeller-Herzog, Ludwig K von Segesser, Friedrich Eckstein and Denis A Berdajs in Perfusion


We aimed to evaluate the diagnostic potential of non-erectile multi-parametric magnetic resonance imaging (mpMRI) for preoperative assessment of primary penile squamous cell carcinoma (SCC).Twenty-five patients who underwent surgery for penile SCC were included. Preoperative mpMRI without artificial erection was performed in all patients. The preoperative MRI protocol consisted of high-resolution morphological and functional sequences (diffusion-weighted imaging and dynamic contrast-enhanced MRI perfusion) covering the penis and lower pelvis. T and N staging, according to the 8th edition of the Union for International Cancer Control TNM classification, as well as the largest diameter and thickness/infiltration depth of the primary lesions were determined in all patients. Imaging data were retrospectively collected and compared with the final histopathology reports.p = 0.002) and good agreement was observed for the involvement of penile urethra and tunica albuginea/corpus cavernosum . Good agreement was observed between MRI and histopathology for overall T staging and weaker, but still good agreement was observed for N staging . A strong and significant correlation was observed between MRI and histopathology for the largest diameter and thickness/infiltration depth of the primary lesions (p < 0.001).Very good agreement was observed between MRI and histopathology for the involvement of corpus spongiosum (Good concordance was observed between MRI and histopathological findings. Our initial findings indicate that non-erectile mpMRI is useful in preoperative assessment of primary penile SCC. •An appropriate preoperative evaluation of penile cancer is important to optimize the clinical management.•Good concordance was observed between imaging and histopathological findings.•Non-erectile mpMRI allows precise assessment of penile tumors. A wide variation is observed in the prevalence of penile cancer, ranging from up to 6.8 per 100,000 in Africa, Asia, and South America to less than 1 per 100,000 in North America and Europe Current clinical guidelines for the diagnosis and staging of penile cancer recommend the use of magnetic resonance imaging (MRI) in cases intended for OSS In recent years, MRI has evolved to become the gold standard in oncological imaging, providing excellent anatomical information as well as molecular and physiological information in vivo using diffusion-weighted imaging (DWI) and dynamic contrast-enhanced (DCE) MRI perfusion In summary, MRI is a promising diagnostic tool for the preoperative staging of penile cancer. This study aimed to assess the concordance between imaging and histopathological features and to evaluate the diagnostic potential of mpMRI without artificial erection in preoperative assessment of primary penile carcinoma.2The study was approved by the Institutional Review Board (IRB) and the requirement for informed consent was waived for the retrospective reviewing of MRI and clinicopathological data of the included patients (IRB identifier: 22/09548). We retrospectively analyzed the imaging and clinicopathological data of 25 patients with primary penile SCC referred to our institution for surgery between May 2020 and June 2022. Preoperative mpMRI without artificial erection was performed in all patients. MRI was performed using a 3-T magnet  with a 32-channel phased-array coil. The imaging protocol consisted of high-resolution morphological and functional sequences covering the penis and lower pelvis , Fig. 2.Surgical approaches to penile and lymph node surgery were discussed within the institutional penile cancer team and performed according to the current guidelines Surgical specimens were fixed in 10 % buffered formalin for at least 48 h, macroscopically examined, grossed, and embedded in the paraffin blocks. Sections were cut to a thickness of 4 µm and routinely stained with hematoxylin and eosin. The specimens were analyzed according to standardized protocols based on the American Joint Committee on Cancer format p-value < 0.05 was considered statistically significant.The data were analyzed using IBM SPSS Statistics . Descriptive statistics including frequency distributions and percentages were used to describe the study population. Nonparametric Spearman rank-order correlation was applied to the continuous data. A correlation coefficient (ρ) of 0.90–1.00 implied a very strong correlation; 0.70–0.89, strong correlation; 0.40–0.69, moderate correlation; 0.10–0.39, weak correlation; and < 0.10, negligible correlation 3The age of the included patients ranged from 48 to 80 years, with a mean age of 68 years. Partial and total penectomies were the most frequent surgical modalities, accounting for 11 (44 %) and five (20 %) of the total cases, respectively. Other surgical approaches included glansectomy in four (16 %), partial glans resection in three (12 %), and radical circumcision in two (8 %) patients. Nineteen (76 %) patients were diagnosed with usual SCC, two (8 %) with sarcomatoid SCC, two (8 %) with basaloid SCC, and one (4 %) with papillary or verrucous SCC. A warty-basaloid SCC was found in one patient (4 %). The tumor was located on the glans in 15 (60 %) patients; on preputium in two (8 %); on preputium and glans in five (20 %), on glans and sulcus coronarius in two (8 %); and on preputium, glans, and sulcus coronarius in one (4 %) patient. According to the TNM classification, three (12 %) cases were classified as pathological (p) stage T1 (pT1), 18 (72 %) as pT2, and four (16 %) as pT3. Fourteen (56 %) patients had no lymph node metastases on histopathological evaluation or had a negative SNB (pN0), three (12 %) were classified as pN1, and three (12 %) as pN3. None of the patients were classified as pN2. Histopathological data were not available for five (20 %) patients (pNx). One (4 %) patient presented with a separate tumor satellite in the proximal corpus cavernosum.p = 0.002). Good agreement was observed between MRI and histopathology for the involvement of penile urethra and tunica albuginea/corpus cavernosum .A significant association was observed between MRI and histopathology for the involvement of anatomical structures . A very p < 0.001)  . Twenty-p = 0.002)  . Histopap < 0.001; tumor thickness/infiltration depth: ρ = 0.752, p < 0.001).A strong and significant correlation was observed between MRI and histopathology for the largest diameter and thickness/infiltration depth of the primary lesions , Fig. 3 4The present study reported the initial experience of non-erectile mpMRI for the preoperative assessment of primary penile carcinoma. We found good agreement between MRI and histopathology for T and N staging, and a strong and significant correlation between MRI and histopathology for the largest tumor diameter and infiltration depth.Few reports have assessed the diagnostic potential of MRI in preoperative evaluation of penile carcinomas; however, inconsistencies in MRI protocols can influence the outcomes and make comparisons difficult Most of the studies assessing MRI for the local staging of penile cancers used standard, morphological sequences alone, and only a few studies included DCE-MRI perfusion or DWI in the preoperative protocols Our results showed a strong and significant correlation between MRI and histopathology for the largest tumor diameter and infiltration depth . A studyD-glucose positron emission tomography/computed tomography (FDG PET-CT) with clinicopathological features has been initiated .The main limitations of this study are its retrospective design and limited sample size. The main strengths of the study are its high technical image quality and thorough histopathologic correlation. Despite the limited number of patients in our pilot analysis, the agreement between imaging and histopathological features was remarkably consistent. To verify these findings, a prospective study on a large dataset of patients with penile cancer, correlating mpMRI and 2-fluoro-2-deoxy-5Good concordance was observed between the MRI and histopathological findings despite the small sample size. Our initial findings indicate that mpMRI without artificial erection can be implemented as a precise diagnostic tool for preoperative assessment of primary penile cancer. However, the results should be verified in a larger and, preferably, prospectively collected dataset.MDS: Conceptualization, Methodology, Formal analysis, Investigation, Writing – original draft, Visualization, Project administration, Writing – review & editing. AH: Methodology, Formal analysis, Investigation, Writing – original draft, Writing – review & editing. SS: Formal analysis, Investigation, Writing – original draft, Writing – review & editing. BB: Investigation, Supervision, Writing – review & editing. FO: Investigation, Writing – review & editing. KB: Writing – review & editing. UA: Investigation, Writing – review & editing. KHH: Investigation, Writing – review & editing, Supervision.The Institutional Review Board approved this retrospective study. The need for written informed consent was waived.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Escherichia coli, resulting in a chaotic lipid rearrangement and rapid disruption of the cell envelope. Its action primarily disturbs the packing order of the hydrophobic moiety of a lipid, which consequently might result in a cascade of multiple effects at a cellular level. Here, we investigated OCT’s impact on two different Gram-positive bacteria, Enterococcus hirae and Bacillus subtilis, and their respective model membranes. In accordance with our previous results, OCT induced membrane disorder in all investigated model systems. Electron and fluorescence microscopy clearly demonstrated changes in cellular structure and membrane integrity. These changes were accompanied by neutralization of the surface charge in both E. hirae andB. subtilis and membrane disturbances associated with permeabilization. Similar permeabilization and disordering of the lipid bilayer was also observed in model membranes. Furthermore, experiments performed on strongly versus partly anionic membranes showed that the lipid disordering effect induced by OCT is a result of maximized hydrophobic over electrostatic forces without distinct neutralization of the surface charge or discrimination between the lipid head groups. Indeed, mutants lacking specific lipid head groups were also susceptible to OCT to a similar extent as the wild type. The observed unspecific mode of action of OCT underlines its broad antimicrobial profile and renders the development of bacterial resistance to this molecule less likely.The antimicrobial killing mechanism of octenidine (OCT), a well-known antiseptic is poorly understood. We recently reported its interaction with Gram-negative bacteria by insertion of OCT into the outer and cytoplasmic membrane of IMPORTANCE OCT is a well-established antiseptic molecule routinely used in a large field of clinical applications. Since the spread of antimicrobial resistance has restricted the use of antibiotics worldwide, topically applied antiseptics like OCT, with a broad spectrum of antimicrobial activity and high safety profile, gain increasing importance for effective infection prevention and therapy. To eliminate a wide spectrum of disease-causing microorganisms, a compound’s antiseptic activity should be unspecific or multitarget. Our results demonstrate an unspecific mechanism of action for OCT, which remained largely unknown for years. OCT disturbs the barrier function of a bacterial cell, a function that is absolutely fundamental for survival. Because OCT does not distinguish between lipids, the building blocks of bacterial membranes, its mode of action might be attributed to all bacteria, including (multi)drug-resistant isolates. Our results underpin OCT’s potent antiseptic activity for successful patient outcome. N, N′- bis-(1-octanamine) dihydrochloride) is a quaternary ammonium compound of the bipyridine family exerting mesomeric distribution of the cationic charge via an amino-pyridine structure drug antimicrobial resistance limit effective therapeutic options for successful patient outcome and constitute an enormous threat within health care facilities. In that regard, topically applied antiseptics represent a very useful tool for infection prevention and therapy. The synthetic antimicrobial molecule octenidine (OCT) has established itself over the last 30 years as a key agent for skin, mucous membrane, and wound antisepsis as well as microbial decolonization of patients –5 due to–6–tructure , such astructure  and lacttructure . Its hyd bilayer  or compo bilayer –17.Enterococcus hirae and Bacillus subtilis, which strongly differ in cell shape  but have a very similar cell envelope architecture. Only the composition of their cytoplasmic membranes differs slightly, as E. hirae mainly contains the anionic phospholipids PG and CL  content versus 67% in E. coli. While the exact PL content may vary depending on growth phase, culture media, and the strain analyzed  and lipoteichoic (LTA) acids, are embedded. Furthermore, their single phospholipid membrane is characterized by the presence of exclusively anionic phosphatidylglycerol (PG) and cardiolipin (CL) . SoG and CL ; whereasL and PE . Besidesanalyzed , 22, 23,es of PL , besidesE. hirae and B. subtilis as model organisms, as well as corresponding model membranes mimicking the cytoplasmic membranes of those bacteria. The membrane architecture of both organisms -teichoic acids, which might attract cationic OCT to the bacterial surface. Therefore, we first tested OCT’s capacity to neutralize the negative surface charge of the enterococcal membrane by measuring the zeta potential of E. hirae cells titrated with increasing concentrations of OCT  but also strongly suggests that surface charge neutralization coincides with the key event of the OCT-mediated killing. As zeta potential did not become positive and under the assumption that interaction with bacterial surface is only electrostatic, we might assume that OCT penetrates directly into the cytoplasmic membrane and does not accumulate on the bacterial surface.Disturbances to the enterococcal cell wall and cytoplasmic membrane were clearly observed using electron microscopy. The question arose whether OCT also interacts with components of the cell wall in addition to cytoplasmic membrane. The cell wall of s of OCT . E. hiraE. hirae cells treated with OCT at or higher than 0.005% .To test whether OCT indeed targets the cytoplasmic membrane, we further assessed the membrane permeability using flow cytometry. For this aim, we used the large, normally membrane-impermeable, DNA intercalating fluorescent dye propidium iodide (PI). Indeed, incubation with OCT strongly increased intracellular PI staining in a concentration-dependent manner, reaching up to 55% of the cells within 20 min . This inE. hirae with the membrane fluorescent dye, Nile Red. As shown in fluorescence microscopy images , the staining was more heterogenous, and Nile Red foci became more distinct and remained only at one cell pole. With 0.001% OCT , the Nile Red signals were increasingly cytoplasmic, and the cells became smaller, indicating loss of turgor upon leakage of cytoplasmic content. Comparable to previous observations in E. coli  of 4 mg/L (data not shown). For 1 × 107 CFU/mL, the IC was calculated with maximum 1 mg/L OCT  fluorescence microscopy to simultaneously investigate the effect of OCT on membrane permeability  fluorescence  fluorescence microscopy . As expected, no lysis was observed microscopically when the cells were treated with a lethal dose of OCT for 30 min . Whilst incubation of ΔlytABCDEF cells with OCT still induced a spotty Nile Red membrane stain, the weakly stained membrane areas were absent in this strain, indicating that this phenomenon is not a direct consequence of OCT. Rather, the emergence of such areas is a secondary phenomenon associated with the cell lysis process.We also observed membrane areas with very low Nile Red staining and, at the highest concentrations, fully lysed cells  . To inveB. subtilis cells that constitutively express high levels of cytoplasmic green fluorescent protein (GFP). In untreated cells, we observed a uniform staining of the membrane, and the cytoplasm was flooded with GFP , thus clearly pointing toward a non-lipid-specific mode of action of OCT.Given that bacteria are relatively diverse in their phospholipid composition which, in turn, may affect OCT activity, we investigated OCT susceptibility to a set of E. hirae (PG/CL) and B. subtilis (PE/PG/CL). To test this, CL was introduced into the various membrane formulations, and their thermotropic behavior upon OCT treatment was examined by differential scanning calorimetry. The results showed, independent of phospholipid composition used, the same thermotropic profile as previously described: the main phase transition of all membranes disappeared with increasing OCT concentration, accompanied by a new phase transition at much lower melting temperatures E. hirae eratures  or PE/PGeratures  membraneSimilar observations were made when we performed leakage experiments with different membrane compositions and properties . AlthougE. hirae , B. subtilis , and E. coli . OCT neutralized surface charge of all membrane models; however, neutralization of highly anionic E. hirae and B. subtilis model membranes started above leakage concentrations. Similar results were observed by Rzycki et al. . Although we did not expect such a profile for zwitterionic surfaces, we assume that, in contrast to PG, PE’s capability for hydrogen bonding with OCT-reactive groups may provoke different organization of OCT on the surface of PE. Nevertheless, our data suggest that hydrophobic interactions are maximized upon OCT interaction with phospholipids. In summary, our in vivo and in vitro data strongly support lipid headgroup-independent targeting of bacterial membranes as the primary mechanism underlying the broad and potent activity.To test if OCT interaction with PLs is electrostatic, we performed zeta potential measurements  with modi et al. . This suIn summary and based on the biophysical and cellular data presented herein, the mode of action of OCT for Gram-positive bacteria can be described as follows. Upon adherence to bacteria, OCT begins to neutralize bacterial surface charge due to electrostatic interactions and immediately penetrates through the thick cell wall of Gram-positive bacteria to reach the cytoplasmic membrane. The hydrocarbon chains of OCT rapidly interfere with the fatty acyl chains of the cytoplasmic membrane, thereby inducing a strong disorder due to a hydrophobic mismatch. On a cellular level, OCT-induced changes to the cytoplasmic membrane are sufficient to depolarize by increasing ion conductivity and even induce membrane leakage of larger molecules. Furthermore, OCT induces extensive disturbance to the membrane structure as evidenced by large membrane invaginations and rupture. On a molecular level, OCT lowers the phase transitions of bacterial phospholipids leading to formation of membrane structures with packing disorder, likely explaining the increased permeability. The described mode of action explains OCT’s lack of selectivity toward microorganisms with varying cell envelope structures and compositions, thus explaining its strong and broad antiseptic activity. Crucially, such a rapid bactericidal mechanism targeting critical and essential membrane properties renders bacterial resistance development toward OCT less likely.3(5), Nile Red, CCCP, nisin, and L-dopamine were purchased from Sigma-Aldrich. Propidium iodide (PI) and Sytox Green were purchased from Thermo Fisher Scientific. The following phospholipids (>99% purity) were ordered from Avanti Polar Lipids (USA): E. coli polar lipid extracts, DPPG ]), DPPE , DMPG ]), DMPE , TMCL , POPG ]), and POPE . ANTS  and DPX (p-xylene-bis-pyridinium bromide) were obtained from Molecular Probes (USA).Octenidine (OCT) dissolved in pure water was provided by Schülke & Mayr GmbH (Austria) and SAAP-148  by Jan Wouter Drijfhout  with the purity of >95% as analyzed by high-pressure liquid chromatography (HPLC) . DAPI, DE. hirae strain for antimicrobial susceptibility testing  was cultivated in brain heart infusion broth (BHIB) . B. subtilis wild-type 168 (600). The cultures were grown until the mid-logarithmic phase, which was reached after 3.5 to 4 h.The FDA-approved type 168  and its E. hirae/B. subtilis in the mid-logarithmic phase was washed once with sodium phosphate-buffered saline (PBS)  and inoculated at 1 × 106 CFU/mL, 1 × 107 CFU/mL, 1 × 108 CFU/mL, and 1 × 109 CFU/mL, respectively, in fresh BHIB or LB in the presence and absence of OCT . Growth curves were recorded automatically for 24 h at 37°C under shaking (300 rpm) by Bioscreen C . MIC for bacterial concentration of ~1 × 106 CFU/mL was estimated as the lowest concentration of OCT in milligrams per liter where no visible bacterial growth was observed. Inhibitory concentration (IC) for higher bacterial loads follows the same description and was estimated in the same concentration range but expressed as corresponding %OCT (1 to 8 mg/L corresponds to 0.0001 to 0.0008% OCT), which allows direct comparison of the data to clinically used concentrations of OCT. Of note, MIC/IC for B. subtilis was estimated only for 1 × 106 CFU/mL and 1 × 107 CFU/mL bacterial loads. For elucidation of bactericidal concentration, different concentrations of bacteria were exposed to OCT for 1 h in PBS at 37°C before they were plated on BHIB agar plates. Lethal concentrations (LC) were estimate as a concentration where no visible colonies were observed after 1 day incubation of the plates at 37°C.Antimicrobial activity was assessed as a function of growth. Briefly, E. coli and published before  and stained simultaneously with either 200 nM Sytox Green and 1 μM DiSC3(5) or with 0.125 μg/mL Nile Red and 0.5 μg/mL DAPI  for 5 min at 37°C upon shaking. A total of 0.5 μL of each sample was immobilized on microscope slides covered with a thin layer of H2O/1.2% agarose and imaged immediately. Microscopy was performed using a Nikon Eclipse Ti equipped with Nikon Plan Apo 100×/1.40 Oil Ph3 objective, CoolLED pE-300 light source, and Photometrics Prime sCMOS camera. Images were captured with MetaMorph 7.7 (Molecular Devices) and analyzed with ImageJ/Fiji (Overnight cultures were grown at 30°C in LB supplemented with 0.2% glucose. l-dopamine as described before (l-dopamine (2 mg/mL freshly dissolved in 1 mM Tris pH 8.0) was added to the coverslip and incubated at room temperature for 30 min. The excess l-dopamine and Tris were then removed by aspiration and submersion in H2O followed by evaporation at 37°C for 30 min. Dual-color two-dimensional (2D)-SIM was performed using Nikon N-SIM equipped with 488- and 561-nm lasers, Nikon CFI SR HP Apochromat TIRF 100×/1.49 oil objective, and Andor iXon DU-897 camera. Image capture and SIM reconstruction was carried out with NIS Elements 5.21 (Nikon).Cells were prepared and immobilized on 1.2% agarose slides as described above. To reduce binding of Nile Red to the coverslip surface, which can suppress the structured illumination pattern, the coverslips were coated with d before . In briePreparation of model membranes , 38, vesAccording to methods published previously , 36, thet test (Microsoft Excel). Unless otherwise stated, error bars represent standard deviation (SD) from three independent biological replicates. Significance for t test was assumed as follows: ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05; n.s., not significant.Statical analysis was assessed using an unpaired two-sided